PROCESSES AND SYSTEMS USING FOR CULTURING CELLS USING FEEDER CELLS IN A HOLLOW FIBER CARTRIDGE

[0001] This application claims the benefit of U.S. Provisional Application No. 63/436,162, filed December 30, 2022, U.S. Provisional Application No. 63/325,954, filed March 31, 2022, and U.S. Provisional Application No. 63/325,883, filed March 31, 2022, the contents of each which are hereby incorporated by reference in their entirety.

[0002] Throughout this application, various publications arc referenced, including referenced in parenthesis. The disclosures of all publications mentioned in this application in their entireties are hereby incorporated by reference into this application in order to provide additional description of the art to which this invention pertains and of the features in the art which can be employed with this invention.

FIELD OF THE INVENTION

[0003] The present invention relates to the fields of cell biology, molecular biology and biotechnology. More particularly, the invention relates to a method of culturing cells using feeder cells in a hollow fiber cartridge.

BACKGROUND OF THE INVENTION

[0004] Eukaryotic cells lack a cell wall, making them more susceptible to cell stress and rupture. Because of their sensitive nature, the use of eukaryotic cell cultures has been primarily limited to the pharmaceutical industry. The pharmaceutical industry practices include the use of moderately-sized bioreactors no bigger than 20 m ³ , which results in low biomass yields. Their developed bioprocesses involving mammalian cells are purposefully designed to facilitate low densities to promote the secretion of high-value proteins. Cell therapy requires only small quantities of mammalian cells since they arc almost always paticnt-spccific, meaning the developed cells can only be utilized by one person. The biopharmaceutical industry supports their goals of producing a small amount of high-value biomass for few consumers because it is able to operate with high margins. As the field of cellular agriculture emerges, bio reactors and mammalian cell proliferation are entering the domain of commodification — but enterprises are now faced with the unsolved issue of scaling, as current cell manufacturing methods cannot keep pace with agriculture’s demanded rate of production. [0005] Tn order to transition mammalian cell manufacturing away from the biopharmaceutical industry and into cellular agriculture, techniques and equipment must be developed to facilitate large-scale production of cells, including bioreactors larger than 20 m ³ . The techniques and equipment should be easily adaptable to all eukaryotic cells that lack a cell wall including, but not limited to, vertebrate, mammalian, avian, and reptilian cells. Currently, complete solutions are lacking and remain to be seen. The use of small plastic beads as microcarriers has been shown to aid mammalian cell expansion by providing a surface for cells to adhere to (allowing for adherent cells to be grown in bioreactors). These microcarriers were also shown by one group to provide a small level of protection from shear forces. Verbruggen et al. (2018). However, these microcarriers have proven inadequate, as their plastic material takes up valuable space in the bioreactors, therefore limiting the volume potential for biomass generation.

[0006] Cells need continuous delivery of oxygen while in bioreactor culture and the rate of oxygen demand increases as the cell mass increases. Thus, aeration, or delivery of oxygen to cells, is an important aspect of a bioreactor environment, especially for large-scale production of cells. However, aeration of the bioreactor environment introduces shear forces that can damage eukaryotic cells, either from rotating blades in the bioreactor or from air bubbles. Walls et al. (2017). Thus, to enable large-scale production of cells, there is a need for culturing systems and processes that allow bioreactors to be better aerated without causing damage to the cells.

[0007] Further, cell culture medium has been identified as the most significant cost driver in the production of cultivated meat, namely the recombinant proteins included in the media’s makeup. Specht et al. (2020); Stout et al. (2022a). FBS (Fetal Bovine Serum) is considered an expensive standard in the pharmaceutical industry, but the cellular agriculture industry has decidedly agreed to eliminate its use in cultivated meat. FBS contains a high concentration of recombinant proteins, with albumin, transferrin and fetuin having particularly important roles.

[0008] Additionally, cell culture media is usually supplemented with other recombinant proteins such as bFGF, IGF, TGF-P, PDGF, as well as various cytokines and other growth factors. Fonoudi et al. (2020); Ludwig et al. (2007); Jang et al. (2022); Stout et al. (2022a); Stout et al. (2022b). Growth factors can be sourced from animals, precision fermentation, or molecular farming, however, each of these production methods has several downfalls. Animal sources vary in quality, while precision fermentation can be costly. Efficacy and quality of recombinant proteins may vary due to differences in refolding and post translational modifications to recombinant proteins. Patil ct al. (2022). Microorganisms used for production may not post translationally modify the proteins in a biologically relevant manner, while downstream processing steps to isolate proteins may interrupt proper folding and protein function. Vieira Gomes et al. (2018). Molecular farming is promising as post translational modifications may more readily mimic animal post translational modifications, however, it is costly to scale up and an unproven technology in the market.

[0009] Feeder cells are traditionally used in biomedical science research as a co-culture system. One example is mouse embryonic fibroblasts (MEFs) for the culture of pluripotent stem cells. Eiselleova et al. (2004). Sanchez et al. (2012). However, the only way to use these feeder cells is by disrupting their capacity to replicate, therefore being outgrown by the stem cells. Otherwise, the MEFs would take over the culture and discriminating between the pluripotent stem cells and the feeder cells would be difficult. Other co-culture systems include the use of transwells of physically separate chambers that do not allow the mixture of cell types. Ronaldson-Bouchard et al. (2022).

[0010] Microcarriers are small, typically spherical beads that are used in cell culture to support growth of cells. U.S. Patent No. 9,340,770 B2 describes a variety of microcarrier compositions that may be useful for the culturing cells. However, microcarriers such as those described in U.S. Patent No. 9,340,770 B2 are used in cell culture to increase the surface area available for cell attachment and growth — thus, such microcarriers typically carry the cultured cells on their surface. In the art, such a use of microcarriers is advantageous because the cultured cells are directly exposed to the cell culture medium. However, because the cells are found on the surface of the microcarriers, they are not protected from shearing forces introduced in the culturing process.

[0011] Thus, there is a need for improved systems and processes for producing and delivering the components cell culture media to cells. SUMMARY OF THE INVENTION

[0012] This invention provides a process for culturing eukaryotic cells, comprising:

(a) co-culturing in a bioreactor:

(i) eukaryotic cells encapsulated in a first set of microcarriers; and

(ii) feeder cells encapsulated in a second set of microcarriers; and

(b) separating the first set of microcarriers from the second set of microcarriers.

[0013] This invention also provides a feeder cell comprising one or more or all of the following codon-optimized recombinant genes incorporated into the genome thereof:

(a) somatotropin;

(b) platelet-derived growth factor (PDGF);

(c) albumin;

(d) insulin-like growth factor (IGF);

(e) transferrin;

(f) insulin;

(g) vascular endothelial growth factor (VEGF);

(h) transforming growth factor beta (TGF-p);

(i) hepatocyte growth factor (HGF);

(j) basic fibroblast growth factor (bFGF);

(k) epidermal growth factor (EGF); and

(l) interleukin 6 (IL-6).

[0014] This invention also provides a co-culturing system comprising:

(a) eukaryotic cells encapsulated in a first set of microcarriers;

(b) feeder cells encapsulated in second set of microcarriers; and

(c) a bioreactor comprising:

(i) an interior and interior surface;

(ii) means to aerate the interior of the bioreactor; and

(iii) a semi-permeable barrier which:

1. separates the interior of the bioreactor into a bottom portion and a top portion; 2. is impermeable to the first set of microcarriers and second set of microcarriers; and

3. is permeable to liquids.

[0015] This invention also provides a culturing system comprising:

(a) eukaryotic cells encapsulated in a first set of microcarriers;

(b) a bioreactor comprising:

(i) an interior and interior surface;

(ii) means to aerate the interior of the bioreactor; and

(iii) a semi-permeable barrier which:

1 . separates the interior of the bioreactor into a bottom portion and a top portion;

2. is impermeable to the first set of microcarriers; and

3. is permeable to liquids; and

(c) one or more hollow fiber cartridges containing feeder cells connected to the bioreactor, preferably removably connected to the bioreactor, more preferably removably connected to the bioreactor via one or more couplings, more preferably wherein the couplings are disposable.

