Compositions and Methods

Abstract

The present disclosure provides, inter alia, a bilayer semipermeable fibrosis-resistant hydrogel polymer enclosure optionally containing cells such as mammalian cells, the enclosure being suitable for administration to a subject in need thereof, and further, the enclosure being expandable in accordance with a large number of delivered cells. The present disclosure further provides methods of making and using the enclosure, for example, in a treatment method, for example, in the treatment of disorders of the liver or other cells and organs treatable with secreting cells and / or catalytic cells.

Description

Technical Field

[0001] The present disclosure relates, in part, to bilayer semipermeable fibrosis-resistant hydrogel polymer enclosures containing both modified and unmodified alginates useful for treating diseases or disorders in a subject, and methods of making and using the same.

[0002] Cross - reference to related applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 365,791, filed on June 3, 2022, the entire contents of which are incorporated herein by reference.

Background Art

[0003] Allogeneic cell therapies have been hampered for multiple reasons, including foreign body response, fibrosis, and host immune response. Conventional drug delivery materials cannot enable the co - growth of cells within the same chamber that serves as a delivery vehicle for implantation during manufacture. Typically, cells require either genetic manipulation to achieve low immunity or a physical barrier to prevent direct contact with the body. Conventional anti - fibrotic alginates are limited in that they do not prevent the foreign body response resulting from signals generated by cells and cannot provide a biocompatible cell enclosure. Furthermore, many regenerative medicine applications (e.g., liver replacement) require large cell numbers (over a billion), but many modalities are not scalable and cannot be adapted for, e.g., 3D suspension, differentiation, and proliferation.

[0004] There is a need for an enclosure suitable for administration to a subject that supports the health of encapsulated cells and, for example, improves or eliminates multiple causes of perifibrotic overgrowth: macrophage adhesion, cell - derived innate immune system responses, and lymphocyte activation. Preferably, such an enclosure should be suitable for large - scale cell production.

Summary of the Invention

Problems to be Solved by the Invention

[0005] Accordingly, the present disclosure provides an enclosure that, among other things, supports both the health and phenotypic control of encapsulated cells and is suitable for administration to a subject by, for example, improving or eliminating cell death signals and by improving or eliminating multiple sources of perifibrotic overgrowth such as macrophage attachment, cell-derived innate immune system responses, and lymphocyte activation, and further enables efficient generation of large-scale cells. In multiple embodiments, the disclosed enclosures, compositions containing them, and methods of using them eliminate or reduce the need to manipulate hypoimmunity by use of semipermeable barriers that function as cell growth and differentiation chambers and conduits for in vivo delivery. Without being bound by a particular theory, this significantly increases the productivity of the manufacturing process and the control of the product, reducing the time and cost of the product, which is a conventional rate-limiting step for the production of cell therapies.

Means for Solving the Problems

[0006] In multiple embodiments, the present disclosure provides a bilayer semipermeable fibrosis-resistant hydrogel polymer enclosure containing both modified and unmodified alginates and containing 10s, 100s, or 1000s of cells (e.g., mammalian cells including human cells, such as hepatocytes including hepatocyte-containing organoids) within the internal aqueous chamber of the enclosure, and the enclosure (and compositions containing them) is suitable for administration to a subject (such as a human) in need thereof, for example, a subject having a liver disorder.

[0007] In multiple embodiments, useful cells within the enclosure generally include secretory cells and / or catalytic cells (including organoids containing such secretory cells and / or catalytic cells, including combinations of different types of secretory cells and / or catalytic cells). In multiple embodiments, the cells are derived from primary cells or stem cells, such as induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), or adipose tissue stem cells (ASCs). In multiple embodiments, non-limiting examples of secretory cells and / or catalytic cells include adipogenic cells, ASCs, adipocytes, hepatocytes (including iPSC-derived hepatocytes, ASC-derived hepatocytes, or ESC-derived hepatocytes), pancreatic islet cells (including iPSC-derived pancreatic islet cells, ASC-derived pancreatic islet cells, or ESC-derived pancreatic islet cells), dopaminergic neurons (including iPSC-derived dopaminergic neurons, ASC-derived dopaminergic neurons, or ESC-derived dopaminergic neurons), endocrine cells (including iPSC-derived endocrine cells, ASC-derived endocrine cells, or ESC-derived endocrine cells), cells from a heterologous source (e.g., pig), cadaveric tissue, a living donor (e.g., hepatocytes), and embryonic stem cells. In multiple embodiments, the secretory cells and / or catalytic cells are engineered cells. In multiple embodiments, the secretory cells and / or catalytic cells are non-engineered cells. In multiple embodiments, non-engineered cells are useful for, for example, endogenous functions of the target cells that include a secretory function and / or a catalytic function (e.g., absorbing, converting, releasing a substrate). In multiple embodiments, the secretory cells prevent and / or reduce the accumulation of metabolic by-products that are not used as a precursor. In multiple embodiments, the secretory cells are specialized cells derived from elements belonging to other tissues. In multiple embodiments, the secretory cells have an endogenous function related to the production and release of molecules that may be useful to the organism in which they occur. In multiple embodiments, the catalytic cells are cells that regulate the enzyme activity and catalytic function of interest. In multiple embodiments, the catalytic cells have an endogenous function related to the absorption of a substrate, the conversion of the substrate, and the release of a desired product or molecule.

[0008] The present disclosure further provides methods of making and using an enclosure in a treatment method, such as treating a liver disorder or a disorder related to the dysfunction of any secretory and / or catalytic cells, including, for example, those exemplified above. The present disclosure also provides methods of making the enclosures and compositions containing them.

Brief Description of the Drawings

[0009]

Figure 1

Figure 2

Figure 3

Mode for Carrying Out the Invention

[0010] The present disclosure provides, inter alia, a bilayer semipermeable fibrosis-resistant hydrogel polymer enclosure optionally containing cells such as mammalian cells, wherein the enclosure is suitable for administration to a subject, including a human subject in need thereof. The present disclosure further provides methods of making and using the enclosure in a treatment method, such as treating a liver disorder. The present disclosure is based, at least in part, on the Applicant's discovery of a fibrosis-resistant enclosure suitable for use in mammalian subjects and does not wish to be bound by theory, but this enclosure can advantageously avoid fibrosis by inhibiting macrophage attachment, cell-derived innate immune system responses, and lymphocyte activation.

[0011] Hydrogel Polymer Enclosure In multiple aspects, the present disclosure provides a bilayer semipermeable fibrosis-resistant hydrogel polymer enclosure comprising: an external hydrogel layer surrounding an enclosure; an internal hydrogel layer disposed within the external hydrogel layer; and an internal aqueous chamber disposed within the internal hydrogel layer, wherein the internal aqueous chamber is suitable for accommodating at least about 10 live mammalian cells, and the hydrogel polymer enclosure is suitable for administration to a human subject. In multiple embodiments, the external hydrogel layer comprises one or more rejection inhibitors that reduce rejection upon implantation into a mammalian subject. In multiple embodiments, the internal hydrogel layer comprises one or more agents that promote cell viability, cell differentiation, reduction and / or prevention and / or inhibition of apoptosis, cell health, and / or cell function. Non-limiting examples of cell differentiation include the differentiation of stem cells into primary cells. In multiple embodiments, the external hydrogel layer and the internal hydrogel layer are separated by a liquid layer. Non-limiting examples of the liquid layer include culture media (e.g., agar, alginate). In multiple embodiments, the internal aqueous chamber has a volume that allows at least about 10 cells to grow within the chamber.

[0012] In multiple embodiments, the cells can be loaded into the hydrogel polymer enclosure as organoids or organoid-like aggregates or spherical assemblies. In multiple embodiments, the organoids or organoid-like aggregates or spherical assemblies can be stained in suspension. In multiple embodiments, organoid-like aggregates or spherical assemblies are used to differentiate cells in 3D. In multiple embodiments, the cells can be loaded into the hydrogel polymer enclosure as mini-organs. In multiple embodiments, the mini-organs have at least two organ-specific cell types. In multiple embodiments, the cells of the mini-organs self-organize in 3D to form a structure similar to the tissue of an organ. In multiple embodiments, the mini-organs can express organ-specific functions. In multiple embodiments, the mini-organs replace the function of an organ in the body of a subject. In multiple embodiments, the mini-organs replace the function of cells in the body of a subject.

[0013] In multiple embodiments, the cells can be loaded into the hydrogel polymer enclosure as single cells. Without being bound by a particular theory, single cells can adhere to the inner surface of the hydrogel polymer enclosure, the surface of any microcarriers within the hydrogel polymer enclosure, or both the inner surface of the hydrogel polymer enclosure and the surface of any microcarriers within the hydrogel polymer enclosure. In multiple embodiments, the cells can be loaded into the hydrogel polymer enclosure as organoids or organoid-like aggregates or spherical assemblies. In multiple embodiments, the organoids or organoid-like aggregates or spherical assemblies can be stained in suspension. In multiple embodiments, organoid-like aggregates or spherical assemblies are used to differentiate cells in 3D. In multiple embodiments, the cells can be loaded into the hydrogel polymer enclosure as mini-organs. In multiple embodiments, the mini-organs have at least two organ-specific cell types. In multiple embodiments, the cells of the mini-organs self-organize in 3D to form a structure similar to the tissue of an organ. In multiple embodiments, the mini-organs can express organ-specific functions. In multiple embodiments, the mini-organs replace the function of an organ in the body of a subject. In multiple embodiments, the mini-organs replace the function of cells in the body of a subject.

[0014] In multiple embodiments, the hydrogel polymer enclosure contains cells within an internal aqueous chamber. In multiple embodiments, the cells are mammalian cells such as human cells, including cells derived from human induced pluripotent stem cells (iPSCs), adipose-derived stem cells (ASCs), or embryonic stem cells (ESCs).

[0015] In multiple embodiments, the cells are selected from one or more of adipogenic cells, iPSC-derived hepatocytes, adipocytes, pancreatic islet cells, iPSC-derived pancreatic islet cells, iPSC-derived dopaminergic neurons, endocrine cells, and cells from a heterologous source (e.g., pig), cadaveric tissue, living donors, adipose-derived stem cells, and embryonic stem cells.

[0016] In multiple embodiments, the cells within the internal aqueous chamber are iPSCs, ASCs, or ESCs. In multiple embodiments, the iPSCs, ASCs, and / or ESCs maintain the ability to divide without significant loss or decrease in genetic stability while being maintained in culture (e.g., minimizing the occurrence of copy number variations (CNVs) or single nucleotide polymorphisms (SNPs)). In multiple embodiments, the ability of iPSCs, ASCs, or ESCs to divide without significant loss or decrease in genetic stability is compared to non-encapsulated iPSCs, ASCs, or ESCs. In multiple embodiments, the ability of cells to divide without significant loss or decrease in genetic stability is evaluated by assays such as karyotyping, restriction endonuclease mapping, ddPCR, and / or DNA sequencing.

[0017] In multiple embodiments, the cells within the internal aqueous chamber are iPSCs, ASCs, or ESCs and can stably differentiate into mesodermal, endodermal, or ectodermal lineages.

[0018] In multiple embodiments, the internal aqueous chamber has a volume suitable for at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20000 or more mammalian cells (such as cells differentiated from any of the aforementioned, such as iPSCs, ASCs, ESCs, or hepatocytes).

[0019] In multiple embodiments, the internal aqueous chamber contains at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20000 or more mammalian cells (such as cells differentiated from any of the aforementioned, such as iPSCs, ASCs, ESCs, or hepatocytes). In multiple embodiments, the mammalian cells are (transiently or stably) genetically transformed, and the genetic transformation is further the expression of a transgene (for example, one or more of a therapeutic protein, such as an enzyme (including a gene editing system), a growth factor, a ligand, an antibody, or a structural protein; or a therapeutic nucleic acid, such as siRNA or an aptamer).

[0020] In multiple embodiments, the cells maintain at least about 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% viability without maintaining copy number variation during culture (for example, in about 1 L to 2000 L, or more) as measured by karyotyping, ddPCR, sequencing, or other assays over doublings at least about 10, 12, 15, 20, 25, 30 or more.

[0021] In multiple embodiments, the cells maintain at least about 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% genetic stability during growth over at least about 10, 12, 15, 20, 25, 30 or more doublings in culture (for example, in about 1 L to 2000 L, or more) as measured by karyotyping, ddPCR, sequencing, or other assays.

[0022] In some embodiments, at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, or more, of the cells are negative for a cell death signal, such as an apoptosis or pre-apoptosis signal (e.g., activated Caspase 3 or Annexin V staining), for example, over at least about 10, 12, 15, 20, 25, 30, or more doublings in culture (e.g., about 1 L to 2000 L, or more), or for at least about 5, 10, 20, 30 days or more, such as 5, 10, 15, 20 weeks, upon administration to a subject. In multiple embodiments, the cell death signal includes signaling activity by cells that promote and / or result in cell death, including, but not limited to, signals such as an apoptosis signal or a pre-apoptosis signal. Non-limiting examples for identifying whether a cell is negative for a cell death signal include activated Caspase 3 or Annexin V staining.

