TISSUE, AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. application serial number 62/443,206 filed January 6, 2017, the disclosure of which is hereby incorporated by reference in its entirety.

BACKGROUND OF THE DISCLOSURE

[0002] Engineering tissue and organ requires the ability to pattern mammalian tissues composed of cells, extracellular matrix, and vasculature with controlled microenvironments that can be used in therapy and/or drug toxicity and novel drug candidate screening methods. To date, bioprinting methods have yielded thin tissues that only survive for short durations. Among the challenges in engineering mammalian organs or tissues have been the

inaccessibility of the extracellular matrix (ECM) ministructure to cells seeded on to an organ or tissue ECM due to the complexity and compactness of the ECM ministructure. The present disclosure provides an improved method of engineering organ or tissue (or a functional part thereof).

BRIEF SUMMARY OF THE DISCLOSURE

[0003] The current disclosure provides, inter alia, an in vitro or ex vivo method of decondensing an extracellular matrix (ECM) structure/composition in an acellular structure of a mammalian organ or tissue, or a bioprinted extracellular matrix scaffold, with one or more metalloproteinase (MMP). In embodiments, the acellular structure of the mammalian organ or tissue, or a bioprinted extracellular matrix scaffold includes ECM components of a kidney, a liver, a bladder, a vagina, a urethra, a trachea, an esophagus, a heart, a lung, a pancreas, a skin, or other bodily organ [0004] In one aspect, the present disclosure provides a method of reconditioning and/or cellularizing a decondensed extracellular matrix (ECM) structure in an acellular structure of a mammalian organ or tissue, or a bioprinted ECM scaffold, comprising: introducing human pluripotent stem cells or human adult multipotent stem cells, or blood comprising progenitor cells to a decondensed ECM structure prepared by introducing one or more metalloproteinase (MMP, e.g., MMP1, MMP2, MMP3, MMP7, MMP9, MMP13, and/or MMP 14) to the ECM structure; and perfusing media for growth and differentiation of the cells, thereby

reconditioning and/or cellularizing the decondensed ECM structure in the acellular structure of a mammalian organ or tissue, or the bioprinted ECM scaffold.

[0005] In one aspect, the present disclosure provides use of a reconditioned and/or cellularized decondensed ECM structure in an in-vitro method of testing toxicity of a compound or a therapeutic agent; screening of a new therapeutic agent; and/or identifying a therapeutic agent candidate for treating a mammalian organ or tissue disease or disorder; the method comprising introducing a compound, a therapeutic agent, a new therapeutic agent, or a candidate therapeutic agent to the reconditioned and/or cellularized decondensed ECM structure, where the testing toxicity includes ex vivo testing for safety, toxicity, on-target and/or off-target adverse effects, and/or production of toxic metabolites. In one aspect, the present disclosure provides use of a reconditioned and/or cellularized decondensed ECM structure prepared by a method of the present invention, in regenerative medicine to repair or replace tissues or organs, or in transplantation or as a graft. [0006] In one aspect, the present disclosure provides an acellular organ or tissue {e.g., a MMP -treated acellular organ or tissue) or a bioprinted polymeric extracellular matrix scaffold, including extracellular matrix (ECM) components/composition of a mammalian organ or tissue {e.g., a kidney, a liver, a bladder, a vagina, a urethra, a trachea, an esophagus, a heart, a lung, a pancreas, a skin, other bodily organ) or a functional part thereof, cellularized with human pluripotent stem cells or human adult multipotent stem cells, or with blood, for use in therapy or for testing toxicity of a compound, a therapeutic agent, screening a new therapeutic agent, and/or identifying a therapeutic agent candidate. The present disclosure provides an organ or tissue (or a functional part thereof) scaffold composition including ECM components/composition and a polymer, cellularized with human pluripotent stem cells or human adult multipotent stem cells for use in therapy.

[0007] In one aspect, the present disclosure provides a method of cellularizing a MMP- treated acellular structure of a mammalian organ or tissue, or a bioprinted extracellular matrix scaffold, in which the MMP treatment facilitates infiltration and migration of cells seeded onto the acellular structure. In certain embodiments, the organ or tissue is a diseased organ or tissue. In certain embodiments, the organ or tissue is characterized as having a tumor or cancer. [0008] In one aspect the present disclosure provides a method of identifying human kidney stem/progenitor cells, the method including detecting the expression of Ephrin receptor 7 (EphA7) in cells obtained from a kidney.

[0009] In one aspect the present disclosure provides a method of testing toxicity of a compound; a therapeutic agent; screening of a new therapeutic agent; and/or identifying a therapeutic agent candidate for treating a mammalian organ or tissue disease or disorder, the method including: (i) introducing a compound, a therapeutic agent, a new therapeutic agent, or a candidate therapeutic agent to a recellularized mammalian organ or tissue, where the recellularized organ or tissue is prepared by a method in which an organ or tissue is decellularized, while retaining an extracellular matrix (ECM) structure, the ECM structure is then treated with one or more metalloproteinase (MMP), and MMP -treated decellularized organ or tissue is then recellularized with human pluripotent stem cells or human adult multipotent stem cells; or (ii) introducing a compound, a therapeutic agent, a new therapeutic agent, or a candidate therapeutic agent to a bioprinted organ or tissue scaffold. The bioprinted organ or tissue scaffold is formed by cellularizing a bioprinted polymeric scaffold with human pluripotent stem cells or human adult multipotent stem cells. In certain embodiments, the recellularized organ or tissue is prepared from a diseased organ or tissue. In certain embodiments, the recellularized organ or tissue is prepared from an organ or tissue characterized as having a tumor or cancer. An organ or a tissue is characterized as having a tumor or cancer by the expression of one or more tumor and/or cancer specific biomarker.

[0010] In embodiments, the recellularized organ or tissue or the bioprinted organ or tissue scaffold may include an ECM structure of a kidney, a liver, a bladder, a vagina, a urethra, a trachea, an esophagus, a heart, a lung, a pancreas, a skin, or other bodily organ. In

embodiments, the recellularized organ or tissue scaffold or the bioprinted organ or tissue scaffold may include cells, tissues, or functional parts of a kidney, a liver, a bladder, a vagina, a urethra, a trachea, an esophagus, a heart, a lung, a pancreas, a skin, or other bodily organ.

[0011] In one aspect the present disclosure provides a method of treating kidney

disease/disorder in a subject with end- stage renal failure including (i)

administering/implanting/transplanting to the subject a recellularized kidney or a functional part thereof, where the kidney or the functional part thereof is prepared following a method disclosed herein, and recellularized with human pluripotent stem cells or human adult multipotent stem cells; or (ii) administering/implanting/transplanting to the subject a bioprinted kidney scaffold cellularized with human pluripotent stem cells or human adult multipotent stem cells.

[0012] In one aspect the present disclosure provides a method of treating chronic renal failure in a subject in need thereof including (i) administering/implanting/transplanting to the subject a recellularized kidney or a functional part thereof, where the kidney or the functional part thereof is prepared following a method disclosed herein, and recellularized with human pluripotent stem cells or human adult multipotent stem cells, or (ii)

administering/implanting/transplanting to the subject a bioprinted kidney scaffold cellularized with human pluripotent stem cells or human adult multipotent stem cells. [0013] In one aspect the present disclosure provides a method of preparing a MMP -treated acellular organ or tissue scaffold, the method including treating an organ or a tissue sample with an ionic and/or a non-ionic detergent. The treatment with the ionic and/or the non-ionic detergent results in an acellular scaffold, which retains the structural extracellular matrix (ECM) components. One or more metalloproteinase (MMP) is then added to the acellular scaffold to de-condense the ECM mini- or microstructure, thereby preparing the MMP- treated acellular organ or tissue scaffold.

[0014] In one aspect the present disclosure provides a method of preparing a functional kidney or a functional part thereof with human pluripotent stem cells or human adult multipotent stem cells, the method including: (a) adding one or more metalloproteinase (MMP) to an acellular kidney scaffold that includes an extracellular matrix (ECM) composition, vascular architecture, glomerular capillaries, and tubular membrane, or adding one or more metalloproteinase (MMP) to a bioprinted polymeric scaffold that includes an ECM matrix composition; (b) seeding human pluripotent stem cells or human adult multipotent stem cells to the MMP -treated kidney scaffold or the MMP -treated bioprinted polymeric scaffold; (c) allowing the cells to attach to the MMP -treated kidney scaffold or the MMP -treated bioprinted polymeric scaffold; (d) perfusing media for growth and

differentiation of the cells to form at least renal proximal tubules; and (e) culturing the cells, thereby differentiating the cells to form at least renal proximal tubules; thereby, preparing a functional kidney or a functional part thereof. [0015] Other features and advantages of the disclosure will be apparent from the following detailed description and claims. [0016] Unless noted to the contrary, all publications, references, patents and/or patent applications reference herein are hereby incorporated by reference in their entirety for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS [0017] FIGs. 1A-1B are gross morphology images of pig kidney. Normal pig kidney showing brown color (FIG. 1 A) and decellularized pig kidney showing a "whitish" colour (FIG. IB). FIG. 1C is a bar graph showing quantification of proteins in decellularized Extra Cellular Matrix. There is a decrease in DNA and GAGs and increases in collagen and elastin in decellularized kidney as compared to normal. [0018] FIGs. 2A-2B show analysis of human fetal kidney progenitor cells (hFKPC). FIG. 2A shows fluorescence activated cell sorting of hFKPCs grown in endothelial specific medium showing positive staining for ULEX, ENDOCAN, CD133, CD34, and VWF. FIG. 2B shows positive staining for CK18, EPCAM and DLK, and a small fraction of positive staining for EPHA6, EPHA7 and EPHB3, on hFKPCs grown in renal epithelial specific medium via fluorescence activated cell sorting.

[0019] FIGs. 3A-3F are a series of immunohistochemical images for the expression of Ephrin receptors (Eph) and stem cell markers in fetal kidneys. FIG. 3A is an image showing immunohistochemical staining for EPHA6 in a fetal kidney. FIG. 3B is an image showing immunohistochemical staining for EPHA7 in a fetal kidney. FIG. 3C is an image showing immunohistochemical staining for EPHB3 in a fetal kidney. In fetal kidney cells, EPHA7 was the most highly and widely expressed among the tested Ephrin receptors. FIG. 3D is an image showing immunohistochemical staining for DLK1 in a fetal kidney. FIG. 3E is an image showing immunohistochemical staining for CD 133 in a fetal kidney. FIG. 3F is an image showing immunohistochemical staining for EPCAM in a fetal kidney. Of the stem cells markers tested, fetal kidneys expressed strongly DLK1 as compared to CD 133 and EPCAM. FIG. 3G is an image showing immunohistochemical staining for EPHA6 in an adult kidney. FIG. 3H is an image showing immunohistochemical staining for EPHA7 in an adult kidney. FIG. 31 is an image showing immunohistochemical staining for EPHB3 in an adult kidney. In contrast to fetal kidney cells, expression of EPHA7 was weak or absent in adult kidneys while EPHB3 continued to be strongly expressed. FIG. 3J is an image showing immunohistochemical staining for DLK1 in an adult kidney. FIG. 3K is an image showing immunohistochemical staining for CD 133 in an adult kidney. FIG. 3L is an image showing immunohistochemical staining for EPCAM in an adult kidney. Expression of DLK1 was weak or absent in adult kidneys as compared to fetal kidneys, while CD 133 and EPCAM continued to be expressed in adult kidneys.

[0020] FIGs. 4A-4H are a series of images of hematoxylin and eosin (HE) staining of decellularized pig kidney extracellular matrix (ECM). FIG. 4A shows before treatment with recombinant metalloproteinases MMP-2 and MMP-9. FIG. 4B shows post-treatment with MMP-2 and MMP-9. FIGs. 4C-4E are images of decellularized pig kidney slices recellularized with human fetal kidney progenitor cells. FIG. 4C shows the presence of cells scattered in the parenchyma. FIG. 4D shows the presence of cells scattered in the glomerulus, and FIG. 4E shows the presence of cells scattered in the tubule. FIG. 4F is an image of an HE stain of decellularized pig kidney slices recellularized with human fetal kidney progenitor cells showing cells growing on top of the scaffold creating new kidney tissue. FIG. 4G is an image of an HE stain of decellularized pig kidney slices recellularized with human fetal kidney progenitor cells showing another area with newly formed kidney tissue by the seeded human cells. FIG. 4H is an image of an HE stain of decellularized pig kidney slices recellularized with human fetal kidney progenitor cells showing tubule-like structures in the neo-tissue formed by the seeded human cells. In certain areas, abundant cells were found on the surface of the decelluarized ECM.

[0021] FIGs. 5A-5C are images showing immunohistochemical staining of the

recellularized kidney for cytokeratin 18. FIG. 5 A shows that the cells formed a tubule-like structure. FIG. 5B shows that the cells formed structures resembling glomeruli. FIG. 5C shows that the cells formed tubule and glomerular-like structures. In the areas where abundant cells were detected, these cells themselves formed tubular and glomerular-like structures. [0022] FIGs. 6A-6F are images showing immunohistochemical detection of epithelial and stem cell markers in pig kidneys recellularized with human fetal kidney progenitor cells. FIG. 6A is the negative control. FIG. 6B shows positive staining for the epithelial cell marker cytokeratin 18. FIG. 6C shows positive staining for the epithelial cell marker cytokeratin 8. FIG. 6D shows positive staining for EphA7 mainly in the blood vessels. FIG. 6E shows positive staining for CD 133 mainly in the blood vessels. FIG. 6F shows that most of the cells were found to be positive for the the stem cell marker DLK-1. [0023] FIG. 7 is a bar plot, which shows quantification of gene expression in pig kidneys recellularized with human fetal kidney progenitor cells. There is an increased expression of important transcription factors in kidney development such as SIX2, EYA1, CITED 1, LHX1, SALL1 and WT1 as compared to only cultured kidney cells. DETAILED DESCRIPTION OF THE DISCLOSURE

[0024] Provided herein, inter alia, is an in vitro or ex vivo method of decondensing an extracellular matrix (ECM) structure in an acellular structure of a mammalian organ or tissue with one or more metalloproteinase (MMP). In one aspect, the present disclosure provides a method of cellularizing a MMP -treated acellular structure of a mammalian organ or tissue, in which the MMP treatment facilitates infiltration and migration of cells seeded onto the acellular structure.

Definitions

[0025] The following definitions are included for the purpose of understanding the present subject matter and for constructing the appended patent claims. Abbreviations used herein have their conventional meaning within the chemical and biological arts.

[0026] The term, "biomaterial" or "engineered biomaterial" as used in this disclosure may refer to any matter, surface, or construct that interacts with living systems, that may be naturally occurring or synthetically made. Thus, the term "biomaterial," as used herein, refers to a substance that has been engineered to take a form which, alone or as part of a complex system, is used to direct, by control of interactions with components of living systems, the course of any therapeutic or diagnostic procedure. The term "one or more of as used in this disclosure refers to a single component or alternatively a combination of two or more components.

[0027] The term "stem cells" refers to cells that have the ability to divide for indefinite periods and to give rise to virtually all of the tissues of the mammalian body, including specialized cells. The stem cells include pluripotent cells, which upon undergoing further specialization become multipotent progenitor cells that can give rise to functional or somatic cells.

[0028] Human pluripotent stem cells are a cell type that can both self-renew indefinitely and differentiate to form any cell type in a human being. Types of pluripotent stem cells include embryonic stem cells, induced pluripotent stem cells (iPSCs), somatic cell nuclear transfer derived embryonic stem cells (ntES), and, parthenogenesis derived pluripotent stem cells (hpSC). iPSCs are derived from differentiated cells isolated from various tissues, such as skin, in which a combination of transcription factors are overexpressed that ultimately reprograms the cells to a pluripotent state. The resulting iPSCs are able to differentiate into cells of any of the three germ layers that form a human being: endoderm, mesoderm and ectoderm. Somatic cell nuclear transfer involves transferring the nucleus of a diploid cell to an enucleated oocyte. Based on culturing in specific conditions, pluripotent stem cells (ntES), which are transcriptionally and functionally indistinguishable from normal embryonic stem cells, are derived from this reconstructed cell. Parthenogenesis is a form of asexual reproduction in which unfertilized eggs develop into embryos. In humans, parthenogenesis can be activated by electrical or chemical stimuli of an oocyte that mimic spermatozoon penetration. Activated oocytes have the ability to develop into parthenogenetic embryos, from which hpSCs are isolated from the inner cell mass of the resulting blastocyst. See Kim JS, Choi HW, Choi S, Do JT. Reprogrammed pluripotent stem cells from somatic cells. Int J Stem Cells. 2011; 4 (l) l-8.

[0029] Examples of stem and progenitor cells include hematopoietic stem cells (adult stem cells; i.e., hemocytoblasts) from the bone marrow that give rise to red blood cells, white blood cells, and platelets; mesenchymal stem cells (adult stem cells) from the bone marrow that give rise to stromal cells, fat cells, and types of bone cells; epithelial stem cells

(progenitor cells) that give rise to the various types of skin cells; neural stem cells and neural progenitor cells that give rise to neuronal and glial cells; and muscle satellite cells (progenitor cells) that contribute to differentiated muscle tissue. Non- limiting examples of stem cells are listed below:

Human Multipotent Stem Cells

Type Source

Haematopoietic stem cells Adult bone marrow, umbilical cord blood

Mesenchymal stem cells Adult bone marrow

Neural stem cells Brain

Epithelial stem cells Lining of digestive tract

Follicular stem cells Basal layer of epidermis, base of hair follicles

Adipose-derived stem cells Adipose tissue

(ASCs) Human Pluripotent Stem Cells

Type Source

Embryonic stem cells Inner cell mass of embryo at blastocyst stage

Embryonic germ cells Epiblast, primordial germ cells of embryo

Induced Pluripotent stem cells Skin cells, fibroblasts

[0030] The matrix metalloproteinases (MMPs) are a family of extracellular matrix (ECM)- degrading enzymes that shares common functional domains and activation mechanisms. These are Ca ²⁺ - and Zn ²⁺ -dependent endopeptidases that are active at neutral pH. These enzymes are synthesized as secreted or transmembrane proenzymes and processed to an active form by the removal of an amino -terminal propeptide. The propeptide is thought to keep the enzyme in latent form by the interaction of a cysteine residue in this peptide with the zinc moiety in the enzyme active site. Disruption of this interaction triggers the cysteine switch mechanism and results in activation of the enzyme. MMPs can be activated by chaotropic agents or by cleavage of the propeptide by members of the MMP family or by other proteases. These enzymes are inhibited by a family of tissue inhibitors of

metalloproteinases, the TIMPs. As a family, MMPs degrade most components of the ECM. There are more than 20 members of the MMP family. There are several distinct subgroups based on preferential substrates or similar structural domains: Collagenases that are active against fibrillar collagen, gelatinases that have high activity against denatured collagens, stromelysins that degrade noncollagen components of the ECM, membrane-type MMPs (MT- MMPs) that are transmembrane molecules, and other less characterized members. The Table below lists known MMPs and their other biological names based on respective functions. From Vu and Werb, Genes & Dev. 2000, 14: 2123-2133. [0031] TABLE 1.