[0016] This invention also provides a process for culturing eukaryotic cells, comprising culturing eukaryotic cells encapsulated in a first set of microcarriers in a culturing system comprising:

(i) a bioreactor comprising:

1. an interior and interior surface;

2. means to aerate the interior of the bioreactor; and

3. a semi-permeable barrier which: a. separates the interior of the bioreactor into a bottom portion and a top portion; b. is impermeable to the first set of microcarriers; and c. is permeable to liquids; and

(ii) one or more hollow fiber cartridges containing feeder cells connected to the bioreactor, preferably removably connected to the bioreactor, more preferably removably connected to the bioreactor via one or more couplings, more preferably wherein the couplings arc disposable.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : A schematic of a bioreactor comprising: (1A) a ridged perimeter or other non-circular perimeter that increase the internal surface area of the reactor, (IB) one or more sensors placed throughout the reactor to provide proper coverage and accurate measurements of the distributions of nutrients and waste products, determine pH, temperature, oxygen, carbon dioxide, and/or detect levels of other compounds critical for cell health and expansion, (1C) a barrier in the foam zone to prevent cells from entering the cytotoxic area of the system, (ID) mixing and/or aeration elements that allow the bioreactor to operate at mixing speeds and/or aeration levels higher than traditional bioreactors used in the biopharmaceutical industry for cells that lack a cell wall, and (1 E), a feedback system that controls the level of pH, temperature, oxygen, carbon dioxide, and other compound relevant to cell health.

[0018] Figure 2 : Microcarriers used to support high cell densities from multiple cells sourced from any eukaryotic cell type, including but not limited to (2A) mollusks, chordates and arthropods. The microcarriers encapsulate cells (2B) and protect them from shear forces while allowing for prevention of waste accumulation in the cellular microenvironment and facilitating higher available oxygen concentration. The microcarriers may be edible and may be made of foodgrade and/or plant-based hydrogel such as alginate or agarose (2C). The microcarriers can allow for the growth of cells in a 3D space, therefore eliminating the traditional loss of volume from plastic microcarriers (2D). The microcarriers can be used in conjunction with plastic microcarriers or any cell scaffold to protect cells from shear forces (2E) and can be used to support high cell densities from multiple cell types including, but not limited to, myoblasts, mesenchymal stem cells, pluripotent stem cells and fibroblasts (2F). The formation of edible microcarriers maybe reversible and does not require the use of complex equipment or enzymes to isolate the biomass (2G).

[0019] Figure 3 : A schematic of a bioreactor comprising (IF) a hollow fiber cartridge comprising feeder cells. The bioreactor may contain one or more or all of the elements of the bioreactor depicted in Figure 1.

[0020] Figure 4 : A co-culture system with recyclable microcarriers. The recyclable microcarriers can be separated from other microcarriers by methods including size exclusion, density gradients or magnetic separation (4A). The recyclable microcarriers may be made from food grade plant-based hydrogel such as agarose or alginate and may be infused with iron to induce a magnetic or density differential from other microcarriers (4B). Feeder cells encapsulated in the recyclable microcarriers arc engineered to secrete recombinant proteins including, for example, somatotropin, platelet-derived growth factor (PDGF), albumin, insulin-like growth factor (IGF), insulin, transferrin, vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF-p), hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and/or interleukin 6 (IL-6). The feeder cells may be immortalized using methodologies such as mitomycin C treatment or gamma radiation. The feeder cell may be allowed to proliferate and are passed between batches. The feeder calls may be made of cell types traditionally known to be contributors to paracrine signaling such as fibroblasts, mesenchymal stem cells, or endothelial cells. Paracrine signaling refers to traditional cell to cell communication within the same tissue or organ whereas endocrine signaling refers to traditional cell to cell communication between separate organs. The feeder cells may be made of cell types traditional known to be contributors to endocrine signaling such as beta cells. The feeder cell may be used to support multiple cell types such as myoblasts, adipocytes, mesenchymal stem cells, pluripotent stem cells and fibroblasts. The feeder cells may be used to support cells sourced from varying animal kingdoms including but not limited to, mollusks, chordates and arthropods. The feeder cells in recyclable microcarriers may be used to provide recombinant and endogenous proteins for cell types used in the production of cell-based consumer goods such as, but not limited to, meat and skin.

[0021] Figure 5: Generation of bFGF feeder cells. Stromal cells were transduced with a lentiviral particle containing an mScarlet protein and the bovine bFGF gene.

[0022] Figure 6 : The price of the bFGF produced, compared to competitors.

[0023] Figure 7: Growth factors diffuse out of biomaterial to meet the demands in cell culture media. Rhodamine labeled 75 kDa dextran was encapsulated into alginate pods and the amount of rhodamine that diffused out of the pods over the course of an hour was measured. Diffusion of the dextran beads was then modeled over the course of three days. Left: the concentration of dextran compared to the required concentration of albumin in Beefy-9. Right: the concentration of dextran compared to the required concentration of bFGF in Beefy-9.

[0024] Figure 8: Conditioned media (Edge media) promotes wound healing, (a) A standard Pl 000 pipette was used to disrupt a confluent layer of fibroblasts, (b) Wound closure was tracked over a period of four days with bright field images of entire wells, (c) Average wound closure for entire fibroblast wells were calculated and a percent change was used to compare the effect of conditioned media (Edge) on cellular proliferation/migration (grey dots represent technical replicates, bars represent averages). T-test, n=3, p <.05

[0025] Figure 9: Genetically Engineered stromal cells express high levels of albumin in a cost effective manner. Stromal cells were transduced with a lentiviral particle containing an mScarlet protein and the bovine albumin gene. Stromal cells are part of connective tissue and generally form supporting biological structures.

[0026] Figure 10: Quantification of the amount of albumin produced per 100,000 stromal cells (solid line; albumin produced, dotted line: the concentration of recombinant albumin in beefy-9, a leading serum free myoblast media).

[0027] Figure 11: The price of albumin produced using the invention compared to competitors.

[0028] Figure 12: Panel A depicts modulation of voltage to precisely control diameter of spherical alginate capsules. Panel B through D depict Alginate beads of various diameters created with an in-house electrostatic bead generator (diameter range approximately 120- 1000pm).

[0029] Figure 13: Panel A depicts cells encapsulated in an alginate bead at rest, with view of the scaffolding formed by the bead matrix. Panel B provides a schematic representation of two potential sources of bead stress due to external hydrodynamic forces: bead deformation and porous flow inside the bead.

DETAILED DESCRIPTION OF THE INVENTION

Processes for culturing eukaryotic cells

[0030] This invention provides a process for culturing eukaryotic cells, comprising:

(a) co-culturing in a bioreactor:

(i) eukaryotic cells encapsulated in a first set of microcarriers; and

(ii) feeder cells encapsulated in a second set of microcarriers; and

(b) separating the first set of microcarriers from the second set of microcarriers.

[0031] In embodiments of the invention, the process further comprises:

(a) removing the eukaryotic cells from the first set of microcarriers;

(b) recovering the second set of microcarriers;

(c) recovering exosomes from the bioreactor; and/or

(d) recovering conditioned media from the bioreactor.

[0032] In embodiments of the invention, the first and/or second set of microcarriers comprise a food-grade hydrogel, preferably a plant-based food-grade hydrogel.

[0033] In embodiments of the invention, the first and/or second set of microcarriers are:

(a) edible; and/or

(b) semi-permeable, preferably permeable to the secretome of the feeder and/or eukaryotic cell and impermeable to the feeder and/or eukaryotic cell itself, more preferably permeable to molecules less than about 150 kDa in size and impermeable to molecules greater than about 150 kDa in size.