[0023] In multiple embodiments, the coefficient of variation of the hydrogel polymer enclosure is less than about 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%.

[0024] In multiple embodiments, at least about 60, 70, 80, 85, 90, 95% or more of the hydrogel polymer enclosure contains at least about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 100, 200, 250, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000 or more mammalian cells.

[0025] The hydrogel polymer enclosures of the present disclosure can be made using any method useful for preparing such enclosures, as will be understood by those of ordinary skill in the art. In a plurality of embodiments, the hydrogel polymer enclosures are made by a method that includes coaxial injection of an external vapor containing a polymer solution and an internal vapor containing cells. In a plurality of embodiments, the method is performed under cGMP conditions (21 CFR Parts 210, 211, 314) and / or International Council on Harmonization (ICH) quality guidelines (such as Q7). In a plurality of embodiments, the hydrogel polymer enclosures of the present disclosure are made from hydrogels prepared by the method of International Publication No. WO 2019 / 169245, which is hereby incorporated by reference in its entirety, and which includes a surfactant such as, for example, poloxamer 188.

[0026] In a plurality of embodiments, non-limiting examples of methods for making the hydrogel polymer enclosures of the present disclosure can be found in International Publication No. WO 2021 / 113751, which is hereby incorporated by reference in its entirety.

[0027] In multiple embodiments, such compositions can contain additional polymers such as non-alginates, as well as one or more agents useful for promoting cell viability, cell function (including, for example, cell differentiation from stem cells to primary cells), and / or reducing and / or preventing and / or inhibiting apoptosis, and / or preventing rejection reactions and / or fibrosis, which reduce (e.g., by 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99% or more relative to an unmodified polymer such as alginate) or substantially eliminate one or more of macrophage attachment, cell-derived innate immune system responses, or lymphocyte activation, including all three. Non-limiting examples of one or more agents include growth factors, transcription factors, proteins, and polypeptides. In multiple embodiments, one or more agents are useful for supporting cell viability such as cell differentiation and / or extracellular matrix (ECM) proteins. In multiple embodiments, the compositions or enclosures of the present disclosure can further include an integrated anti-inflammatory agent, including those described in International Publication No. WO 2012 / 12982, which is hereby incorporated by reference in its entirety.

[0028] In certain embodiments, one or more agents that promote cell viability and / or useful synergistic functions with the present disclosure (e.g., on an inner hydrogel layer) are polypeptides or fragments thereof (e.g., peptides of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more contiguous residues).

[0029] In certain embodiments, the polypeptide or fragment thereof can comprise one or more of the following: collagen V A1, collagen V A2, collagen VI A1, collagen VI A2, collagen VI A3, fibronectin, laminin, fibrin, fibrinogen alpha, fibrinogen beta, fibrinogen gamma, factor XIII a chain, factor XIII b chain, basement membrane-specific heparan sulfate proteoglycan core protein, elastin (including the foregoing combinations). Exemplary sequences of these proteins from which suitable fragments can be derived include, but are not limited to, the sequences provided by Uniprot accession: P02452, P08123, P02461, P53420, P20908, P05997, P12109, P12110, P12111, P02751, P02671, P02675, P02679, P00488, P05160, P98160, P15502, Q16787, P07942, P24043, P55268, O15230, Q13751, A4D0S4, P11047, Q13753, Q16363, P08865, P25391 and Q9Y6N6, each of which is incorporated herein by reference in its entirety.

[0030] In embodiments where the enclosure is made of a polymer (e.g., alginate) modified with a polypeptide, the composition is evaluated for quality, for example, by the method of WO 2020 / 069429, which is incorporated herein by reference in its entirety.

[0031] In a plurality of embodiments, non-limiting examples of methods for making the hydrogel polymer enclosures of the present disclosure can be found in WO 2021 / 113751, which is incorporated herein by reference in its entirety.

[0032] In multiple embodiments, a bilayer semipermeable fibrosis-resistant hydrogel polymer enclosure, an external hydrogel layer encompassing the enclosure, the external hydrogel layer optionally containing one or more rejection inhibitors that reduce rejection reactions upon implantation into a mammalian subject; an internal hydrogel layer disposed within the external hydrogel layer, the internal hydrogel layer containing alginate and one or more agents that promote cell viability, cell differentiation, cell health, cell function, and / or reduction and / or prevention and / or inhibition of apoptosis, and / or prevention of rejection reactions and / or fibrosis, and further optionally, the external hydrogel layer and the internal hydrogel layer are separated by a liquid layer, such as a culture medium; and an internal aqueous chamber disposed within the internal hydrogel layer, the internal aqueous chamber containing at least about 10 viable induced pluripotent stem cells (iPSCs), providing a hydrogel polymer enclosure that is suitable for administration to a human subject.

[0033] In multiple embodiments, a bilayer semipermeable fibrosis-resistant hydrogel polymer enclosure, an external hydrogel layer encompassing the enclosure, the external hydrogel layer optionally containing one or more rejection inhibitors that reduce rejection reactions upon implantation into a mammalian subject; an internal hydrogel layer disposed within the external hydrogel layer, the internal hydrogel layer containing alginate and one or more agents that promote cell viability, cell differentiation, cell health, cell function, and / or reduction and / or prevention and / or inhibition of apoptosis, and / or prevention of rejection reactions and / or fibrosis, and further optionally, the external hydrogel layer and the internal hydrogel layer are separated by a liquid layer, such as a culture medium; and an internal aqueous chamber disposed within the internal hydrogel layer, the internal aqueous chamber containing at least about 10 viable embryonic stem cells (ESCs), providing a hydrogel polymer enclosure that is suitable for administration to a human subject.

[0034] In multiple embodiments, a bilayer semipermeable fibrosis-resistant hydrogel polymer enclosure, an external hydrogel layer encompassing the enclosure, the external hydrogel layer optionally containing one or more rejection inhibitors that reduce rejection reactions upon implantation into a mammalian subject; an internal hydrogel layer disposed within the external hydrogel layer, the internal hydrogel layer containing alginate and one or more agents that promote reduction and / or prevention and / or inhibition of apoptosis and / or rejection reactions and / or fibrosis, and / or promotion of cell viability, cell differentiation, cell health, and / or cell function, and further optionally, the external hydrogel layer and the internal hydrogel layer are separated by a liquid layer, such as a culture medium; and an internal aqueous chamber disposed within the internal hydrogel layer, the internal aqueous chamber containing at least about 10 adipose-derived stem cells (ASCs), providing a hydrogel polymer enclosure suitable for administration to a human subject.

[0035] In multiple embodiments, the volume enables at least about 10 cells to grow within the chamber. In multiple embodiments, one or more agents are or include extracellular matrix (ECM) proteins. In multiple embodiments, the cells can stably differentiate into mesodermal, endodermal, or ectodermal lineages.

[0036] Hydrogel As will be understood by those of ordinary skill in the art, any hydrogel and / or hydrogel polymer useful within the enclosure of the present disclosure, including the external hydrogel layer and / or the internal hydrogel layer, is contemplated herein. As used herein, the term "hydrogel" refers to a stable biocompatible composition that contains one or more polysaccharides and swells and maintains stability in the presence of water. In a plurality of embodiments, the hydrogel is known to those of ordinary skill in the art, and non-limiting exemplary hydrogels include alginate, acrylate (e.g., methacrylate), and / or a combination of alginate and acrylate (e.g., methacrylate), whether mixed in one or both layers or in separate layers within the same enclosure.

[0037] In a plurality of embodiments, the hydrogel is a naturally-derived hydrogel. Non-limiting examples of naturally-derived hydrogels include, but are not limited to, DNA-based gels; protein-based gels (e.g., collagen, fibrin, gelatin, elastin-like peptides, fibrinogen, self-assembling peptides, elastin-like polypeptides); polysaccharide-based gels (e.g., alginate, alginate-co-gelatin, styrenated gelatin, chitosan, chondroitin sulfate, hyaluronic acid, chitin); and modified gels thereof. Non-limiting examples of modified gels include gels containing one or more polyethylene glycol (PEG) moieties and / or one or more RGD oligopeptides. In a plurality of embodiments, the hydrogel is a synthetic hydrogel. Non-limiting examples of synthetic hydrogels include, but are not limited to, biodegradable PEG-based gels (e.g., macromers include triblock copolymers of poly(α-hydroxyester)-b-poly(ethylene glycol)-b-poly(α-hydroxyester) end-capped with (meth)acrylate functional groups (e.g., PLA, poly(ε-caprolactone) (PCL)); polyfumarate-based hydrogels (e.g., macromers containing poly(lactide-co-ethylene oxide-co-fumarate) and MMP-diacrylate); and phosphoester-based hydrogels (e.g., containing poly(6-aminohexylpropylene phosphate)-acrylate).

[0038] In multiple embodiments, a hydrogel polymer enclosure that includes an external hydrogel layer and / or its internal hydrogel layer includes one or more polysaccharide gels, one or more acrylates (e.g., methacrylates), or any combination thereof. In multiple embodiments, a hydrogel polymer enclosure that includes its external hydrogel layer and / or internal hydrogel layer includes alginate. Non-limiting examples of polysaccharide gels include alginate, alginate-co-gelatin, styrenated gelatin, chitosan, chondroitin sulfate, hyaluronic acid, and chitin. Non-limiting examples of acrylates include acrylate, methacrylate, methyl (meth)acrylate, methyl ethyl acrylate, ethyl (meth)acrylate, n-propyl (meth)acrylate, i-propyl (meth)acrylate, n-butyl (meth)acrylate, i-butyl (meth)acrylate, sec-butyl (meth)acrylate, tert-butyl (meth)acrylate, 2-pentyl (meth)acrylate, 3-pentyl (meth)acrylate, isopentyl (meth)acrylate, neopentyl (meth)acrylate, n-octyl (meth)acrylate, 1,1,3,3-tetramethylbutyl (meth)acrylate, ethylhexyl (meth)acrylate, n-nonyl (meth)acrylate, n-decyl (meth)acrylate, n-undecyl (meth)acrylate, tridecyl (meth)acrylate, myristyl (meth)acrylate, pentadecyl (meth)acrylate, palmityl (meth)acrylate, heptadecyl (meth)acrylate, nonadecyl (meth)acrylate, arachinyl (meth)acrylate, behenyl (meth)acrylate, lignoceranyl (meth)acrylate, cerotinyl (meth)acrylate, melissinyl (meth)acrylate, palmitoleinyl (meth)acrylate, oleyl (meth)acrylate, linolyl (meth)acrylate, linolenyl (meth)acrylate, stearyl (meth)acrylate, lauryl (meth)acrylate, phenoxyethyl (meth)acrylate, 4-tert-butylcyclohexyl acrylate, cyclohexyl (meth)acrylate, ureido (meth)acrylate, tetrahydrofurfuryl (meth)acrylate, and mixtures thereof.

[0039] In multiple embodiments, "alginate" encompasses both "unmodified alginate" (a polysaccharide-containing copolymer made from (1→4)-linked β-D-mannuronate and α-L-guluronate) and "modified alginate" (including covalent modification to one or more monomers of the alginate polysaccharide).

[0040] In multiple embodiments, the external and internal hydrogel layers each independently include a polysaccharide-based gel, acrylate, or a combination thereof. In multiple embodiments, the internal and external hydrogel layers each independently include alginate, methacrylate, or a combination thereof. In multiple embodiments, the internal and / or external hydrogel layer includes alginate or methacrylate.

[0041] In multiple embodiments, without limitation, the hydrogel polymer enclosure including the external hydrogel layer and / or the internal hydrogel layer is further functionalized and / or modified to include additional functional groups and / or additional polymers. Non-limiting examples of additional functional groups include zwitterionic groups such as phosphobetaine, sulfobetaine, carboxybetaine, cysteine, sulfopyridinium betaine, phosphorylcholine, or sulfobetaine siloxane. Non-limiting examples of additional polymers include neutral polymers such as poly(ethylene glycol) (PEG), polysaccharides, poly(acid methacryl) (PAAm), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(N-vinylpyrrolidone) (PVP), and poly(2-methyl-2-oxazoline) (PMOXA).

[0042] As used herein, the term "zwitterion" refers to an ionic molecule whose net charge is neutral but which contains both positively and negatively charged functional groups. Exemplary zwitterionic groups include, but are not limited to, phosphobetaine, sulfobetaine, carboxybetaine, cysteine, sulfopyridinium betaine, phosphorylcholine, or sulfobetaine siloxane.

[0043] In multiple embodiments, the internal and / or external hydrogel layer comprises alginate and / or methacrylate, and the alginate and / or methacrylate comprises one or more additional functional groups and / or one or more additional polymers. In multiple embodiments, the internal and / or external hydrogel layer comprises alginate and / or methacrylate, and the alginate and / or methacrylate comprises one or more zwitterionic groups such as phosphobetaine, sulfobetaine, carboxybetaine, cysteine, sulfopyridinium betaine, phosphorylcholine, or sulfobetaine siloxane.