MMP designation Name

collagenase-1, interstitial collagenase, fibroblast

MMP-1 collagenase

MMP-2 gelatinase A, 72-kD gelatinase

MMP-3 stromelysisn-1, transin-1

MMP-7 matrilysin, matrin, PUMP-1

MMP-8 neutrophil collagenase

MMP-9 gelatinase B, 92-kD gelatinase

MMP- 10 stromelysin-2, transin-2

MMP-11 stromelysin-3 MMP-12 metalloelastase, macrophage elastase

MMP-13 collagenase-3

MMP-14 MT1-MMP, membrane-type MMP

MMP-15 MT2-MMP, MT-MMP-2

MMP-16 MT3-MMP, MT-MMP-3

MMP-17 MT4-MMP, MT-MMP-4

MMP-18 collagenase-4 (Xenopus)

Matrix metalioproteina.se RASI (RASI- 1 , occasionally

MMP-19 referred to as stromelysin-4)

MMP-20 Enamelysin

MMP-21 Matrix metalloproteinase-21

MMP-22 Matrix metal loptOteinase-22

MMP-23 Matrix metal loproteina.se-23

MMP-24 MT5-MMP, MT-MMP-5

MMP-25 MT6-MMP, MT-MMP-6, leukolysin

[0032] The term, "gel" as used in this disclosure may refer to a material which is not a readily flowable liquid and not a solid, i.e., semi-solid. Gels may be formed from naturally occurring or synthetic materials. In embodiments, the gel may be a formed starting from a skin decellularized to prepare a powder, which includes extracellular matrix components.

[0033] The term "Extracellular Matrix" or "ECM" may refer to natural or artificial scaffolding for cell growth. Natural ECMs (ECMs found in multicellular organisms, such as mammals and humans) are complex mixtures of structural and non- structural bio molecules, including, but not limited to, collagens, elastins, laminins, glycosaminoglycans,

proteoglycans, antimicrobials, chemoattractants, cytokines, and growth factors. In mammals, ECM often comprises about 90% collagen, in its various forms. In certain embodiments, the extracellular matrix material of the cell-laden filaments and the extracellular matrix composition that at least partially surrounds the tissue and vascular patterns may include a synthetic or naturally derived biocompatible material. The extracellular matrix material and the extracellular matrix composition may include the same or different biocompatible materials.

[0034] The composition and structure of ECMs vary depending on the source of the tissue. For example, small intestine submucosa (SIS), urinary bladder matrix (UBM) and liver stroma ECM each differ in their overall structure and composition due to the unique cellular niche needed for each tissue, and further characteristics and details as described in US Patent 8,361 ,503, incorporated herein by reference. For example, kidney ECM has fibrinogen, gelatin, and two enzymes (thrombin and transglutaminase). The dual enzyme scheme enables rapid solidification of the ECM around printed features, through thrombin action on fibrinogen to make fibrin. The second enzyme, transglutaminase, provides a slower crosslinking of gelatin with fibrin, enabling a seamless integration of the upper and lower ECM layers during assembly.

[0035] Components of the ECM may be isolated and/or purified from an organ or tissue (or a part thereof), e.g., a decellularized organ or tissue (or a part thereof), of a mammal (e.g., human) or generated using recombinant DNA technology involving gene or gene fragments of a mammal (e.g., human) encoding the respective ECM component (e.g., collagens, gelatin, fibrinogen, elastins, laminins, glycosaminoglycans, proteoglycans, chemoattractants, cytokines, enzymes (thrombin and transglutaminase) and growth factors) and expressed in from a suitable expression system (prokaryotic or eukaryotic (e.g., yeast, insect or mammalian cells)), and subsequently isolated and/or purified by suitable method in the art. [0036] The term "decellularized" or "acellular" is used interchangeably in this disclosure, and used to mean that physical, chemical, or enzymatic means, or any combination thereof, has removed the cellular component of an organ or a tissue thereof. The remaining decellularized organ or tissue includes the extracellular matrix of the native organ or tissue and may include, but is not limited to, elastin, collagen, fibrin, and other extracellular proteins or non-proteinaceous molecules found in the organ or tissue; or any combination thereof.

[0037] The term "polymer" as referred to in this disclosure is meant as a molecule, or macro molecule, composed of many repeated subunits. Because of their broad range of properties, both synthetic and natural polymers play an essential and ubiquitous role in everyday life. Polymers range from familiar synthetic plastics such as polystyrene to natural biopolymers such as DNA and proteins that are fundamental to biological structure and function. Polymers, both natural and synthetic, are created via polymerization of many small molecules, known as monomers. Their consequently large molecular mass relative to small molecule compounds produces unique physical properties, including toughness,

viscoelasticity, and a tendency to form glasses and semicrystalline structures rather than crystals. Exemplary natural polymeric materials include shellac, amber, wool, silk, rubber, and cellulose. Non-natural (e.g., synthetic) polymers include synthetic rubber, phenol formaldehyde resin, neoprene, nylon, polyvinyl chloride, polystyrene, polyethylene, polypropylene, and silicone.

[0038] Additional synthetic polymers that can be used include biodegradable polymers such as poly(lactide) (PLA), poly(glycolic acid) (PGA), poly(lactide-co-glycolide) (PLGA), poly(caprolactone), polycarbonates, polyamides, polyanhydrides, polyamino acids, polyortho esters, polyacetals, polycyanoacrylates and degradable polyurethanes, and non-erodible polymers such as polyacrylates, ethylene-vinyl acetate polymers and other acyl substituted cellulose acetates and derivatives thereof, non-erodible polyurethanes, polystyrenes, polyvinyl chloride, polyvinyl fluoride, poly(vinyl imidazole), chlorosulphonated polyolifms, polyethylene oxide, polyvinyl alcohol, teflon®, and nylon. A non-absorbable polyvinyl alcohol sponge is available commercially as IVALON™, from Unipoint Industries. Methods for making this material are described in U.S. Pat. Nos. 2,609,347 to Wilson; 2,653,917 to Hammon, 2,659,935 to Hammon, 2,664,366 to Wilson, 2,664,367 to Wilson, and 2,846,407 to Wilson, the teachings of which are incorporated by reference herein. [0039] The tissue construct may also include one or more functional chemical substances selected from among drugs, toxins, proteins and/or hormones, including, but not limited to: growth factors, growth inhibitors, cytokines, steroids, and/or morphogens. Some cell specific examples include: bone morphogenic protein, vascular endothelial growth factor, fibroblast growth factors, including but not limited to VEGF, EGF, TGF-β. The one or more functional chemical substances may be deposited with the cell-laden filament(s) and/or the sacrificial filaments and may diffuse into the surrounding extracellular matrix composition.

[0040] "Patient," "subject," "patient in need thereof," and "subject in need thereof are used interchangeably in this disclosure, and refer to a living organism suffering from or prone to a disease or condition that can be treated by administration using the methods and compositions provided in this disclosure. Non-limiting examples include humans, other mammals, bovines, rats, mice, dogs, monkeys, goat, sheep, cows, deer, and other

non-mammalian animals. In some embodiments, a patient is human. Tissues, cells and their progeny of a biological entity obtained in vitro or cultured in vitro are also contemplated.

[0041] The terms "treat," "treating," or "treatment," and other grammatical equivalents as used in this disclosure, include alleviating, abating, ameliorating, or preventing a disease, condition or symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the disease or condition, and are intended to include prophylaxis. The terms further include achieving a therapeutic benefit and/or a prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient may still be afflicted with the underlying disorder. [0042] "Control" or "control experiment" is used in accordance with its plain ordinary meaning and refers to an experiment in which the subjects or reagents of the experiment are treated as in a parallel experiment except for omission of a procedure, reagent, or variable of the experiment. In some instances, the control is used as a standard of comparison in evaluating experimental effects. In some embodiments, a control is the measurement of the activity of a phenotype or outcome in the absence of a composition as described in this disclosure (including embodiments and examples).

[0043] "Contacting" is used in accordance with its plain ordinary meaning and refers to the process of allowing at least two distinct species (e.g. chemical compounds including bio molecules or cells) to become sufficiently proximal to react, interact or physically touch. It should be appreciated; however, the resulting reaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents which can be produced in the reaction mixture. In some embodiments contacting includes allowing a composition described in this disclosure to interact with a subject. [0044] Throughout the description and claims of this specification the word "comprise" and other forms of the word, such as "comprising" and "comprises," means including but not limited to, and is not intended to exclude, for example, other components.

[0045] A weight percent of a component, unless specifically stated to the contrary, is based on the total weight of the composition in which the component is included. [0046] The term "about" refers to any minimal alteration in the concentration or amount of an agent that does not change the efficacy of the agent in preparation of a formulation and in treatment of a disease or disorder. In embodiments, the term "about" may include ±15% of a specified numerical value or data point.

[0047] Ranges can be expressed in this disclosure as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it is understood that the particular value forms another aspect. It is further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed in this disclosure, and that each value is also disclosed as "about" that particular value in addition to the value itself. It is also understood that throughout the application, data are provided in a number of different formats and that this data represent endpoints and starting points and ranges for any combination of the data points. For example, if a particular data point "10" and a particular data point "15" are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

METHODS OF PREPARATION OF A BIOENGINEERED GRAFT

[0048] In one aspect, the present disclosure provides an in vitro or ex vivo method of decondensing an extracellular matrix (ECM) structure in a decellularized mammalian organ or tissue {e.g., a kidney, a liver, a bladder, a vagina, a urethra, a trachea, an esophagus, a heart, a lung, a pancreas, a skin, or other bodily organ) in need thereof by introducing one or more metalloproteinase (MMP, e.g., MMP1, MMP2, MMP3, MMP7, MMP9, MMP13, and/or MMP14). The organ or tissue sample with the ECM is the treated with

metalloproteinase (MMP, e.g., MMP1, MMP2, MMP3, MMP7, MMP9, MMP 13, and/or MMP14). The MMP used for decondensing ECM depends on the type of organ or tissue. For example, MMP-9 may be used in kidney or bone tissue; MMP-2 may be used in kidney; MMP- 13 may be used for bone tissue; and MMP- 14 may be used in skeletal muscle and related tissue, etc. The MMP may be purified from an organ or tissue, or recombinantly generated, isolated, and purified. [0049] The MMP decondenses a compact ECM ministructure and/or microstructure. In embodiments, decondensing the ECM structure of a decellularized organ or tissue with tumor or cancer is achieved by introducing one or more metalloproteinase (MMP, e.g., MMP1, MMP2, MMP3, MMP7, MMP9, MMP13, and/or MMP 14). [0050] In one aspect, the present disclosure provides a method of preparing an ECM decondensed decellularized organ or tissue (e.g., a kidney, a liver, a bladder, a vagina, a urethra, a trachea, an esophagus, a heart, a lung, a pancreas, a skin, or other bodily organ) or a functional part thereof, involving decondensing the extracellular matrix (ECM) ministructure in a decellularized mammalian organ or tissue. In embodiments, an organ or a tissue sample (or a part thereof) is treated with an ionic and/or a non- ionic detergent to produce an acellular scaffold, which retains the structural extracellular matrix (ECM) proteins, but removes DNA. The detergent treated organ or tissue retains an ECM structure including mini- or

microstructure. In embodiments, the organ or tissue (or a part thereof) for decellularization and recellularization is obtained from an allogeneic or autologous donor. In embodiments, the decellularized organ or the tissue (or a part thereof) is prepared from an organ or tissue obtained from a pig, a cow, a lamb, a goat, a sheep, a chimpanzee, a monkey, a human or other primate.

[0051] In embodiments, the present disclosure provides a method of preparing an ECM decondensed decellularized organ or tissue that is not from a healthy organ or tissue. In embodiments, a method is provided for preparing an ECM decondensed decellularized organ or tissue from an organ or tissue characterized as having a tumor or cancer. Examples of organ or tissue characterized as having a tumor or cancer includes, but not limited to, kidney, liver, breast, lung, pancreas, skin, bladder, and urethra.

[0052] In certain embodiments, the MMP is introduced to a decellularized organ or tissue under static culture condition. For example, the MMP is added to a decellularized organ or tissue in a tissue culture plastic container or a bioreactor without moving (e.g., rotating, vibrating, or shaking). The MMP is added to the decellularized organ or tissue before any subsequent treatment of the samples, e.g., adding cells to recellularize the decellularized organ or tissue. The present disclosure further provides that the decondensing the ECM structure of a decellularized organ or tissue with one or more MMP under static culture conditions improves the decondensing and subsequent recellularization process. In embodiments, MMPs breakdown proteins and tissue surrounding an ECM and facilitate migration. In embodiments, a decellularized organ or tissue is or may be treated with human recombinant MMP2 and MMP9 to open up the compact ECM microstructure.

[0053] The MMP treatment of a decellularized organ or tissue decondenses the ECM structure by degrading or cleaving ECM proteins, thereby opening condensed and complex architecture. The decondensed ECM structure allows improved infiltration, migration, and/or attachment of the cells (e.g., human pluripotent stem/progenitor cells, human multipotent stem/progenitor cells, or human fetal kidney precursor cells (hFKPC)), seeded or introduced to the MMP -treated decellularized organ or tissue, and thereby improves the recellularization of the decellularized organ or tissue to prepare or manufacture an organ or tissue grafts. [0054] In certain embodiments, the MMP treatment in combination with incubation of a decellularized organ or tissue in a transwell improves infiltration of cells under static culture conditions. In certain embodiments, optimal distribution of the seeded cells (e.g., human pluripotent stem/progenitor cells, human multipotent stem/progenitor cells, human fetal kidney precursor cells (hFKPC)) into a decellularized organ or tissue may be obtained. For example, in a decellularized kidney scaffold, which is pre-treated with one or more MMPs (e.g., MMP2 or MMP9), seeded cells infiltrate and migrate through the decondensed ECM to distribute through the microstructure of individual glomeruli, blood vessels, and renal parenchyme. These distributed cells within the decondensed ECM of a decellularized organ or tissue may express epithelial, podocytic, and endothelial lineages. In embodiments, genes up-regulated in the recellularized grafts may be transcription factors specifying the kidney progenitor cells including WT1, PAX2, LIM1, SIX2, EYA1, SALL1, and CITEDl .

Expression of SIX2, SALL1, and WT1 are considered to be markers of the metanephric cap mesenchyme. For example, the seeded cells (e.g., human pluripotent stem/progenitor cells, human multipotent stem/progenitor cells, human fetal kidney precursor cells (hFKPC)), which recapitulate the in vivo metanephric development efficiently generate mature kidney cells.

[0055] In certain embodiments, acellular porcine scaffolds can optimally be recellularized with human pluripotent stem/progenitor cells, human multipotent stem/progenitor cells, or human fetal kidney precursor cells (hFKPC) that would facilitate ex vivo expansion and localized differentiation after scaffold seeding. The human pluripotent stem/progenitor cells, human multipotent stem/progenitor cells, or human fetal kidney precursor cells (hFKPC) can then be directed toward mature epithelial, endothelial, mesodermal, or mesenchymal cell phenotypes by exogenous growth factors and intrinsic matrix-derived cues.

[0056] In certain embodiments, MMP -treated decellularized organ or tissue (or a part thereof) may be used to prepare an organ or tissue pattern including or is defined by a two- or three-dimensional arrangement of one or more cell-laden filaments, and each tissue pattern (and thus each arrangement of cell-laden filaments) may include a different subset of the predetermined cell types. In addition to the viable cells, the one or more cell-laden filaments may include a synthetic or naturally-derived biocompatible material that may be referred to as an extracellular matrix material. Each of the one or more cell-laden filaments may also or alternatively include one or more functional chemical substances (e.g., drugs, toxins, proteins and/or hormones). Each tissue pattern may include one layer or multiple layers of the cell- laden filament(s), which may at least be partially coalesced at regions of contact there between. For example, adjacent layers formed from one or more cell-laden filaments may be partially or fully coalesced depending on filament composition and the deposition (or post- deposition) conditions.

[0057] The arrangement of the cell-laden filaments in the tissue construct may be continuous or discontinuous. In a continuous arrangement, the cell-laden filaments of an exemplary tissue pattern (and including one or more predetermined cell types) may form a single interconnected tissue network in the tissue construct. For example, a single cell-laden filament including viable cells of the predetermined cell type(s) may be deposited in a single layer or in multiple layers to form the continuous arrangement. Alternatively, a plurality of cell-laden filaments including viable cells of the predetermined cell type(s) may be deposited in a single layer or in multiple layers to form the continuous arrangement, where each of the cell-laden filaments is in physical contact with, and possibly at least partially coalesced with, another cell-laden filament comprising the same predetermined cell type(s).

[0058] In a discontinuous arrangement of cell-laden filaments including one or more predetermined cell types, a single interconnected tissue network of the predetermined cell type(s) is not formed within the tissue construct. Instead, the cell-laden filaments including the predetermined cell type(s) may be dispersed uniformly or non-uniformly throughout the tissue construct. Consequently, the cells corresponding to the predetermined cell type(s) may also be dispersed uniformly or non-uniformly (e.g., in clumps) throughout the tissue construct. All or none of the cell-laden filaments of a given tissue pattern and cell type(s) may be in physical contact with another cell-laden filament comprising cells of the same cell type(s).