[0034] In embodiments of the invention, the first and/or second set of microcarriers comprise:

(a) alginate, preferably RGD modified alginate;

(b) agarose; and/or

(c) one or more extracellular components, preferably gelatin, vitronectin and/or RGD.

[0035] In embodiments of the invention, the first and/or second set of microcarriers further comprise:

(a) one or more plastic microcarriers; and/or

(b) one or more cell scaffolds. [0036] In embodiments of the invention, the first set of microcarriers and/or second set of microcarriers arc microbcads or microsphcrcs.

[0037] In embodiments of the invention, the first set of microcarriers and/or second set of microcarriers are:

(a) between about 100 pm and about 500 pm in diameter, or

(b) between about 200 pm and about 400 pm in diameter.

[0038] In embodiments of the invention, the first set of microcarriers differs from the second set of microcarriers in density, size, and/or magnetic property such that the first set of microcarriers can be separated from the second set of microcarriers based on their respective densities, sizes, and/or magnetic properties.

[0039] In embodiments of the invention, the first set of microcarriers and the second set of microcarriers are of different densities, so as to allow the first set of microcarriers to be separated from the second set of microcarriers based on their respective densities.

[0040] In embodiments of the invention, the step of separating the first set of microcarriers from the second set of microcarriers comprises density gradient separation or centrifugation.

[0041] In embodiments of the invention, the first and/or second set of micro carriers are infused with magnetic particles, preferably iron particles, so as to allow the first set of microcarriers to be separated from the second set of microcarriers by magnetic separation.

[0042] In embodiments of the invention, the step of separating the first set of microcarriers from the second set of microcarriers comprises magnetic separation.

[0043] In embodiments of the invention, the first set of microcarriers are smaller in size than the second set of microcarriers, or wherein the first set of microcarriers are larger in size than the second set of micro carriers.

[0044] In embodiments of the invention, the first set of microcarriers and second set of microcarriers have size distributions that do not overlap, so as to allow the first set of microcarriers to be separated from the second set of microcarriers by size exclusion.

[0045] In embodiments of the invention, the first set of microcarriers are of uniform size and/or wherein the second set of microcarriers are of uniform size. [0046] Tn embodiments of the invention, the step of separating the first set of microcarriers from the second set of microcarriers comprises size exclusion.

[0047] In embodiments of the invention, the bioreactor used in the process comprises:

(i) an interior and interior surface;

(ii) means to aerate the interior of the bioreactor; and

(iii) a semi-permeable barrier which:

1. separates the interior of the bioreactor into a bottom portion and a top portion;

2. is impermeable to the first set of microcarriers and second set of microcarriers; and

3. is permeable to liquids.

[0048] In embodiments of the invention, the bioreactor further comprises one or more sensors.

[0049] In embodiments of the invention, the one or more sensors comprise:

(a) one or more pH sensors;

(b) one or more temperature sensors; and/or

(c) one or more carbon dioxide sensors.

[0050] In embodiments of the invention, the one or more sensors are distributed throughout the interior surface of the bioreactor.

[0051] In embodiments of the invention, the bioreactor further comprises a feedback system that controls one or more of:

(a) pH;

(b) temperature;

(c) oxygen;

(d) carbon dioxide;

(e) glucose;

(f) ammonia;

(g) other compounds relevant to cell health.

[0052] In embodiments of the invention, the means to aerate the interior of the bioreactor comprises: (a) a sparger-based aeration system; or

(b) an impeller or paddle-based aeration system.

[0053] In embodiments of the invention, the bioreactor further comprises a mixer.

[0054] In embodiments of the invention:

(a) the eukaryotic cells encapsulated in the first set of microcarriers were produced by:

(i) mixing eukaryotic cells with a carrier substance to produce a microcarrier mixture; and

(ii) electrostatic bead generation of the microcarrier mixture; and/or

(b) the feeder cells encapsulated in the second set of microcarriers were produced by:

(i) mixing feeder cells with a carrier substance to produce a microcarrier mixture; and

(ii) electrostatic bead generation of the microcarrier mixture.

[0055] In embodiments of the invention, the process comprises, prior to the co-culturing step:

(a) producing the eukaryotic cells encapsulated in a first set of microcarriers by:

(i) mixing eukaryotic cells with a carrier substance to produce a microcarrier mixture; and

(ii) electrostatic droplet formation of the microcarrier mixture;

(b) producing the feeder cells encapsulated in a second set of microcarriers by:

(i) mixing feeder cells with a carrier substance to produce a microcarrier mixture; and

(ii) electrostatic droplet deposition of the microcarrier mixture; and/or

(c) obtaining feeder cells encapsulated in a second set of microcarriers by recycling feeder cells encapsulated in microcarriers that were used in a prior culturing process, preferably a prior culturing process according to the present invention.

[0056] In embodiments of the invention, the carrier substance comprises:

(a) alginate, preferably RGD modified alginate;

(b) agarose; and/or (c) one or more extracellular components, preferably gelatin, vitronectin and/or RGD.

[0057] In embodiments of the invention, the eukaryotic cells and/or feeder cells are encapsulated in the microcarriers in the form of clumps of cells or single cell suspensions.

[0058] In embodiments of the invention, the eukaryotic cells lack a cell wall.

[0059] In embodiments of the invention, the eukaryotic cells are cells from the kingdom Animalia, preferably from the phylum Chordata, Mollusca, or Arthropoda, more preferably of the superclass Agnatha or Gnathosomata or class Amphibia, Reptilia, Mammalia or Aves.

[0060] In embodiments of the invention, the eukaryotic cells are cells from the family Bovidae or Phasianidae, preferably the species Bos taurus or Gallus gallus domesticus.

[0061] In embodiments of the invention, the eukaryotic cells are of one or more of the following cell types:

(a) myoblasts;

(b) mesenchymal stem cells;

(c) pluripotent stem cells;

(d) fibroblasts;

(e) Adipocytes; and

(f) Adipogenic stem cells.

[0062] The feeder cells of the invention may also be cells the kingdom Animalia, from the phylum Chordata, Mollusca, or Arthropoda, of the superclass Agnatha or Gnathosomata or class Amphibia, Reptilia, Mammalia or Aves. Further, the feeder cells of the invention may be from the family Bovidae or Phasianidae, preferably the species Bos taurus or Gallus gallus domesticus.

[0063] In embodiments of the invention, the feeder cells are one or more of the following cell types:

(a) fibroblasts;

(b) hepatocytes;

(c) mesenchymal stem cells;

(d) endothelial cells;

(e) hematopoietic stem cells; and (f) beta cells.

[0064] In embodiments of the invention, the feeder cells recombinantly express and secrete one or more of the following:

(a) somatotropin;

(b) platelet-derived growth factor (PDGF);

(c) albumin;

(d) insulin-like growth factor (IGF);

(e) transferrin;

(f) insulin;

(g) vascular endothelial growth factor (VEGF);

(h) transforming growth factor beta (TGF-p);

(i) hepatocyte growth factor (HGF);

(j) basic fibroblast growth factor (bFGF);

(k) epidermal growth factor (EGF); and

(l) interleukin 6 (IL-6).

[0065] In embodiments of the invention, the feeder cells are immortalized.

[0066] In embodiments of the invention, the bioreactor in step (a) contains a cell culturing medium comprising one or more or all of the following:

(a) A basal media, preferably DMEM/F12;

(b) 2-Phospho-L-ascorbic acid trisodium salt;

(c) Insulin (human, recombinant), preferably at a concentration of 20 pg/mL;

(d) Transferrin (human, recombinant), preferably at a concentration of 20 pg/mL;

(e) Sodium selenite, preferably at a concentration of 20 ng/mL;

(f) Fibroblast growth factor (FGF-2), preferably at a concentration of 40 ng/mL;

(g) Neuregulin (NRG1), preferably at a concentration of 0.1 ng/mL;

(h) Transforming growth factor (TGF[J3), preferably at a concentration of 0.1 ng/mL;

(i) Albumin, preferably at a concentration of 0.800-11.2 mg/mL;

(j) y-aminobutyric acid (GABA);

(k) lithium chloride (LiCl); (l) pipecolic acid;

(m) olcic acid; and

(n) palmitic acid.