[0044] In multiple embodiments, the hydrogel is as follows: a) Optionally, a hydrogel derived from nature selected from the following: i. DNA-based gel; ii. Protein-based gel (e.g., collagen, fibrin, gelatin, elastin-like peptide, fibrinogen, self-assembling peptide, elastin-like polypeptide); iii. Polysaccharide-based gel (e.g., alginate, alginate-co-gelatin, styrenated gelatin, chitosan, chondroitin sulfate, hyaluronic acid, chitin); and iv. A modified gel described in any one of i. to iii. above (e.g., containing one or more polyethylene glycol (PEG) moieties and / or one or more RGD oligopeptides); or b) Optionally, a synthetic hydrogel selected from the following: i. Biodegradable PEG-based gel (e.g., as macromers, triblock copolymers of poly(α-hydroxyester)-b-poly(ethylene glycol)-b-poly(α-hydroxyester) (e.g., PLA, poly(ε-caprolactone) (PCL)) end-capped with (meth)acrylate functional group poly(α-hydroxyester)) can be mentioned) ii. Polyfumarate-based hydrogel (e.g., a macromer containing poly(lactide-co-ethylene oxide-co-fumarate) and MMP-diacrylate); and iii. Phosphoester-based hydrogels (e.g., poly(6-aminohexylpropylene phosphate)-acrylate) selected from

[0045] For further examples of hydrogels useful in the present disclosure, see Nicodemus and Bryant, Tissue Engineering Part B: Reviews 14(2008), which is hereby incorporated by reference in its entirety.

[0046] In a plurality of embodiments, the hydrogel includes a stiffness range of from about 0.1 to about 500 kPa, such as from about 0.1 to about 10 kPa, from about 0.5 to about 15 kPa, from about 1 to about 15 kPa, from about 5 to about 20 kPa, from about 10 to about 50 kPa, from about 20 to about 100 kPa, from about 150 to about 300 kPa, from about 100 to about 400 kPa, from about 200 to about 450 kPa, or from about 250 to about 500 kPa. In a further aspect, each cell containing a hydrogel capsule is characterized by a stiffness of about 10 kPa, about 15 kPa, about 20 kPa, about 25 kPa, about 30 kPa, about 35 kPa, about 40 kPa, about 45 kPa, about 50 kPa, about 55 kPa, about 60 kPa, about 65 kPa, about 70 kPa, about 75 kPa, about 80 kPa, about 85 kPa, about 90 kPa, or about 95 kPa or about 100 kPa. In a plurality of embodiments, the hydrogel includes a water content of greater than about 20% w / w, about 30% w / w, about 40% w / w, about 50% w / w, about 60% w / w, about 70% w / w, about 80% w / w, about 90% w / w, or about 95% w / w.

[0047] In multiple embodiments, the alginate is a modified alginate or includes a modified alginate. In multiple embodiments, the alginate is chemically modified to include, for example, zwitterionic groups that include combinations of the aforementioned modifications. In multiple embodiments, the alginate composition includes both modified and unmodified alginate in various ratios including, but not limited to, ratios of about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, or about 1:9. In multiple embodiments, an alginate that includes a zwitterionic monomer can be mixed with unmodified alginate in a ratio of about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, or about 1:9.

[0048] In multiple embodiments, a hydrogel polymer enclosure, including but not limited to an external hydrogel layer and / or an internal hydrogel layer, contains one or more rejection reaction inhibitors. As will be understood by those skilled in the art, rejection reaction inhibitors are useful for preventing unwanted protein absorption that can lead to the accumulation of unwanted proteins on the hydrogel surface. In multiple embodiments, the rejection reaction inhibitor can form a hydration shell to prevent unwanted protein absorption. In multiple embodiments, including the rejection reaction inhibitor in the hydrogel (e.g., within the external hydrogel layer) reduces unwanted foreign body reactions including but not limited to macrophage adhesion and lymphocyte activation. As will be understood by those skilled in the art, a foreign body reaction (FBR) is a process in which the implantation and implantation process damage the tissue surrounding the foreign body, which induces an inflammatory process that can develop into a fibrotic reaction, which surrounds and separates the implant material. In multiple embodiments, the external hydrogel layer prevents or reduces the inflammatory reaction during the implantation process. In a non-limiting example, a hydrogel, including but not limited to an external hydrogel layer and / or an internal hydrogel layer, can be modified and / or functionalized to include an anti-fouling functional group and / or an anti-fouling polymer, providing a hydrogel with anti-fouling properties. Examples of anti-fouling functional groups include, but are not limited to, zwitterionic chemical groups such as sulfobetaine, carboxybetaine, and phosphocholine. Examples of anti-fouling polymers include, but are not limited to, poly(ethylene glycol) (PEG), polysaccharides, poly(acid methacryl) (PAAm), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(N-vinylpyrrolidone) (PVP), and poly(2-methyl-2-oxazoline) (PMOXA). In multiple embodiments, the external hydrogel layer contains one or more rejection reaction inhibitors. In multiple embodiments, one or more rejection reaction inhibitors are zwitterionic groups. In multiple embodiments, the zwitterionic group is selected from sulfobetaine, carboxybetaine, phosphocholine, or other anti-fouling polymers including combinations of the foregoing.

[0049] In multiple embodiments, a hydrogel polymer enclosure that includes, but is not limited to, an external hydrogel layer and / or an internal hydrogel layer includes one or more polyalkylene glycol-based linkers. Non-limiting examples of polyalkylene glycol-based linkers include polyethylene glycol (PEG), polypropylene glycol (PPG), triethylene glycol, tetraethylene glycol, pentaethylene glycol, heptamethylene glycol, nonaethylene glycol, poly(ethylene glycol) methyl ether, poly(ethylene glycol) diacrylate (PEGDA), poly(ethylene glycol) diacrylate, (poly(ethylene glycol) diacrylate, poly(ethylene glycol) dimethacrylate (PEGDMA), and poly(ethylene glycol) dimethacrylate. In multiple embodiments, the external hydrogel includes a polyethylene glycol (PEG) linker.

[0050] In multiple embodiments, a hydrogel polymer that includes an external hydrogel layer and / or an internal hydrogel layer includes, but is not limited to, one or more extracellular matrix (ECM) components. In non-limiting examples, the inclusion of ECM components provides a hydrogel having the ability to mimic the natural extracellular matrix of various tissues. Any ECM component is contemplated by the present disclosure. Non-limiting examples of ECM components include reconstituted basement membrane (e.g., Matrigel), collagen, fibronectin, laminin, fibrin, nestin, perlecan, or signaling domains such as RGD (arginylglycylaspartic acid) (including combinations thereof).

[0051] Any hydrogel polymer enclosure shape that may be useful for encapsulating the cells of the present disclosure is contemplated herein. In a plurality of embodiments, the hydrogel polymer enclosure comprises one layer. In a plurality of embodiments, the hydrogel polymer enclosure comprises a plurality of layers. In a plurality of embodiments, the plurality of layers comprises one or more inner layers, one or more intermediate layers, and / or one or more outer layers. In a plurality of embodiments, the hydrogel polymer enclosure comprises one or more inner layers, one or more intermediate layers, and one or more outer layers. In a plurality of embodiments, the hydrogel polymer enclosure shape can be modified to increase the ratio of surface area to volume, as will be understood by those skilled in the art. In a plurality of embodiments, the hydrogel polymer enclosure is or comprises a coil or a cylinder. In a plurality of embodiments, the hydrogel polymer enclosure comprises a sandwich of two or more layers comprising the materials described herein.

[0052] In a plurality of embodiments, the outer hydrogel layer has an average thickness of about 5 - 50 μm, such as about 10 - 50 μm, about 10 - 20 μm, such as about 5, 10, 15, 20, 5, 30, 35, 40, 45, 50, 55, or 60 μm.

[0053] In a plurality of embodiments, the inner hydrogel layer has an average thickness of about 5 - 50 μm, such as about 10 - 50 μm, about 10 - 20 μm, such as about 5, 10, 15, 20, 5, 30, 35, 40, 45, 50, 55, or 60 μm.

[0054] In a plurality of embodiments, the inner hydrogel layer and / or the outer hydrogel layer has an average pore size of about 1 - 20 μm, such as about 0.1, 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μm, or about 1 - 10 μm, such as about 5 μm.

[0055] In other embodiments, the total of the external hydrogel layer and the internal hydrogel layer is about 10 to 100 μm, for example, about 20 to 100, for example, about 20 to 40, for example, about 10, 15, 20, 5, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 μm.

[0056] In a plurality of embodiments, the internal hydrogel layer and / or the external hydrogel layer have an average pore size that allows molecules less than about 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900 or 1000 kDa to pass through, but does not allow larger molecules to pass through.

[0057] In certain embodiments, the pores are sized to allow gases, fluids, and small solutes (e.g., small molecules, smaller proteins) to flow through.

[0058] In a plurality of embodiments, the hydrogel polymer enclosure is substantially spherical. In a plurality of embodiments, the substantially spherical enclosure has a diameter of about 200 to 1000 μm, for example 300 to 800 μm, 400 to 600 μm, 450 to 550 μm.

[0059] In a plurality of embodiments, sphericity is defined as an aspect ratio close to 1 (e.g., less than about 1.4, 1.3, 1.2, 1.19, 1.18, 1.17, 1.16, 1.15, 1.14, 1.13, 1.12, 1.11, 1.10, 1.09, 1.08, 1.07, 1.06, 1.05, 1.04, 1.03), where the aspect ratio is the ratio of the length to the width of the sphere (e.g., determined by SEM).

[0060] In a plurality of embodiments, the distribution of sphericity can be calculated as the coefficient of variation (CV) (e.g., less than about 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%) over a plurality of spheres (e.g., n ≧ 50 spheres). In certain embodiments, substantially spherical is a CV of 5% or less and an aspect ratio of 1.10.

[0061] In multiple embodiments, the alginates for use in the enclosures, compositions, and methods of the present disclosure include those described in one or more of the following: WO 2012 / 112982, WO 2012 / 167223, WO 2016 / 019391, WO 2017 / 075631, WO 2019 / 090309, WO 2018 / 067615, WO 2019 / 169333, WO 2021 / 062263, WO 2021 / 062273, WO 2022 / 031862, WO 2021 / 119522, WO 2019 / 169245, WO 2019 / 195055, WO 2020 / 069429, WO 2021 / 113751, WO 2018 / 140834, U.S. Pat. No. 10,730,983, U.S. Pat. No. 9,867,781, U.S. Pat. No. 10,278,922, U.S. Pat. No. 10,709,667, U.S. Pat. No. 10,709,818, and U.S. Pat. No. 10,426,735.

[0062] In multiple embodiments, the composition or enclosure of the present disclosure may further include an integrated anti-inflammatory agent as described in WO 2012 / 12982, which is hereby incorporated by reference in its entirety.

[0063] In multiple embodiments, the hydrogel polymer enclosure includes a modified alginate comprising one or more covalently modified monomers defined by Formula I as described in WO 2012 / 167223, which is hereby incorporated by reference in its entirety.

[0064] In multiple embodiments, the hydrogel polymer enclosure comprises a multi-modified alginate polymer having a structure according to Formula III as described in WO 2016 / 019391 pamphlet, which is hereby incorporated by reference in its entirety.

[0065] In multiple embodiments, the hydrogel polymer enclosure comprises a modified alginate polymer having a structure according to Formula I as described in WO 2017 / 075631 pamphlet, which is hereby incorporated by reference in its entirety.

[0066] In multiple embodiments, the hydrogel polymer enclosure has a low molecule containing a chemical moiety of Formula XII as described in WO 2019 / 090309 pamphlet or at least one polymer containing a chemical moiety of Formula XII bonded to one or more of its surfaces, which is hereby incorporated by reference in its entirety.

[0067] In multiple embodiments, the hydrogel polymer enclosure comprises a compound of Formula (II-C) as described in WO 2018 / 067615 pamphlet or a pharmaceutically acceptable salt thereof, which is hereby incorporated by reference in its entirety.

[0068] In multiple embodiments, the hydrogel polymer enclosure comprises a polymer modified with a compound of Formula (II-b) or a pharmaceutically acceptable salt thereof as described in WO 2019 / 169333 pamphlet, which is hereby incorporated by reference in its entirety.

[0069] In multiple embodiments, the hydrogel polymer enclosure of the present disclosure is made from a hydrogel prepared by the method of WO 2019 / 169245 pamphlet number, including a surfactant such as poloxamer 188, which is hereby incorporated by reference in its entirety. In multiple embodiments, a method for evaluating a polymer composition containing a polymer modified with a compound of Formula (I-b) as described in WO 2021 / 062273 pamphlet is provided.

[0070] In multiple embodiments, the hydrogel polymer enclosure comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof as described in WO 2022 / 031862 pamphlet, which is hereby incorporated by reference in its entirety.

[0071] In multiple embodiments, the hydrogel polymer enclosure comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof as described in WO 2021 / 119522 pamphlet, which is hereby incorporated by reference in its entirety.

[0072] In embodiments where the enclosure is made of a polymer (e.g., alginate) modified with a polypeptide, the composition is evaluated for quality, for example, by the method of WO 2021 / 062263 pamphlet, which is hereby incorporated by reference in its entirety.

