Technical field

The present invention relates to the bioengineering of animal pelts and anti-counterfeit of prime bioengineered pelts and to bioengineered pelts with inseparable molecular signatures for authenticity, timestamping and provenance. The present invention relates to a bioengineering process to derive animal pelt from the in vitro disposition and

differentiation of interfollicular epidermis cells, hair follicle cells, fibroblast-like cells and extracellular matrix into a bioengineered pelt tissue. The present invention also relates to the process of cell specific enrichment, genetic engineering, differentiation and disposition of said cells in the manufactured tissue. The present invention further relates to pre-coding and use of a combination of genetic signature combinations as an anti-counterfeit mechanism and as proof of authenticity, timestamp and provenance.

Description

Means and methods to derive bioengineered animal pelt with anti-counterfeit properties

[1] Bioengineering definition: Application of engineering and biological principles for purposefully defining cellular behavior and or cellular disposition within an engineered synthetic tissue and or organ. Furthermore, the designed modification of genetic information to code altered cellular functions with beneficial impact on biologically derived materials and or cellular behavior. In addition, the use of engineering and biological principles to create novel tissue and or organs and or biologically derived materials.

[2] The use of vertebrata adult progenitors or adult stem cells has enabled the derivation of a variety of cell types within said adult progenitor tissue. In addition, vertebrata embryonic stem cells which count with a higher differentiation potential enable deriving all adult cell types in said vertebrata species, particularly adult stem cells and terminally differentiated cells.

Furthermore, the reprogramming process from differentiated cells towards higher pluripotency states (primed or naive) provides an inexhaustible cell source without the need for early embryo isolation.

[3] The reprogramming process from differentiated cells towards cells of higher pluripotency state may be achieved through the exogenous synthetic expression of transcriptional factors and or epigenetic modifiers that reinforce the core transcriptional network of said pluripotent state. The simplicity of this process has placed reprogrammed stem cells as a convenient cell source for bioengineering and regeneration.

[4] Differentiation of stem cells can be achieved through the use of culture conditions with defined combinations of nutrients, co-factors, growth factors and or small molecules.

Differentiation protocols can rely on external signals or intrinsic signals, and the differentiation propensity of adult cell types varies from one cell type to another and is dependent on its hierarchy within the pluripotency level and linage commitment level.

[5] However, though the differentiation of stem cells towards tissue specific cells types is widely and efficiently applicable, the derivation of complex tissues composed of multiple cells types and with characteristic structures is nevertheless highly variable and unsuitable for industrial manufacturing processes. In fact, the current protocols for complex tissue generation rely on the self-organizing properties wired in the developmental program of the used species and hence inherently highly variable (Lancaster et al., Nat. 19, 373-379 (2013), Kadoshima et al., PNAS 110, 20284-20289 (2013)).

[6] Engineered cells are capable of presenting unique features not present in natural counterparts, such features include the ability to continuously proliferate, sustainably proliferate, proliferate at a faster rate, express exogenous proteins, express native or altered proteins at a higher expression level and or present increased telomeric stability.

[7] Up today there is a variety of differentiation protocols to derive some components of vertebrata skin (Blanpain et al., Cell 118, 635-648 (2004), Itoh et al., PNAS 108, 8797- 8802 (2011)), however none of them have successfully developed a complete and completely ex-vivo vertebrata skin or dermis or hair bearing epidermis or pelt. Furthermore, up to today, there is no protocol capable of reliably generating manufacturing quality bioengineered animal pelt. This imposes a production pressure on natural sources, highlighting the requirements for sustainable sources. Hence, there is still a need for means and methods to derive bioengineered animal pelt having the desired mechanical properties, or biomaterial properties, or textile quality, or cellular composition and disposition, or tissular properties. The present application addresses this need and thus provides means and methods to derive bioengineered animal pelt having the desired mechanical properties, biomaterial properties, textile quality, cellular composition and/or disposition, and/or tissular properties. Furthermore, the presented bioengineered pelt may be inseparably attached to a combination of nucleic acid molecular signature combinations that allows determining its authenticity, and assist the manufacturing process by enabling genetically encoded batch labels, product type labels, genetic timestamping, proof of provenance and anti-counterfeit measures.

[8] Accordingly, the means and methods of the present application allow deriving vertebrata pelt with prime manufacturing quality in a standardized and homogeneous manner.

[9] As used in the description herein and throughout the claims that follow, the meaning of“a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of“in” includes“in” and“on” unless the context clearly dictates otherwise.