[0067] In embodiments of the invention, the bioreactor in step (a) contains a cell culturing medium in which one or more of the following are not added prior to or during step (a):

(a) DMEM/F12;

(b) 2-Phospho-L-ascorbic acid trisodium salt;

(c) Insulin (human, recombinant);

(d) Transferrin (human, recombinant);

(e) Sodium selenite;

(f) Fibroblast growth factor (FGF-2);

(g) Neuregulin (NRG1);

(h) Transforming growth factor (TGFp3);

(i) Albumin;

(j) y-aminobutyric acid (GABA);

(k) lithium chloride (LiCl);

(l) pipecolic acid;

(m) oleic acid; and

(n) palmitic acid.

[0068] In embodiments of the invention, step (a) comprises aerating the bioreactor, preferably with a sparging or impeller-based system.

[0069] In embodiments of the invention, step (a) comprises mixing the bioreactor.

[0070] In embodiments of the invention, step (a) comprises controlling one or more of the following parameters in the bioreactor:

(a) pH;

(b) temperature;

(c) oxygen;

(d) carbon dioxide;

(e) glucose;

(f) ammonia; and (g) other compounds relevant to cell health.

Feeder cells

[0071] This invention also provides a feeder cell comprising one or more or all of the following codon-optimized recombinant genes incorporated into the genome thereof:

(a) somatotropin;

(b) platelet-derived growth factor (PDGF);

(c) albumin;

(d) insulin-like growth factor (IGF);

(e) transferrin;

(f) insulin;

(g) vascular endothelial growth factor (VEGF);

(h) transforming growth factor beta (TGF-p);

(i) hepatocyte growth factor (HGF);

(j) basic fibroblast growth factor (bFGF);

(k) epidermal growth factor (EGF); and

(l) interleukin 6 (IL-6).

[0072] In embodiments of the invention, the feeder cell is a fibroblast, hepatocyte, mesenchymal stem cells, hematopoietic stem cells, endothelial, or beta cell.

[0073] This invention also provides a microcarrier, feeder cell combination comprising:

(a) a microcarrier; and

(b) any one of the feeder cells of the invention encapsulated in the microcarrier.

[0074] In embodiments of the invention, the microcarrier:

(a) comprises a food-grade hydrogel, preferably a plant-based food-grade hydrogel;

(b) is edible;

(c) semi-permeable, preferably permeable to the secretome of the feeder cell and impermeable to the feeder cell itself, more preferably permeable to molecules less than about 150 kDa in size and impermeable to molecules greater than about 150 kDa in size;

(d) comprises alginate and/or agarose, preferably RGD modified alginate; (e) comprises one or more extracellular components, preferably gelatin, vitronectin and/or RGD;

(f) comprises one or more plastic microcarriers

(g) comprises one or more scaffolds;

(h) is a microbead or microsphere;

(i) is between about 100 pm and about 500 pm in diameter, preferably between about 200 pm and about 400 pm in diameter.

Co-culturing systems

[0075] This invention also provides a co-culturing system comprising:

(a) eukaryotic cells encapsulated in a first set of microcarriers;

(b) feeder cells encapsulated in second set of microcarriers; and

(c) a bioreactor comprising:

(i) an interior and interior surface;

(ii) means to aerate the interior of the bioreactor; and

(hi) a semi-permeable barrier which:

1. separates the interior of the bioreactor into a bottom portion and a top portion;

2. is impermeable to the first set of microcarriers and second set of microcarriers; and

3. is permeable to liquids.

[0076] In embodiments of the co-culturing system, the bioreactor further comprises one or more sensors.

[0077] In embodiments of the co-culturing system, the one or more sensors comprise:

(a) one or more pH sensors;

(b) one or more temperature sensors; and/or

(c) one or more carbon dioxide sensors.

[0078] In embodiments of the co-culturing system, the one or more sensors are distributed throughout the interior surface of the bioreactor. [0079] Tn embodiments of the co-culturing system, the bioreactor further comprises a feedback system that controls one or more of:

(a) pH;

(b) temperature;

(c) oxygen;

(d) carbon dioxide;

(e) glucose;

(f) ammonia;

(g) other compounds relevant to cell health.

[0080] Tn embodiments of the co-culturing system, the means to aerate the interior of the bioreactor comprises:

(a) a sparger-based aeration system; or

(b) an impeller or paddle-based aeration system.

[0081] In embodiments of the co-culturing system, the bioreactor further comprises a mixer.

[0082] In embodiments, the co-culturing system further comprises a cell culture medium in the bioreactor.

[0083] In embodiments, the cell culture medium comprises:

(a) A basal media, preferably DMEM/F12;

(b) 2-Phospho-L-ascorbic acid trisodium salt;

(c) Insulin (human, recombinant), preferably at a concentration of 20 pg/mL;

(d) Transferrin (human, recombinant), preferably at a concentration of 20 pg/mL;

(e) Sodium selenite, preferably at a concentration of 20 ng/mL;

(f) Fibroblast growth factor (FGF-2), preferably at a concentration of 40 ng/mL;

(g) Neuregulin (NRG1), preferably at a concentration of 0.1 ng/mL;

(h) Transforming growth factor (TGF[13), preferably at a concentration of 0.1 ng/mL;

(i) Albumin, preferably at a concentration of 0.800-11.2 mg/mL;

(j) y-aminobutyric acid (GABA);

(k) lithium chloride (LiCl);

(l) pipecolic acid; (m) oleic acid; and

(n) palmitic acid.

[0084] In embodiments, one or more or all of the following are not added to the cell culture medium:

(a) A basal media, preferably DMEM/F12;

(b) 2-Phospho-L-ascorbic acid trisodium salt;

(c) Insulin (human, recombinant), preferably at a concentration of 20 pg/mL;

(d) Transferrin (human, recombinant), preferably at a concentration of 20 pg/mL;

(e) Sodium selenite, preferably at a concentration of 20 ng/rnL;

(f) Fibroblast growth factor (FGF-2), preferably at a concentration of 40 ng/mL;

(g) Neuregulin (NRG1), preferably at a concentration of 0.1 ng/mL;

(h) Transforming growth factor (TGF03), preferably at a concentration of 0.1 ng/mL;

(i) Albumin, preferably at a concentration of 0.800-11.2 mg/mL;

(j) y-aminobutyric acid (GABA);

(k) lithium chloride (LiCl);

(l) pipecolic acid;

(m) oleic acid; and

(n) palmitic acid.

[0085] In embodiments, the first set of microcarriers and second set of microcarriers are contained in the bottom portion of the bioreactor.

[0086] In embodiments of the co-culturing system, the first and/or second set of microcarriers comprise a food-grade hydrogel, preferably a plant-based food-grade hydrogel.

[0087] In embodiments of the co-culturing system, the first and/or second set of microcarriers are:

(a) edible; and/or

(b) semi-permeable, preferably permeable to the secretome of the feeder and/or eukaryotic cell and impermeable to the feeder and/or eukaryotic cell itself, more preferably permeable to molecules less than about 150 kDa in size and impermeable to molecules greater than about 150 kDa in size. [0088] Tn embodiments of the co-culturing system, the first and/or second set of microcarriers comprise:

(a) alginate, preferably RGD modified alginate;

(b) agarose; and/or

(c) one or more extracellular components, preferably gelatin, vitronectin and/or RGD.

[0089] In embodiments of the co-culturing system, the first and/or second set of microcarriers further comprise:

(a) plastic microcarriers;

(b) one or more cell scaffolds.

[0090] In embodiments of the co-culturing system, the first set of microcarriers and/or second set of microcarriers are microbeads or microspheres.