[0073] In multiple embodiments, the hydrogel polymer enclosure comprises a) a first compartment, b) a second compartment, and c) particles comprising a compound of formula (I-a) as described in WO 2019 / 195055 pamphlet, which is hereby incorporated by reference in its entirety.

[0074] In some embodiments, the hydrogel polymer enclosure comprises alginate, and the alginate is high guluronic acid (G) alginate, containing about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, more than 90% or more guluronic acid (G). In some embodiments, the alginate is high mannuronic acid (M) alginate, containing more than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% mannuronic acid (M). In some embodiments, the M:G ratio is about 1. In some embodiments, the M:G ratio is less than 1. In some embodiments, the M:G ratio is greater than 1. In some embodiments, the alginate has an approximate molecular weight of less than 75 kDa and optionally a G:M ratio of 1.5 or more. In some embodiments, the alginate has an approximate molecular weight of 75 kDa to 150 kDa and optionally a G:M ratio of 1.5 or more. In some embodiments, the alginate has an approximate molecular weight of 150 to 250 kDa and optionally a G:M ratio of 1.5 or more. In the alginate-containing particle fluid, the amount of alginate (e.g., the weight % or volume % of the fluid relative to the actual weight of the alginate) can be 0.5%, e.g., at least 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12.5%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more, e.g., w / w%; less than 50%, e.g., 40%, 30%, 25%, 20%, 15%, 10%, 5%, or less. In one embodiment, the particle fluid comprises a single type of polymer. In other embodiments, the particle fluid comprises two or more types of polymers, e.g., two types of polymers or three types of polymers. In some embodiments, the particle fluid contains alginate. In some embodiments, the particle fluid contains alginate and a second type of polymer (e.g., a polysaccharide, e.g., hyaluronate or chitosan). The polymers in the particle fluid may be chemically modified, for example, with low molecules, peptides or proteins.In one embodiment, the polymer (e.g., alginate) in the particle fluid is modified with a compound containing an amide, carboxyl, ester, amine, aryl ring, heteroaryl ring, cycloalkyl ring, heterocyclyl ring, or polyethylene glycol moiety. In one embodiment, the polymer (e.g., alginate) in the particle fluid is modified with a compound described in any one of WO 2012 / 112982, WO 2017 / 075630, WO 2017 / 075631, WO 2017 / 218507, or WO 2018 / 067615. In one embodiment, the compound has the following structure:. [Chemical formula] is a low molecular weight compound having.

[0075] In multiple embodiments, the hydrogel polymer enclosure comprises a monomer of formula (I) as described in WO 2018 / 140834, which is hereby incorporated by reference in its entirety.

[0076] In multiple embodiments, the hydrogel polymer enclosure comprises a biocompatible polymer comprising one or more monomer subunits A and B, the polymer optionally further comprising one or more monomer subunits C; wherein each A is a zwitterionic monomer; each B is a monomer having a reactive side chain, the reactive side chain being of formula IV: d-R1-Y and comprising an amide bond and further an ester, ether, acylhydrazine, carbamate, ketone, carbonate, sulfone, sulfoxide, thioether, azo, or aldimine; wherein each C is independently a hydrophobic monomer or a neutral hydrophilic monomer, and d is the covalent attachment point of the reactive side chain to the polymer backbone.

[0077] In multiple embodiments, the hydrogel polymer enclosure comprises a modified alginate polymer comprising one or more covalently modified monomers as described in Formula I of U.S. Patent No. 10,709,818, which is hereby incorporated by reference in its entirety.

[0078] In multiple embodiments, the hydrogel polymer enclosure comprises a multi-modified alginate polymer having the structure described in Formula III of U.S. Patent No. 10,426,735, which is hereby incorporated by reference in its entirety.

[0079] In multiple embodiments, a mixture of these hydrogels containing the aforementioned alginate is used to supply one of the three inlets of a microfluidic nozzle used in coaxial injection, as shown in Figure 3. One of the other inlet streams contains extracellular matrix molecules that form an intermediate layer in contact with the cells. The third inlet contains a cell solution. The three streams are arranged such that the cell solution is in the center, the intermediate layer surrounds the cell solution, and the alginate solution encompasses the intermediate layer and the cell solution. When this material is injected into a calcium-containing solution bath, cross-linking occurs, stabilizing the bilayer hydrogel-containing cells. Methods useful for making multilayer enclosures are described, for example, in International Publication No. WO 2018 / 096277 (see also U.S. Patent Application Publication No. US 2019 / 330589), International Publication No. WO 2018 / 115723, International Publication No. WO 2019 / 101734 (see also U.S. Patent Application Publication No. US 2020 / 360888 and U.S. Patent No. 11,511,254), International Publication No. WO 2019 / 224467 (see also U.S. Patent Application Publication No. US 2021 / 123013), International Publication No. WO 2022 / 263601, and International Publication No. WO 2022 / 238485, each of which is hereby incorporated by reference in its entirety.

[0080] In multiple embodiments, there is provided a cell microcompartment continuously organized around a lumen, comprising at least one layer of human pluripotent cells; an extracellular matrix layer; and an outer hydrogel layer.

[0081] In multiple embodiments, a chamber for culturing and imaging a biological sample by optical microscopy is provided. The culturing and imaging chamber includes a support and a cover. The support includes at least one internal housing, and at least one microscope slide is disposed opposite an opening formed in the support or cover facing the internal housing; a seal disposed between the support and the cover in contact with the at least one microscope slide, the seal defining at least one other opening facing the microscope slide; a hydrogel layer disposed within the other opening of the seal, the hydrogel layer including an array of open wells formed at a predetermined three-dimensional position within an orthogonal frame connected to the support, each well being adapted to receive a biological sample; and the support, the cover, and the seal are configured to be fixed to form a culture chamber and an imaging seal for fluids.

[0082] In multiple embodiments, a method for manufacturing a plurality of capsules each including a crosslinked hydrogel outer shell surrounding a central core, the method comprising: a hydrogel solution and an aqueous composition of interest are designed to form the central core and are coextruded concentrically to form a mixture droplet including a layer of the hydrogel solution surrounding a droplet of the composition of interest, and in the coextrusion step, the layer of the hydrogel solution is carried out over a crosslinking aerosol such that the layer of the hydrogel solution contacts the aerosol and at least partially crosslinks around the droplet of the composition of interest, and the capsules are spherical and have a diameter of 50 to 500 μm. A manufacturing method is provided.

[0083] Cell In multiple aspects, the present disclosure provides cells useful within the hydrogel polymer enclosures of the present disclosure. Non-limiting examples of cells include adipogenic cells, ASCs, adipocytes, induced pluripotent stem cells (iPSCs), iPSC-derived hepatocytes, iPSC-derived pancreatic islet cells, iPSC-derived dopaminergic neurons, endocrine cells, cells from a heterologous source (e.g., porcine), cadaveric tissue, living donors (e.g., hepatocytes), and embryonic stem cells. In multiple embodiments, the cells can stably differentiate into mesodermal, endodermal, or ectodermal lineages, including but not limited to iPSCs, ASCs, or ESCs.

[0084] In multiple embodiments, the cells are allogeneic. In multiple embodiments, allogeneic cells include cells obtained from a donor different from the subject being treated. In multiple embodiments, the secretory cells and / or catalytic cells are autologous.

[0085] In multiple embodiments, the cells are substantially pure. In multiple embodiments, substantially pure means that greater than about 80%, or greater than about 85%, greater than about 90%, or greater than about 95%, or greater than about 97%, or greater than about 98%, or greater than about 99% of the cells exhibit the same or similar characteristics (e.g., therapeutic effect, potency, differentiation ability, mitotic activity, proliferation ability, morphology, cell surface markers, and combinations of the foregoing).

[0086] In multiple embodiments, the cells are cultured and expanded. Culture methods are described herein and will be understood by those skilled in the art. In multiple embodiments, the cells are cultured and expanded to a desired quantity of cells. In multiple embodiments, the secretory cells and / or catalytic cells are newly prepared and / or harvested. In multiple embodiments, the secretory cells and / or catalytic cells are thawed from a cryopreserved stock. In multiple embodiments, the secretory cells and / or catalytic cells are suitable for cryopreservation with a cryoprotectant including, for example, DMSO, albumin (e.g., human serum albumin), and / or saline.

[0087] In multiple embodiments, the cells are isolated from any source, as understood by those skilled in the art. In multiple embodiments, the cells are isolated from adipose tissue. In multiple embodiments, the cells are isolated from peripheral blood. In multiple embodiments, the cells are isolated from human peripheral blood. In multiple embodiments, the cells are mammalian cells. In multiple embodiments, the cells are human cells. In multiple embodiments, the cells are suitable for use in a human subject.

[0088] In multiple embodiments, the cells are non-immunogenic. In multiple embodiments, the cells do not induce and / or substantially do not induce an innate immune response in a subject. Non-limiting methods for identifying an innate immune response include measuring the levels of factors indicative of an innate immune response, including but not limited to TNFα, IFNγ, IL1β, IL6, IL10, and IL2, using any method as understood by those skilled in the art. In multiple embodiments, the cells of the present disclosure do not result in and / or substantially do not result in upregulation of one or more factors selected from TNFα, IFNγ, IL1β, IL6, IL10, and IL2 in a subject. In multiple embodiments, the cells of the present disclosure result in a decrease and / or suppression of the levels of one or more factors selected from TNFα, IFNγ, IL1β, IL6, IL10, and IL2 in a subject as compared to a subject showing an innate immune response.

[0089] In multiple embodiments, the cells comprise or consist of adipogenic cells. Any adipogenic cells are contemplated by the present disclosure. Non-limiting examples of adipogenic cells include adipocytes, adipose-derived stem cells (ASCs), and CD34 + cells are included. In multiple embodiments, the cells are ASCs. In multiple embodiments, the cells are derived from ASCs. Non-limiting examples of cells derived from ASCs include adipocytes.

[0090] In multiple embodiments, the adipogenic cells are allogeneic. Allogeneic cells include cells obtained from a donor different from the subject being treated. In multiple embodiments, the adipogenic cells are autologous.

[0091] In multiple embodiments, the cells comprise or consist of embryonic stem cells (ESCs). In multiple embodiments, the cells are derived from ESCs.

[0092] In multiple embodiments, the cells comprise or consist of iPSCs. Any iPSCs are contemplated by the present disclosure. In multiple embodiments, the cells are derived from iPSCs. Non-limiting examples of cells derived from iPSCs include iPSC-derived hepatocytes, iPSC-derived pancreatic islet cells, and iPSC-derived dopaminergic neurons. Non-limiting examples of pancreatic islet cells include alpha cells, beta cells, delta cells, and PP (gamma or F cells) cells.

[0093] In multiple embodiments, the cells comprise or consist of endocrine cells. Any endocrine cells are contemplated by the present disclosure. In non-limiting embodiments, the endocrine cells can be derived from the pancreas, thyroid, parathyroid, pineal gland, pituitary gland, hypothalamus, ovaries, and / or testes.

[0094] In multiple embodiments, pancreatic cells include or consist of alpha cells (e.g., capable of secreting glucagon hormone), delta cells (e.g., capable of secreting somatostatin hormone), and / or beta cells (e.g., capable of secreting insulin). In multiple embodiments, pancreatic cells include or consist of thyroid cells including follicular cells of the thyroid and / or C cells of the thyroid (e.g., capable of producing calcitonin). In multiple embodiments, parathyroid cells include or consist of chief cells (e.g., capable of secreting parathyroid hormone). In multiple embodiments, pinealocytes include or consist of pinealocytes (e.g., capable of secreting melatonin). In multiple embodiments, pituitary cells include or consist of thyroid-stimulating hormone-producing cells (e.g., capable of secreting thyrotropin), mammotropic hormone-producing cells (e.g., capable of secreting prolactin), adrenocorticotropic hormone-producing cells (e.g., capable of secreting adrenocorticotropic hormone (ACTH)), growth hormone-producing cells (e.g., capable of secreting growth hormone), and gonadotropin hormone-producing cells (e.g., capable of secreting gonadotropins such as luteinizing hormone and follicle-stimulating hormone). In multiple embodiments, hypothalamic cells include or consist of secretory neurons (e.g., capable of secreting antidiuretic hormone and oxytocin). In multiple embodiments, endocrine cells include or consist of endocrine cells of the ovaries and testes.

[0095] In multiple embodiments, the cells include or are cells derived from a heterologous (xenograft) source (e.g., pig), cadaveric tissue, a living donor (e.g., hepatocytes), and / or embryonic stem cells (ESCs). Any cells derived from a heterologous source, cadaveric tissue, living donor, and / or ESCs are contemplated by the present disclosure. Non-limiting examples of heterologous sources include pigs and goats.

[0096] Treatment methods In multiple embodiments, a method or process for delivering one or more cells is provided that includes contacting a biological tissue with the hydrogel polymer enclosure or composition of the present disclosure, where the hydrogel polymer enclosure or composition includes one or more cells, such as mammalian cells.