[0059] Each of the one or more cell-laden filaments includes at least one viable cell and may include a large number of viable cells. For example, each of the cell-laden filaments may have a cell concentration of at least about 100 cells/ml, at least about 1000 cells/ml, at least about 10 ⁴ cells/ml, at least about 10 ⁵ cells/ml, at least about 10 ⁶ cells/ml, at least about 10 ⁷ cells/ml, or at least about 10 ⁸ cells/ml. Consistent with this, the one or more tissue patterns of an organ or a tissue construct may have a cell concentration of at least about 100 cells/ml, at least about 1000 cells/ml, at least about 10 ⁴ cells/ml, at least about 10 ⁵ cells/ml, at least about 10 ⁶ cells/ml, at least about 10 ⁷ cells/ml, or at least about 10 ⁸ cells/ml. The cell concentration in the tissue pattern may be no higher than about 10 ⁹ cells/ml or no higher than about 10 ⁸ cells/ml.

[0060] The cell concentration may be substantially uniform (e.g., within ±10%, within ±5%, or within ±1%) throughout each of the cell-laden filaments, and the cell concentration may also be substantially uniform throughout each of the tissue pattern(s). Alternatively, it is possible to deposit cell-laden filaments that include aggregates or clusters of cells that may range in size from about 10 cells/cluster to about 1000 cells/cluster, or from about 10 cells/cluster to about 100 cells/cluster. Such clusters may be dispersed uniformly or non- uniformly within the cell- laden filaments (and thus uniformly or non-uniformly throughout the one or more tissue patterns). Overall, the cell concentration may be substantially uniform throughout the tissue construct, or the cell concentration may include predetermined inhomogeneities within the tissue construct that may be defined by the location and morphology of the one or more tissue patterns, and/or by the cell distribution within the one or more tissue patterns. [0061] The vascular network that interpenetrates the one or more tissue patterns is a two- or three-dimensional interconnected arrangement of vascular channels. The network may include one or more-furcations (e.g., bifurcations, trifurcations, etc.) from a parent vascular channel to a plurality of branching vascular channels. The network may have a hierarchical branching structure, where larger diameter channels branch into smaller diameter channels. Some or all of the vascular channels may follow a curved path, and thus may be considered to be curvilinear. All of the vascular channels in the network may have the same diameter, or at least one, some, or all of the vascular channels may have a different diameter. In some cases, one or more of the vascular channels may have a non-uniform diameter along a length thereof.

[0062] It is beneficial for the cells of the tissue construct to be close enough to the interpenetrating network of vascular channels to remain viable. One major problem with previous attempts to create tissue and organ-like structures is that necrotic regions could develop in areas without accessible perfusable vasculature. In the present work, each cell- laden filament, and thus each cell, may be placed in a location near to the vascular network, or near to where the vascular network may be formed by using MMP -treated decellularized organ or tissue (or a part thereof). One or more of the cell-laden filaments and thus at least some of the viable cells may be deposited so as to be in direct contact with a vascular channel.

METHODS OF PREPARATION OF A BIOPRINTED GRAFT

[0063] In one aspect, the present disclosure provides a method of preparing a bioprinted organ or tissue (e.g., a kidney, a liver, a bladder, a vagina, a urethra, a trachea, an esophagus, a heart, a lung, a pancreas, a skin, or other bodily organ) scaffold using a bioprinted polymeric scaffold including MMP -treated extracellular matrix (ECM) material and/or the extracellular matrix (ECM) composition of a mammalian organ or tissue. The bioprinted organ or tissue (of a functional part thereof) scaffold is prepared on a polymer (natural or synthetic), which may be in a gel form, sponge form, foam form, patch form, or a semi- liquid/fluid form.

[0064] In certain embodiments, the ECM components may be added to the polymer in a powder form. In the present disclosure the powder for preparing the composition of the present disclosure is prepared by treating a tissue with a chemical, freeze-drying the chemical treated tissue, and homogenization of freeze-dried tissue. In certain embodiments, the powder is filamentous.

[0065] In certain embodiments, the extracellular matrix material and/or the extracellular matrix composition on a bioprinted polymeric scaffold may include gelatin and fibrin. The gelatin and fibrin may form an interpenetrating polymer network that mimics natural extracellular matrix (ECM) and may be optimized for cell attachment, bioprinting, transparency, and biocompatibility. The fibrin-gelatin interpenetrating polymer network may be created by mixing solutions of fibrinogen and gelatin with transglutaminase (TG), a slow- acting Ca ²⁺ dependent enzyme, to create a gel-precursor solution that may later be mixed with thrombin to create a fibrin gel backbone. Fibrin may be made from a concentrated fibrinogen solution that has been activated by thrombin and calcium chloride. Fibrin is a rapidly coagulating phase that permits rapid, controllable gelation of a printed structure.

Advantageously, fibrin and gelatin can be welded together via mobile surface chain entanglement, while forming a strong interface. Creating monolithic gels of this nature is possible due to the slow crosslinking kinetics of transglutaminase (TG). Although thrombin rapidly induces fibrin gel formation, the gelatin present allows one to print ink on an already cast layer, and, ultimately, to encapsulate with liquid gelatin-fibrin. The two phases may weld together, creating a monolithic gel. This material system can be readily tailored to modify gelation kinetics, interface adhesion, mechanical properties, optical properties, and cell-material interactions.

[0066] To form the extracellular matrix composition, a microgel (e.g., a poly(acrylic acid) (PAA) microgel) may be used as a rheological modifier and blended with one or more extracellular matrix materials, such as gelatin methacrylate. A semi- interpenetrating polymer network (semi-IPN) may be formed. Microgels may be understood to include colloidal gel particles that are composed of chemically cross-linked three-dimensional polymer networks. Microgels may act as sterically stabilized colloids with only a shell and no core, andean vary in composition and may include, e.g., PAA, polystyrenes, PEG, and/or other biomaterials. In certain embodiments, a natural extracellular matrix or biomaterial may be converted into a microgel form to impart the ideal rheology. Examples of suitable biomaterials include hyaluron, collagen, alginate, fibrin, albumin, fibronectin, elastin, or matrigel. In certain embodiments,, synthetic materials such as PEG, acrylates, urethanes, or silicones may be modified in a similar manner to prepare a extracellular matrix composition. [0067] The present disclosure provides pre-treating the ECM composition/microgel/gel on a bioprinted polymeric scaffold with one or more metalloproteinase (MMP, e.g., MMP1, MMP2, MMP3, MMP7, MMP9, MMP 13, and/or MMP 14) before seeding cells (e.g., human pluripotent stem/progenitor cells, human multipotent stem/progenitor cells, or human fetal kidney precursor cells (hFKPC)) for preparing a bioprinted organ or tissue (or a functional part thereof). Thus, in certain embodiments, a bioprinted polymeric scaffold (e.g., 3-D printed scaffold) with extracellular matrix material and/or the extracellular matrix

composition is perfused with one or more metalloproteinase (MMP, e.g., MMP1, MMP2, MMP3, MMP7, MMP9, MMP 13, and/or MMP 14) to de-condense the ECM mini- or microstructure. The present disclosure further provides that the decondensing the ECM structure formed on a bioprinted polymeric scaffold with one or more MMP under static culture conditions results in improved de-condensation of the structure (and/or mini- or microstructure), which improves subsequent cellularization of cells seeded on the bioprinted polymeric scaffold.

[0068] In certain embodiments, cells are seeded onto the MMP -treated biopolymer including ECM composition/microgel/gel. Subsequently, growth and/or differentiation media is perfused through the cell seeded scaffold for proliferation and/or differentiation of the seeded cells. Pre-treatment with MMP substantially improves infiltration and migration of the seeded cells through an ECM mini- or microstructure. In certain embodiments, MMPs breakdown proteins and tissue surrounding an ECM and facilitate cell migration. In certain embodiments, the bioprinted organ or tissue of the present disclosure is or may be prepared without the MMP treatment.

[0069] In certain embodiments, components of the ECM are or may be isolated and/or purified from a tissue of a mammal (e.g. , a pig, a cow, a lamb, a goat, a sheep, a chimpanzee, a monkey, a human, or other primate) or generated using recombinant DNA technology involving gene or gene fragments of a mammal (e.g. , a pig, a cow, a lamb, a goat, a sheep, a chimpanzee, a monkey, a human, or other primate) encoding the respective ECM component (e.g., collagens, elastins, laminins, glycosaminoglycans, proteoglycans, antimicrobials, chemoattractants, cytokines, and growth factors) and expressed in from a suitable expression system (prokaryotic or eukaryotic (e.g. , yeast, insect, or mammalian cells)), and subsequently isolated and/or purified by suitable method in the art. In certain embodiments, one or more of the ECM components may be added to the polymeric scaffold for preparing a bioprinted polymeric scaffold. CULTURE CONDITIONS

[0070] In certain embodiments, during recellularization, the cell seeded ECM is perfused with a medium, which may be serum- free or serum containing medium. In certain embodiments, the medium may include constituents of keratinocyte medium and/or thioglycolate. In certain embodiments, nutrients may be added to the media, nutrients including one or more of: amino acid, monosaccharide, vitamin, inorganic ion and trace element, and/or salt. In certain embodiments, the nutrient may be an amino acid. In certain embodiments, the nutrient may be a monosaccharide. In certain embodiments, the nutrient may be a vitamin. In certain embodiments, the nutrient may be an inorganic acid.

[0071] In certain embodiments, the medium is serum containing, reduced serum, or non- serum containing medium. In certain embodiments, the growth factor is one or more of: Recombinant 4-lBBL, Recombinant 6Ckine, 6Ckine Recombinant Human Protein, ANGPT2 (ANG2), ANGPTL5, Activin A, Activin Rib, BAFF, BAMBI, CXCL13, BDNF, BLC, BMP2, BMP4, BMP5, BMP7, BMPR1A, CCL1, CCL17, CCL20 (MIP-3), CCL21, CD40, GM-CSF, IL-3, IL-4, NT-6, HB-GAM, MK, IP-10, PF-4, MCP-1, RANTES, IL-8, IGFs, FGF 1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, TGF-β, VEGF.

PDGF-A, PDGF-B, HB-EGF, HGF, TNF-a, IGF-I, or any combination(s) thereof.

[0072] In certain embodiments, the nutrients may include an amino acid, a

monosaccharide, a vitamin, an inorganic ion and a trace element, and/or a salt. In certain embodiments, the amino acid included as a nutrient may be one or more of: L-ArginineHCl, L-Cystine2HCl, L-CystineHCl H ₂ 0, L-HistidineHCl H ₂ 0, L-Isoleucine, L-Leucine, L- LysineHCl, L-Methionine, L-Phenylalanine, L-Threonine, L-Tryptophan, L-Tyrosine2H ₂ 0, L- Valine, L- Alanine, L-Asparagine, L-Aspartic acid, L-Glutamic acid, Glycine, L-Proline, L- Serine, and/or L-Hydroxyproline. Vitamine included as a nutrient may be one or more of: K- Ca-Pantothenate, Choline Chloride, Folic acid, i-Inositol, Niacinamide, Pyridoxal HC1, Pyridoxine HC1, Riboflavin, Thiamine HC1, Biotin, Vitamin B 12, Para-aminobenzoic acid, Niacin, Ascorbic acid, a-Tocopherol phosphate, Calciferol, Menadione, Vitamin A.

[0073] In certain embodiments, other compounds may be present as a nutrient, for example, one or more of: D-Glucose, Phenol red, HEPES, Sodium pyruvate, Glutathione (reduced), Hypoxantine.Na, Thymidine, Lipoic acid, Putrescine 2HC1, Bacto-peptone, Thymine, Adenine sulphate, Adenosine-5 -triphosphate, Cholesterol, 2-deoxy-D-ribose, Adenosine-5 -phosphate, Guanine HC1, Ribose, Sodium acetate, Tween 80, Uracil, and/or Xanthine Na. Inorganic salts added as nutrient may be one or more of: CaCl ₂ , KCl, MgS04, NaCl, NaHCOs, NaHP0 ₄ , KN0 ₃ , NaSe0 ₃ , Ca(N0 ₃ ) ₂ , CuS0 ₄ , NaHP0 ₄ , MgCl ₂ , Fe(N0 ₃ ) ₃ , CuS0 ₄ , FeS0 ₄ , and/or KH ₂ P0 ₄ .

[0074] In certain embodiments, for the ECM components and/or the seeded cells to attach, grow, and/or differentiate on an organ or tissue scaffold or a bioprinted polymeric scaffold and thereby cellularizing the scaffold, one or more growth or differentiation factor may be used. In certain embodiments, the growth or differentiation factor used in the method of cellularization of an organ or tissue scaffold or a bioprinted polymeric scaffold of the present disclosure is one or more of: Recombinant 4-1BBL, Recombinant 6Ckine, 6Ckine

Recombinant Human Protein, ANGPT2 (ANG2), ANGPTL5, Activin A, Activin Rib, BAFF, BAMBI, CXCL13, BDNF, BLC, BMP2, BMP4, BMP5, BMP7, BMPR1A, CCL1, CCL17, CCL20 (MIP-3), CCL21, CD40, PF-4, MCP-1, RANTES, IL-8, IGFs, granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin (IL)-3, IL-4, neutrophin (NT)- 6, pleiotrophin (HB-GAM), midkine (MK), interferon inducible protein- 10 (IP- 10), platelet factor (PF)-4, monocyte chemotactic protein- 1 (MCP-1), RANTES (CCL-5, chemokine (C-C motif) ligand 5), IL-8, IGFs, fibroblast growth factor (FGF)-l, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, transforming growth factor (TGF)-P, VEGF, platelet-derived growth factor (PDGF)-A, PDGF-B, HB-EGF, hepatocyte growth factor (HGF), tumor necrosis factor (TNF)-a, insulin-like growth factor (IGF)-I, or any combination(s) thereof. In certain embodiments, the growth or differentiation factor used in the method of the present disclosure is or may be GM-CSF. In certain embodiments, the growth or differentiation factor used in the method of the present disclosure is or may be IL-3. In certain

embodiments, the growth or differentiation factor used in the method of the present disclosure is or may be IL-4. In certain embodiments, the growth factor in the composition of the present disclosure is or may be NT-6. In certain embodiments, the growth or

differentiation factor used in the method of the present disclosure is or may be HB-GAM. In certain embodiments, the growth or differentiation factor used in the method of the present disclosure is or may be MK. The growth or differentiation factor used in the method of the present disclosure is or may be IP- 10. In certain embodiments, the growth or differentiation factor used in the method of the present disclosure is or may be PF-4. In certain

embodiments, the growth factor used in the method of the present disclosure is or may be MCP-1. In certain embodiments, the growth or differentiation factor used in the method of the present disclosure is or may be CCL-5. In certain embodiments, the growth factor used in the method of the present disclosure is or may be IL-8. In certain embodiments, the growth or differentiation factor used in the method of the present disclosure is or may be IGFs. In certain embodiments, the growth or differentiation factor used in the method of the present disclosure is or may be (FGF)-l, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, or FGF-9. In certain embodiments, the growth or differentiation factor used in the method of the present disclosure is or may be transforming growth factor (TGF)-p. In certain

embodiments, the growth factor used in the method of the present disclosure is or may be VEGF. In certain embodiments, the growth or differentiation factor used in the method of the present disclosure is or may be platelet-derived growth factor (PDGF)-A. In certain embodiments, the growth or differentiation factor used in the method of the present disclosure is or may be PDGF-B. In certain embodiments, the growth or differentiation factor used in the method of the present disclosure is or may be HB-EGF. In certain embodiments, the growth or differentiation factor used in the method of the present disclosure is or may be hepatocyte growth factor (HGF). In certain embodiments, the growth or differentiation factor used in the method of the present disclosure is or may be tumor necrosis factor (TNF)-a. In certain embodiments, the growth or differentiation factor used in the method of the present disclosure is or may be insulin- like growth factor (IGF)-I.

METHODS OF IDENTIFYING KIDNEY PROGENITOR CELLS

[0075] In one aspect, the present disclosure provides a method of identifying a human kidney stem/progenitor cells. In this method, the expression of Ephrin receptor 7 (EphA7) is assayed in cells obtained from a kidney. In certain embodiments, identification of the human kidney stem/progenitor cells is performed by a method including: staining the cell with an anti-EphA7 antibody, staining the cell with an anti-EphA7 antibody for use in

immunohistochemistry, and/or by polymerase chain reaction (RT-PCR) of EphA7.

[0076] For example, the amount of EphA7 in a kidney tissue sample, or a fraction of a kidney tissue sample of a known volume may be determined by immunochemistry. Methods of determining EphA7 expression by immunochemistry include, but are not limited to,

Western blotting, ELISA, and immunostaining methods. In certain embodiments, a method of determining EphA7 expression by immunochemistry is performed using an antibody that can bind to EphA7 expression of interest, for instance, an anti-EphA7 antibody or an antibody directed against the protein. Assaying EphA7 expression by immunochemistry requires, for example, at least one antibody against an EphA7 protein or an antigenic fragment thereof, e.g., at least one anti-EphA7 antibody. A primary antibody may be tagged with a detectable label, e.g., a fluorescent marker. Alternatively, a secondary antibody tagged with a detectable label, e.g., a fluorescent marker, that binds specifically to the species isotype of the primary antibody may be used to perform immunochemistry. Methods of determining EphA7 expression by immunochemistry may also involve the use of buffers, blocking reagents, unconjugated primary antibodies, and primary and/or secondary antibodies conjugated to tags that allow for antibody detection, such as fluorescent probes or substrate- specific enzymes.