[0091] In embodiments of the co-culturing system, the first set of microcarriers and/or second set of microcarriers are:

(a) between about 100 pm and about 500 pm in diameter, or

(b) between about 200 pm and about 400 pm in diameter.

[0092] In embodiments of the co-culturing system, the first set of microcarriers differs from the second set of microcarriers in density, size, and/or magnetic property such that the first set of microcarriers can be separated from the second set of microcarriers based on their respective densities, sizes, and/or magnetic properties.

[0093] In embodiments of the co-culturing system:

(a) the first set of microcarriers and the second set of microcarriers are of different densities, so as to allow the first set of microcarriers to be separated from the second set of microcarriers based on their respective densities;

(b) wherein the first and/or second set of microcarriers are infused with magnetic particles, preferably iron particles, so as to allow the first set of microcarriers to be separated from the second set of microcarriers by magnetic separation; or

(c) wherein the first set of microcarriers and second set of microcarriers have size distributions that do not overlap, so as to allow the first set of microcarriers to be separated from the second set of microcarriers by size exclusion, preferably wherein the first set of microcarriers are of uniform size and/or the second set of microcarriers arc of uniform size.

[0094] In embodiments of the co-culturing system, the semi-permeable barrier comprises pores that are smaller than the size of the first set of microcarriers and second set of microcarriers.

[0095] In embodiments of the co-culturing system, the semi-permeable barrier comprises:

(a) mesh;

(b) a perforated metal sheet; and/or

(c) fabric.

[0096] In embodiments of the co-culturing system, the interior surface of the bioreactor comprises ridges.

[0097] This invention also provides a culturing system comprising:

(a) eukaryotic cells encapsulated in a first set of microcarriers;

(b) a bioreactor comprising:

(i) an interior and interior surface;

(ii) means to aerate the interior of the bioreactor; and

(iii) a semi-permeable barrier which:

1. separates the interior of the bioreactor into a bottom portion and a top portion;

2. is impermeable to the first set of microcarriers; and

3. is permeable to liquids; and

(c) one or more hollow fiber cartridges containing feeder cells connected to the bioreactor, preferably removably connected to the bioreactor, more preferably removably connected to the bioreactor via one or more couplings, more preferably wherein the couplings are disposable.

[0098] In embodiments of the culturing system, the bioreactor further comprises one or more sensors.

[0099] In embodiments of the culturing system, the one or more sensors comprise:

(a) one or more pH sensors;

(b) one or more temperature sensors; and/or (c) one or more carbon dioxide sensors.

[0100] In embodiments of the culturing system, the one or more sensors are distributed throughout the interior surface of the bioreactor.

[0101] In embodiments of the culturing system, the bioreactor further comprises a feedback system that controls one or more of:

(a) pH;

(b) temperature;

(c) oxygen;

(d) carbon dioxide;

(e) glucose;

(f) ammonia;

(g) other compounds relevant to cell health.

[0102] In embodiments of the culturing system, the means to aerate the interior of the bioreactor comprises:

(a) a sparger-based aeration system; or

(b) an impeller or paddle-based aeration system.

[0103] In embodiments of the culturing system, the bioreactor further comprises a mixer.

[0104] In embodiments of the culturing system, the culturing system further comprises a cell culture medium in the bioreactor.

[0105] In embodiments of the culturing system, the cell culture medium comprises:

(a) A basal media, preferably DMEM/F12;

(b) 2-Phospho-L-ascorbic acid trisodium salt;

(c) Insulin (human, recombinant), preferably at a concentration of 20 g/mL;

(d) Transferrin (human, recombinant), preferably at a concentration of 20 pg/mL;

(e) Sodium selenite, preferably at a concentration of 20 ng/mL;

(f) Fibroblast growth factor (FGF-2), preferably at a concentration of 40 ng/mL;

(g) Neuregulin (NRG1), preferably at a concentration of 0.1 ng/mL;

(h) Transforming growth factor (TGF[13), preferably at a concentration of 0.1 ng/mL; (i) Albumin, preferably at a concentration of 0.800-1 1 .2 mg/mL;

(j) y-aminobutyric acid (GABA);

(k) lithium chloride (LiCl);

(l) pipecolic acid;

(m) oleic acid; and

(n) palmitic acid.

[0106] In embodiments of the culturing system, one or more or all of the following are not added to the cell culture medium:

(a) A basal media, preferably DMEM/F12;

(b) 2-Phospho-L-ascorbic acid trisodium salt;

(c) Insulin (human, recombinant), preferably at a concentration of 20 pg/mL;

(d) Transferrin (human, recombinant), preferably at a concentration of 20 pg/mL;

(e) Sodium selenite, preferably at a concentration of 20 ng/mL;

(f) Fibroblast growth factor (FGF-2), preferably at a concentration of 40 ng/mL;

(g) Neuregulin (NRG1), preferably at a concentration of 0.1 ng/mL;

(h) Transforming growth factor (TGFfG), preferably at a concentration of 0.1 ng/mL;

(i) Albumin, preferably at a concentration of 0.800-11.2 mg/mL;

(j) y-aminobutyric acid (GABA);

(k) lithium chloride (LiCl);

(l) pipecolic acid;

(m) oleic acid; and

(n) palmitic acid.

[0107] In embodiments of the culturing system, the first set of microcarriers are in the bottom portion of the bioreactor.

[0108] In embodiments of the culturing system:

(a) a screen separates the one or more hollow fiber cartridges from the bioreactor;

(b) a pump controls flow of cell culture media between the bioreactor and the hollow fiber cartridge; (c) the feeder cells contained in the hollow fiber cartridge have proliferated to conflucncy;

(d) the feeder cells contained in the hollow fiber cartridge condition cell culture media that is perfused through the hollow fiber cartridge; and/or

(e) the feeder cells are adhered to fibers of the hollow fiber cartridge.

[0109] In embodiments of the culturing system, the first set of microcarriers comprise a foodgrade hydrogel, preferably a plant-based food-grade hydrogel.

[0110] In embodiments of the culturing system, the first set of microcarriers are:

(a) edible; and/or

(b) semi-permeable, preferably permeable to the secretome of the feeder and/or eukaryotic cell and impermeable to the feeder and/or eukaryotic cell itself, more preferably permeable to molecules less than about 150 kDa in size and impermeable to molecules greater than about 150 kDa in size.

[0111] In embodiments of the culturing system, the first set of microcarriers comprise:

(a) alginate, preferably RGD modified alginate;

(b) agarose; and/or

(c) one or more extracellular components, preferably gelatin, vitronectin and/or RGD.

[0112] In embodiments of the culturing system, the first set of microcarriers further comprise:

(a) plastic microcarriers;

(b) one or more cell scaffolds.

[0113] In embodiments of the culturing system, the first set of microcarriers are microbeads or microspheres.

[0114] In embodiments of the culturing system, the first set of microcarriers are:

(a) between about 100 pm and about 500 pm in diameter, or

(b) between about 200 pm and about 400 pm in diameter.

[0115] In embodiments of the culturing system, the semi-permeable barrier comprises pores that are smaller than the size of the first set of microcarriers and second set of microcarriers.

[0116] In embodiments of the culturing system, the semi-permeable barrier comprises: (a) mesh;

(b) a perforated metal sheet; and/or

(c) fabric.

[0117] In embodiments of the culturing system, the interior surface of the bioreactor comprises ridges.