[0097] In multiple embodiments, the hydrogel polymer enclosure of the present disclosure supports the health and phenotypic control of encapsulated cells suitable for administration to a subject, and further enables efficient generation of large-scale cells, for example, by improving or eliminating multiple sources of perifibroblastic overgrowth, such as macrophage adhesion, cell-derived innate immune system responses, and lymphocyte activation, by improving or eliminating cell death signals.

[0098] In multiple embodiments, the tissue is liver tissue and the one or more cells are hepatocytes, including organoid-related hepatocytes, such as iPSC-derived, ASC-derived, or ESC-derived hepatocytes.

[0099] In multiple embodiments, the biological tissue is present in a mammalian subject. In multiple embodiments, the mammalian subject has or is suspected of having a liver disease. Non-limiting examples of liver diseases include acute liver failure (ALF; including those caused by or associated with inborn metabolic diseases [IMD]), chronic liver failure, and acute-on-chronic liver failure (ACLF).

[0100] In multiple embodiments, the mammalian subject has or is suspected of having a disease that results in a deficiency of liver function. Non-limiting examples of diseases that result in a deficiency of liver function include: a) diseases that result in a deficiency of protein synthesis by the liver (e.g., α1-antitrypsin deficiency, Wilson's disease) or multiple proteins; b) diseases that result in a deficiency of the metabolic function of the liver (e.g., ornithine transcarbamylase deficiency, Crigler-Najjar syndrome); or c) diseases that result in a deficiency of multiple functions of the liver (e.g., various forms of liver failure, acute, chronic, and acute-on-chronic).

[0101] In multiple embodiments, the mammalian subject has, or is suspected of having, a disease that results in a deficiency in liver function, adipogenic cell (including ASC or adipocytes) function, pancreatic islet cell function, or dopaminergic neuron function selected from the following: a) a disease that results in a deficiency in the synthesis of a protein or multiple proteins by the following: the liver (e.g., α1-antitrypsin deficiency, Wilson's disease, coagulation factor deficiency, acute intermittent porphyria, and familial amyloidotic polyneuropathy), adipogenic cells (e.g., lack of secretion of adipokines in leptin deficiency, etc., or lack of secretion of proteins such as lipoprotein lipase in familial chylomicronemia syndrome), pancreatic islet cells (e.g., lack of insulin secretion in type I or type II diabetes, and glucagon deficiency) or dopaminergic neurons; b) a disease that results in the following metabolic function deficiencies: the liver (e.g., ornithine transcarbamylase deficiency, Crigler-Najjar syndrome, branched-chain amino acid metabolism disorders (e.g., maple syrup urine disease), urea cycle disorders, familial hypercholesterolemia, glycogen storage diseases, hyperlipidemia, fatty acid transport disorders, disorders caused by mitochondrial defects (e.g., mitochondrial oxidation), peroxisomal biosynthesis disorders, hemochromatosis-hemosiderosis, organic acidemias, phenylketonuria, primary oxalosis, and tyrosinemia), adipogenic cells (e.g., branched-chain amino acid metabolism disorders (e.g., maple syrup urine disease), urea cycle disorders, hyperlipidemia, fatty acid transport disorders, disorders caused by mitochondrial defects (e.g., mitochondrial oxidation), peroxisomal biosynthesis disorders, organic acidemias, lipidogenesis disorders, lipid storage disorders, lipolysis disorders), pancreatic islet cells or dopaminergic neurons (e.g., diseases caused by a decrease in the production or release of dopamine such as neurodegenerative diseases including Parkinson's disease); c) a disease that results in a deficiency in multiple functions of the following: the liver (e.g., acute liver failure, acute alcoholic hepatitis, acute and chronic liver failure, chronic liver failure, end-stage liver disease, viral hepatitis, autoimmune hepatitis, neonatal hepatitis, congenital hepatic fibrosis, cirrhosis, graft-versus-host disease, Budd-Chiari syndrome, cystic fibrosis, biliary atresia, benign or malignant neoplasms of the liver, progressive familial intrahepatic cholestasis), adipogenic cells (e.g., congenital generalized lipodystrophy,Congenital partial lipodystrophy, acquired generalized lipodystrophy and acquired partial lipodystrophy), pancreatic islet cells (e.g., type I and type II diabetes), or dopaminergic neurons (e.g., neurodegenerative diseases that damage dopaminergic neurons such as Parkinson's disease).

[0102] In multiple embodiments, the hydrogel polymer enclosure is useful for treating, but not limited to, acute liver failure, acute and chronic liver failure (ACLF), generalized lipodystrophy (GLD), partial lipodystrophy (PL), diabetes, hypothyroidism (including, but not limited to, genetic causes (e.g., congenital hypothyroidism), hypertriglyceridemia, autoimmune diseases (e.g., Hashimoto's disease, atrophic thyroiditis), and / or hypothyroidism due to surgical removal and / or radiotherapy (e.g., used to treat parathyroid tumors)), hypoparathyroidism (including, but not limited to, hypoparathyroidism due to genetic causes (e.g., hereditary hypoparathyroidism) and / or hypoparathyroidism due to surgical removal and / or radiotherapy (e.g., used to treat parathyroid tumors)), hormonal deficiency disorders (e.g., growth hormone deficiency), hypothalamic obesity, adrenal insufficiency, and diseases or disorders including hypogonadotropin.

[0103] Manufacturing and cell culture In multiple embodiments, provided is a method of culturing cells, comprising culturing the hydrogel polymer enclosure or composition of the present disclosure within a bioreactor, wherein the hydrogel polymer enclosure or composition contains one or more cells.

[0104] In multiple embodiments, the composition is sterile (free of fungal, bacterial or archaeal cells; free of viral particles), pyrogen-free, substantially free of debris, or any or all combinations of the foregoing.

[0105] In multiple embodiments, a bioreactor cell culture system is provided that includes a closed chamber containing a plurality of floating cell microcompartments, each microcompartment including an outer hydrogel layer that provides a cavity containing a set of self-organizing cells and an extracellular matrix or extracellular matrix substitute.

[0106] As will be understood by those skilled in the art, microcarriers can be used in the methods of culturing the cells of the present disclosure. In multiple embodiments, the cells are dispersed on the microcarriers (e.g., during culture). In multiple embodiments, the cells are pre-cultured on the microcarriers (e.g., before encapsulation within a hydrogen polymer enclosure). In multiple embodiments, the cells are pre-cultured in vitro (e.g., before encapsulation within a hydrogen polymer enclosure). In multiple embodiments, the cells are dispersed within an enclosure (e.g., a semi-permeable enclosure such as an alginate enclosure). In multiple embodiments, the semi-permeable enclosure is separated (i.e., the cells are released) before the cells are dispersed within the hydrogel polymer enclosure. In multiple embodiments, the cells are cryopreserved. In multiple embodiments, the cells are cultured using any combination of the foregoing. By way of non-limiting example, the cells are cultured on microcarriers within an enclosure, then the semi-permeable enclosure is dissociated to release the cells, and then the cells are re-encapsulated within a hydrogel polymer enclosure.

[0107] In multiple embodiments, the cells are dispersed on the microcarriers; the semi-permeable enclosure is separated, the cells are recovered, and pre-cultured within the semi-permeable enclosure (such as an alginate enclosure) before re-encapsulating the cells within a hydrogel polymer enclosure of any of the preceding embodiments; recovered from cryopreservation; or a combination thereof.

[0108] In multiple embodiments, the cells are encapsulated by solid microcarriers. In multiple embodiments, the microcarriers are coated with various bioactive materials such as collagen, gelatin, fibrin, fibronectin, albumin, hyaluronic acid, elastin, chitosan, alginate, silk, its copolymers, or combinations thereof.

[0109] In multiple embodiments, the microcarriers include non-absorbable polymers (e.g., polyethylene, polyethylene oxide, polyethylene terephthalate, polyester, polymethyl methacrylate, polyacrylonitrile, silicone, polyurethane (PU), polycarbonate, polyether ketone, polyether ether ketone, polyether imide, polyamide, polystyrene, polyether sulfone, polysulfone, polyvinyl acetate, polytetrafluoroethylene, polyvinylidene fluoride, copolymers thereof, or combinations thereof); absorbable polymers (e.g., polycaprolactone, poly(lactide-co-caprolactone), poly(lactide-co-glycolide), polyglycolide, polylactic acid, such as, but not limited to, poly(lactic acid) (L-lactic acid), poly(lactic acid) (D,L-lactic acid), polyglycolic acid, polydioxanone, poly(3-hydroxybutyrate-co-3-hydroxyvalerate), trimethylene carbonate, polydiol, polyester, polyethylene terephthalate (PET), poly(butylene terephthalate) (PBT), polyurethane, polyethylene, polyethylene oxide, polymethyl methacrylate, polyacrylonitrile, silicone, polycarbonate, polyether ketone, polyether ether ketone, polyether imide, polyamide, polystyrene, polyether sulfone, polysulfone, polyvinyl acetate, polytetrafluoroethylene, polyvinylidene fluoride, polyglycolic acid, polydioxanone, collagen, gelatin, fibrin, fibronectin, albumin, hyaluronic acid, elastin, chitosan, alginate, or combinations thereof).In multiple embodiments, the absorbent polymer is selected from poly(lactide-co-glycolide), polyglycolide, poly(L-lactic acid), its copolymers, and combinations thereof; natural polymers (collagen, gelatin, fibrin, fibronectin, albumin, hyaluronic acid, elastin, chitosan, alginate, silk (e.g., silk fibroin), its copolymers, or combinations thereof); polyvinyl alcohol (PVA); nylon; and 1D polymer nanofibers (e.g., polyurethane, polyurethane copolymer, cellulose acetate, cellulose, acetate butyrate, cellulose derivatives, styrene-acrylonitrile (SAN), polyacrylonitrile (PAN), poly(vinyl acetate) (PVAc), polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), polyacrylic acid (PAA), hydroxypropyl cellulose (HPC), polymethyl methacrylate (PMMA), polyfurfuryl alcohol (PFA), polystyrene (PS), polystyrene copolymer, polyaniline (PANT), polyvinyl chloride (PVA), polypropylene (PP)), and polyimide, or combinations thereof).

[0110] For further examples of microcarrier culture, see Ashok, P., Fan, Y., Rostami, M. R., & Tzanakis, E. S. (2016). Aggregate and Microcarrier Cultures of Human Pluripotent Stem Cells in Stirred-Suspension Systems. Methods in Molecular Biology, 1502, 35-52, and 「General Guidelines for Corning Microcarriers」, www.corning.com / catalog / cls / documents / protocols / protocol_CLS_AN_241_microcarriers.pdf (which are hereby incorporated by reference in their entirety).

[0111] In multiple embodiments, the present disclosure provides compositions useful for encapsulation of the compositions and / or cells of the present disclosure, such as microencapsulation. In multiple embodiments, the compositions and / or cells of the present disclosure are modified by disposal and / or encapsulation within the hydrogel polymer enclosures disclosed herein.

[0112] In multiple embodiments, the encapsulation shape is spherical. In multiple embodiments, the encapsulation shape is non-spherical, such as a teardrop (i.e., a spherical and an elongated teardrop). In multiple embodiments, the encapsulation shape is designed for manufacture. In multiple embodiments, the encapsulation shape is formed into a scaffold, a pouch, and / or a tube, or a tubular lattice.

[0113] In multiple embodiments, the cell / organoid is removed inside the composition of the present disclosure using chemical or biological means to dissociate the hydrogel without adversely affecting the cell / organoid. In multiple embodiments, the chemical or biological means includes using a calcium chelating agent or an alginate lyase. In multiple embodiments, non-limiting examples of calcium chelating agents include EDTA, EGTA, and sodium citrate.

[0114] For further examples of using sodium citrate to dissociate hydrogels, see Maguire, T., Novik, E., Schloss, R., & Yarmush, M. (2006). Alginate-PLL microencapsulation: effect on the differentiation of embryonic stem cells into hepatocytes. Biotechnology and Bioengineering, 93(3), 581-591 and Belhaj, M. (2018). Enhancements in Alginate Microencapsulation Technology & Impacts on Cell Therapy Development. (Doctoral dissertation). Obtained from Scholarcoms.sc.edu / etd / 4475 (entirely incorporated herein by reference).

[0115] For further examples of using sodium citrate, EDTA, and EGTA to dissociate hydrogels, see Rodriguez, S., Lau, H., Corrales, N., Heng, J., Lee, S., Stiner, R., Alexander, M., & Lakey, J.R.T. (2020). Characterization of chelator-mediated recovery of pancreatic islets from barium-stabilized alginate microcapsules. Xenotransplantation, 27(1), e12554 (entirely incorporated herein by reference).

[0116] For a further example of using EDTA to dissociate a hydrogel, see Lehnert, S., & Sikorski, P. (2022). Application of Temporary, Cell-Containing Alginate Microcarriers to Facilitate the Fabrication of Spatially Defined Cell Pockets in 3D Collagen Hydrogels. Macromolecular Bioscience, 22(1), e2100319 (which is hereby incorporated by reference in its entirety).