[10] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g.“such as”) provided with respect to certain embodiments herein is intended merely to better clarify the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[11] The description“fur follicle” and“hair follicle” are used indistinguishably form each other and the use of one includes the use of the other. The words“barcode” refers to a nucleic acid sequence of at least one nucleotide and a barcode presents 4 ^L h combinations where n is the arbitrary length of said barcode and four correspond to the main nucleic acid bases present in vertebrata and reducing the epigenetic modifications or chemical modifications to its principal purine or pyrimidine core.

[12] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all groups used in the appended claims.

[13] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The methods and techniques of the present invention are generally performed according to conventional methods well- known in the art. Generally, nomenclatures used in connection with techniques of

biochemistry, enzymology, molecular and cellular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art.

[14] The methods and techniques of the present invention are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited and discussed throughout the present

specification unless otherwise indicated. See, e. g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.

Y. (2001 ); Ausubel et al., Current Protocols in Molecular Biology, J, Greene Publishing Associates (1992, and Supplements to 2002); Handbook of Biochemistry: Section A Proteins, Vol I 1976 CRC Press; Handbook of Biochemistry: Section A Proteins, Vol II 1976 CRC Press. The nomenclatures used in connection with, and the laboratory procedures and techniques of, molecular and cellular biology, protein biochemistry, enzymology and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art.

[15] A cell when used herein may preferably be a vertebrata cell. A vertebrata cell may preferably be a cell from an antelope, antilopini, beaver, buffalo, caiman, caracal, cat, cheetah, chinchilla, cow, crocodille, deer, eland, elephant, ermine, faux, fisher, fox, genet, giraffe, goat, golden jackal, hedgehog, horse, leopard, lynx, lion, marten, mink, monkey, ape, nutria, otter, rabbit, rhinoceros, sable, serval, sheep, shrew, snake, stoat, swine, wolf, australian brushtail possum, mouse, rat, Camelidae, their subspecies and Pantholopinae. A preferred vertebrata cell is a stem cell or induced pluripotent stem cell or adult stem cell or differentiated cell. The vertebrata cells may be obtained from a healthy individual. The vertebrata cell may be from a cell line, e.g. a deposited cell line or a commonly available cell line. Object of the invention

[1] Some of the objects of the present disclosure, are as listed herein below.

[2] It is an object of the present disclosure to provide for means and methods to manufacture bioengineered pelt to be a source for textile industry and pelt industry.

[3] It is an object of the present disclosure to provide for a system and method to derive bioengineered pelt avoiding the need for conventional pelt farming.

[4] It is another object of the present disclosure to provide a bioengineered pelt with a unique pre-coded genetic signature combination that distinguish it from other pelt sources including pelt farming or poached pelts.

[5] It is another object of the present disclosure to provide a bioengineered pelt with a unique pre-coded genetic signature that distinguish it and its product-derivatives from counterfeited pelts and or counterfeited product-derivatives.

[6] It is another object of the present disclosure to provide for a system and method to sustainably and with minimal impact to the environment derive a bioengineered pelt product, and that is continuously available to all its users such as various constituents/entities of the supply chain of a product and its end customers and is very easy to deploy and implement.

[7] It is yet another object of the present disclosure to provide a bioengineered pelt material with a characteristic genetic fingerprint that allows the identification of manufactured parts that cannot be subjected to any counterfeit and secures anti-counterfeit measures of other items permanently attached or integrated to the bioengineered pelt.

[8] It is yet another object of the present disclosure to provide a bioengineered pelt bearing a genetic anti-counterfeit signature combination that is homogeneous at will within the bioengineered pelt batches, and derived product types that use such bioengineered pelt.

[9] It is yet another object of the present disclosure to provide a bioengineered pelt bearing a genetic signature combination that can be used as manufacturing timestamp of the bioengineered pelts, and derived product types that use such bioengineered pelt. [10] It is still another object of the present disclosure to provide a methodology for the identification of original products manufactured using the bioengineered pelt, and that does not require the expensive development of additional advanced technology or technologies.

Items

[1] A nucleic acid molecule containing at least one nucleic acid barcode, and comprising at least one nucleotide sequence encoding a selection marker indicating homologous or heterologous recombination when integrated in the genome of a vertebrata cell, wherein the selection markers when being expressed are optically discriminable, e.g. in FACS or any fluorescence guided capture, and wherein the nucleotide sequence encoding a selection marker indicating homologous or heterologous recombination in a vertebrata cell is flanked 5' and/or 3' by nucleotide sequences that are homologous to nucleotide sequences of a nucleic acid sequence present in the vertebrata cell.