[0118] In embodiments of the culturing system, the feeder cells:

(a) are one or more of the following cell types:

(i) fibroblasts;

(ii) hepatocytes;

(iii) mesenchymal stem cells;

(iv) endothelial cells;

(v) hematopoietic stem cells; and

(vi) beta cells;

(b) recombinantly express and secrete one or more of the following:

(i) somatotropin;

(ii) platelet-derived growth factor (PDGF);

(iii) albumin;

(iv) insulin-like growth factor (IGF);

(v) transferrin;

(vi) insulin;

(vii) vascular endothelial growth factor (VEGF);

(viii) transforming growth factor beta (TGF-P);

(ix) hepatocyte growth factor (HGF);

(x) basic fibroblast growth factor (bFGF);

(xi) epidermal growth factor (EGF); and

(xii) interleukin 6 (IL-6); and/or

(c) are immortalized. Alternative processes for culturing eukaryotic cells

[0119] This invention also provides a process for culturing eukaryotic cells, comprising culturing eukaryotic cells encapsulated in a first set of microcarriers in a culturing system comprising:

(a) a bioreactor comprising:

(i) an interior and interior surface;

(ii) means to aerate the interior of the bioreactor; and

(iii) a semi-permeable barrier which:

1. separates the interior of the bioreactor into a bottom portion and a top portion;

2. is impermeable to the first set of microcarriers; and

3. is permeable to liquids; and

(b) one or more hollow fiber cartridges containing feeder cells connected to the bioreactor, preferably removably connected to the bioreactor, more preferably removably connected to the bioreactor via one or more couplings, more preferably wherein the couplings are disposable.

[0120] In embodiments of the process for culturing eukaryotic cells, the first set of microcarriers comprise a food-grade hydrogel, preferably a plant-based food-grade hydrogel.

[0121] In embodiments of the process for culturing eukaryotic cells, the first set of microcarriers are:

(a) edible; and/or

(b) semi-permeable, preferably permeable to the secretome of the feeder and/or eukaryotic cell and impermeable to the feeder and/or eukaryotic cell itself, more preferably permeable to molecules less than about 150 kDa in size and impermeable to molecules greater than about 150 kDa in size.

[0122] In embodiments of the process for culturing eukaryotic cells, the first set of microcarriers comprise:

(a) alginate, preferably RGD modified alginate;

(b) agarose; and/or (c) one or more extracellular components, preferably gelatin, vitronectin and/or RGD.

[0123] In embodiments of the process for culturing eukaryotic cells, the first set of microcarriers further comprise:

(a) one or more plastic microcarriers; and/or

(b) one or more cell scaffolds.

[0124] In embodiments of the process for culturing eukaryotic cells, the first set of microcarriers are microbeads or microspheres.

[0125] In embodiments of the process for culturing eukaryotic cells, the first set of microcarriers arc:

(a) between about 100 pm and about 500 pm in diameter, or

(b) between about 200 pm and about 400 pm in diameter.

[0126] In embodiments of the process for culturing eukaryotic cells, the bioreactor further comprises one or more sensors.

[0127] In embodiments of the process for culturing eukaryotic cells, the one or more sensors comprise:

(a) one or more pH sensors;

(b) one or more temperature sensors; and/or

(c) one or more carbon dioxide sensors.

[0128] In embodiments of the process for culturing eukaryotic cells, the one or more sensors are distributed throughout the interior surface of the bioreactor.

[0129] In embodiments of the process for culturing eukaryotic cells, the bioreactor further comprises a feedback system that controls one or more of:

(a) pH;

(b) temperature;

(c) oxygen;

(d) carbon dioxide;

(e) glucose;

(f) ammonia; (g) other compounds relevant to cell health.

[0130] In embodiments of the process for culturing eukaryotic cells, the means to aerate the interior of the bioreactor comprises:

(a) a sparger-based aeration system; or

(b) an impeller or paddle-based aeration system.

[0131] In embodiments of the process for culturing eukaryotic cells, the bioreactor further comprises a mixer.

[0132] In embodiments of the process for culturing eukaryotic cells, the bioreactor contains a cell culture medium.

[0133] In embodiments of the process for culturing eukaryotic cells, the cell culture medium comprises:

(a) A basal media, preferably DMEM/F12;

(b) 2-Phospho-L-ascorbic acid trisodium salt;

(c) Insulin (human, recombinant), preferably at a concentration of 20 g/mL;

(d) Transferrin (human, recombinant), preferably at a concentration of 20 pg/mL;

(e) Sodium selenite, preferably at a concentration of 20 ng/mL;

(f) Fibroblast growth factor (FGF-2), preferably at a concentration of 40 ng/mL;

(g) Neuregulin (NRG1), preferably at a concentration of 0.1 ng/mL;

(h) Transforming growth factor (TGFJG), preferably at a concentration of 0.1 ng/mL;

(i) Albumin, preferably at a concentration of 0.800-11.2 mg/mL;

(j) y-aminobutyric acid (GABA);

(k) lithium chloride (LiCl);

(l) pipecolic acid;

(m) oleic acid; and

(n) palmitic acid.

[0134] In embodiments of the process for culturing eukaryotic cells, one or more or all of the following are not added to the cell culture medium:

(a) A basal media, preferably DMEM/F12;

(b) 2-Phospho-L-ascorbic acid trisodium salt; (c) Insulin (human, recombinant), preferably at a concentration of 20 pg/mL;

(d) Transferrin (human, recombinant), preferably at a concentration of 20 pg/mL;

(e) Sodium selenite, preferably at a concentration of 20 ng/mL;

(f) Fibroblast growth factor (FGF-2), preferably at a concentration of 40 ng/mL;

(g) Neuregulin (NRG1), preferably at a concentration of 0.1 ng/mL;

(h) Transforming growth factor (TGFp3), preferably at a concentration of 0.1 ng/mL;

(i) Albumin, preferably at a concentration of 0.800-11.2 mg/mL;

(j) y-aminobutyric acid (GABA);

(k) lithium chloride (LiCl);

(l) pipecolic acid;

(m) oleic acid; and

(n) palmitic acid.

[0135] In embodiments of the process for culturing eukaryotic cells, the first set of microcarriers are in the bottom portion of the bioreactor.

[0136] In embodiments of the process for culturing eukaryotic cells, the step of culturing eukaryotic cells comprises:

(a) aerating the bioreactor;

(b) mixing th e bi oreactor ;

(c) perfusing culture media between the hollow fiber cartridge and the bioreactor; and/or

(d) controlling one or more of the following parameters in the bioreactor:

(i) pH;

(ii) temperature;

(iii) oxygen.

(iv) carbon dioxide.

[0137] In embodiments of the process for culturing eukaryotic cells:

(a) a screen separates the one or more hollow fiber cartridges from the bioreactor; and/or (b) a pump controls flow of cell culture media between the bioreactor and the hollow fiber cartridge;

(c) the feeder cells contained in the hollow fiber cartridge have proliferated to confluency;

(d) the feeder cells contained in the hollow fiber cartridge condition cell culture media that is perfused through the hollow fiber cartridge; and/or

(e) the feeder cells are adhered to fibers of the hollow fiber cartridge.

[0138] In embodiments of the process for culturing eukaryotic cells, the first set of microcarriers comprise a food-grade hydrogel, preferably a plant-based food-grade hydrogel.

[0139] In embodiments of the process for culturing eukaryotic cells, the first set of microcarriers are:

(a) edible; and/or

(b) semi-permeable, preferably permeable to the secretome of the feeder and/or eukaryotic cell and impermeable to the feeder and/or eukaryotic cell itself, more preferably permeable to molecules less than about 150 kDa in size and impermeable to molecules greater than about 150 kDa in size.

[0140] In embodiments of the process for culturing eukaryotic cells, the first set of microcarriers comprise:

(a) alginate, preferably RGD modified alginate;

(b) agarose; and/or

(c) one or more extracellular components, preferably gelatin, vitronectin and/or RGD.

[0141] In embodiments of the process for culturing eukaryotic cells, the first set of microcarriers further comprise:

(a) plastic microcarriers;

(b) one or more cell scaffolds.

[0142] In embodiments of the process for culturing eukaryotic cells, the first set of microcarriers are microbeads or microspheres. [0143] Tn embodiments of the process for culturing eukaryotic cells, the first set of microcarriers arc:

(a) between about 100 pm and about 500 pm in diameter, or

(b) between about 200 pm and about 400 pm in diameter.