[0117] For a further example of using alginate lyase to dissociate a hydrogel, see Zhang, H., Cheng, J., & Ao, Q. (2021). Preparation of Alginate-Based Biomaterials and Their Applications in Biomedicine. Marine Drugs, 19(5), and Saenz Del Burgo, L., Ciriza, J., Espona-Noguera, A., Illa, X., Cabruja, E., Orive, G., Hernandez, R. M., Villa, R., Pedraz, J. L., & Alvarez, M. (2018). 3D Printed porous polyamide macrocapsule combined with alginate microcapsules for safer cell-based therapies. Scientific Reports, 8(1), 8512 (which is hereby incorporated by reference in its entirety).

[0118] In a plurality of embodiments, a non-limiting protocol for using EDTA to dissociate a hydrogel involves using EDTA at a concentration of about 10 - 1000 mM, or about 50 - 100 mM, in a buffer solution (i.e., phosphate buffered saline), and incubating the hydrogel at room temperature (i.e., about 37 °C) for less than about 20 minutes, or less than 10 minutes.

[0119] In multiple embodiments, a non-limiting protocol for using sodium citrate to dissociate a hydrogel involves using sodium citrate at a concentration of about 10 - 1000 mM, or about 55 - 100 mM, a buffer solution (i.e., 10 mM HEPES on 10 mM MOPS and 27 mM NaCl), and incubating the hydrogel with shaking at room temperature (i.e., about 37 °C) for about 5 minutes to 2 hours, or 5 minutes to 30 minutes.

[0120] In multiple embodiments, a non-limiting protocol for using alginate lyase to dissociate a hydrogel involves using alginate lyase at a concentration of about 500 μg / mL.

[0121] Without being bound by a particular theory, this dissociation would enable re-encapsulation of cells / organsoids if a multi-step process is required for iPSC proliferation and cell differentiation.

[0122] In multiple embodiments, cryopreservation is a process by which cell organelles, cells, tissues, extracellular matrices, organs, or any other biological construct that is vulnerable to damage caused by unregulated chemical kinetics are preserved by cooling to a very low temperature (typically -80 °C using solid carbon dioxide or -196 °C using liquid nitrogen). At a sufficiently low temperature, enzymatic or chemical activities that could potentially cause damage to the biological material in question are effectively halted. Cryopreservation methods aim to reach low temperatures without causing additional damage caused by ice formation during freezing.

[0123] In multiple embodiments, methods are provided that include cooling the compositions of the present disclosure to about -30, -40, -50, -60, -70, -80, -90, -100, -110, -120, -130, -140, -150, -160, -170, -180, -190 °C.

[0124] In multiple embodiments, non-limiting methods of cryopreservation include gradually increasing the dimethyl sulfoxide (DMSO) concentration prior to freezing, followed by using a highly controlled supercooling process (e.g., in the range of about -0.25 °C / min to about -5 °C / min).

[0125] In multiple embodiments, the hydrogel polymer enclosure is suitable for cryopreservation and further includes one or more of the following: DMSO, glycerol, anti-apoptotic compounds (e.g., ZVAD), iron chelating agents (e.g., desferrioxamine), serum albumin (e.g., human serum albumin), or combinations of the foregoing.

[0126] In multiple embodiments, the hydrogel polymer enclosure is cryopreserved and enables thawing while maintaining cell viability. In multiple embodiments, the hydrogel polymer enclosure is recovered from cryopreservation. In multiple embodiments, the cells are suitable for cryoprotection with a cryoprotectant that includes, for example, DMSO, albumin (e.g., human serum albumin), and / or saline.

[0127] In multiple embodiments, non-limiting methods of cryopreservation include using a slow cooling rate process. In multiple embodiments, the slow cooling rate for cryopreservation of mature isolated hepatocytes utilizes DMSO as a cryoprotectant and various cooling rates in the range of about -1 °C to about -5 °C / min, or up to about -40 °C or about -80 °C, prior to storage at -196 °C in liquid nitrogen.

[0128] For further examples of cryopreservation methods, see Whaley, D., Damyar, K., Witek, R. P., Mendoza, A., Alexander, M., & Lakey, J. R. (2021). Cryopreservation: An Overview of Principles and Cell-Specific Considerations. Cell Transplantation, 30, 963689721999617, Huang, H., Yarmush, M. L., & Usta, O. B. (2018). Long-term deep-supercooling of large-volume water and red cell suspensions via surface sealing with immiscible liquids. Nature Communications, 9(1), 3201, and Jitraruch, S., Dhawan, A., Hughes, R. D., Filippi, C., Lehec, S. C., Glover, L., & Mitry, R. R. (2017). Cryopreservation of Hepatocyte Microbeads for Clinical Transplantation. Cell Transplantation, 26(8), 1341-1354 (which are hereby incorporated by reference in their entirety).

[0129] In a plurality of embodiments, non-limiting examples of methods for making the hydrogel polymer enclosures of the present disclosure can be found in International Publication No. WO 2021 / 113751, which is hereby incorporated by reference in its entirety.

[0130] In a plurality of aspects, the present disclosure provides dosage forms and / or compositions thereof of a therapeutically effective amount of the hydrogel polymer enclosures of the present disclosure. In a plurality of embodiments, the dosage forms of a therapeutically effective amount of the hydrogel polymer enclosures of the present disclosure and / or compositions thereof contain cells, such as human cells, such as the secretory cells or catalytic cells disclosed herein.

[0131] In multiple embodiments, the dosage form is a container such as a prefilled syringe. In multiple embodiments, the prefilled syringe is sterile. In multiple embodiments, the dosage form further comprises a pharmaceutically acceptable carrier, diluent, excipient, or vehicle. In multiple embodiments, the dosage form is a syringe comprising one or more hydrogel polymer enclosures and / or the compositions of the present disclosure. In some embodiments, the syringe is prefilled with a fixed amount of the composition.

[0132] Subject and / or animal In multiple embodiments, the subject and / or animal is a mammal, such as a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep, or non-human primate such as a monkey, chimpanzee or baboon. In other embodiments, the subject and / or animal is a non-mammal, such as a zebrafish. In multiple embodiments, the subject and / or animal may comprise fluorescently tagged cells (e.g., with GFP). In multiple embodiments, the subject and / or animal is a transgenic animal comprising fluorescent cells, such as RPE cells and / or immune cells. In multiple embodiments, the subject and / or animal is a human. In multiple embodiments, the human is a pediatric human. In multiple embodiments, the human is an infant or child. In multiple embodiments, the human is an adult human. In multiple embodiments, the human is an elderly human. In other embodiments, the human can be referred to as a patient.

[0133] In multiple embodiments, the human ranges in age from about 0 months to about 6 months, about 6 to about 12 months, about 6 to about 18 months, about 18 to about 36 months, about 1 to about 5 years, about 5 to about 10 years, about 10 to about 15 years, about 15 to about 20 years, about 20 to about 25 years, about 25 to about 30 years, about 30 to about 35 years, about 35 to about 40 years, about 40 to about 45 years, about 45 to about 50 years, about 50 to about 55 years, about 55 to about 60 years, about 60 to about 65 years, about 65 to about 70 years, about 70 to about 75 years, about 75 to about 80 years, about 80 to about 85 years, about 85 to about 90 years, about 90 to about 95 years, or about 95 to about 100 years.

[0134] In other embodiments, the subject is a non-human animal, and thus the present disclosure relates to veterinary use. In certain embodiments, the non-human animal is a household pet. In other certain embodiments, the non-human animal is a livestock animal.

[0135] Numbered embodiments The features of the enclosure, composition, or method of making and using them can include one or more of the embodiments listed below.

[0136] Embodiment 1: A bilayer semipermeable fibrosis-resistant hydrogel polymer enclosure, An external hydrogel layer encompassing the enclosure, the external hydrogel layer optionally comprising one or more rejection inhibitors that reduce rejection reactions upon implantation into a mammalian subject; An internal hydrogel layer disposed within the external hydrogel layer, the internal hydrogel layer optionally comprising one or more agents that promote cell viability, cell differentiation, cell health, cell function, and / or reduction and / or prevention and / or inhibition of apoptosis, and / or prevention of rejection reactions and / or fibrosis, and further optionally, the external hydrogel layer and the internal hydrogel layer are separated by a liquid layer, such as a culture medium; and An internal aqueous chamber disposed within the internal hydrogel layer, the internal aqueous chamber being suitable for containing at least about 10 live mammalian cells, and optionally, the volume enables growth of the at least about 10 cells within the chamber, A hydrogel polymer enclosure suitable for administration to a human subject.

[0137] Embodiment 2: The hydrogel polymer enclosure according to Embodiment 1, wherein the internal and external hydrogel layers independently comprise alginate, methacrylate, or a combination thereof.

[0138] Embodiment 3: The hydrogel polymer enclosure according to Embodiment 1, wherein the internal and / or the external hydrogel layer contains alginate or methacrylate, and optionally, the alginate or methacrylate contains one or more zwitterionic groups such as phosphobetaine, sulfobetaine, carboxybetaine, cysteine, sulfopyridinium betaine, phosphorylcholine, or sulfobetaine siloxane.

[0139] Embodiment 4: The hydrogel polymer enclosure according to any one of the preceding embodiments, wherein the internal aqueous chamber contains cells, and optionally, the cells are mammalian cells, such as human cells, such as hepatocytes, adipocytes, and pancreatic islet cells, or cells derived from human induced pluripotent stem cells (iPSCs), adipose-derived stem cells (ASCs), or embryonic stem cells (ESCs).

[0140] Embodiment 5: The hydrogel polymer enclosure according to Embodiment 4, wherein the cells in the internal aqueous chamber are iPSCs, ASCs, or ESCs, and maintain the ability to divide without significantly losing genetic stability (e.g., minimizing the occurrence of copy number variations (CNVs) or single nucleotide polymorphisms (SNPs) during culture, as evaluated by assays such as optionally karyotyping, restriction endonuclease mapping, ddPCR, and / or DNA sequencing).

[0141] Embodiment 6: The hydrogel polymer enclosure according to Embodiment 4, wherein the cells in the internal aqueous chamber are iPSCs, ASCs, or ESCs, and can stably differentiate into mesodermal, endodermal, or ectodermal lineages.

[0142] Embodiment 7: The inner hydrogel layer contains one or more agents that promote cell viability and / or function. Optionally, the one or more agents that promote cell viability and / or function are polypeptides having a biological function that promotes genetic stability and / or differentiation, and one or more of the extracellular matrix (ECM) components, for example: reconstituted basement membrane (e.g., Matrigel), collagen, fibronectin, laminin, fibrin, nestin, perlecan, or one or more of the signal transduction domains such as RGD (including combinations thereof), the hydrogel polymer enclosure according to any one of the preceding embodiments.

[0143] Embodiment 8: The outer hydrogel layer contains one or more rejection inhibitors and is a zwitterionic chemical group (e.g., modification of the hydrogel). Optionally, the zwitterionic chemical group prevents unwanted protein adsorption, for example, through the formation of a hydration shell, the hydrogel polymer enclosure according to any one of the preceding embodiments.

[0144] Embodiment 9: The zwitterionic chemical group is selected from sulfobetaine, carboxybetaine, phosphocholine, or other anti-fouling polymers including combinations of the foregoing, the hydrogel polymer enclosure according to Embodiment 8.

[0145] Embodiment 10: The outer hydrogel contains a polyethylene glycol (PEG) linker, the hydrogel polymer enclosure according to Embodiment 8.

[0146] Embodiment 11: The outer hydrogel layer reduces an unwanted foreign body reaction including macrophage adhesion and lymphocyte activation, the hydrogel polymer enclosure according to Embodiment 8.

[0147] Embodiment 12: The hydrogel polymer enclosure according to any one of the preceding embodiments, comprising one or more extracellular matrix (ECM) components, such as the following: reconstituted basement membrane (e.g., Matrigel), collagen, fibronectin, laminin, fibrin, nestin, perlecan, or a signaling domain such as RGD (including combinations thereof).

[0148] Embodiment 13: The hydrogel polymer enclosure according to any one of the preceding embodiments, which is substantially spherical.

[0149] Embodiment 14: The hydrogel polymer enclosure according to Embodiment 13, wherein the substantially spherical enclosure has a diameter of about 200 - 1000 μm, such as about 300 - 800 μm, 400 - 600 μm, or 450 - 550 μm, for example about 500 μm.

[0150] Embodiment 15: The hydrogel polymer enclosure according to any one of the preceding embodiments, wherein the internal aqueous chamber has a volume suitable for at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20000 or more mammalian cells (such as cells differentiated from any of the aforementioned, such as iPSC, ASC, ESC, or hepatocytes).

[0151] Embodiment 16: The internal aqueous chamber contains at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20000 or more mammalian cells (e.g., iPSCs, ASCs, ESCs, or cells differentiated from any of the foregoing, such as hepatocytes), optionally, the mammalian cells are (transiently or stably) genetically transformed, and further, the genetic transformation is the expression of a transgene (e.g., a therapeutic protein, such as: one or more of an enzyme (including a gene editing system), a growth factor, a ligand, an antibody, or a structural protein; or a therapeutic nucleic acid, such as siRNA or an aptamer), the hydrogel polymer enclosure according to any one of the preceding embodiments.