[2] The nucleic acid barcode of item 1 , wherein said nucleic acid barcode is not present in the genome of the target vertebrata cell and encodes data regarding characteristics of a item 32, its manufacturing process, and its upstream and downstream production chain.

[3] The nucleic acid molecule of items 1 , wherein said nucleotide sequence encoding a selection marker indicating homologous or heterologous recombination comprises a promoter driving expression of said selection markers.

[4] The nucleic acid molecule of item 3, wherein said promoter is constitutive or inducible.

[5] The nucleic acid molecule of item 1 , wherein said nucleic acid molecule comprises a chemical resistance selection marker selected from neomycin resistance, hygromycin resistance, HPRT1 , puromycin resistance, puromycin N-acetyl-transferase, blasticidin resistance, G418 resistance, phleomycin resistance, nourseothricin resistance or chloramphenicol resistance.

[6] The nucleic acid molecule of item 1 , wherein said nucleotide sequences that are homologous to nucleotide sequences of a nucleic acid sequence present in the target vertebrata cell allow homologous or heterologous recombination with nucleotide sequences of a nucleic acid sequence present in said vertebrata cell. [7] The nucleic acid molecule of item 1 , wherein homologous or heterologous recombination allows depositing a nucleic acid barcode into the genome of the target vertebrata cell.

[8] The nucleic acid molecule of item 1 , wherein homologous or heterologous recombination occurs at one or multiple loci comprised by said vertebrata cell.

[9] The nucleic acid molecule of any one of the preceding items, wherein homologous or heterologous recombination is induced by random integration, TALENs, ZFNs, meganucleases, or CRISPR nuclease.

[10] The nucleic acid molecule of item 1 , wherein said vector is circular or linearized.

[11] The nucleic acid molecule of any one of the preceding items, wherein said optical discriminability is different emission wavelength. The nucleic acid molecule of any one of the preceding items, wherein said selection marker indicating homologous or heterologous recombination is a fluorescent protein.

[12] The nucleic acid molecule of item 11 , wherein said fluorescent protein is selected from Sirius, SBFP2, Azurite, EBFP2, mKalamal , mTagBFP2, Aquamarine, ECFP, Cerulean, mCerulean3, SCFP3A, mTurquoise2, CyPet, AmCyanl , mTFP1 , MiCy, iLOV, AcGFPI , sfGFP, mEmerald, EGFP, mAzamiGreen, cfSGFP2, ZsGreen, mWasabi, SGFP2, Clover, mClover2, EYFP, mTopaz, mVenus, SYFP2, mCitrine, YPet, ZsYellowl , mPapayal , mKO, mOrange, mOrange2, mK02, TurboRFP, mRuby2, eqFP61 1 , DsRed2, mApple, mStrawberry, FusionRed, mRFP1 , mCherry, mCherry2, dTOMATO, tdTOMATO, tagBFP, photoactivatable or photoswitchable fluorescent protein.

[13] At least one nucleic acid barcode of at least one nucleotide length that creates a composition of matter comprising a mixture of at least one different nucleic acid barcode, each comprising a different nucleotide sequence encoding a selection marker indicating homologous or heterologous recombination in said vertebrata cell.

[14] A fibroblast-like cell derived from a vertebrata, wherein said vertebrata cell is selected from antelope, antilopini, beaver, buffalo, caiman, caracal, cat, cheetah, chinchilla, cow, crocodille, deer, eland, elephant, ermine, faux, fisher, fox, genet, giraffe, goat, golden jackal, hedgehog, horse, leopard, lynx, lion, marten, mink, monkey, ape, nutria, otter, rabbit, rhinoceros, sable, serval, sheep, shrew, snake, stoat, swine, wolf, australian brushtail possum, mouse, rat Camelidae, their subspecies and Pantholopinae such as: Dromedary Camel (Camelus Dromedarius), Bactrian Camel (Camelus Bactrianus) and it’s wildtype, Llama (Llama glama), Alpaca (Vicugna Pacos), Vicuna (Vicugna Vicugna), Guanaco (Lama Guanicoe) and Tibetan Antilope (Pantholops Hodgsonii)

[15] A fur follicle stem cell derived from a vertebrata, wherein said vertebrata cell is selected from antelope, antilopini, beaver, buffalo, caiman, caracal, cat, cheetah, chinchilla, cow, crocodille, deer, eland, elephant, ermine, faux, fisher, fox, genet, giraffe, goat, golden jackal, hedgehog, horse, leopard, lynx, lion, marten, mink, monkey, ape, nutria, otter, rabbit, rhinoceros, sable, serval, sheep, shrew, snake, stoat, swine, wolf, australian brushtail possum, mouse, rat Camelidae, their subspecies and Pantholopinae such as: Dromedary Camel (Camelus Dromedarius), Bactrian Camel (Camelus Bactrianus) and it’s wildtype, Llama (Llama glama), Alpaca (Vicugna Pacos), Vicuna (Vicugna Vicugna), Guanaco (Lama Guanicoe) and Tibetan Antilope (Pantholops Hodgsonii)

[16] The vertebrata fur follicle stem cell of item 15, wherein said vertebrata cell markers is selected from Cd34, Lgr, Lgr5, integrin alpha-6, Lgr6, GIH , Lrig1 , Krt, or combinations of the above.