[0144] In embodiments of the process for culturing eukaryotic cells, the semi-permeable barrier comprises pores that are smaller than the size of the first set of microcarriers.

[0145] In embodiments of the process for culturing eukaryotic cells, the semi-permeable barrier comprises:

(a) mesh;

(b) a perforated metal sheet; and/or

(c) fabric.

[0146] In embodiments of the process for culturing eukaryotic cells, the interior surface of the bioreactor comprises ridges.

[0147] In embodiments of the process for culturing eukaryotic cells, the feeder cells:

(a) are one or more of the following cell types:

(i) fibroblasts;

(ii) hepatocytes;

(iii) mesenchymal stem cells;

(iv) endothelial cells;

(v) hematopoietic stem cells; and

(vi) beta cells;

(b) recombinantly express and secrete one or more of the following:

(i) somatotropin;

(ii) platelet-derived growth factor (PDGF);

(iii) albumin;

(iv) insulin-like growth factor (IGF);

(v) transferrin;

(vi) insulin;

(vii) vascular endothelial growth factor (VEGF);

(viii) transforming growth factor beta (TGF-[3); (ix) hepatocyte growth factor (HGF);

(x) basic fibroblast growth factor (bFGF);

(xi) epidermal growth factor (EGF); and

(xii) interleukin 6 (IL-6); and/or

(c) are immortalized.

Processes for producing conditioned media and/or products purified from the conditioned media

[0148] This invention also provides a process for producing conditioned media and/or products purified from the conditioned media, preferably exosomes, comprising

(a) culturing eukaryotic cells

(i) according to any of the processes of the invention;

(ii) in the presence of any of the feeder cells, or microcarrier-feeder cell combinations of the invention;

(iii) using any one of the co-culturing systems of the invention; and

(b) obtaining culture media produced by the eukaryotic cells and/or feeder cells;

(c) optionally purifying one or more products from the culture media, preferably purifying exosomes from the culture media.

[0149] In embodiments, the process further comprises centrifugation and/or lyophilization of the conditioned media and/or products purified from the conditioned media, preferably wherein the conditioned media and/or products purified from the conditioned media are subjected to no other processing step.

[0150] This invention also provides a process for producing a consumer good comprising:

(a) producing conditioned media and/or products purified from the conditioned media, preferably exosomes, according to any of the processes of the invention described above;

(b) adding the conditioned media and/or purified products to one or more other ingredients.

[0151] This invention also provides conditioned media and/or products purified from conditioned media, preferably exosomes, produced according to any of the processes of the invention described above. [0152] Tn alternative embodiments of processes for producing conditioned media and/or products purified from the conditioned media, feeder cells may be cultured according to the invention as described herein in the absence of the eukaryotic cells of the invention. Conversely, eukaryotic cells may be cultured according to the invention as described herein in the absence of the feeder cells of the invention. In other words, this invention contemplates that the processes and systems described herein may be modified to use only the feeder cells or only eukaryotic cells of the invention to produce conditioned media and/or products purified from the conditioned media.

Electrostatic Bead Generation

[0153] In an embodiment of the invention, microcarriers of the invention are formed using electrostatic bead generation. Such a method of microcarrier formation allows for the size of the microcarriers to be controlled. Electrostatic bead generation is described in Klokk et al. (2002), the entire contents of which is hereby incorporated by reference.

Hollow Fiber Cartridge

[0154] In embodiments of the invention, a hollow fiber cartridge containing feeder cells is connected to a bioreactor containing cells to be cultured. In such embodiments of the invention, the culturing system may be set up analogously to an alternating tangential flow (ATF) system as described in Karst et al. (2016) except that feeder cells are restricted solely to the hollow fiber cartridge and do not enter the bioreactor. The contents of Karst et al. (2016) are hereby incorporated by reference for its description of an alternating tangential flow (ATF) system.

Microcarriers

[0155] As used herein, “microcarrier” refers to a small particle or bead that is used in cell culture to support the grown of cells. Microcarriers of the invention are preferably spherical in shape, in which case such microcarriers may be referred to as microbeads or microspheres. Whereas in the art cells are often seeded on the surface of microcarriers, the invention provides microcarriers in which cells are encapsulated. Encapsulation of the cells protects the cells from shear forces. Microcarriers of the invention may be coated in a matrix and said matrix may comprise extracellular matrix components, and/or one or more of hyaluronic acid, laminin, fibronectin, vitronectin, collagen, elastin, heparan sulphate, dextran, dextran sulphate, chondroitin sulphate, or a mixture of laminin, collagen I, heparan sulfate proteoglycans, and entactin 1. [0156] Tn an embodiment, microcarrier particles of the invention may comprise any of the microcarricr particles described in U.S. Patent No. 9,340,770 B2, the contents of which is specifically incorporated-by-reference for its description of microcarrier particles. Similarly, the microcarrier particles of the invention may be coated in any matrix described in U.S. Patent No. 9,340,770 B2, the contents of which is specifically incorporated-by-reference for its description of matrix coatings of microcarriers.

Definitions

[0157] As used herein, “means to aerate” a bioreactor include a sparger, bubbler, diffuser, blower, paddle stirrer, agitator blades, or magnetic stirrer. In a preferred embodiment, the means to aerate involves mechanically stirring the culture medium with a paddle stirrer, agitator blades, or magnetic stirrer.

[0158] As used herein, a cell “secretome” refers to the complete collection of proteins, extracellular vesicles and metabolites secreted by a cell.

[0159] As used herein, “conditioned media” refers to media that has been exposed to cells grown in culture for a time sufficient to include at least one additional component in the media, produced by the cells, that was not present in the starting media. Conditioned media of the invention may contain the secretome of the cells grown in culture.

[0160] As used herein, “exogenous” in the context of a polynucleotide or polypeptide refers to the polynucleotide or polypeptide when present in a cell which does not naturally comprise the polynucleotide or polypeptide. Such a cell is referred to herein as a “recombinant cell” or a “transgenic cell”. In an embodiment, the exogenous polynucleotide or polypeptide is from a different genus to the cell comprising the exogenous polynucleotide or polypeptide. In another embodiment, the exogenous polynucleotide or polypeptide is from a different species. The exogenous polynucleotide or polypeptide may be non-naturally occurring, such as for example, a synthetic DNA molecule which has been produced by recombinant DNA methods. The DNA molecule may, preferably, include a protein coding region which has been codon-optimised for expression in the cell, thereby producing a polypeptide which has the same amino acid sequence as a naturally occurring polypeptide, even though the nucleotide sequence of the protein coding region is non-naturally occurring. [0161] As used herein an “engineered cell” refers to a cell which has been modified to express polynucleotides or polypeptides that the cell type docs not normally express. These expressed polynucleotides or polypeptides may be polynucleotides or polypeptides that are normally expressed in (1) a different cell type of the same species, (2) the same cell type of a different species, or (3) a different cell type of a different species. In embodiments, an engineered cell has been genetically modified to express polynucleotides or polypeptides that the cell type does not normally express, using any known method for genetically modifying cells such as (without limitation) lentivirus systems, TALENs, or programmable nuclease systems such as CRISPR. Feeder and/or eukaryotic cells of the invention may be engineered cells and may recomb inantly express one or more polynucleotides or polypeptides that the cell type does not normally express as discussed above. In this context, “recombinantly express” refers to expression of polynucleotides or polypeptides introduced using techniques of genetic engineering such as those discussed above.

[0162] As used herein, “feeder cell” refers to cells which provide extracellular secretions to help another cell to proliferate. Llames et al. (2015) provides a general description of feeder cells and is hereby incorporated by reference. As used in the art, feeder cells often do not divide. However, as used herein, feeder cells of the invention may be capable of dividing, and may divide in the systems and processes described herein.

[0163] As used herein, “recyclable” in the context of microcarriers means that microcarriers and/or the cells in the microcarriers can be reused towards subsequent batches instead of used for harvest.