[0077] Methods of determining EphA7 expression by nucleotide analysis include, but are not limited to, methods of analyzing EphA7 mRNA transcript levels such as Northern blotting and polymerase chain reaction methods, for example, quantitative polymerase chain reaction methods. Nucleotide analysis may be performed using an oligonucleotide probe that binds an EphA7 nucleotide sequence or a pair of oligonucleotide primers capable of amplifying an EphA7 nucleotide sequence via a polymerase chain reaction, for example, by a quantitative polymerase chain reaction. Oligonucleotide probes and oligonucleotide primers may be linked to a detectable tag, such as, for example, a fluorescent tag. In determining EphA7 expression by nucleotide analysis, the practitioner may evaluate EphA7 mRNA transcript concentration in a sample. Alternatively, in determining EphA7 concentration by nucleotide analysis, the practitioner may establish a correlation between EphA7 mRNA transcript abundance and the EphA7 protein abundance in order to extrapolate EphA7 protein concentration based on a measure of EphA7 mRNA transcript abundance.

[0078] Methods of the present disclosure include steps that may be carried out in vitro. For instance, it is contemplated that the steps of measuring EphA7 and/or other biomarker levels, determining the levels of EphA7 and/or other biomarkers in a sample may be carried out in vitro. For example, the level of EphA7 and/or another biomarker in a sample may be determined by performing immunochemistry or nucleotide analysis on the sample in vitro. Examples of contemplated in vitro assays include, but are not limited to, time-resolved fluorescence resonance energy transfer assays (TR-FRET; e.g., Cisbio HTRF ^® ), surface plasmon resonance assays {e.g., Biacore ^® surface plasmon resonance array systems), realtime polymerase chain reaction (RT-PCR), and cell-based assays {e.g., reporter cell-based assays).

[0079] An anti-EphA7 antibody suitable for ELISA is commercially available, such as, for example, from Biorbyt, Cambridge, UK.

Enzyme-Linked Immunosorbent Assay

[0080] In certain embodiments, EphA7 concentration may be determined by Enzyme- linked immunosorbent assay (ELISA). Specifically, levels of EphA7 in a sample, especially a kidney sample, for example, can be determined by ELISA. Assaying EphA7 concentration by ELISA requires at least one antibody against EphA7 protein, e.g., at least one anti-EphA7 antibody, and/or at least one secondary antibody, e.g., at least one labeled secondary antibody. In certain embodiments, the anti-EphA7 antibody is labeled with, e.g., a fluorescent label. In certain embodiments, the anti-EphA7 antibody is not labeled and a secondary antibody capable of binding the species isotype of the primary antibody is labeled, e.g., with a fluorescent probe or enzyme capable of reacting with a specific substrate, thereby providing a detectable signal.

[0081] Performing an ELISA requires at least one capture antibody, at least one detection antibody, and/or at least one enzyme-linked or fluorescent labeled secondary antibody. For example, assaying EphA7 levels by ELISA may require an anti-EphA7 antibody as the capture antibody. The anti-EphA7 antibody is immobilized on a solid support such as a polystyrene microtiter plate. A sample, for example, a kidney sample is then added and allowed to complex with the bound antibody. Unbound serum components are removed with a wash. A detection antibody, e.g., a different anti-EphA7 antibody, e.g., an anti-EphA7 antibody that binds to a different portion of the EphA7 protein than the capture antibody, is added and is allowed to bind to the captured EphA7. The detection antibody is linked to a detectable tag, such as an enzyme, either directly or indirectly, e.g., through a secondary antibody that specifically recognizes the detection antibody. Typically between each step, the plate, with bound protein, is washed with a wash buffer, e.g., a mild detergent solution.

Typical ELISA protocols also include one or more blocking steps, which involve use of a non-specifically-binding protein such as bovine serum albumin to block unwanted nonspecific binding of protein reagents to the plate. After a final wash step, the plate is developed by addition of an appropriate enzyme substrate, to produce a visible signal, which indicates the amount of EphA7 protein in the sample. The substrate can be, e.g., a chromogenic substrate or a fluorogenic substrate. ELISA methods, reagents and equipment are well-known in the art and commercially available.

[0082] An anti-EphA7 antibody suitable for ELISA is commercially available, such as, for example, from Biorbyt, Cambridge, UK.

Nucleotide Analysis [0083] In certain embodiments, EphA7 concentration may be determined by performing a "nucleotide analysis." A nucleotide analysis may include analysis of EphA7 nucleotide transcript levels (e.g., EphA7 mR A transcript levels) in a sample, for example, a kidney sample. EphA7 transcript levels may be determined by Northern blot, for example, a quantitative Northern blot; or polymerase chain reaction, for example, a quantitative polymerase chain reaction. Reagents necessary to perform Northern blot include

oligonucleotide probes, for example, oligonucleotide probes linked to a detectable label. Detectable labels may include fluorescent labels or enzymes capable of reacting with a specific substrate. Reagents necessary to perform polymerase chain reaction include oligonucleotide primers capable of specifically binding to a EphA7 mRNA transcript and amplifying the number of EphA7 mRNA transcripts by polymerase chain reaction.

Oligonucleotide primers may be linked to a detectable label to enable, for example, quantitative polymerase chain reaction. Other reagents necessary to perform quantitative polymerase chain reaction include, but are not limited to, primers capable of amplifying a control transcript signal, for instance, a β-tubulin transcript signal. Buffers, reagents (including oligonucleotide primers and probes), techniques, and equipment necessary for performing Northern blotting and polymerase chain reactions are readily available and are well-known in the art.

TEST KITS

[0084] The present disclosure provides a test kit including certain components for performing the methods disclosed herein. A test kit may enhance convenience, speed and reproducibility in the performance of the disclosed methods. An exemplary kit may include detailed instructions for measuring a EphA7 expression in a kidney sample. Alternatively or optionally, the test kit may include reagents and instructions for testing toxicity and identifying therapeutic agents described in the following section.

[0085] An exemplary immunochemistry-based test kit may contain materials for determining EphA7 protein levels by immunochemistry, for example, by ELISA or by Western blotting. An immunochemistry kit, for example, may contain a primary antibody against EphA7 and a secondary antibody conjugated to a reporter enzyme, e.g., horseradish peroxidase, or a fluorescent probe. In other embodiments, the test kit contains not only antibodies, but also buffers, reagents and detailed instructions for determining EphA7 concentration in a sample. Such a kit may include detailed instructions for measuring EphA7 concentration in a kidney sample.

[0086] In certain embodiments, a test kit may contain materials for determining EphA7 mRNA transcript levels by polymerase chain reaction, for example, by quantitative polymerase chain reaction, or by Northern blotting, for example, by quantitative Northern blotting. A kit for determining EphA7 mRNA transcript levels by Northern blotting may include oligonucleotide probes, for example, oligonucleotide probes linked to a detectable label. Detectable labels may include fluorescent labels or enzymes capable of reacting with a specific substrate. A kit for determining EphA7 mRNA transcript levels by polymerase chain reaction may include oligonucleotide primers capable of specifically binding to a EphA7 mRNA transcript and amplifying the number of EphA7 mRNA transcripts by polymerase chain reaction. Oligonucleotide primers may be linked to a detectable label to enable, for example, quantitative polymerase chain reaction. Other reagents necessary to perform quantitative polymerase chain reaction including, but not limited to, primers capable of amplifying a control transcript signal, for instance, a beta tubulin transcript signal, may be included in the kit. In other embodiments, the kit for determining EphA7 mRNA transcript levels contains not only oligonucleotide primers and/or oligonucleotide probes, but also buffers, reagents and detailed instructions for determining EphA7 mRNA transcript levels in a sample. Such a kit may include detailed instructions for measuring EphA7 mRNA transcript level in a kidney sample.

METHOD OF TESTING TOXICITY OR IDENTIFYING THERAPEUTIC AGENTS

[0087] In one aspect the present disclosure provides a method of testing toxicity of a compound, a therapeutic agent; screening of a new therapeutic agent; and/or identifying a therapeutic agent candidate for treating a mammalian organ or tissue disease or disorder, the method including: (i) introducing a compound, a therapeutic agent, a new therapeutic agent, or a candidate therapeutic agent to a recellularized mammalian organ or tissue, where the recellularized organ or tissue is prepared by a method in which an organ or tissue is decellularized, while retaining an extracellular matrix (ECM) structure, the ECM structure is then treated with one or more metalloproteinase (MMP), and MMP -treated decellularized organ or tissue is then recellularized with human pluripotent stem cells or human adult multipotent stem cells; or (ii) introducing a compound, a therapeutic agent, a new therapeutic agent, or a candidate therapeutic agent to a bioprinted organ or tissue scaffold. The bioprinted organ or tissue scaffold is formed by cellularizing a bioprinted polymeric scaffold with human pluripotent stem cells or human adult multipotent stem cells. In certain embodiments, the recellularized organ or tissue is prepared from a diseased organ or tissue. In certain embodiments, the recellularized organ or tissue is prepared from an organ or tissue characterized as having a tumor or cancer. [0088] In one aspect, the present disclosure provides use of a reconditioned and/or cellularized decondensed ECM structure in an in-vitro method of testing toxicity of a compound or a therapeutic agent; screening of a new therapeutic agent; and/or identifying a therapeutic agent candidate for treating a mammalian organ or tissue disease or disorder; the method comprising introducing a compound, a therapeutic agent, a new therapeutic agent, or a candidate therapeutic agent to the reconditioned and/or cellularized decondensed ECM structure, where the testing toxicity includes ex vivo testing for safety, toxicity, on-target and/or off-target adverse effects, and/or production of toxic metabolites.

[0089] Examples of tumor or cancer of organs or tissues include, but not limited to, kidney, liver, breast, lung, pancreas, skin, bladder, and urethra. In embodiments, the recellularized ECM structure is differentiated to form a tumor or a cancer. In embodiments, the new therapeutic agent or the therapeutic agent candidate is for treating a tumor and/or cancer.

[0090] In embodiments, the recellularized organ or tissue or the bioprinted organ or tissue scaffold may include an ECM structure of a kidney, a liver, a bladder, a vagina, a urethra, a trachea, an esophagus, a heart, a lung, a pancreas, a skin, or other bodily organ. In

embodiments, the recellularized organ or tissue scaffold or the bioprinted organ or tissue scaffold may include cells, tissues, or functional parts of a kidney, a liver, a bladder, a vagina, a urethra, a trachea, an esophagus, a heart, a lung, a pancreas, a skin, or other bodily organ.

[0091] In certain embodiments, toxicity of a therapeutic agent is tested ex vivo testing for safety, toxicity, on-target and/or off-target adverse effects, and/or production of toxic metabolites.

[0092] Cellular toxicity can occur through a diverse range of mechanisms that disrupt cellular integrity. Membrane soluble or pore-forming compounds may act directly on the cytoplasmic membrane and prevent the cell maintaining homeostatic integrity, leading to necrosis. Other compounds may act indirectly to disrupt the cell's biochemical, synthetic, or signaling integrity, leading to apoptosis. Further compounds may act directly or indirectly to damage the cell's genetic integrity, resulting in inheritable mutation, disruption of

proliferative integrity, or apoptosis.

Membrane integrity [0093] Cell toxicity and death caused by drugs can occur through necrosis or apoptosis. In some cases these events may occur sequentially or in parallel depending on the dose and duration of exposure of cells to a test compound. There are several morphological and biochemical differences between necrosis and apoptosis and these may be detected using high-content analysis (HCA) markers. Necrosis typically occurs when cells are exposed to an injury that damages the plasma membrane and prevents the cell from maintaining

homeostasis. Necrosis can be readily detected by imaging the uptake of cell-impermeable fluorescent dyes such as propidium iodide into cells with damaged plasma membranes. In contrast to passive necrosis, apoptosis is an active energy requiring process that occurs under normal physiological conditions where cells are triggered to self-destruct.

[0094] Apoptotic cells show characteristic morphological and biochemical features including nuclear and cytoplasmic condensation, membrane blebbing, and membrane inversion. In the early stages of apoptosis the anionic lipid phosphatidylserine (PS) translocates from the inner side of the plasma membrane to the outer layer. This inversion can be imaged using fluorescently labeled annexin-V (a calcium dependent phospholipid- binding protein) as a marker for early apoptosis. Analysis of cell morphology is a powerful and informative adjunct to the use of fluorescent dyes for investigating toxic action of candidate drugs. Analysis using cytoplasmic and nuclear shape descriptors allows rapid quantitation of cells exhibiting normal and aberrant morphology as a measure of drug effects on cellular integrity.

Proliferative integrity [0095] The cell cycle is of key importance to many areas of drug discovery. This fundamental process provides on the one hand the opportunity to discover new targets for anticancer agents and improved chemotherapeutics, and on the other hand requires the testing of drugs and targets in other therapeutic areas for undesirable effects on the cell cycle.

Measurement of DNA content by flow cytometry of fixed cells stained with fluorescent dyes such as propidium iodide is a standard method of analyzing cell cycle distribution.

Performing the same analysis using high-throughput imaging provides a significant increase in throughput coupled with the ability to multiplex cell cycle analysis determined by DNA content with other parameters. Conventional immmunodetection procedures for detecting 5- bromo-2'-deoxyuridine (BrdU) incorporation into the DNA of replicating cells use acid or alkali denaturation to allow access of the anti-BrdU antibody. However, these methods can significantly alter cell morphology and preclude the use of additional cellular probes. To enable the use of BrdU assays for HCA, nuclease treatment is applied during incubation with monoclonal anti-BrdU to allow antibody access without adversely affecting cell morphology or compromising the signal from multiplexed fluorescent probes. Detection with a Cy™ 5- labeled second antibody allows BrdU incorporation to be multiplexed with GFP or analyzed with other cellular markers. For further in-depth analysis of the effects of compounds on cell cycle and proliferation, GE Healthcare has developed two dynamic cell cycle sensors based on EGFP fusion proteins. Coupled with automated image analysis modules, these G2/M and Gl/S Cell Cycle Phase Markers (CCPMs) allow detailed cell-by-cell analysis for effects of candidate drugs on cell cycle checkpoint progression, delay, and arrest. Imaging of CCPMs can be multiplexed with imaging of DNA content and BrdU incorporation to yield a highly informative picture of drug effects on the cell cycle.

Organelle integrity

[0096] Changes in the shape, distribution, or other characteristics of subcellular organelles can be an important indicator of toxicity in cellular assays. For example, swelling of mitochondria accompanies homeostatic disruption in the early stages of cell necrosis, and leakage of proteins and other factors from mitochondria is an early indicator of apoptosis.

Nuclear integrity

[0097] Changes in the number, size, and shape of nuclei in HCA images are a simple but powerful indicator of toxic effects in cells exposed to test compounds. Decreases in nuclear number/image may indicate inhibitory effects on the cell cycle or may be due to loss of cells through lysis depending on the duration of exposure. Similarly, changes in nuclear size may be indicative of cell cycle blockage in G2 (increase in nuclear size) or apoptotic cell death (decrease in nuclear size with chromatin condensation). In the advanced stages of apoptosis many nuclei will show clear breakdown into two or more fragments. These parameters can readily be quantitated by HCA using a range of IN Cell Analyzer Image Analysis Modules and can be applied to any assay using a nuclear stain to gain valuable additional information on compound toxicity.

Genetic integrity

[0098] Micronucleus induction is a key characteristic of genotoxic compounds. Analysis of micronucleus formation is an important component of toxicology evaluation of new drug candidates and other chemicals and materials, such as food dyes and cosmetics that are intended for human consumption or use, or which may be indirectly or accidentally consumed or ingested. Micronuclei formation occurs during cell division of cells exposed to genotoxic compounds either as a result of DNA strand breakage (clastogenic compounds) or through interference with chromosome segregation (aneugenic compounds) by interference with components of the cell's chromosome separation machinery, such as tubulin. Manual scoring of micronucleus assays is time consuming and subject to operator variance, bias, and error. Automated analysis of micronucleus assays allows significantly faster analysis and consistently objective scoring. The IN Cell Analyzer Micronuclei Formation Analysis Module enables fast automated scoring of micronucleus assays. The software allows the user to set parameters to identify nuclei, segregate mono-nucleate and bi-nucleate cells (for cytokinesis block protocols) based on nuclear DNA content and symmetry, and to define a search area around each nucleus to identify micronuclei. The software is compatible with either single-channel imaging (DNA staining only) or with two-channel imaging (DNA and cytoplasm staining). Additionally the software provides the option to use a third imaging channel in combination with live-cell staining to detect and reject cells with damaged cytoplasmic membranes from assays where cytotoxicity is present. In a typical cytokinetic block assay, exposure of cells to increasing concentrations of compounds of known genotoxicity results in an increase in the percentage of binucleate cells with micronuclei. As cells are exposed to higher doses of compounds, cell cycle inhibition and cytotoxicity results in cell arrest prior to mitosis. This prevents micronuclei formation, with a resulting drop in micronuclei frequency at higher compound doses.

Intracellular signaling integrity

[0099] In addition to effects on the physical integrity of cells, candidate drugs may also interfere with essential cell signaling pathways. To allow evaluation of possible interactions with key intracellular signaling pathways GE Healthcare has developed an extensive range of GFP translocation and nitroreductase (NTR) live-cell reporter gene assays packaged ready to use in adenoviral vectors. Ad-A-Gene Vectors are validated for function, provided in a convenient, ready to use format, and give high-efficiency transduction in both established and primary cell types. Used alone or in combination with other cell integrity readouts in HCA, Ad-A-Gene Vectors provide a powerful toolbox for detailed investigation of toxic effects of candidate drugs on cellular integrity. For further details of Ad-A-Gene Vectors and signaling pathway coverage, visit www.gehealthcare.com/ad-a-gene. METHODS OF TREATMENT OR USE

[00100] In one aspect, the present disclosure provides use of a reconditioned and/or cellularized decondensed ECM structure prepared by a method of the present invention, in regenerative medicine to repair or replace tissues or organs, or in transplantation or as a graft. In one aspect, the present disclosure provides a method of treating kidney disease/disorder in a subject with end-stage renal failure. In one aspect, the present disclosure provides a method of treating chronic renal failure in a subject in need thereof. In embodiments, a recellularized or a bioprinted kidney scaffold in implanted or transplanted to the subject in need of treatment. The implanted or transplanted recellularized or bioprinted kidney scaffold is prepared following the method provided in the present disclosure.