[17] A pluripotent stem cell derived from a vertebrata, wherein said vertebrata cell is selected from antelope, antilopini, beaver, buffalo, caiman, caracal, cat, cheetah, chinchilla, cow, crocodille, deer, eland, elephant, ermine, faux, fisher, fox, genet, giraffe, goat, golden jackal, hedgehog, horse, leopard, lynx, lion, marten, mink, monkey, ape, nutria, otter, rabbit, rhinoceros, sable, serval, sheep, shrew, snake, stoat, swine, wolf, australian brushtail possum, mouse, rat, Camelidae, their subspecies and Pantholopinae such as: Dromedary Camel (Camelus Dromedarius), Bactrian Camel (Camelus Bactrianus) and it’s wildtype, Llama (Llama glama), Alpaca (Vicugna Pacos), Vicuna (Vicugna Vicugna), Guanaco (Lama Guanicoe) and Tibetan Antilope (Pantholops Hodgsonii).

[18] The vertebrata pluripotent stem cell of item 17, wherein said pluripotent stem cell type is selected from embryonic stem cell or induced pluripotent stem cell.

[19] A extracellular matrix blend, wherein said matrix blend components is selected from laminin, fibronectin, collagen, heparan sulfate, chondroitin sulfate, keratan sulfate, hyaluronic acid, elastin, integrin, cadherin, selectin, connexins, claudins, occludins, and chemically modified extracellular matrix proteins, or combinations of the above.

[20] A nucleic acid molecule different from item 1 , wherein said nucleic acid molecule comprises at least one nucleotide sequence encoding at least one fitness gene, selection marker indicating homologous or heterologous recombination when integrated in the genome of a vertebrata cell, wherein the selection markers when being expressed confers chemical resistance or is optically discriminable, e.g. in FACS or any fluorescence guided capture, and wherein the nucleotide sequence encoding a selection marker indicating homologous or heterologous recombination in a vertebrata cell is flanked 5' and or 3' by nucleotide sequences that are homologous to nucleotide sequences of a nucleic acid sequence present in the vertebrata cell.

[21] The nucleic acid molecule of item 20, wherein said nucleic acid molecule comprises a chemical resistance selection marker selected from neomycin resistance, hygromycin resistance, HPRT1 , puromycin resistance, puromycin N-acetyl-transferase, blasticidin resistance, G418 resistance, phleomycin resistance, nourseothricin resistance or chloramphenicol resistance.

[22] The nucleic acid molecule of item 20, wherein said optical discriminability is different emission wavelength. The nucleic acid molecule of item 20, wherein said selection marker indicating homologous or heterologous recombination is a fluorescent protein.

[23] The nucleic acid molecule of item 22, wherein said fluorescent protein is selected from Sirius, SBFP2, Azurite, EBFP2, mKalamal , mTagBFP2, Aquamarine, ECFP, Cerulean, mCerulean3, SCFP3A, mTurquoise2, CyPet, AmCyanl , mTFP1 , MiCy, iLOV, AcGFPI , sfGFP, mEmerald, EGFP, mAzamiGreen, cfSGFP2, ZsGreen, mWasabi, SGFP2, Clover, mClover2, EYFP, mTopaz, mVenus, SYFP2, mCitrine, YPet, ZsYellowl , mPapayal , mKO, mOrange, mOrange2, mK02, TurboRFP, mRuby2, eqFP61 1 , DsRed2, mApple, mStrawberry, FusionRed, mRFP1 , mCherry, mCherry2, dTOMATO, tdTOMATO, tagBFP, photoactivatable or photoswitchable fluorescent protein.