[0164] As used herein, the term “about”, unless stated to the contrary, refers to +/- 10%, more preferably +/- 5%, more preferably +/- 2%, more preferably +/- 1%, even more preferably +/- 0.5%, of the designated value.

[0165] As used herein in the context of the size of microcarriers, “uniform size” refers to microcarriers with a size distribution such that at least 75%, more preferably 80%, more preferably 85%, more preferably 90%, even more preferably 95% of the microcarriers are about the same size. General

[0166] For the foregoing embodiments, each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiment.

[0167] As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections. All combinations of the various elements disclosed herein are within the scope of the invention.

[0168] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which arc not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0169] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0170] Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only.

EXAMPLES

[0171] A scaled-down model is used to (1) explore the maximum achievable density after generating microcarriers with a low starting concentration of cells and (2) demonstrate how these microcarriers respond to industrial shear forces. In the following Examples, (i) microcarriers are optimized for enhanced cell expansion, (ii) an reporter cell line is used to assess the viability and density of cells within the Edge Pod, and (iii) a bench-scale bioreactor is adapted with high shear forces equivalent to those found in 100m ³ stirred tank biorcactors.

[0172] Of particular interest are the use of two types of microcarriers that differ in physical characteristics, such as size. The use of two types of microcarriers allows for the creation of a novel co-culture bioprocess. This co-culture feeder cell system uses one bioreactor to house both stem cell and feeder cell populations, while the biomaterial allows for their discrimination. Namely, feeder cells are encapsulated in a first set of microcarriers and muscle stem cells are encapsulated in a second set of microcarriers. These feeder cells will reduce the need to introduce recombinant proteins into the cell culture medium from external sources, similar to the use of MEFs with iPSCs/ESCs. Upon harvest, size exclusion is used to separate the two biomaterial types. Muscle stem cells from this bioprocess are used for demonstration purposes, but in principle any type of feeder or co-culture cell may be used.

Example 1

[0173] In this Example, (1) a scaled-down model of an industrial bioreactor with supraphysiological shear force is engineered, (2) an alginate-based biomaterial is optimized to facilitate enhanced cell growth, (3) and the shear-protecting attributes of alginate biomaterials is explored.

[0174] Outcomes: Tn-silico and in-vitro models are generated to determine the shear forces in a stirred tank bioreactor. Alginate modifications which affect cellular proliferation are identified. Cell growth curves, ECM deposition, and cell viability are used to validate alginate biomaterials as a substrate to enhance cell manufacturing.

[0175] A scaled-down model of industrial shear forces is created. Small agitator blades are used at an appropriate intensity/speed to match the shear forces of a 100,000L bioreactor. An off- the-shelf bioreactor adapted with an Arduino. Methods of programming and customizing the Arduino are described in Kii^ukaga et aL, 2022 (which is hereby incorporated by reference) and are used to adapt the bioreactor to the requirements of this Example. The shear force in the fluid as a function of the rotational speed is calculated, and a computational model of shear force in the bioreactor is developed as described in Sanchez Perez et al., 2006 (which is hereby incorporated by reference). Briefly, a bioreactor with a up to 100mm sized blades are used at 60 RPM - 360 RPM (a speed used in traditional bacteria cultures). Shear force at varying locations in the biorcactor arc modeled with COMSOL.

[0176] As many groups have reported the successful adaptation of stromal cells from various species, we explore alginate with and without RGD (arginine-glycine-aspartate-peptide) modifications.

[0177] First, CHO cells, a well-established suspension cell line used for the development of biologies, are used to explore how suspension-adapted cells behave in the microcarriers. RGD (arginine-glycine-aspartate-peptide) modified alginate is also used to explore how adherentdependent cells behave in the microcarriers.

[0178] The (1) size of the microcarriers, and (2) composition of alginate biomatcrial, that sufficiently protects against shear forces, without interfering with the diffusion limit of nutrients, waste, and oxygen is determined. Microcarriers within the sizes of 100-500pm, with a stiffness of between 10-45 kPa that protects against shear forces, without interfering with the diffusion limit of nutrients, waste, and oxygen are produced. See Ogneva et al., 2010. Using a 96-well plate, varying concentrations and alginate modifications are tested. After 3 days in culture a plate reader is used to measure luminescence and quantify the total cell density in each well. Biomaterials with enhanced cellular proliferation are validated using the bioreactor described above to introduce physiological shear force and for use in subsequent experiments.

[0179] The selected biomaterials are used to encapsulate suspension-adapted and adherent cells. Cell-laden microcarriers are then introduced into the bioreactor at varying speeds. After 3 days in culture, cell density is measured using luminescence. LDH is measured every 24 hours to measure cellular viability over time. Cell-laden biomaterials undergo fixation, and pentachrome staining to visualize collagen deposition and microcarrier integrity.

[0180] As an alternative to the customized Arduino-based system described above, a stirred tank reactor that operates over a magnetic stirrer is used. This type of stirred tank reactor is relatively cheap and allows for testing more than one condition using standard laboratory equipment. Variability in the cellular behavior in response to biomaterial stiffness, microcarrier size, and composition is observed. [0181] Tn one outcome, a soft microcarrier may provide optimal conditions for enhancing cellular proliferation during static culture, but bursts under shear force. Thus, the most proliferation-enabling biomaterials is selected with varying stiffness and is use for subsequent experimentation to identify an microcarrier that enhances proliferation under industrial shear force. In one example, suspension adapted cells perform well in the microcarriers without substantial modification. In another example, adherent cells are modified to the encapsulation protocol to ensure that adherent cells are dense enough to form clumps and maintain viability.

Example 2

[0182] In this Example, stromal cells are engineered to support stem cells by secreting exogenous albumin and bFGF.

[0183] Outcomes: Recombinant protein production in feeder cells over time is quantified. Cell growth curves from stem cells grown in serum free conditions composed of feeder-cell recombinant proteins are also determined. Efficient separation of feeder cells from stem cells is demonstrated.

[0184] Feeder cells are generated for mesenchymal stem cells and fibroblasts using a lentivirus construct. Mesenchymal stem cells are believed to secrete more endogenous growth factors that promote the growth of stem cells and, therefore, may be a more suitable feeder cell. See Lee et al., 2012; Sanchez et al., 2012, both incorporated by reference. ELIS As are used to measure the yield of recombinant protein, and conditioned media is generated using the engineered cells. Stem cells are dosed with conditioned media or commercially purchased recombinant proteins and cell growth is compared. Additionally, cells are grown in a co-culture system using a transwell and cell proliferation is compared to commercially added recombinant proteins. Finally, feeder cells and stem cells are seeded into alginate biomaterials and co-cultured as described above. PCR analysis is used after harvest to validate the efficiency of size/exclusion when separating the two sets of microcarriers.

[0185] In one outcome, the separation of feeder cell and stem cell populations during harvest is not efficient enough to rid the wild type stem cells from engineered feeder cells. Tn this case an ATF system is used, where stem cells are cultured in a stirred tank bioreactor as above, however, the feeder cells are housed in a hollow fiber bioreactor. A hollow fiber bioreactor allows for extremely high cell densities (>10 ^A 9/mL) and is designed to keep cells adhered to the fibers, while recombinant proteins can be easily perfused out.

[0186] Further, in another outcome, when generating the feeder cells, it is possible that albumin bioavailability becomes an issue. Notably, albumin is a carrier protein and is most effective in the presence of fatty acids. To ensure the appropriate activity from albumin, initial tests are conducted with and without the supplementation of fatty acids. To address the limited lifetime of feeder cells, various monoclonal populations are selected and followed over a series of passages to ensure the selection of a stable cell line. Lastly, a direct genetic engineering approach (for example using a programmable nuclease such as CRISPR) is used to insert the recombinant plasmid into a safe harbor locus of the feeder cell for the generation of a stable cell line.

[0187] In this Example, the protective attributes of alginate is demonstrated, along with the ability to reduce the need for supplementing cultures with costly and wasteful recombinant proteins.

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