[00101] In embodiments, the method of treating kidney disease/disorder may include treating all of the nephropathic diseases, such as primary renal diseases, nephropathies in systemic diseases, congenital renal diseases, renal infections, nephropathies induced by any nephrotoxic substance and obstructive urinary diseases. Specific examples of the causal disease include, but are not limited to, chronic glomerulonephritis, diabetic nephropathy, chronic pyelonephritis, acute progressive nephritis, gestosis, cystic kidney, nephrosclerosis, malignant hypertension, aephropathies accompanied by various collagen diseases such as SLE, amyloid kidney, gouty kidney, disbolic renal failure, tuberculosis, renal calculosis, malignant tumor in the kidney and urinary tracts, obstructive urinary tract diseases, myeloma and renal hypoplasia.

[00102] The renal failure to be treated with the bioengineered recellularized kidney or the bioprinted kidney of the present disclosure is not particularly limited to either of acute or chronic type. However, the bioengineered recellularized kidney or the bioprinted kidney may be effective on chronic renal failure for which no effective therapy has currently been established and can delay the entrance into dialysis. Even when entered into dialysis, the bioengineered recellularized kidney or the bioprinted kidney may be effective for the preservation of functions of the remained kidney.

[00103] In certain embodiments the method is performed on a human subject having, or at risk of having renal inflammation or renal disease. As herein defined, the term "renal inflammation and disease" extends to all conditions which are substantially characterized by the occurrence of inflammation within the kidney, or where the occurrence of inflammation in the kidney is caused by a disease or an inflammatory condition which primarily affects a site in the body other than the kidney. In particular, inflammation may occur at a site including, but not limited to; the glomerulus, Bowman's capsule or Bowman's space.

Typically, the inflammation results in at least partial impairment of kidney function and/or kidney failure. [00104] Furthermore, the term "renal inflammation and disease" may further include

"kidney disease," in which the term kidney disease generally refers to a disorder of at least one kidney in a human, in which the disorder involves or impairs the function of the kidney(s), this being characterized physiologically by, for example, the leakage of protein into the urine, or by the excretion of nitrogenous waste. The Kidney disease may also result from a primary pathology of the kidney, such as injury to the glomerulus or tubule, or from damage to another organ, such as the pancreas, which adversely affects the ability of the kidney to perform biological functions, such as the retention of protein. Thus, kidney disease in the human can be the direct or indirect effect of a disease condition which may affect other organs. Examples of diseases which affect the kidneys, but which do not specifically target the kidneys are diabetes and systemic lupus. The terms renal disease and kidney disease are used interchangeably herein with the phrase "diseases of the kidney." The kidney disease can, for example, result from, or be a consequence of any change, damage, or trauma to the glomerulus, tubules or interstitial tissue in either the kidney cortex or kidney medulla.

[00105] The kidney disease may also be a progressive kidney disease. The term "progressive kidney disease" as used herein refers to any disease of the kidney that over time (e.g., days, weeks, months, or years) leads to a loss of kidney function. As herein defined, the term "kidney function" generally refers to a physiological property of the kidney, such as the ability to retain protein thereby preventing proteinuria (e.g. , albuminuria). Kidney function can be assessed, for example, by glomerular filtration rate (e.g., creatinine clearance), excretion of protein in urine e.g. albuminuria, blood urea nitrogen, serum or plasma creatinine, or any combination thereof.

[00106] Examples of specific conditions which fall within the meaning of the term "renal inflammation and disease" include, but are not limited to: renal disorders which include, but are not limited to: chronic renal failure, acute renal failure, heterologous nephrotoxic nephritis, glomerulonephritis, sclerosis of the glomerulus, systemic lupus erythematosus (SLE), diabetic nephropathy, diabetic nephropathy wherein the diabetic nephropathy accompanies sclerosis of the liver, and glomerulonephritis wherein the glomerulonephritis is accompanied by sclerosis of the liver.

[00107] In certain further embodiments, renal inflammation and disease may relate to an immune-mediated disease which affects the cells of the kidney and/or kidney function. Such conditions may include, but are not limited to: Immunoglobulin A nephropathy,

membranoproliferative glomerulonephritis, mesangial proliferative glomerulonephritis, nonproliferative glomerulonephritis, membranous glomerulonephritis, minimal-change disease, primary focal segmental glomerulosclerosis (FSGS), fibrillary glomerulonephritis, immunotactoid glomerulonephritis, proliferative glomerulonephritis, progressive

glomerulonephritis, anti-GBM disease, kidney ischemia, kidney vasculitis, including disease associated with anti-neutrophil cytoplasmic antibodies (ANCA) (e.g., Wegener

granulomatosis), lupus nephritis cryoglobulinemia-associated glomerulonephritis, bacterial endocarditis, Henoch-Schonlein purpura, postinfectious glomerulonephritis, Hepatitis C, diabetic nephropathy, myloidosis, hypertensive nephrosclerosis, light-chain disease from multiple myeloma, secondary focal glomerulosclerosis, and hypertensive nephrosclerosis.

[00108] The term "renal inflammation and disease" also encompasses acute renal failure. Acute renal failure ("ARF") refers to the clinical conditions associated with rapid, steadily increasing azotemia, with or without oliguria (<500 mL/day). The cause of ARF can be grouped into three diagnostic categories: prerenal (inadequate renal perfusion); postrenal (obstruction); and renal. The pathophysiology of ARF is complex and multifactorial. Current concepts suggest that ARF may result from direct renal tubular injury, renal ischemia or intra-tubular obstruction. Clinically, ARF results in diminished glomerular filtration and reduced secretion of metabolic waste products, water, and electrolytes. Fluid overload, electrolyte imbalances and the uremic syndrome result in organ dysfunction. Organ dysfunction may ultimately result in death.

[00109] In certain embodiments, the method of treating liver disease/disorder may include treating liver cancer and liver metastases. Since diamidines (e.g., pentamidine) preferentially accumulate in the liver in therapeutic concentrations following oral administration, they could be used orally for the treatment liver cancer or liver metastasis. In certain embodiments, the liver cancer is intrahepatic bile duct cancer or hepatocarcinoma. In certain embodiments, the liver metastasis is liver dominant cancer metastasis or liver limited cancer metastasis. [00110] In oncology, most metastasis occurs in the liver, a vital organ. Rapidly, the primary cancer that has metastasized in the liver becomes life-threatening. In certain embodiments, there is provided, the uses or methods as defined herein, for treating liver dominant colorectal cancer metastasis. Liver dominant cancer metastasis refers to metastases that are mainly located in the liver (e.g., determination of size, number and type of lesions). Liver limited cancer metastasis refers to metastases that are only located in the liver (e.g., determination of size, number and type of lesions).

[00111] In certain embodiments, the cancer condition or status of the patient is determined in accordance with the Response Evaluation Criteria in Solid Tumors (RECIST). See for example EUROPEAN JOURNAL OF CANCER 45 (2009) 228-247. In one embodiment there is provided, the uses or methods as defined herein, for treating metastasized cancer. In certain embodiments, the patient has one or more of the following conditions: a. Inoperable liver tumors, minor lung or bone metastasis or abnormal hepatic enzyme level.

[00112] In certain embodiments there is provided the uses or methods as defined herein, wherein the primary cancer originates from squamous cell carcinoma cells, larger cell carcinoma of the lymph node cells, breast cancer cells, colon cancer cells, lung carcinoma cells, melanoma cells, pancreatic cancer cells, leukemia cells, non-small cell lung cancer cells, colon cancer cells, central nervous system (CNS) cancer cells, ovarian cancer cells, renal cancer cells or prostate cancer cells. [00113] In certain embodiments there is provided, the use or method of as defined herein wherein the primary cancer originates from pancreatic cancer cells, colon cancer cells, breast cancer cells or ovarian cancer cells. In one embodiment the diamidine analogues (e.g., pentamidine) are used in combination with standard chemotherapy after the subject is transplanted with a bioengineered liver or a bioprinted liver prepared by the methods of the present disclosure.

[00114] In certain embodiments, the method of treating liver disease/disorder may include treating other liver conditions or diseases such as alcoholic liver disease (including acute alcoholic hepatitis), cirrhosis, cysts, primary biliary cirrhosis, fatty liver disease (NAFLD), fibrosis, jaundice, primary sclerosing cholangitis (PSC), hemochromatosis, primary biliary cirrhosis, or Alpha- 1 Antitrypsin Deficiency. Liver damage is determined by standard liver function tests and or by imaging (CT, X-Ray, MRI etc.). Liver function tests include bilirubin, ammonia, gamma-glutamyl transferase (GGT), alanine aminotransferase (ALT or SGPT), aspartate aminotransferase (AST or SGOT), and alkaline phosphatase (ALP).

Non-Alcoholic Fatty Liver Disease (NAFLD) and Non-Alcoholic Steatohepatitis (NASH)

[00115] NAFLD and its more severe form NASH are associated with several diseases (obesity, type 2 diabetes, dyslipidemia and hypertension), having insulin resistance as the common factor. These conditions cluster to form the insulin resistance or metabolic syndrome, carrying a high risk for cardiovascular complications. NASH itself, as well as pure fatty liver, is an insulin-resistant state, not only in subjects with additional metabolic disorders, but also in lean subjects. Because the histopathology of NASH resembles that of alcohol- induced steatohepatitis (ASH), these 2 conditions share common pathogenic aspects. Immunological mechanisms play a pivotal role in the pathogenesis of ASH. This has been well demonstrated by studies of patients and experimental animals. In hospitalized patients with severe ASH and NASH, serum levels of several pro -inflammatory cytokines, including TNF-. alpha., are increased significantly. Cytokine levels correlate well with liver disease severity.

Alcoholic Liver Disease (ALD)

[00116] Alcoholic liver disease occurs after years of heavy drinking. Alcohol can cause inflammation in the liver. ALD has three stages: 1) alcoholic fatty liver disease; 2) alcoholic hepatitis and 3) Cirrhosis. Alcoholic hepatitis (not related to infectious hepatitis) is the second, more serious stage of ALD. It occurs when alcohol misuse over a longer period causes the tissues of the liver to become inflamed. Damage caused by alcoholic fatty liver disease or Alcoholic hepatitis can usually be reversed if the use of alcohol is stopped.

[00117] Cirrhosis is the final stage of alcohol-related liver disease, which occurs when the liver becomes significantly scarred. Cirrhosis is generally not reversible, but stopping drinking alcohol can prevent further damage and significantly increase life expectancy. In one aspect the ALD is diagnosed with blood test, liver biopsy or imagery (ultrasound scan, computerized tomography (CT) scan).

Cirrhosis

[00118] Cirrhosis is scarring of the liver caused by many forms of liver diseases and conditions, such as hepatitis and chronic alcohol abuse. In certain embodiments, the ALD is diagnosed with blood test, liver biopsy or imagery (ultrasound scan, computerized tomography (CT) scan).

[00119] In certain embodiments, in the uses and methods as described herein the cirrhosis patient will be treated orally with the diamidine analogue in order to prevent, control or reduce liver damage.

DECELLULARIZATION METHODS

[00120] The disclosure provides for methods and materials to decellularize an organ or tissue. Physical methods and chemical and biologic agents are used in combination to lyse cells, often followed by a rinsing step to remove cell remnants and debris. Effective decellularization is dictated by factors such as tissue density and organization, geometric and biologic properties desired for the end product, and the targeted clinical application.

[00121] One or more cellular disruption solutions may be used to decellularize an organ or tissue. A cellular disruption solution generally includes at least one detergent, such as SDS, PEG, or Triton X. A particularly preferred detergent is Triton X. A cellular disruption solution can include water such that the solution is osmotically incompatible with the cells. Alternatively, a cellular disruption solution can include a buffer (e.g., PBS) for osmotic compatibility with the cells. Cellular disruption solution also can include enzymes such as, without limitation, one or more collagenases, one or more dispases, one or more DNases, or a protease such as trypsin. In certain embodiments, cellular disruption solution also or alternatively can include inhibitors of one or more enzymes (e.g., protease inhibitors, nuclease inhibitors, and/or collagenase inhibitors).

[00122] In certain embodiments, the renal artery and ureter of a pig kidney may be cannulated using arteriotomy cannulas and perfused with decellularization solutions using peristaltic pump, e.g., at about 10 ml/min. The kidney may first be perfused with distilled water for about 72 h followed by an ionic detergent, e.g., sodium deoxycholate (SDC), a non- ionic detergent, and a DNAse for a few hours with each solution. After treatment with each detergent solution, the kidney may be washed overnight with distilled water to remove cell debris. The cycle from ionic to DNase may be repeated until complete decellularization. At the end of decellularization, the kidneys may be washed with distilled water. Distilled water and non-ionic detergent may be used throughout protocol. In certain embodiments, the detergent may include contained about 0.2% sodium azide. After complete decellularization, several biopsies may be collected from all kidneys and processed for histology, DNA quantification, collagen quantification, elastin quantification and glycosaminoglycans quantification.

[00123] The decellularization method of the present disclosure may result in kidneys in which cellular compartments may be completely removed, leaving behind an acellular ECM scaffold, which may retain its complex 3D structure. The acellular scaffold may retain structural ECM proteins with a complete removal of DNA. The retention of ECM

components is critical for attachment and proliferation of cells seeded for cellularization in subsequent steps. Preservation of intact tubular, vascular, and glomerular structure with the ECM scaffold with native signals of the method of the present disclosure facilitates the process of recellularization by the precursor cells.

[00124] Sequential treatment may include repeating treatment with at least one of the cellular disruption solutions in the treatment sequence. In certain embodiments, the organ or tissue may be treated by decellularization cycles comprising the sequential treatment of one or more cellular disruption solutions in the same order until the desired level of

decellularization is achieved. In certain embodiments, the number of decellularization cycles may be at least or equal to 2, at least or equal to 3, at least or equal to 4, at least or equal to 5, at least or equal to 6, at least or equal to 7, at least or equal to 8, at least or equal to 9, at least or equal to 10, at least or equal to 11, at least or equal to 12, at least or equal to 13, at least or equal to 14, at least or equal to 15, at least or equal to 16, at least or equal to 17, at least or equal to 19, or at least or equal to 20 cycles.

[00125] In certain embodiments, each cellular disruption solution may further include additional components, such as antibiotics (i.e., penicillin, streptomycin, and amphotericin), ethylenediaminetetraaceticacid (EDTA) disodium salt dehydrate (EDTA), and/or phenyl methyl sulfonyl fluoride (PMSF). For example, a cellular disruption solution that comprises DNase I may also include calcium chloride and magnesium chloride (A12858, Life

Technologies) to activate the enzyme.

[00126] In certain embodiments, perfusion methods may be used to treat the organ or tissue with cellular disruption solutions for decellularization of the organ or tissue. Alternating the direction of perfusion (e.g., antegrade and retrograde) can help to effectively decellularize the organ or tissue. Depending upon the size and weight of the tissue and the particular detergent(s) and concentration of detergent(s) in the cellular disruption solution, an organ or tissue generally may be perfused from about 2 to about 12 hours per gram of organ or tissue with cellular disruption medium. Including washes, an organ or tissue may be perfused for up to about 12 to about 72 hours per gram of tissue. Perfusion generally is adjusted to physiologic conditions including pulsatile flow, rate and pressure. Perfusion decellularization as described herein can be compared to immersion decellularization as described, for example, in U.S. Pat. Nos. 6,753,181 and 6,376,244 (relevant parts of which are incorporated herein).

[00127] As indicated herein, a decellularized organ or tissue may retain the extracellular matrix (ECM) components of the vascular tree. ECM components can include any or all of the following: fibronectin, fibrillin, laminin, elastin, members of the collagen family (e.g., collagen I, III, and IV), glycosaminoglycans, ground substance, reticular fibers and thrombospondin, which can remain organized as defined structures such as the basal lamina. Successful decellularization is defined as the absence of detectable myofilaments, endothelial cells, smooth muscle cells, and nuclei in histologic sections using standard histological staining procedures.

[00128] Upon the initial decellularization process, the morphology and the architecture of the ECM may be maintained (e.g., ECM ministructure remain substantially intact) during and following the process of decellularization. "Morphology," as used herein, refers to the overall shape of the organ or tissue or of the ECM, while "architecture," as used herein, refers to the exterior surface, the interior surface, and the ECM there between. The morphology and architecture of the ECM can be examined visually and/or histologically to verify that the decellularization process has not compromised the three-dimensional structure and bioactivity of the ECM scaffold. Histological analysis by staining (i.e., H&E, MT or VVG) may be useful to visualize decellularized blood vessel architecture and preservation of ECM components, such as collagen I, collagen IV, laminin and fibronectin. Other methods and assays known in the art may be useful for determining the preservation of ECM components, such as glycosaminoglycans and collagen Importantly, residual angiogenic or growth factors remain associated with the ECM scaffold after decellularization. Examples of such angiogenic or growth factors include, but are not limited to VEGF-A, FRF-2, PLGF, G-CSF, FGF-1, Follistatin, HGF, Angiopoietin-2, Endoglin, BMP-9, HB-EGF, EGF, VEGF-C, VEGF-D, Endothelin-1, Leptin, and other angiogenic or growth factors known in the art. METHOD OF CELLULARIZING AN ORGAN/TISSUE SCAFFOLD

[00129] In one aspect, the present disclosure provides a method of preparing a recondition and/or recellularized organ or tissue (e.g., a kidney, a liver, a heart, a lung, a pancreas, a skin, or any solid organ) scaffold. In embodiments, a decellularized kidney sample, which includes an extracellular matrix (ECM) vascular architecture, glomerular capillaries, and tubular membrane, is recellularized with human pluripotent stem cells or human adult multipotent stem cells. In this method a decellularized kidney sample is prepared following the method provided in the present disclosure, and then treated with one or more

metalloproteinase (MMP, e.g., MMP1, MMP2, MMP3, MMP7, MMP9, MMP 13, and/or MMP14). MMP-treated kidney scaffold is then seeded with human pluripotent stem cells or human adult multipotent stem cells. The scaffold is then perfused one or more times with a media for growth and differentiation of the cells. The differentiated cells form a

recellularized kidney.