[24] The nucleic acid molecule of item 20, wherein said fitness gene is selected from telomerase, Ras, Abl, Akap13, Araf, Tim, Atf, Axl, Bel, Braf, Brea, Brip, Cbl, Csflr, Dapk, Dek, Dusp, Egf, Egfr, Erbb, Erg, Ets, Ewsr, Fes, Fgf, Fgfr, Flcn, Fos, Frap, Fus, Hras, Gli, Gpc, Neu, Hgf, Irf, Junb, Kit, Kras, Lck, Leo, Mapk, Mcf, Mdm2, Met, Mlh, Mmd, Mos, Mras, Msh, Myb, Myc, Lmyc, Nmyc, Ele1 , Nf 1 , Trk, Can, Ovc, Tp53, Palb2, Pax3, Pdgfb, Pim, Pml, Pms, Wip, Pten, Pvt, Raf, Craf, Rb, Rras, Met, Smad, Smurf, Src, Stat, Tdgf, Tgfbr, Erba, Tgf, Tif, Tnc, Trk, Tusc, Usp, Wnt, Wt, Vhl, or combinations of the above.

[25] The nucleic acid molecule of items 24, wherein said nucleotide sequence encoding a fitness gene comprises a promoter driving expression of said selection markers.

[26] The nucleic acid molecule of item 25, wherein said promoter is constitutive or inducible.

[27] The nucleic acid molecule of any one of the preceding items, wherein homologous or heterologous recombination is induced by random integration, TALENs, ZFNs, meganucleases, or CRISPR nuclease.

[28] The vertebrata fibroblast-like cell of item 14, wherein said fibroblast-like cell is engineered to contain at least one nucleic acid molecule of item 20, at least one chemical selection marker of item 21 and or at least one optically active selection marker of item 22 and 23, at least one fitness gene of item 24, and at least one nucleic acid molecule of item 1 containing at least one nucleic acid barcode of item 13.

[29] The vertebrata fur follicle stem cell of item 15, wherein said fur follicle stem cell is engineered to contain at least one nucleic acid molecule of item 20, at least one chemical selection marker of item 21 and or at least one optically active selection marker of item 22 and 23, at least one fitness gene of item 24, and at least one nucleic acid molecule of item 1 containing at least one nucleic acid barcode of item 13.

[30] The vertebrata pluripotent stem cell of item 17, wherein said pluripotent stem cell is engineered to contain at least one nucleic acid molecule of item 20, at least one chemical selection marker of item 21 and or at least one optically active selection marker of item 22 and 23, at least one fitness gene of item 24, and at least one nucleic acid molecule of item 1 containing at least one nucleic acid barcode of item 13.

[31] A nucleic acid barcode supra-combination, wherein said nucleic acid barcode supra- combination is comprised of the combinations of barcodes in items 28, item 29 and item 30.

[32] Encapsulated fur follicle stem cells from item 15, wherein said cells are encapsulated in extracellular matrix from item 19. [33] A lower cellular layer, wherein said lower layer is composed of fibroblast-like cells of item 14 and extracellular matrix of item 19.

[34] An intermediate cellular layer, wherein said intermediate layer is composed of the fur follicle cells of item 15 and interfollicular epidermis cells derived from cells of item 17.

Alternatively, an intermediate cellular layer, wherein said intermediate layer is composed of the encapsulated fur follicle cells of item 32 and interfollicular epidermis cells derived from cells of item 17.

[35] An upper layer, wherein said upper layer is composed of fur and or hair filaments protruding from the intermediate layer of item 34.

[36] A polymer surface, wherein said surface is selected from poly-acrylates, poly ornithine, poly-olefines, hydrogels, poly-amino acids, poly-peptides, or other polymer surfaces.

[37] A bioengineered pelt, wherein said bioengineered pelt is composed of the layers in item 33, item 34 and item 35.

[38] An in vitro method for manufacturing and deriving bioengineered pelt, wherein said bioengineered pelt is composed of the layers in item 33, item 34 and item 35.

(a) Subjecting population of engineered fibroblast-like cells of item 14 to liquid merging with extracellular matrix of item 19 and seeding on surface item 36.

(b) Culturing the lower layer of item 33 within one hour and seven days.

(c) Subjecting populations of engineered fur follicle cells of item 15 and interfollicular epidermis cells derived from cells of item 17 to liquid merging and plating onto lower layer of item 33. The ratio of cells of item 15 and item 17 is adjusted within 1 :1 to 1 :1x10e6.

Alternatively, populations of interfollicular epidermis derived from cells of item 17 can be merged in colloidal solution with encapsulated fur follicle cells of item 32.

(d) Partly exposing the intermediate layer of item 34 to normoxic atmosphere, white light, mechanical shearing and or shedding, magnetic shearing and or shedding, and or combinations of the above. (e) Culturing the combined superstructure of lower layer of item 33 with the intermediate layer of item 34 within seven days and thirty days.