[0152] Embodiment 17: The cells maintain a viability of at least about 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% without maintaining copy number variation during culture (e.g., about 1 L to 2000 L, or more) as measured by karyotyping, ddPCR, sequencing, or other assays over doublings at at least about 10, 12, 15, 20, 25, 30 or more, the hydrogel polymer enclosure according to Embodiment 16.

[0153] Embodiment 18: At least about 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, or more, of the cells are at least about 10, 12, 15, 20, 25, 30, or more, over doublings during culture (e.g., about 1 L to 2000 L, or more), or upon administration to a subject, negative for a cell death signal, such as apoptosis or a pre-apoptosis signal (e.g., activated Caspase 3 or Annexin V staining), for at least about 5, 10, 20, 30 days or more, e.g., 5, 10, 15, or 20 weeks. The hydrogen gel polymer enclosure according to claim 16 or 17.

[0154] Embodiment 19: The cells maintain at least about 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% genetic stability as measured by karyotype analysis, ddPCR, sequencing, or other assays during growth over at least about 10, 12, 15, 20, 25, 30 or more doublings during culture (e.g., about 1 L to 2000 L, or more). The hydrogel polymer enclosure according to embodiments 16 - 18.

[0155] Embodiment 20: Upon implantation into a mammalian subject, induces a decrease in one or more levels of the following compared to a control (non - encapsulated cells or an enclosure not resistant to fibrosis): macrophage attachment, cell - derived innate immune system response, or lymphocyte activation. The hydrogel polymer enclosure according to any one of embodiments 16 - 19.

[0156] Embodiment 21: Does not induce an immune response upon implantation into a mammalian subject. The hydrogel polymer enclosure according to any one of embodiments 16 - 20.

[0157] Embodiment 22: The outer hydrogel layer has an average thickness of about 5 - 50 μm, e.g., about 10 - 50 μm, e.g., about 10 - 20 μm, e.g., about 5, 10, 15, 20, 5, 30, 35, 40, 45, 50, 55, or 60 μm. The hydrogel polymer enclosure according to any one of the preceding embodiments.

[0158] Embodiment 23: The hydrogel polymer enclosure according to any one of the preceding embodiments, wherein the external hydrogel layer has an average thickness of about 5 to 50 μm, for example, about 10 to 50 μm, for example about 10 to 20 μm, for example, about 5, 10, 15, 20, 5, 30, 35, 40, 45, 50, 55, or 60 μm.

[0159] Embodiment 24: The hydrogel polymer enclosure according to any one of the preceding embodiments, wherein the internal hydrogel layer and / or the external hydrogel layer has an average pore size of about 1 to 20 μm, for example, about 0.1, 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μm, or about 1 to 10 μm, for example about 5 μm.

[0160] Embodiment 25: The hydrogel polymer enclosure according to any one of the preceding embodiments, wherein the internal hydrogel layer and / or the external hydrogel layer has an average pore size that allows molecules less than about 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, or 1000 kDa to pass through, but does not allow larger molecules to pass through.

[0161] Embodiment 26: The hydrogel polymer enclosure according to any one of the preceding embodiments, which is produced by a method comprising a coaxial jet comprising an external vapor containing a polymer solution and an internal vapor containing cells, and optionally, the method is carried out under cGMP conditions (21 CFR Parts 210, 211, 314) and / or International Council on Harmonization (ICH) quality guidelines (such as Q7).

[0162] Embodiment 27: The hydrogel polymer enclosure according to any one of the preceding claims, wherein the cell differentiation includes differentiation from stem cells to primary cells.

[0163] Embodiment 28: A composition comprising a plurality of hydrogel polymer enclosures according to any one of the preceding embodiments.

[0164] Embodiment 29: The composition according to embodiment 28, wherein the coefficient of variation of the hydrogel polymer enclosure is less than about 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%.

[0165] Embodiment 30: The composition according to embodiment 28 or 29, wherein at least about 60, 70, 80, 85, 90, 95% or more of the hydrogel polymer enclosures contain at least about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 100, 200, 250, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000 or more mammalian cells, cells or more.

[0166] Embodiment 31: The composition according to any one of embodiments 28 to 30, which is sterile (free of fungal, bacterial or archaeal cells; free of virus particles), pyrogen-free, substantially free of debris, or any or all of the foregoing combinations.

[0167] Embodiment 32: A method of delivering one or more cells, comprising contacting a biological tissue with a hydrogel polymer enclosure or composition according to any one of the preceding embodiments, wherein the hydrogel polymer enclosure or composition contains one or more cells.

[0168] Embodiment 33: The method according to embodiment 32, wherein the biological tissue is within a mammalian subject.

[0169] Embodiment 34: A method of treating a disease or disorder, comprising providing to a tissue of a subject in need thereof a therapeutically effective amount of the hydrogel polymer enclosure or composition according to any one of the preceding embodiments, wherein the hydrogel polymer enclosure or composition comprises one or more cells.

[0170] Embodiment 35: The method according to embodiment 34, wherein the tissue is liver tissue and the one or more cells are hepatocytes (including organoid-related hepatocytes), for example, hepatocytes derived from iPSCs, ASCs or ESCs.

[0171] Embodiment 36: The method according to embodiment 34 or 35, wherein the mammalian subject has or is suspected of having a liver disease, such as acute liver failure (ALF; including those caused by or related to inborn metabolic diseases (IMD)), chronic liver failure, and acute-on-chronic liver failure (ACLF).

[0172] Embodiment 37: The method according to embodiment 34 or 35, wherein the mammalian subject has or is suspected of having a disease that results in a deficiency in liver function, adipogenic cell (including ASCs or adipocytes) function, pancreatic islet cell function, or dopaminergic neuron function selected from: a) Diseases that result in the synthesis deficiency of a protein or multiple proteins by: The liver (e.g., α1-antitrypsin deficiency, Wilson's disease, coagulation factor deficiency, acute intermittent porphyria, and familial amyloid polyneuropathy), Adipogenic cells (e.g., lack of adipokine secretion in familial chylomicronemia syndrome, lack of secretion of proteins such as leptin), Pancreatic islet cells (e.g., lack of insulin secretion in type I or type II diabetes, and glucagon deficiency), Or dopaminergic neurons b) Diseases that result in the following incomplete metabolic functions: The liver (e.g., ornithine transcarbamylase deficiency, Crigler-Najjar syndrome, branched-chain amino acid metabolism disorders (e.g., maple syrup urine disease), urea cycle disorders, familial hypercholesterolemia, glycogen storage diseases, hyperlipidemia, fatty acid transport disorders, disorders caused by mitochondrial defects (e.g., mitochondrial oxidation), peroxisome biosynthesis disorders, hemochromatosis-hemosiderosis, organic acidemias, phenylketonuria, primary oxalosis, and tyrosinemia), Adipogenic cells (e.g., branched-chain amino acid metabolism disorders (e.g., maple syrup urine disease), urea cycle disorders, hyperlipidemia, fatty acid transport disorders, disorders caused by mitochondrial defects (e.g., mitochondrial oxidation), peroxisome biosynthesis disorders, organic acidemias, lipidogenesis disorders, lipid storage disorders, lipolysis disorders), Pancreatic islet cells, or Dopaminergic neurons (e.g., diseases caused by a decrease in the production or release of dopamine, such as neurodegenerative diseases including Parkinson's disease) c) Diseases that result in the deficiency of the following multiple functions: The liver (e.g., acute liver failure, acute alcoholic hepatitis, acute and chronic liver failure, chronic liver failure, end-stage liver disease, viral hepatitis, autoimmune hepatitis, neonatal hepatitis, congenital hepatic fibrosis, cirrhosis, graft-versus-host disease, Budd-Chiari syndrome, cystic fibrosis, biliary atresia, benign or malignant neoplasms of the liver, progressive familial intrahepatic cholestasis), Adipogenic cells (e.g., congenital generalized lipodystrophy, congenital partial lipodystrophy, acquired generalized lipodystrophy, and acquired partial lipodystrophy), Pancreatic islet cells (e.g., type I diabetes and type II diabetes), or Dopaminergic neurons (e.g., neurodegenerative diseases that damage dopaminergic neurons, such as Parkinson's disease).

[0173] Embodiment 38: A method for culturing cells, comprising culturing the hydrogel polymer enclosure or composition according to any one of the preceding embodiments in a bioreactor, wherein the hydrogel polymer enclosure or composition contains one or more cells.

[0174] Embodiment 39: The cells are dispersed on a microcarrier; the semipermeable enclosure is separated, the cells are recovered, and pre-cultured inside the semipermeable enclosure (such as an alginate enclosure) before re-encapsulating the cells into the hydrogel polymer enclosure of any of the preceding embodiments; recovered from cryopreservation; or a combination thereof, the composition according to Embodiment 30 or 31 or the method according to any one of Embodiments 32 to 38.

[0175] Embodiment 40: The composition according to Embodiment 30 or 31 or the method according to any one of Embodiments 32 to 39, wherein the hydrogel polymer enclosure is recovered from cryopreservation.

[0176] Embodiment 41: The composition is suitable for cryopreservation, and the composition optionally further comprises one or more of the following: DMSO, glycerol, anti-apoptosis compound (e.g., ZVAD), iron chelating agent (e.g., desferrioxamine), serum albumin (e.g., human serum albumin), or a combination of the foregoing, the hydrogel polymer enclosure according to any one of Embodiments 1 to 27, the composition according to any one of Embodiments 28 to 31, 39, or 40.

[0177] Embodiment 42: A method comprising cooling the enclosure or composition according to Embodiment 41 to about -20, -30, -40, -50, -60, -70, -80, -90, -100, -110, -120, -130, -140, -150, -160, -170, -180, -190 °C.

[0178] Embodiment 43: A double-layer semipermeable fibrosis-resistant hydrogel polymer enclosure, An external hydrogel layer encompassing the enclosure, optionally containing one or more rejection inhibitors that reduce rejection reactions upon implantation into a mammalian subject; An internal hydrogel layer disposed within the external hydrogel layer, containing alginate and one or more agents that promote cell viability, cell differentiation, cell health, cell function, and / or reduction and / or prevention and / or inhibition of apoptosis, and / or prevention of rejection reactions and / or fibrosis, and further optionally, the external hydrogel layer and the internal hydrogel layer are separated by a liquid layer, such as a culture medium; and An internal aqueous chamber disposed within the internal hydrogel layer, containing an internal aqueous chamber containing at least about 10 viable induced pluripotent stem cells (iPSCs), A hydrogel polymer enclosure suitable for administration to a human subject.

[0179] Embodiment 44: A bilayer semipermeable fibrosis-resistant hydrogel polymer enclosure, An external hydrogel layer encompassing the enclosure, optionally containing one or more rejection inhibitors that reduce rejection reactions upon implantation into a mammalian subject; An internal hydrogel layer disposed within the external hydrogel layer, containing alginate and one or more agents that promote cell viability, cell differentiation, cell health, cell function, and / or reduction and / or prevention and / or inhibition of apoptosis, and / or prevention of rejection reactions and / or fibrosis, and further optionally, the external hydrogel layer and the internal hydrogel layer are separated by a liquid layer, such as a culture medium; and An internal aqueous chamber disposed within the internal hydrogel layer, containing an internal aqueous chamber containing at least about 10 viable embryonic stem cells (ESCs), A hydrogel polymer enclosure suitable for administration to a human subject.

[0180] Embodiment 45: A bilayer semipermeable fibrosis-resistant hydrogel polymer enclosure, an outer hydrogel layer encompassing the enclosure, the outer hydrogel layer optionally containing one or more rejection inhibitors that reduce rejection reactions upon implantation into a mammalian subject; an inner hydrogel layer disposed within the outer hydrogel layer, the inner hydrogel layer containing alginate and one or more agents that promote cell viability, cell differentiation, cell health, cell function, and / or reduction and / or prevention and / or inhibition of apoptosis, and / or prevention of rejection reactions and / or fibrosis, and further optionally, the outer hydrogel layer and the inner hydrogel layer are separated by a liquid layer, such as a culture medium; and an inner aqueous chamber disposed within the inner hydrogel layer, the inner aqueous chamber containing at least about 10 adipose-derived stem cells (ASCs), a hydrogel polymer enclosure suitable for administration to a human subject.

[0181] Embodiment 46: The hydrogel polymer enclosure according to any one of Embodiments 43 to 45, wherein the volume enables at least about 10 cells to grow within the chamber.

[0182] Embodiment 47: The hydrogel polymer enclosure according to any one of Embodiments 43 to 45, wherein the one or more agents are or include extracellular matrix (ECM) proteins.

[0183] Embodiment 48: The hydrogel polymer enclosure according to any one of Embodiments 43 to 45, wherein the cells can stably differentiate into mesodermal, endodermal, or ectodermal lineages.

[0184] Embodiment 49: A dosage form of a therapeutically effective amount of the hydrogel polymer enclosure according to any one of Embodiments 1 to 27, or the composition according to any one of Embodiments 28 to 31, 39, or 40.

[0185] Embodiment 50: The dosage form according to Embodiment 49, which is a container, optionally a syringe.

[0186] Embodiment 51: The dosage form according to Embodiment 49 or 50, wherein the hydrogel polymer enclosure or composition further comprises a pharmaceutically acceptable carrier, diluent, excipient, or vehicle.