[00130] In one aspect, the present disclosure provides a method of reconditioning and/or cellularizing a decondensed extracellular matrix (ECM) structure in an acellular structure of a mammalian organ or tissue, or a bioprinted ECM scaffold, comprising: introducing human pluripotent stem cells or human adult multipotent stem cells, or blood comprising progenitor cells to a decondensed ECM structure prepared by introducing one or more metalloproteinase (MMP, e.g., MMP1, MMP2, MMP3, MMP7, MMP9, MMP 13, and/or MMP 14) to the ECM structure; and perfusing media for growth and differentiation of the cells, thereby

reconditioning and/or cellularizing the decondensed ECM structure in the acellular structure of a mammalian organ or tissue, or the bioprinted ECM scaffold.

[00131] During recellularization, the MMP-treated decellularized organ or tissue may be maintained under conditions in which at least some of the seeded cells can multiply and/or differentiate within and on the MMP-treated decellularized organ or tissue. Those conditions include, without limitation, the appropriate temperature and/or pressure, electrical and/or mechanical activity, force, the appropriate amounts of 0 ₂ and/or CO2, an appropriate amount of humidity, and sterile or near-sterile conditions. During recellularization, the MMP-treated decellularized organ or tissue and the cells attached thereto are maintained in a suitable environment. For example, the cells may require a nutritional supplement (e.g., nutrients and/or a carbon source such as glucose), exogenous hormones or growth factors, and/or a particular pH. [00132] The present disclosure also provides for a bioreactor for recellularizing a MMP- treated decellularized organ or a tissue under the appropriate conditions, as described herein. Specifically, the bioreactor may be a completely closed chamber that is large enough to fit the organ or tissue to be recellularized and can be sterilized, and may include a tube or channel connected to a pumping mechanism (e.g. , a peristaltic pump) for supplying cells and/or media through the organ or tissue within the bioreactor.

[00133] The population of cells utilized for recellularization may be isolated from a heterogeneous population of cells. In certain embodiments, the present disclosure provides that population of cells may be pluripotent stem or progenitor cells, e.g., isolated from a solid organ (e.g., kidney or liver) or adult multipotent stem cells. Methods for isolating particular populations of cells from a population are known in the art. Such methods include

lymphotrap, density gradients, differential centrifugation, affinity chromatography, and FACS flow cytometry. Markers known in the art that identify particular populations of cells of interest may be used to isolate the cells from the heterogeneous population. For example, CD 133 is known to be expressed on the surface of stem cells or stem- like cells derived from the bone marrow. Selection for CD 133+ cells can be achieved by utilization of MACs beads and specific antibodies that recognize CD 133. Markers specific for endothelial progenitor or smooth muscle cell progenitor cells can also be utilized to purify the population of cells of interest. [00134] In certain embodiments, the population of cells may be cultured in vitro prior to introduction to the MMP -treated decellularized organ or tissue. The purpose of culturing in vitro includes expanding cell numbers and differentiating cells to specific cell lineages of interest. The present disclosure provides that population of cells may be first isolated from a heterogeneous population prior to culturing in vitro. The present disclosure provides that population of cells may be bone marrow-derived stem or progenitor cells (e.g., CD 133+ cells) and may be differentiated in vitro prior to introduction to the acellular organ or tissue scaffold. Various differentiation protocols are known in the art and include, for example, growing cells in growth media supplemented with factors, agent, molecules or compounds that induce differentiation into endothelial cells or smooth muscle cells. [00135] The number of cells that is introduced to a MMP -treated decellularized organ or tissue in order to generate a recellularized or a bioprinted organ or tissue may be dependent on the size (i.e., length, diameter, or thickness) of the organ or tissue and the types of cells used for recellularization (i.e., stem cells vs. more differentiated cells, such as whole blood). Different types of cells may have different tendencies as to the population density those cells will reach. By way of example, a decellularized organ or tissue can be "seeded" with at least or equal to about 1,000 (e.g., at least or equal to about 10,000, at least or equal to about 100,000, at least or equal to about 1,000,000, at least or equal to about 10,000,000, or at least or equal to about 100,000,000) cells; or can have from about 1,000 cells/mg tissue (wet weight, i.e., prior to decellularization) to about 10,000,000 cells/mg tissue (wet weight) attached thereto.

[00136] The population of cells can be introduced ("seeded") into a MMP -treated decellularized organ or tissue by injection into one or more locations. In addition, more than one type of cell (i.e., endothelial cells or smooth muscle cells) can be introduced into a MMP- treated decellularized organ or tissue. For example, the population of cells can be introduced to a MMP -treated decellularized organ or tissue by perfusion. After perfusion of the cells, expansion and/or differentiation media may be perfused through the MMP -treated

decellularized organ or tissue to induce growth and/or differentiation of the seeded cells. The present disclosure provides that anti-coagulant agents, such as heparin, may be administered prior to and/or simultaneously to the introduction the population of cells.

[00137] Expansion and differentiation media, as used in the present disclosure, includes cell growth medium containing supplements and factors required for proliferation of endothelial cell or smooth muscle cell, and differentiation to endothelial cell or smooth muscle cell. The present disclosure provides that differentiation medium for endothelial cells may be the same as the growth/proliferation medium for endothelial cells. For example, additional factors or supplements present in endothelial growth or differentiation media may include, but are not limited to: ascorbic acid, hydrocortisone, transferrin, insulin, recombinant human VEGF, human fibroblast growth factor, human epithelial growth factor, heparin and gentamycin sulfate. The present disclosure provides that differentiation medium for smooth muscle cells may be the same as the growth/proliferation medium for smooth muscle cells. For example, additional factors or supplements present in endothelial growth or differentiation media may include, but are not limited to: smooth muscle growth supplement, smooth muscle

differentiation supplement, MesenPro, and transforming growth factor βΐ . At minimum, growth and differentiation media comprise a base media (i.e., MCDB131, M231, or DMEM) heat inactivated serum (for example, at 10%), glutamine and antibiotics (i.e., penicillin, streptomycin, amphotericin). COMPOSITION

[00138] In one aspect, the present disclosure provides an acellular organ or tissue (e.g., a MMP -treated acellular organ or tissue, or a bioprinted polymeric scaffold) including extracellular matrix (ECM) components/composition of a mammalian organ or tissue (e.g. , a kidney, a liver, a bladder, a vagina, a urethra, a trachea, an esophagus, a heart, a lung, a pancreas, a skin, or other bodily organ) or a functional part thereof, cellularized with human pluripotent stem cells or human adult multipotent stem cells for use in therapy or for testing toxicity of a compound, a therapeutic agent, screening a new therapeutic agent, and/or identifying a therapeutic agent candidate. The present disclosure provides an organ or tissue (or a functional part thereof) scaffold composition including ECM components/composition and a polymer, cellularized with human pluripotent stem cells or human adult multipotent stem cells for use in therapy.

[00139] The composition of the present disclosure (e.g. , a MMP -treated acellular organ or tissue, or a bioprinted polymeric scaffold) includes an ECM composition. In embodiments, the ECM composition may be in gel form.

[00140] The mammalian tissue of the composition of the present disclosure may include epithelial and/or connective tissue. The organ or tissue may be obtained from a mammal, (e.g., a pig, a cow, a lamb, a goat, a sheep, a chimpanzee, a monkey, a human, or other primate). The organ or tissue may be autologous or allogenic. The composition is or may include mammalian organ or tissue and/or extracellular matrix (ECM)

components/composition of mammalian organ or tissue, one or more buffers, and one or more of: nutrients, growth factors, and/or polymers. The buffer is or may be a complete cell culture medium.

[00141] In certain embodiments, the ECM components may be added to the biopolymer in a powder form. In the present disclosure the powder for preparing the composition of the present disclosure is prepared by treating a tissue with a chemical, freeze-drying the chemical treated tissue, and homogenization of freeze-dried tissue. In embodiments, the powder is filamentous.

[00142] In certain embodiments, the extracellular matrix material and/or the extracellular matrix composition on an organ or tissue scaffold or a bioprinted polymeric scaffold may include gelatin and fibrin. The gelatin and fibrin may form an interpenetrating polymer network that mimics natural extracellular matrix (ECM) and may be optimized for cell attachment, bioprinting, transparency, and biocompatibility. The fibrin-gelatin

interpenetrating polymer network may be created by mixing solutions of fibrinogen and gelatin with transglutaminase (TG), a slow-acting Ca ²⁺ dependent enzyme, to create a gel- precursor solution that may later be mixed with thrombin to create a fibrin gel backbone. Fibrin may be made from a concentrated fibrinogen solution that has been activated by thrombin and calcium chloride. Fibrin is a rapidly coagulating phase that permits rapid, controllable gelation of a printed structure. Advantageously, fibrin and gelatin can be welded together via mobile surface chain entanglement, while forming a strong interface. Creating monolithic gels of this nature is possible due to the slow crosslinking kinetics of

transglutaminase (TG). Although thrombin rapidly induces fibrin gel formation, the gelatin present allows one to print ink on an already cast layer, and, ultimately, to encapsulate with liquid gelatin- fibrin. The two phases may weld together, creating a monolithic gel. This material system can be readily tailored to modify gelation kinetics, interface adhesion, mechanical properties, optical properties, and cell-material interactions.

[00143] To form the extracellular matrix composition, a microgel (e.g., a poly(acrylic acid) (PAA) microgel) may be used as a rheological modifier and blended with one or more extracellular matrix materials, such as gelatin methacrylate. A semi-interpenetrating polymer network (semi-IPN) may be formed. Microgels may be understood to include colloidal gel particles that are composed of chemically cross-linked three-dimensional polymer networks. Microgels may act as sterically stabilized colloids with only a shell and no core. They can vary in composition and may include PAA, polystyrenes, PEG, and/or other biomaterials. In certain embodiments, a natural extracellular matrix or biomaterial may be converted into a microgel form to impart the ideal rheology. Examples of suitable biomaterials include hyaluron, collagen, alginate, fibrin, albumin, fibronectin, elastin, or matrigel. Alternatively, synthetic materials such as PEG, acrylates, urethanes, or silicones may be modified in a similar manner.

[00144] The recellularized organ or tissue (or a functional part thereof) prepared from a MMP -treated acellular organ or tissue scaffold, or the bioprinted organ or tissue (or a functional part thereof) scaffold of the present disclosure may be used in ex vivo testing for safety, toxicity, on-target and/or off-target adverse effects, and/or production of toxic metabolites of a therapeutic agent; screening of a new therapeutic agent; or identifying a therapeutic agent candidate for treating an organ or tissue disease or disorder of a subject in need thereof.

[00145] The recellularized organ or tissue (or a functional part thereof) prepared from a MMP -treated acellular organ or tissue scaffold, or the bioprinted organ or tissue (or a functional part thereof) scaffold of the present disclosure may be used in treating an organ or tissue disease or disorder of a subject in need thereof.

[00146] The recellularized organ or tissue (or a functional part thereof) prepared from a MMP -treated acellular organ or tissue scaffold, or the bioprinted organ or tissue (or a functional part thereof) scaffold of the present disclosure may be used as an organ or tissue for transplantation in a subject.

[00147] The recellularized organ or tissue (or a functional part thereof) prepared from a MMP -treated acellular organ or tissue scaffold, or the bioprinted organ or tissue (or a functional part thereof) scaffold of the present disclosure may be used in treating chronic renal failure in a subject in need thereof. [00148] The composition of the present disclosure includes or may include cells such as, but not limited to, hematopoietic stem cells (adult stem cells; i.e., hemocytoblasts) from the bone marrow that give rise to red blood cells, white blood cells, and platelets; mesenchymal stem cells (adult stem cells) from the bone marrow that give rise to stromal cells, fat cells, and types of bone cells; epithelial stem cells (progenitor cells) that give rise to the various types of skin cells; neural stem cells and neural progenitor cells that give rise to neuronal and glial cells; and muscle satellite cells (progenitor cells) that contribute to differentiated muscle tissue, umbilical cord derived stem cells, multipotent adult progenitor cells (listed in paragraph [0027]), whole-blood derived stem or progenitor cells such as endothelial stem cells, endothelial progenitor cells, smooth muscle progenitor cells, whole blood, peripheral blood, and any cell populations that can be isolated from whole blood. The progenitor cells are defined as cells that are committed to differentiate into one type of cells. For example, endothelial progenitor cells means cells that are programmed to differentiate into endothelial cells; smooth muscle progenitor cells means cells that are programmed to differentiate into smooth muscle cells. Progenitor cells in whole blood or peripheral blood includes population of uncommitted and/or committed cells, such as pluripotent cells or totipotent cells. [00149] The composition of the present disclosure may also include a growth factor. The growth factor in the composition of the present disclosure is or may be one or more of:

granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin (IL)-3, IL-4, neutrophin (NT)-6, pleiotrophin (HB-GAM), midkine (MK), interferon inducible protein- 10 (IP- 10), platelet factor (PF)-4, monocyte chemotactic protein- 1 (MCP- 1 ), RANTES (CCL-5 , chemokine (C-C motif) ligand 5), IL-8, IGFs, fibroblast growth factor (FGF)-l, FGF-2, FGF- 3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, transforming growth factor (TGF)-P,

VEGF, platelet-derived growth factor (PDGF)-A, PDGF-B, HB-EGF, hepatocyte growth factor (HGF), tumor necrosis factor (TNF)-a, insulin-like growth factor (IGF)-I, and any combination(s) thereof. The growth factor in the composition of the present disclosure is or may be GM-CSF. The growth factor in the composition of the present disclosure is or may be IL-3. The growth factor in the composition of the present disclosure is or may be IL-4. The growth factor in the composition of the present disclosure is or may be NT-6. The growth factor in the composition of the present disclosure is or may be HB-GAM. The growth factor in the composition of the present disclosure is or may be MK. The growth factor in the composition of the present disclosure is or may be IP- 10. The growth factor in the composition of the present disclosure is or may be PF-4. The growth factor in the composition of the present disclosure is or may be MCP-1. The growth factor in the composition of the present disclosure is or may be CCL-5. The growth factor in the composition of the present disclosure is or may be IL-8. The growth factor in the

composition of the present disclosure is or may be IGFs. The growth factor in the composition of the present disclosure is or may be (FGF)-l, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, or FGF-9. The growth factor in the composition of the present disclosure is or may be transforming growth factor (TGF)-p. The growth factor in the composition of the present disclosure is or may be VEGF. The growth factor in the composition of the present disclosure is or may be platelet-derived growth factor (PDGF)-A. The growth factor in the composition of the present disclosure is or may be PDGF-B. The growth factor in the composition of the present disclosure is or may be HB-EGF. The growth factor in the composition of the present disclosure is or may be hepatocyte growth factor (HGF). The growth factor in the composition of the present disclosure is or may be tumor necrosis factor (TNF)-a. The growth factor in the composition of the present disclosure is or may be insulin-like growth factor (IGF)-I. [00150] In certain embodiments, the ECM components in the composition (e.g., a bioprinted polymeric scaffold) may include collagen, elastin, and/or sulfated glycosaminoglycans (GAGs). In certain embodiments, the composition (e.g., a bioprinted polymeric scaffold) may include ribbon- like fibers. The fibers may be about 1 μιη - about 40 μιη (e.g., about 1 μιη - about 40 μιη, about 1 μιη - about 35 μιη, about 1 μιη - about 30 μιη, about 1 μιη - about 25 μιη, about 1 μιη - about 20 μιη, about 1 μιη - about 15 μιη, about 1 μιη - about 10 μιη, about 1 μιη - about 5 μιη, about 5 μιη - about 40 μιη, about 10 μιη - about 40 μιη, about 15 μιη - about 40 μιη, about 20 μιη - about 40 μιη, about 25 μιη - about 40 μιη, about 30 μιη - about 40 μιη, about 35 μιη - about 40 μιη, about 1 μιη, about 2 μιη, about 3 μιη, about 4 μιη, about 5 μm, about 6 μιη, about 7 μιη, about 8 μm, about 9 μιη, about 10 μm, about 1 1 μιη, about 12 μιη, about 13 μm, about 14 μιη, about 15 μιη, about 16 μm, about 17 μιη, about 18 μm, about 19 μιη, about 20 μιη, about 21 μm, about 22 μιη, about 23 μm, about 24 μιη, about 25 μιη, about 26 μm, about 27 μιη, about 28 μm, about 29 μιη, about 30 μιη, about 31 μιη, about 32 μιη, about 33 μm, about 34 μιη, about 35 μm, about 36 μιη, about 37 μιη, about 38 μm, about 39 μιη, or about 40 μιη) in width; about 0.1 μιη - about 10 μιη (e.g., about 0.1 μηι - about 10 μιη, about 0.1 μιη - about 9 μιη, about 0.1 μm - about 8 μιη, about 0.1 μιη - about 7 μm, about 0.1 μιη - about 6 μιη, about 0.1 μm - about 5 μιη, about 0.1 μιη - about 4 μιη, about 0.1 μm - about 3 μιη, about 0.1 μιη - about 2 μm, about 0.1 μιη - about 1 μιη, about 0.5 μm - about 10 μιη, about 1 μιη - about 10 μιη, about 2 μm - about 10 μιη, about 3 μιη - about 10 μιη, about 4 μm - about 10 μιη, about 5 μιη - about 10 μm, about 6 μιη - about 10 μιη, about 7 μm - about 10 μιη, about 8 μιη - about 10 μιη, about 9 μm - about 10 μιη, about 0.1 μιη, about 0.5 μm, about 1.0 μιη, about 1.5 μm, about 2.0 μιη, about 2.5 μιη, about 3.0 μm, about 3.5 μιη, about 4.0 μm, about 4.5 μιη, about 5.0 μιη, about 5.5 μm, about 6.0 μιη, about 6.5 μm, about 7.0 μιη, about 7.5 μιη, about 8.0 μm, about 8.5 μιη, about 9.0 μιη, about 9.5 μm, or about 10.0 μιη) in thickness; and/or about >70 μm - about <4000 μm (e.g., about 70 μιη, about 75 μm, about 80 μιη, about 85 μιη, about 90 μm, about 95 μιη, about 100 μm, about 125 μιη, about 150 μιη, about 175 μm, about 200 μιη, about 225 μm, about 250 μιη, about 275 μιη, about 300 μm, about 325 μιη, about 350 μm, about 375 μιη, about 400 μιη, about 425 μm, about 450 μιη, about 475 μm, about 500 μιη, about 525 μιη, about 550 μm, about 575 μιη, about 600 μιη, about 625 μm, about 650 μιη, about 675 μm, about 700 μιη, about 725 μιη, about 750 μm, about 775 μιη, about 800 μm, about 825 μιη, about 850 μιη, about 875 μm, about 900 μιη, about 925 μm, about 950 μιη, about 975 μιη, about 1000 μm, about 1250 μιη, about 1500 μm, about 1750 μιη, about 2000 μιη, about 2250 μηι, about 2500 μηι, about 2750 μηι, about 3000 μηι, about 3250 μηι, about 3500 μηι, about 3750 μηι, or about 4000 μηι) in length. In certain embodiments, the fibers may be of about 5 μιη - about 30 μιη in width. In certain embodiments, the fibers may be of about 0.5 μιη - about 5 μιη in thickness. In certain embodiments, the fibers may be of about >75 μιη - about <800 μιη in length. The present disclosure includes all intervening numbers of the ranges indicated.