(f) Within the co-culture of lower and partly exposed intermediate layer as in (e) permit the development of fur and hair filaments sprouted from the intermediate layer and which constitute the upper layer.

(g) Collection of the three layered bioengineered pelt of item 37.

[38] A mean and method for determining the authenticity of the bioengineered pelt of item 37 and its derivative products.

(h) The bioengineered pelt of item 37 carries an inseparable molecular signature or molecular fingerprint composed by the combination of combinations of nucleic acid barcodes of item 13 held within the genome of cells item 14, item 15 and item 17.

(i) Part of the bioengineered pelt of item 37 and holder of the properties in (h) can be analyzed by DNA sequencing to detect the presence, composition and dose of the nucleic acid barcodes of item 13.

(j) The readout in (i) constitutes a binary-readout of the authenticity of the bioengineered pelt of item 37 and its derivative products. The readout enables demonstrating the source, production batch, production time and excluding counterfeit or adulteration of item 37, its derived products and items irreversibly attached to item 37.

(k) The properties of (h), analysis of (i) and readout of (j) constitutes a mean of authenticity and a mechanism of anti-counterfeit, proof of provenance and allow differentiating item 37 from other pelts derived from pelt farming or pelt poaching.

Brief Description Of The Drawings

The accompanying drawings are included to provide a further understanding of the present disclosure and are incorporated in and constitute a part of this specification. The drawings illustrate exemplary embodiments of the present disclosure and, together with the description, serve to explain the principles of the present disclosure.

In the figures, similar components and/or features may have the same reference label.

Further, various components of the same type may be distinguished by following the reference label with a second label that distinguishes among the similar components. If only the first reference label is used in the specification, the description is applicable to any one of the similar components having the same first reference label irrespective of the second reference label.

FIG. 1 illustrates a proposed bioengineered pelt in accordance with an exemplary embodiment of the present disclosure.

a item 35 upper layer

b item 34 intermediate layer

c item 33 lower layer

d hair and or fur fillament

e item 19 extracellular matrix

f item 14 vertebrata fibroblast-like cell

g interfollicular epidermis cells derived of item 17

h fur follicle cells derived of item 15 fur follicle stem cells or item 32

FIG. 2 illustrates a proposed anti-counterfeit genetic signature combination intrinsically integrated in the bioengineered pelt, and derived product, and product batch, and product types, and timestamp. The genetic signature is inseparably connected to the bioengineered pelt and its derived products

a item 37 bioengineered pelt

b product derived from bioengineered pelt by manufacturing

c data extracted and decoded from item 2 barcodes

d combination of combinations of item 2 barcodes storing encoded information of item 32 and its upstream and downstream production chain

(barcode 1 SEQ ID NO:1-2; barcode 2, SEQ ID NO:3-4; barcode 3, SEQ ID NO:5-6;

timestamp, SEQ ID NO:7-8)

FIG. 3 Aggregates of HFDPC at day 33 in two different samples and 3 different wells, in sample 2 the aggregates are between 126-192 pm in diameter and in sample 1 the aggregates are between 169-322 pm in diameter. (Fig.3: Macro images of HFDPC aggregates at day 33 from above with measurements of the aggregates. 10x magnification, scale bar 100pm).

FIG. 4 Measurements of aggregate and pore size over the culture period. FIG. 5 Collection of brightfield images taken from below during the culture period. ( Table 3. Images from bottom of constructs over the culture time for different samples. 4x

magnification, scale bar 200pm. )

FIG. 6 Viability analysis for HDF (fibroblasts), HFDPC (human follicle dermal papilla cells) respectively HEK (keratinocytes) over the culture period. Represented in bar graph in the top row and as lines in the bottom row.

FIG. 7 Cross-section of 3D constructs after 1 day of co-culture of all three cell types. Top images: viable cells stained with Calcein (green), bottom images: brightfield images of the same position. The right and left column have same position but different depth focus. Cluster of follicle dermal papilla cells is highlighted with orange circle.

FIG. 8 Viability images over the culturing time period. Green: viable cells stained with calcein. Red: dead cells stained with PI. 10x magnification, scalebar 100pm.

FIG. 9 Actin staining. Collection of actin images over the culturing time period. (Fig 9 Images of actin staining over the culturing time period. Green: actin. Blue: NucBlue staining of nucleuses. 10x magnification, scalebar 100pm.)

FIG. 10 Collection of H&E images over the culturing time period. The reason the samples are highly orange, instead of the traditional purple-pink is due to manual fabrication where small time differences in the staining steps mature the pink color into become more reddish. (FIG. 10: images of H&E staining over the culturing time period. 10x magnification, scale bar 100pm. 4x magnification, scale bar 200pm.)

FIG. 11 Collection of immunofluorescence images of versican (VCAN) and K75 over the culturing time period. (FIG. 11 Immunofluorescence staining of versican (VCAN) and k75 over the culturing time period. Merged of VCAN/K75 (red) and DAPI (blue) counter staining. 10x magnification, scalebar 100pm.)

FIG. 12 Collection of immunofluorescence images of Keratin 14 (KRT14), Keratin 5 (KRT5), laminin 5 (LAM5), filaggrin (FLG), vimentin (VIM) and collagen type 1 (COL1) over the culturing time period. (FIG 12 Images of immunofluorescence staining over the culturing time period. Merged images of keratin 14 (KRT14) or vimentin (VIM) (green) and keratin 5 (KRT5) or collagen type 1 (COL1) (red), marked for each column, and DAPI (blue) counter staining. 10x magnification, scalebar 100pm.)

FIG 13 Immunofluorescence images of native skin, positive control. (FIG 13

Immunofluorescence staining of native skin. Merged images of keratin 14 (KRT14) or vimentin (VIM) (green) and keratin 5 (KRT5) or collagen type 1 (COL1) (red), marked for each column, and DAPI (blue) counter staining. 10x magnification, scalebar 100pm.)

Experimental section

Feasibility study

3D Bioprinting is a novel technique that shows great promise for the field of biofabrication. The long-term goal is to use 3D bioprinting to construct a 3D bioprinted artificial mink fur that can be used by the fashion industry. A feasibility study is performed using mesenchymal stem cells (MSC ^' s), dermal papillae cells (DP ^' s) and mink induced pluripotent stem cells (iPSC ^' s) to derive 3D bioprinted models, followed by tissue culture to induce the formation of hair follicles.

The aim of the study is to derive artificial mink fur prototypes. Besides examining how the morphology and viability of cells, the study will aim to identify the appropriate scaffold materials.

The experimental design consists of two parts, A and B. Where part A cover the creation, culture and maturation of the 3D artificial fur models and part B includes evaluation and analyses the artificial fur models.

Bioink: CELLINK GelMA, ColMA and Colli in combination with CELLINK bioink.

Cells: Mesenchymal stem cells, dermal papillae and induced pluripotent stem cells.

Culture timeline:

Print: dO

Cell culture: up to 32 days

Dimensions of construct:

• Dimensions: 20x20x2.0-2.4mm

• Layer height: 0.4mm (22G nozzle)

Part A:

• 3D bioprinting of MSC ^' s and DP ^' s followed by seeding of iPSC ^' s in CELLINK’s GelMA, ColMa and Coin , in combination with CELLINK bioink for stability reasons. • Cell concentration will be discussed with the client

• Maturation of models (up to 32 days).

Part B:

• Collect samples at chosen, relevant time points.

• Preform analyses and analyze collected data.

Analyses include:

• Viability

• Cell morphology

o Actin/nuclei staining

Sampling of models are taken regularly and will be analysed at specified timepoint. High resolution images of the scaffold are prepared during the experimental sequences.

Materials and Methods

Bioink: Specially formulated bioinks designed to have enhanced durability and suitability towards hair and fur model creation. The bioink for the dermis is based on GelMA and the bioink for the epidermis is based on ColMA.

Cells: Human dermal fibroblasts (PromoCell, C-12300), human follicle dermal papilla cells (PromoCell, C-12005) and human epidermal keratinocytes (PromoCell, C-12005) were purchased from PromoCell. Each cell type was expanded in Fibroblast growth medium (PromoCell, C-23010), Follicle Dermal Papilla Cell Growth Medium (PromoCell, C-26501) respectively keratinocyte growth medium 2 (PromoCell, C-20011) and used at passage 5-6. Establishment of 3D models: Expansion of cells was started 14 days prior to print. At day -1 the dermis, with 1x10 ⁶ HDF/ml bioink, was bioprinted as a skin patch with 4 layers of a 16x16 grid 1 structure on top of a solid bottom layer. The grid structure had 2 layers of brim to reinforce the edges at the top of the construct. The dimensions of the skin patches were 20x20 mm. Constructs were crosslinked by 10 sec photocuring with 405 nm at 5 cm distance and submerged in HDF medium for at least 1h prior to addition of HFDPC. The HDF medium was replaced with HFDPC medium, added in a volume to precisely reach the top edge of the constructs, and 10OmI cell suspension with 7.68x10 ⁵ HFDPC (3 000 HFDPC per well) was added on top of each construct. Constructs were left in incubator overnight to sediment. Following day HFDPC medium were replaced with NHEK medium and 4x10 ⁶ ColMA embedded NHEK were seeded on top of each construct to form the epidermis. The epidermis was crosslinked 2 times by 30 sec photocuring with 405nm at 5cm distance. The 3D bioprinted hair models were kept submerged in NHEK medium for up to 33 days. All bioprinting were performed with a BIO X bioprinter (CELLINK).