EXAMPLES

[00151] Example 1: Decellularization of Porcine Kidney Organ retrieval [00152] Porcine kidneys (n=9) were retrieved from healthy pigs (40- 50 kg) from the animal laboratory-EBM (Sahlgrenska, Gothenburg). The abdominal cavity was opened through a long midline incision and the ureter, renal artery and renal vein were separated clearly and cut with scissor. The renal artery was cannulated with 4mm DLP arteriotomy cannula

(Medtronic, USA) to allow antegrade arterial perfusion and perfused with distilled water containing 0.2% Sodium Azide (SA) (Sigma, Germany) and 1.86% ethylene diamine tetra acetic acid (EDTA) (Alfa Aesar, Germany) to remove the blood. Later, the organs were rinsed twice with phosphate-buffered saline (PBS) and stored at -20° C. until use. Two biopsies of 2 cm ³ were collected from 3 new kidneys and one snap frozen in liquid nitrogen and stored at -80° C. for later analysis and the other was fixed in formalin for

immunohistochemical analysis.

Decellularized pig kidney

[00153] The renal artery and ureter were cannulated using arteriotomy cannulas and perfused with decellularization solutions using peristaltic pump (Oina, Sweden) at 10 ml/min. The kidney was first perfused with distilled water for 72 h followed by 4% sodium

deoxycholate (SDC) (Sigma, Germany), 4% triton-xl00 (Alfa Aesar, Germany) and 40 IU/ml deoxyribo nuclease I (DNase I) (Worthington, USA) for 4 h with each solution. After treatment with each detergent solution, the kidney was washed overnight with distilled water to remove cell debris. The cycle from SDC to DNase was repeated until complete

decellularization. At the end of decellularization, the kidneys were washed for 72 h with distilled water. Distilled water and Triton X 100 used throughout protocol contained 0.2% sodium azide and 1.86% EDTA. The detergent SDC contained only 0.2% sodium azide. The enzyme DNase was prepared in Dulbecco's PBS containing CaCb and MgCl ₂ (Sigma, Germany). After complete decellularization, 5 biopsies were collected from all kidneys and processed for histology, DNA quantification, Collagen quantification, elastin quantification and glycosaminoglycans quantification. [00154] The decellularized pig kidney was pale-white and retained overall kidney shape

(FIGs. 1 A and IB). Histological staining using HE showed presence of nucleated cells in the normal pig kidney but no basophilic staining of the nuclear material in decellularized kidney . Masson's Trichrome of normal pig kidney showed dark pink cytoplasmic staining, black nuclei and blue staining for collagen, while the decellularized tissue showed complete removal of nuclei but retained collagen, staining of decellularized kidney confirmed that collagen was preserved. Immunohistochemistry staining of normal kidneys showed prominent staining of fibronectin in blood vessels and around the glomeruli, which was also retained in the decellularized kidney. Similarly, laminin was detected in both the normal and decellularized kidney. Verification of decellularization and characterization of the extracellular matrix

[00155] DNA quantification of normal and decellularized samples showed significant removal of DNA in decellularized samples (FIG. 1C) from 1923.31±609.18 ng/mg tissue in normal to 26.35±7.63 ng/mg tissue in decellularized. The quantification of extracellular matrix proteins collagen and elastin showed an increase in the amount of elastin and collagen in decellularized kidneys as compared to normal (FIG. 1C) even though the increase is not significant. The quantification of glycosaminoglycans showed a decrease in decellularized kidneys in comparison to normal kidneys (FIG. 1C).

[00156] The experiments here show that the cellular compartment of porcine kidneys was completely removed, leaving behind an acellular ECM scaffold, which retains its complex 3D structure using a milder protocol involving the use of ionic and non- ionic detergents as compared to protocols involving the use of anionic detergents such as SDS. The acellular scaffold retained structural ECM proteins with a complete removal of DNA. The retention of ECM components is critical for attachment and proliferation of incoming cells. Thus, preservation of intact tubular, vascular and glomerular structure with the ECM scaffold with native signals would facilitate the process of recellularization by the precursor cells. [00157] Example 2: Characterization of human fetal kidney cells Derivation and Culture of Human Fetal Kidney Cells

[00158] Human fetal kidneys (hFK) (n=7) were isolated from aborted fetuses at 9-11 weeks of gestation in accordance with the Swedish guidelines and with approval by the local ethics committee. Gestational age was estimated according to specific anatomical markers.

Gestational age is given as menstrual age. The abortions were performed in pregnancies with no apparent abnormalities, and no fetuses with anomalies were included. All women donating fetal tissue had been serologically screened for syphilis, toxoplasmosis, rubella, HIV-1, cytomegalovirus, hepatitis B and C, parovirus and herpes simplex types 1 and 2. Biopsies from adult kidneys not used for transplantation (n=3) obtained from cadaver organ donors after appropriate consent were also used for analysis.

[00159] Human fetal kidney progenitor cells were isolated from two hFK. For this, hFK from two different fetuses were placed in a sterile tube containing DMEM medium (Gibco, Invitrogen Corp. UK) and then disintegrated into a single cell suspension by passage through a 70μιη nylon mesh. The single cell suspension was centrifuged at 200 g for 10 min to pellet the cells. hFK precursor cells (hFKPC) were then seeded on cell culture flasks pre-coated with 0.1% w/v collagen (Sigma- Aldrich, Germany). Approximately, 10±2 x 10 ⁶ cells were obtained from a single fetus. The cells were divided equally into two, and cultured either using a commercially available endothelial medium (ECM) (clonetics, USA) or renal epithelial medium (REM) (PromoCell, Germany).

Determination of stem cell markers by immunohistochemistry of hFKs

[00160] Five isolated human fetal kidneys were embedded in paraffin and sectioned. The sections were stained by standard immunohistochemistry using primary antibodies to-DLK-1 (Delta-like lhomolog) (Abeam, Cambridge, UK), -EphB2 (AbD Serotec, Taby, Sweden), - EphB3 (AbD Serotec), -EphA6 (AbD Serotec), -EphA7 (Biorbyt, Cambridge, UK), -CD 133 (Abd Serotec). EPCAM (Abeam), CK8 (Santa Cruz, Heidelberg, Germany) and CK18 (Santa Cruz). The expression of these markers in all five fetuses was compared and the

presence/absence was recorded.

Flow cytometry based characterization of hFKPCs [00161] Three of 3x10 ⁶ cells passages were taken in FACS tubes and centrifuged at 1 00 rpm for a minute to pellet cells and washed twice with PBS. The cells grown in ECM were stained using primary antibodies anti-CD 133 (BD Stockholm, Sweden), -CD34 (BD), -Ulex Europaeus (Sigma, Stockholm, Sweden), -CD31 (BD), -von Williebrand factor, - vascular endothelial growth factor receptor-2, -Endocan, Tyrosine kinase receptor (R&D Systems, Oxford, UK), - Delta-like lhomolog and -CD271 (BD). Primary antibodies used for cells grown in REM were anti-Epithelial Cell Adhesion Molecule (Santa Cruz), -DLK-1, -EphB2, -EphB3, -EphA6, -CD34 (BD), -CK7, -CK8, and CK18 (all from Santa Cruz). The cells were stained with primary antibodies for 30 minutes at 4° C. The unconjugated primary antibodies were further stained with respective FITC-conjugated goat anti-mouse, mouse anti-goat, or mouse anti-rabbit secondary antibodies for 30 minutes at 4° C. For staining intracellular markers CK7, CK8 and CK18, the cells membrane was permeabilized by treatment using 5% saponin before staining with primary antibody for 10 min and washed twice with PBS for 5 min.

Immunocytochemistry ofhFKPCs

[00162] The cells grown in ECM and REM were also characterized by

immunocytochemistry. Cells were seeded into each well of an 8 chamber slide and cultured for 1 day. The cells were fixed using 30% acetone-70% methanol for 2 minutes, washed thrice with PBS and blocked in 1% Bovine Serum Albumin (BSA). The cells were stained overnight at 4° C. using the same primary antibodies as in flow cytometry, washed with PBS thrice and stained with goat-anti-mouse or goat-anti-rabbit or donkey-anti-goat secondary antibodies for 30 min at room temperature on a shaker. The staining step was followed by further washing with PBS thrice and mounted using DAPI mounting medium (Vector labs, USA).

Characterization of human fetal cells and tissue

[00163] The hFKPC grown in ECM showed an elongated growth. Characterization of these cells by flow cytometer showed the expression of endothelial specific markers CD31 , vWF, endocan, ulex and endothelial progenitor/precursor cell markers VEGFR2, Tie2, CD133, CD34 and DLK-1 (FIG. 2A). The characterization of same cells by immunocytochemistry gave reproducible results showing the expression of mature endothelial cell marker CD31 and endothelial progenitor cell markers CD133, CD34, Tie-2 and DLK-1, which showed that the cultured endothelial cells were a mixture of mature endothelial and progenitor cells. The cells grown in REM showed the characteristic polygonal shape of epithelial cells. These cells characterized by flow cytometry showed the expression of phenotypic epithelial cell markers CK18 and EPCAM, the Ephrin receptors EphA6, EphA7, EphB3 and the stem cell marker DLK-1 (FIG. 2B). Once again the immunocytochemistry staining gave reproducible results demonstrating the expression of CK18, EphA6, EphA7, EphB3 and DLK-1. The cells did not show positivity for EphB2, CD34, c-kit and CK20 by either of the methods.

5 [00164] TABLE 2 A: Markers expressed by kidney endothelial cells isolated from human fetal kidneys

[00165] TABLE 2B: Markers expressed by kidney epithelial cells isolated from human fetal kidneys

+= > 10-25, ++= >26-50, +++= >51-75 mean fluorescence channels as compared to negative I o control (only secondary antibodies). Identification of stem cells markers in human fetal and adult kidneys

[00166] In fetal kidneys, the Ephrin receptor 6 (EphA6) was found to be expressed mainly in the capsule and in some interstitial cells in the cortex, as well as in the proximal and distal tubules (FIG. 3 A), while intense staining of EphA7 was found in all the cells of the fetal kidney, the Bowman's capsule, podocytes, and the epithelial cells of the proximal tubules (FIG. 3B). EphB3 was found mainly in the distal and proximal convoluted tubules (FIG. 3C) of the fetal kidney. Intense staining of DLK-1 was mainly found in the kidney capsule, cap mesenchyme and renal vesicles and collecting ducts of the fetal kidney. Some distal tubules of the fetal kidney also expressed DLK-1 (FIG. 3D). CD 133 positive cells were found localized in the tubular epithelium, renal papilla, interstitial space and Bowman's capsule of the fetal kidney (FIG. 3E). EPCAM was found in the epithelium of proximal and distal tubules as well as ducts of the fetal kidney (FIG. 3F). These data are summarized in Table 3A and 3B.

[00167] In adult kidneys, the EphA6 was found in the tubules (FIG. 3G), while weak or no expression of EphA7 was found (FIG. 3H). By contrast, strong expression of EphB3 was found in the tubules as well as the glomerulus of the adult kidney (FIG. 31). DLK-1 was found weakly or not expressed in the adult kidney (FIG. 3 J), while CD 133 was found in blood vessels (FIG. 3K), and EPCAM was positive in the tubules of the adult kidney (FIG. 3L). [00168] TABLE 3A: Expression of stem cell markers and ephrin receptors on human fetal kidneys

[00169] TABLE 3B: Localization of stem cell markers and ephrin receptors on human fetal kidneys

[00170] Considering the complex cellular nature of human kidney, and the morphological and functional heterogeneity of renal cell types, human fetal kidney cells were used as the precursor population since they possess a high proliferative and differentiation capacity into renal specific cell types. At the same time, the expression of certain putative stem cell markers expressed on fetal kidney cells were of interest, as they could give an insight into which cell populations might be potential candidates for future clinical kidney regeneration. Of particular interest was the expression of certain Ephrin receptors reported as stem cell markers in neurogenesis and colonic cells, for which there is limited knowledge of their expression in kidney development. Expression of Ephrin A6 was observed in the interstitial cells, and EphB3 expression was observed in the renal tubules. A very intense and reproducible staining for EphA7 was observed in the fetal kidney capsule, renal mesenchyme, tubules, glomerulus and the collecting ducts of all fetal kidneys tested. Based on the expression of EphA7 in the human kidneys, EphA might be a new interesting marker for renal stem/precursor cell population. No expression of EphB2 was observed in any of the fetal kidneys. In addition, the expression of renal stem cell markers DLKl, CD 133 and EPCAM in human fetal kidneys was confirmed. DLKl staining was intense and was found in locations very similar to EphA7, while CD 133 was less intense and EPCAM was found only in the epithelia of tubules and ducts. Characterization of the isolated hFKPC

demonstrated the continued expression of all the stem cell markers tested such as DLK-1, CD133, EPCAM, EphA6, -A7, -and -B3 as evidenced by both flow cytometric and immunocytochemical analysis suggesting that these markers are stably expressed on these cells. [00171] Example 3: Recellularization of sliced decellularized kidney scaffolds with human fetal kidney cells

Recellularization of kidney slices

[00172] Decellularized kidneys were cut into slices of 3 mm thickness (Histocenter, Gothenburg). The slices were sterilized by agitation in 0.1% per acetic acid in PBS for 2 h at 37° C. and then washed in PBS containing 0.5% penicillin and streptomycin (PSA) for 72 hrs. and treated with metalloproteinases (MMP; activated 2.5 μg MMP-2 and 2.5 μg MMP9) for 24 h at room temperature on a shaker and washed with distilled water containing 20mM EDTA and antibiotics. The washed tissue slices were placed on transwells and a total of approximately 30xl0 ⁶ cells grown in ECM and REM were suspended in 200 μΐ medium and seeded using 10 μΐ pipette tip and then incubated for an hour to allow cell attachment. Later,

2 ml medium was added gently along the sides of the well and cultured for 2 weeks in transwell plates. The medium (50% ECM and 50% REM) was changed once a week.

Characterization of recellularized tissue [00173] The recellularized tissue was fixed in formalin and characterized by standard immunohistochemistry using hematoxylin and eosin and stained for expression of CK8, CK18, EphA7, CD 133 and DLK-1 using antibodies. The tissues were also characterized by gene expression using qPCR.

Gene Analysis of recellularized tissue [00174] Extraction: RNA from two cell samples (renal fetal cells, passage 9° and 11°), two decellularized pig kidney tissue samples and a negative control (water) were extracted using Qiagen RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) adding the following steps. Before addition to the column each sample was added to a 2 ml tube with a stainless steel bead and 600 μΐ RLT buffer. The samples were homogenized in a TissueLyser (Qiagen GmbH, Hilden, Germany) at 25 Hz for 2 x 5 min. The samples were centrifuged 16,000 g for

3 min at 4° C. and the supernatants were added to the kit columns. After binding and washing the samples were DNase treated on column by adding 80 μΐ DNase I solution to each sample and incubate at room temperature for 15 min. After washing, the samples were eluted in 30 μΐ RNase free water. [00175] Reverse transcription: The five extracted samples and a commercial human kidney total RNA sample (Thermo Fisher, Cat. No. AM7976) were reverse transcribed using TATAA GrandScript cDNA Synthesis Kit (Tataa Biocenter, Goteborg, Sweden). 10 μΐ R A of each sample was used in a total volume of 20 μΐ in a Bio-Rad T100 Thermal Cycler (Bio- Rad Laboratories, Inc) using a temperature protocol of 5 min at 22° C, 30 min at 42° C, 5 min at 85° C. and hold at 4° C. [00176] Preamplification: Four μΐ of each cDNA sample and 4 μΐ of a negative PreAmp control (water) were preamp lifted in a total volume of 20 μΐ using TATAA GrandMaster Probe MasterMix (Tataa Biocenter, Goteborg, Sweden) and 50 nM of each of 27 of the 28 primer pairs of the analyzed assays (the primers for the 18S assay was not included to avoid preamplification of 18S rRNA due to the natural high abundance). Amplification was performed in a Bio-Rad T100 Thermal Cycler (Bio-Rad Laboratories, Inc) using a temperature protocol of enzyme activation 1 min at 95° C. followed by 17 cycles of 2 min at 60° C. and 1 min at 72° C. Samples were flash frozen directly from 72° C. after the last cycle. The preamp lifted samples were thawed by adding 140 μΐ RNase free water to each sample and mixing thoroughly by pipetting. The diluted samples plus a NTC (water) were analyzed in duplicates for 28 genes in a Bio-Rad cfx384 Real-Time System (Bio-Rad Laboratories, Inc). Each reaction contained 2 μΐ sample in a total volume of 10 μΐ using TATAA GrandMaster Probe MasterMix (Tataa Biocenter, Goteborg, Sweden) with 400 nM primers and 200 nM probe. The amplification protocol included enzyme activation for 30 seconds at 95° C. followed by 40 cycles of 30 seconds at 60° C. and 10 seconds at 72° C. [00177] Data analysis: All negative controls were negative or had Cq-values at least 5 higher than the highest Cq for a positive sample. Samples were evaluated for excessive amount of genomic DNA using the ValidPrime approach. The 12 candidate reference genes (TATAA Human Reference Gene Panel, Cat. No. AlOlp, Tataa Biocenter AB, Goteborg, Sweden) were evaluated with the GeNorm and Normfmder algorithms to find the most stable reference genes. Both methods found TBP and YWHAZ to be the most stable reference genes. For remaining genes, Cq-values for qPCR replicates were averaged, normalized with the two reference genes (delta-Cq were calculated for each sample using the average of Cq-values for TBP and YWHAZ). Relative quantities (2 ^A delta-delta-Cq) were calculated normalized to the average of the two renal fetal cell samples, and log2 values were calculated. All data analyses were performed using GenEx version 6.1.0.757 software (MultiD Analysis AB, Goteborg, Sweden). [00178] The decellularized kidney ECM looked compact and condensed, but post-treatment with MMP-2 and -9, the ECM looked uncompressed and less dense (FIGs. 4A and 4B). FIG. 4A shows before treatment with recombinant metalloproteinases MMP-2 and MMP-9; FIG. 4B shows post-treatment with MMP-2 and MMP-9. Hematoxylin and eosin staining of the recellularized kidney slices cultured in transwells for 2 weeks showed several colonies of cells spread throughout the tissue, in the parenchyma, several glomeruli and some tubules (FIGs. 4C - 4E). FIG. 4C shows the presence of cells scattered in the parenchyma; FIG. 4D shows the presence of cells scattered in the glomerulus; and FIG. 4E shows the presence of cells scattered in the tubule. In some areas of the recellularized kidney slices, large numbers of cells were found growing adjacent to the ECM (FIG. 4F - 4H). FIG. 4F is an HE stain of decellularized pig kidney slices recellularized with human fetal kidney progenitor cells showing cells growing on top of the scaffold creating new kidney tissue. FIG. 4G is an HE stain of decellularized pig kidney slices recellularized with human fetal kidney progenitor cells showing another area with newly formed kidney tissue by the seeded human cells. FIG. 4H is an HE stain of decellularized pig kidney slices recellularized with human fetal kidney progenitor cells showing tubule-like structures in the neo-tissue formed by the seeded human cells.

[00179] Interestingly, at two weeks, the cells in these areas formed tubule and glomeruli-like structures, observed with immunohistochemical staining with cytokeratin. FIG. 5A shows that the cells formed a tubule-like structure; FIG. 5B shows that the cells formed structures resembling glomeruli; and FIG. 5C shows that the cells formed tubule and glomerular-like structures. The immunohistochemical analysis of the recellularized kidney slices showed positive staining for the epithelial cell markers CK8 (FIG. 6C) and CK18 (FIG. 6B), endothelial marker CD 133 in the blood vessels (FIG. 6E), the stem cell markers EphA7 in the blood vessels (FIG. 6D) and DLK-1 staining in most cells (FIG. 6F).

[00180] The q-PCR analysis of recellularized tissue showed an increased expression of transcription factors SIX2 and EYA1 that are involved in kidney development when compared to the seeded cells alone (FIG. 7). A few other transcriptional factors involved in kidney development such as WT1, CITED 1, LHX1 and SALL1 were also slightly increased in comparison to cells alone (FIG. 7). In addition, an increased expression of DLK1 and

EphB3 the transcriptional factors that are important in growth and differentiation of epithelial cells was seen in recellularized tissue in comparison to cells alone (FIG. 7). All these results together show the proliferation and differentiation of cells in the recellularized kidney tissue. [00181] Initial attempts to recellularize porcine kidney slices with hFKPC under static culture indicated that pre-treatment of the tissue before cell seeding was necessary to improve infiltration. Since human cells produce MMPs to breakdown surrounding tissue to facilitate migration, the tissue was treated with human recombinant MMP 2 & 9 to open up the compact ECM microstructure. This treatment in combination with incubation of the tissue in a transwell improved infiltration of cells under static culture conditions. With this approach, good though not uniform distribution of the seeded hFKPC cells into the porcine renal tissue was obtained, as confirmed by immunohistochemical analysis of cells found inside individual glomeruli, blood vessels and renal parenchyme. The cells expressed epithelial, podocytic, and endothelial lineages. Among genes up-regulated in the recellularized grafts, were the transcription factors specifying the kidney progenitor cells including WT1, PAX2, LIM1, SIX2, EYA1, SALLl, and CITEDl . There was an increased level of all the tested genes in the recellularized tissue pieces as compared to isolated hFKPC. Expression of SIX2, SALLl, and WT1 are considered to be markers of the metanephric cap mesenchyme. Thus, the hFKPC, which recapitulate the in vivo metanephric development are most likely to efficiently generate mature kidney cells.

[00182] Hence, acellular porcine scaffolds can optimally be recellularized with human fetal kidney progenitor cells that would facilitate ex vivo expansion and localized differentiation after scaffold seeding. The hFKPC can then be directed toward mature epithelial cell phenotypes by exogenous growth factors and intrinsic matrix-derived cues. That kidney specification in the hFKPC is enhanced in cell-matrix culture compared with differentiation in traditional culture was demonstrated.

[00183] In conclusion the implemented decellularization protocol successfully removed all cellular components from porcine kidneys in short time, preserved ECM, vasculature architecture, glomerular capillaries, and tubular membrane. The successful rapid

recellularization of acellular porcine scaffolds with hFKPC paves the ground for more extensive experimental investigations with prolonged culture to achieve uniform

recellularization within the tubular compartments of the sliced matrix, and it possibly induces further differentiation of the cells into mature human renal cells. The matrix slice model allows for testing of specific cell populations, variable seeding techniques, and biomimetic culture conditions in a reproducible fashion. Matrix reseeding efficacy at various stages of differentiation can also be tested to assess expansion requirements. The efficient

differentiation of hFKPC toward mature cell populations is also beneficial for the study of cell biology and the screening of drugs for toxicity testing. Furthermore, a demand for kidney progenitors is increasing because of a severe shortage of donor organs for orthotopic kidney transplantation. Because dialysis and kidney transplantation currently are the only successful therapies for patients suffering chronic renal failure, cell therapy with renal progenitors offers an alternative approach for therapies of kidney diseases.

[00184] There is continued need for alternate and novel therapies for patients with end- stage kidney disease. In the advancing field of organ engineering, a requirement for xenogeneic scaffolds remains. Although advances in organ preservation, recipient management, and immunosuppression represent significant therapeutic advancements, kidney transplantation remains the only effective treatment option. Decellularization and regeneration would impart new value to these organs from xenograft donors with a structurally undamaged matrix.

[00185] Example 4

[00186] An organ (e.g., a kidney, a liver, a heart, a lung, a pancreas, or any mammalian solid organ) is decellularized by a combination of perfusion and then recellularized by perfusing with human pluripotent stem/progenitor cells or human adult multipotent stem cells followed by perfusion with cells growth media respectively to prepare a bioengineered organ. A bioprinted organ (e.g., a kidney, a liver, a heart, a lung, a pancreas, or any mammalian solid organ) is prepared by cellularizing a bio-polymeric scaffold by perfusing the scaffold with human pluripotent stem/progenitor cells or human adult multipotent stem cells followed by perfusion with cells growth media respectively. Before recellularization to prepare a bioengineered organ or cellularization to prepare a bioprinted organ, the sample is treated with one or more MMP. Successful recellularization or cellularization is confirmed by the presence of biomarker of cells specific to an organ. A subject in need of the transplanted organ or tissue is selected, and the engineered or the bioprinted organ or tissue prepared by the methods of the present disclosure is implanted/transplanted into the subject. The subject's prognosis and recovery post-transplantation is monitored.

Bioreactor

[00187] A bioreactor is used for the recellularization of a MMP -treated decellularized organ (e.g., a kidney, a liver, a heart, a lung, a pancreas, or any mammalian solid organ (i.e., any organ that does not contain a cavity or lumen and that is not gaseous, is an organ which has parenchyma and stroma. The latter often arranged as trabeculae or surrounding groups of parenchymatous cells to provide support)). The bioreactor may include an enclosed setup of polypropylene tube connected to polyethylene and silicon tubes. Bioreactor and tubes may be sterilized in an autoclave before use. Cell culture media may be perfused using a peristaltic pump. [00188] The bioreactor may be designed as disclosed in US Patent No. 9,433,706, or modified as-needed based on the organ or tissue for recellularization.

Recellularization of Solid Organs

[00189] The entire recellularization process is performed under very sterile conditions and all perfusions are carried out in an incubator at 37° C. supplied with 5% C0 ₂ . Before recellularization, the MMP -treated decellularized organ or tissue structure/scaffold/graft is perfused with heparin at a concentration of 50 IU/ml PBS for 2 h. The heparin is drained off and cells (human pluripotent stem/progenitor cells or human adult multipotent stem cells) are immediately perfused. The cells are then drained off and the organ or tissue is washed with PBS containing 1% penicillin-streptomycin-amphotericin for 3-5 min or until blood was completely removed. The organ or tissue is subsequently perfused for one or more days with media. For example, endothelial medium is prepared using MCDB131 (10372, Life technologies, Sweden) basal medium supplemented with 10% heat inactivated human AB serum (34005100, Life technologies, Sweden), 1% glutamine (25030, Lonza, Denmark), 1% penicillin- streptomycin-amphotericin, and EGM2 single quote kit (CC-4176, Lonza, Denmark) that contains ascorbic acid, hydrocortisone, transferrin, insulin, recombinant human VEGF, human fibroblast growth factor, human epithelial growth factor, heparin and gentamycin sulfate. The medium may also be prepared using 500 ml Medium 231 (M231 , Life technologies, Sweden) supplied with 10% heat inactivated human AB serum, 1% penicillin-streptomycin amphotericin and 20 ml smooth muscle growth supplement (SMGS) (S-007-25, Life Technologies, Sweden) and 5 ml smooth muscle differentiation supplement (SMDS) (S-008-5, Life technologies).

[00190] Numbered clauses of the present disclosure are:

1. An in vitro or ex vivo method of decondensing an extracellular matrix (ECM) structure in an acellular structure of a mammalian organ or tissue in need thereof, the method comprising introducing a metalloproteinase (MMP) to the acellular structure of the mammalian organ or tissue, thereby decondensing the ECM structure. 2. The method of claim 1 , wherein the MMP is MMPl , MMP2, MMP3, MMP7, MMP9, MMP13, and/or MMP14.

3. The method of claim 1, wherein the MMP decondenses a compact ECM

microstructure or microstructure.

4. The method of claim 1, wherein the MMP is introduced under static culture condition.

5. The method of any one of claims 1-4, wherein the acellular structure of the mammalian organ or tissue comprises ECM components of a kidney, a liver, a bladder, a vagina, a urethra, a trachea, an esophagus, a heart, a lung, a pancreas, a skin, or other bodily organ.

6. The method of any one of claims 1-5, wherein the acellular structure of the mammalian organ or tissue is not from a healthy organ or tissue.

7. The method of claim 6, wherein the acellular structure of the mammalian organ or tissue is from an organ or tissue characterized as having a tumor or cancer.

8. A method of identifying a human kidney stem/progenitor cells, the method

comprising detecting the expression of Ephrin receptor 7 (EphA7) in cells obtained from a kidney.

9. The method of claim 6, wherein the identification of the human kidney

stem/progenitor cells is performed by a method selected from the group consisting of:

staining the cell with an anti-EphA7 antibody, staining the cell with an anti-EphA7 antibody for use in immunohistochemistry, and polymerase chain reaction (RT-PCR) of EphA7.

10. A method of testing toxicity of a compound, a therapeutic agent; screening of a new therapeutic agent; and/or identifying a therapeutic agent candidate for treating a mammalian organ or tissue disease or disorder, the method comprising:

(i) introducing a compound, a therapeutic agent, a new therapeutic agent, or a candidate therapeutic agent to a recellularized mammalian organ or tissue, wherein the recellularized organ or tissue is prepared by a method comprising decellularizing the organ or tissue, while retaining an extracellular matrix (ECM) structure, treating the ECM structure with one or more metalloproteinase (MMP), and recellularizing the MMP -treated ECM structure of the decellularized organ or tissue with human pluripotent stem cells or human adult multipotent stem cells; or

(ϋ) introducing a compound, a therapeutic agent, a new therapeutic agent, or a candidate therapeutic agent to a bioprinted organ or tissue scaffold, wherein the bioprinted organ or tissue scaffold is formed with human pluripotent stem cells or human adult multipotent stem cells.

11. The method of claim 10, wherein the testing toxicity comprises ex vivo testing for safety, toxicity, on-target and/or off-target adverse effects, and/or production of toxic metabolites.

12. The method of claim 10, wherein the recellularized organ or tissue or the bioprinted organ or tissue scaffold comprises an ECM structure of a kidney, a liver, a bladder, a vagina, a urethra, a trachea, an esophagus, a heart, a lung, a pancreas, a skin, or other bodily organ.

13. The method of claim 10, wherein the recellularized organ or tissue or the bioprinted organ or tissue scaffold comprises cells, tissues, or functional parts of a kidney, a liver, a bladder, a vagina, a urethra, a trachea, an esophagus, a heart, a lung, a pancreas, a skin, or other bodily organ.

14. The method of claim 13, wherein the MMP -treated decellularized organ or tissue is not from a healthy organ or tissue. 15. The method of claim 14, wherein the MMP -treated decellularized organ or tissue is from an organ or tissue characterized as having a tumor or cancer.

16. The method of claim 15, wherein the human pluripotent stem cells or human adult multipotent stem cells are differentiated to form tissues characterized as having a tumor or cancer. 17. The method of claim 16, wherein the new therapeutic agent or the therapeutic agent candidate is for treating a tumor and/or cancer.

18. A method of treating kidney disease/disorder in a subject with end- stage renal failure comprising (i) administering/implanting/transplanting to the subject a recellularized kidney or a functional part thereof, wherein the kidney or the functional part thereof is prepared following the method of any one of claims 1-4, and recellularized with human pluripotent stem cells or human adult multipotent stem cells; or (ii)

administering/implanting/transplanting to the subject a bioprinted kidney scaffold or a functional part thereof, cellularized with human pluripotent stem cells or human adult multipotent stem cells.

19. A method of treating chronic renal failure in a subject in need thereof comprising (i) administering/implanting/transplanting to the subject a recellularized kidney or a functional part thereof, wherein the kidney or the functional part thereof is prepared following the method of any one of claims 1-4, and recellularized with human pluripotent stem cells or human adult multipotent stem cells, or (ii) administering/implanting/transplanting to the subject a bioprinted kidney scaffold or a functional part thereof, cellularized with human pluripotent stem cells or human adult multipotent stem cells.

20. A method of preparing a metalloproteinase (MMP)-treated acellular organ or tissue scaffold, the method comprising: (i) introducing an ionic and/or a non-ionic detergent to an organ or a tissue sample, wherein the ionic and/or the non-ionic detergent removes cellular compartments of the organ or tissue sample, while the acellular extracellular matrix (ECM) structure is retained, and (ii) introducing one or more metalloproteinase (MMP), wherein the MMP decondenses the acellular ECM structure, thereby preparing the MMP -treated acellular organ or tissue scaffold.

21. The method of claim 20, wherein the MMP is MMP1, MMP2, MMP3, MMP7, MMP9, MMP13, and/or MMP14.

22. The method of claim 20, wherein the MMP decondenses a compact ECM

microstructure. 23. The method of claim 20, wherein the MMP is introduced under static culture condition.

24. The method of any one of claims 20-23, wherein the organ or the tissue is a kidney, a liver, a bladder, a vagina, a urethra, a trachea, an esophagus, a heart, a lung, a pancreas, a skin, or other bodily organ. 25. The method of any one of the above claims, wherein the organ or tissue for decondensing or decellularizing is obtained from a pig, a cow, a lamb, a goat, a sheep, a chimpanzee, a monkey, a human, or other primate.

26. A method of preparing a functional kidney or a functional part thereof with human pluripotent stem cells or human adult multipotent stem cells, the method comprising:

a. introducing one or more metalloproteinase (MMP) to an acellular kidney scaffold comprising extracellular matrix (ECM) composition, vascular architecture, glomerular capillaries, and tubular membrane, or to a bioprinted polymeric scaffold comprising ECM matrix composition;

b. seeding human pluripotent stem cells or human adult multipotent stem cells to the MMP -treated kidney scaffold or the MMP -treated bioprinted polymeric scaffold; c. allowing the cells to attach to the MMP -treated kidney scaffold or the MMP- treated bioprinted polymeric scaffold; d. perfusing media for growth and differentiation of the cells to form at least renal proximal tubules; and e. culturing the cells, thereby differentiating the cells to form at least renal proximal tubules; thereby, preparing a functional kidney or a functional part thereof.

27. The method of claim 26, wherein the one or more MMP is MMP1, MMP2, MMP3, MMP7, MMP9, MMP13, and/or MMP14.

28. The method of claim 26, wherein the MMP decondenses a compact ECM

microstructure.

29. The method of claim 26, wherein the MMP is introduced under static culture condition. 30. A method of recellularizing an acellular structure of a mammalian organ or tissue, the method comprising introducing human pluripotent stem cells or human adult multipotent stem cells to a decondensed ECM structure prepared by the method of any one of claims 1-7; allowing the cells to attach to the decondensed ECM structure of the acellular structure of the organ or tissue; perfusing media for growth and differentiation of the cells to form a functional part of the organ or tissue; and cultering the cells; thereby recellularizing the acellular structure of a mammalian organ or tissue.

31. The method of claim 30, wherein the acellular structure of the mammalian organ or tissue comprises ECM components of a kidney, a liver, a bladder, a vagina, a urethra, a trachea, an esophagus, a heart, a lung, a pancreas, a skin, or other bodily organ.

32. The method of claim 30 or 31, wherein the organ or tissue is obtained from a pig, a cow, a lamb, a goat, a sheep, a chimpanzee, a monkey, a human, or other primate.

OTHER EMBODIMENTS

[00191] It is to be understood that while the disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.