Viability assay: Viability analysis was performed by taking samples from two different constructs at each timepoint. Staining of live cells were performed with Calcein AM Viability Dye (Invitrogen™ eBioscience™) followed by staining of dead cells with propidium iodide (Sigma Aldrich, 81845). The viability was analyzed regularly from day one of the culture period using calcein and PI staining for viable respectively dead cells. At each time point two technical measurements of duplicates were analyzed, giving four measuring points per cell type for every time point.

Pore size and aggregate measurements: Pore and aggregate size was measured with Image J on brightfield images of the constructs over the culture period.

Actin staining: Actin staining was performed on fixed samples from two different constructs at each timepoint with ActinGreen™ 488 ReadyProbes™ Reagent (ThermoFisher, R37110). Samples were counter stained with NucBlue™ Fixed Cell ReadyProbes™ (ThermoFisher, R37606) for staining of the nucleus. Fixation was performed with 4% PFA for approximately 24h.

H&E staining: Hematoxylin and Eosin staining was performed on fixed and sectioned samples using Hematoxylin and Eosin Stain Kit (Vector Laboratories, H-3502). Fixation was performed with 4% PFA for approximately 24h. Constructs were dehydrated, embedded in paraffin and sectioned into 5-12 pm thick slices.

Immunofluorescence: 10pm sections were blocked in 3% BSA solution and stained with following primary antibodies over night at 4°C; Anti-Col1A1 (Atlas, HPA011795), Anti-KRT10 (Atlas, HPA012014), Cytokeratin 5 Polyclonal Antibody (Fisher Scientific, PA5-32465), K75 Polyclonal Antibody (Fisher Scientific, PA5-67414), Versican Monoclonal Antibody (Abeam, ab177480) and Vimentin Monoclonal Antibody (V9) (Fisher Scientific, 11344533). Followed by secondary antibody staining for 1 h at room temperature with Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Thermo Fisher, A-11029) and Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 (Thermo Fisher, A-31572). Samples were counter stained with DAPI (ThermoFisher, 62248) for staining of the nucleus.

Viability analysis. In addition to quality control of microwells and cell aggregates, the effect of customized bioink on the viability of cells was evaluated. To do this, the cells was stained with Calcein and PI at different time points as indicated in Figure 3. As shown in Figure 6, HDF cells showed minor changes in total cell count as well as viability indicating that the bioink does not affect the health of cells. In contrary to HDFs, HFDPCs and HEKs showed variability in cell count and viability at different time points. HFDPC analysis showed increased cell count in the first 3 weeks of culture with a sudden drop on day 28, whereas HEK showed very steep increase in cell count in first week with gradual decrease in subsequent weeks of the culture (Figure 6). Although total cell count of HFDPC and HEK showed variability, the viability of both cell types gradually decreased reaching steady state at day 21 and 28 of culture. The decrease in viability of HFDPC and HEK can be explained by the inherent short life span that in normal cases should end in hard respectively soft cornification (1 ,2).

According to the provider, these HFDPCs and HEKs have limited proliferative capacity of 10 and 15 population doubling time respectively.

As quantification of viability in 3D matrix could be challenging due to inability to analyze all cells in one plane, the viability data was rather used as a qualitative measurement of overall cell health in the customized bioink scaffold. For example, Calcein stain (green) showed different intensity when imaged at the same position but in different plane as also visible from the corresponding brightfield image.

Table 1. Collection of aggregate and pore size measurements over the culture period and calculations of average measurement. *Average of aggregate size for the specific timepoint. **Average over all timepoints.

28 262,5 283.9 316,1 287.5 832,3

33 195.2 295.2 243,6 244.6 906.5

AVERAGE** 277.3 868.7

Bibliography

1. Eckhart, L, Lippens, S., Tschachler, E. & Declercq, W. Cell death by cornification.

Biochim. Biophys. Acta - Mol. Cell Res. 1833, 3471-3480 (2013).

2. Rinnerthaler, M., Streubel, M. K., Bischof, J. & Richter, K. Skin aging, gene expression and calcium. Exp. Gerontol. 68, 59-65 (2015).