[0187] Embodiment 52: The hydrogel polymer enclosure according to any one of claims 1 to 27, 41, or 43 to 48, the composition according to any one of claims 28 to 31, 39, or 40, or the method according to any one of claims 32 to 38, 42, wherein the hydrogel polymer enclosure is produced by microfluidic coaxial injection.

[0188] For all numerical boundaries that describe some of the parameters in this application, such as "about", "at least", "less than", and "more than", it should be understood that the description necessarily includes any range bounded by the recited values. Thus, for example, the description "at least 1, 2, 3, 4, or 5" describes, inter alia, the ranges 1 - 2, 1 - 3, 1 - 4, 1 - 5, 2 - 3, 2 - 4, 2 - 5, 3 - 4, 3 - 5, and 4 - 5, etc.

[0189] For all patents, applications, or other references cited in this specification, such as non-patent literature and reference sequence information, it should be understood that they are all incorporated by reference in their entirety for all purposes and the proposals recited. In the event of a conflict between the document incorporated by reference and this application, this application shall prevail. For example, all information related to the reference gene sequences disclosed in this application, such as GeneID or accession numbers (typically referring to NCBI or Uniprot accession numbers), including genomic loci, genomic sequences, functional annotations, allelic variants, and reference mRNAs (including exon boundaries or response elements, for example) and protein sequences (such as conserved domain structures), and chemical references (such as PubChem compounds, PubChem substances, or PubChem Bioassay entries, including annotations therein such as structures and assays), are incorporated by reference in their entirety into this specification.

[0190] The headings used in this application are for convenience only and do not affect the interpretation of this application.

[0191] Each preferred feature of each aspect provided by the present disclosure is applicable with all necessary modifications to all other aspects of the present disclosure, and is not limiting, but is exemplified by the dependent claims (or embodiments), and also includes combinations and substitutions of individual features (e.g., elements including numerical ranges and exemplary embodiments) of specific embodiments and aspects of the present disclosure including examples. For example, the specific experimental parameters exemplified in the examples can be gradually adapted for use in the claimed disclosure without departing from the present disclosure. For example, although specific references to various individual and collective combinations and permutations of these compounds for the disclosed materials may not be explicitly disclosed, each is specifically contemplated and described herein. Thus, if classes of elements A, B, and C and classes of elements D, E, and F are disclosed and an example of a combination of elements A-D is disclosed, each is individually and collectively contemplated even if not individually listed. Thus, in this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F is specifically contemplated and should be considered disclosed from the disclosure of A, B, and C; D, E, and F and the exemplary combination A-D. Similarly, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, subgroups of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from the disclosure of A, B, and C; D, E, and F; and the exemplary combination A-D. This concept applies to all aspects of this application, including elements of a composition of matter and steps of a method of making or using a composition.

[0192] The foregoing aspects of the present disclosure can be claimed in any combination substitution as long as they are new and non-obvious over the prior art as will be recognized by those of ordinary skill in the art in accordance with the teachings herein, and thus can be excluded from the claimed disclosure by negative conditions or denials of particular features or combinations of features, especially as long as the elements are described in one or more references known to those of ordinary skill in the art.

Examples

[0193] Example 1: Production of Double-Layer Hydrogel Encapsulation of Cells This example demonstrates, among other things, the ability to produce a double-layer hydrogel polymer enclosure containing iPSCs or ESCs for the purposes of growth and / or differentiation and delivery as a treatment for disease.

[0194] Treatment Design: iPSCs or ESCs are cultured and prepared as individual cells, and the solution containing these cells is pumped through a microfluidic nozzle surrounded concentrically by a second flow. The second flow contains a hydrogel containing proteins that support cell differentiation and / or cell viability, such as extracellular matrix (ECM) proteins. The design optionally includes a third concentric flow of cell culture medium. The fourth (or third in the absence of the above-described third flow) concentric flow that flows through the microfluidic nozzle and surrounds the aforementioned flows contains a modified alginate solution in which alginate is modified to reduce the immune response when administered. These flows are pumped through the microfluidic nozzle at a rate such that a flow of double-layer hydrogel spheres containing cells for 10 s to 100 s is extruded into a cell culture medium containing calcium to polymerize an external alginate sphere.

[0195] The encapsulated cells are cultured in a culture medium for 10 to 40 days to support differentiation into hepatocytes. Without being bound by a particular theory, the encapsulated cells can be dissociated and re-encapsulated to support a high proliferation number or to allow for changes in the differentiation signals of the inner layer.

[0196] Example 2: In Vitro Testing of Double-Layer Hydrogel Encapsulation of iPSC-Derived Hepatocytes This example demonstrates, among other things, the in vitro activity of the encapsulated cells after differentiation into iPSC-derived hepatocytes.

[0197] Treatment Design: Evaluate the viability, ammonia metabolism, and CYP450 activity of the encapsulated cells prepared in Example 1.

[0198] Materials / Methods: The trypan blue exclusion method (TPE; ThermoFisher, 15250061) was utilized to determine cell viability as described in Gramignoli, et al., 2012 (Cell Transplant. 21(6):1245 - 1260; 2012). Most of the encapsulated cells (more than 90%) examined by microscopy were not stained by trypan blue, indicating a cell viability of more than 90%.

[0199] Ammonia metabolism was evaluated by a commercially available quantitative colorimetric ammonia metabolism assay (Millipore Sigma, AA0100). A 100 μL encapsulated cell solution, a negative control containing 100 μL of water, and a sample containing 100 μL of L - glutamate dehydrogenase solution were operated according to the assay protocol. The encapsulated cell solution showed significant ammonia metabolism.

[0200] CYP450 activity was evaluated using a commercially available P450 - Glo assay (Promega, V9001) as described in Gramignoli, et al., 2014 (Cell Transplant. 23(9):1143 - 1151; 2014). Luciferase - IPA solution was added to wells containing encapsulated cells, negative control (cell culture medium only), and positive control (P450 enzyme solution), and incubated for 30 - 90 minutes. P450 - Glo reagent was added to all wells and equilibrated for 20 minutes. Then, luminescence was measured as a direct indicator of CYP450 activity. The encapsulated iPSC - derived hepatocytes showed CYP450 activity exceeding that of the negative control.

[0201] Example 3: In Vivo Test of Double - Layer Hydrogel - Encapsulated iPSC - Derived Hepatocytes This example demonstrates, among other things, the in vivo activity of encapsulated cells after differentiation into iPSC - derived hepatocytes.

[0202] Treatment design: Evaluate the in vivo activity of the encapsulated cells prepared in Example 1 in a mouse model of acetaminophen - induced liver injury.

[0203] Materials / Methods: The mouse model is created as described by Mossanen and Tacke (Laboratory Animals 49(1):30-36; 2015) and Viswanathan et al. (FASEB J. 35(4):e21471; 2021). Male and female C57BL / 6 mice, 10 to 12 weeks old, are fasted for 12 to 16 hours and allowed free access to water. At the end of fasting, the mice are administered an intraperitoneal dose of 300 mg / kg body weight of acetaminophen (N-acetyl-p-aminophenol [APAP]).

[0204] Two to three hours after APAP administration, a solution containing 50 million total encapsulated cells prepared in Example 1 is administered intraperitoneally to each mouse. Sham mice are administered the APAP dose but an empty double-layer hydrogel sphere is administered. At the 24-hour and 48-hour time points, the survival rates of the treated mice and sham mice are compared. Mice administered the encapsulated cells have improved survival rates at 24 hours and 48 hours compared to sham mice.

[0205] Example 4: Production of Double-Layer Hydrogel Encapsulation of Human ASCs This example demonstrates, among other things, the ability to produce a double-layer hydrogel polymer enclosure containing ASCs for the purposes of proliferation and characterization.

[0206] In this example, ASCs derived from healthy donors are obtained and prepared as individual cells. A solution containing these cells is pump-transported through a microfluidic nozzle surrounded concentrically by a second flow, as described in Example 1. The second flow includes a hydrogel containing proteins that support cell differentiation and / or cell viability, such as extracellular matrix (ECM) proteins. The design optionally includes a third concentric flow of cell culture medium. A fourth (or third in the absence of the aforementioned third flow) concentric flow that flows through the microfluidic nozzle and surrounds the aforementioned flows includes a modified alginate solution in which alginate is modified to reduce the immune response when administered. These flows are pump-transported through the microfluidic nozzle at a rate such that a flow of double-layer hydrogel spheres containing cells for 10 s to 100 s is extruded into a cell culture medium containing calcium to polymerize an external alginate sphere. The encapsulated cells are then grown in a culture medium. To support a high growth number, the encapsulated cells can optionally be dissociated and re-encapsulated.

[0207] The isolated and grown cells are de-encapsulated and characterized for ASC surface markers using flow cytometry analysis. Specifically, the cells are stained with directly conjugated antibodies against CD29, CD73, CD90, CD105, CD31, CD45, and CD34. The isolated cells are expected to show high expression of CD29, CD73, CD90, and CD105, low expression of CD31 and CD45, and variable expression of CD34.

[0208] Overall, this example demonstrates, among other things, the ability to produce a double-layer hydrogel polymer enclosure containing ASCs for the purposes of growth and characterization.

[0209] Example 5: Production of Double-Layer Hydrogel Encapsulation of Adipocytes This example demonstrates, among other things, the ability to produce a double-layer hydrogel polymer enclosure containing ASCs for the purposes of growth, differentiation into adipocytes, and characterization.

[0210] In this example, ASCs derived from healthy donors are obtained. The ASCs are prepared, encapsulated, and grown as described in Example 4. Then, differentiation into adipocytes begins. An adipocyte induction medium is prepared in DMEM / F12 (Thermo Fisher, 10565-018) containing 3% FBS (Gemini, 100-106), 1× penicillin-streptomycin (Thermo Fisher, 15140-122), 33 μM biotin (Fisher Scientific, BP232-1), 17 μM pantothenate (Fisher Scientific, AAA1660922), 1 μM insulin (sigma, I9278), 187.5 μM IBMX (Fisher Scientific, AAJ64598MC), 200 μM indomethacin (Fisher Scientific, AAA1991006), and 1 μM dexamethasone (Fisher Scientific, D16911G), and then sterile filtered through a 0.22 μM PES filter bottle. Next, the ASCs are exposed to the newly prepared adipocyte induction medium and cultured for 3 days. After 3 days, a sufficient adipocyte maintenance medium is prepared in DMEM / F12 (Thermo Fisher, 10565-018) containing 3% FBS (Gemini, 100-106), 1× penicillin-streptomycin (Thermo Fisher, 15140-122), 33 μM biotin (Fisher Scientific, BP232-1), 17 μM pantothenate (Fisher Scientific, AAA1660922), 1 μM insulin (sigma, I9278), (Fisher Scientific, AAA1991006), and 1 μM dexamethasone (Fisher Scientific, D16911G), and then sterile filtered through a 0.22 μM PES filter bottle. Then, the cells are exposed to the newly prepared adipocyte maintenance medium and cultured for 4 days.

[0211] Seven days after differentiation, the cells are characterized for adipogenic differentiation. Adipogenic differentiation is evaluated by the presence of intracellular lipid droplets by Oil Red O staining. Specifically, the cells are fixed in 10% (v / v) neutral buffered formaldehyde (Sigma, HT501128) for 1 hour and stained with 60% (v / v) Oil Red O solution (Fisher, AAA1298914) for 10 minutes. The differentiation rate is represented as the ratio of the number of Oil Red O-positive cells to the total number of cells.

[0212] The efficiency of adipogenic differentiation is also quantified by flow cytometry analysis. Specifically, LipidTOX Deep Red (Fisher, H34477) is added to the cell suspension at a 1:200 dilution and gently mixed. The cells are incubated at room temperature for 30 minutes. The cells are then analyzed by a flow cytometer. Differentiated adipocytes are expected to be stained for LipidTOX at a higher level compared to ASCs.

[0213] The levels of adipocyte-specific gene expression in differentiated cells are quantified by qPCR. Total RNA is isolated, cDNA is generated, and qRT-PCR is performed for adipogenic genes including adiponectin, Pparg, leptin, lipoprotein lipase, FABP4. GAPDH and actin are used as controls.

[0214] Overall, this example demonstrates, among other things, the ability to produce a bilayer hydrogel polymer enclosure containing ASCs for the purposes of, inter alia, proliferation, differentiation into adipocytes, and characterization.

[0215] Example 6: In Vitro Testing of Bilayer Hydrogel Encapsulation of Adipocytes This example demonstrates, among other things, the in vitro activity of encapsulated ASCs after differentiation into adipocytes by measuring, inter alia, leptin secretion, lipoprotein lipase secretion, and BCAA catabolism.

[0216] The encapsulated cells prepared in Example 5 are evaluated for in vitro leptin secretion by leptin ELISA (BioLegend, catalog number: 444304) and lipoprotein lipase (LPL) secretion by LPL ELISA (ABclonal, catalog number: RK06714). The determination of branched-chain α-keto acid dehydrogenase activity is performed on the cell lysates of the cells prepared in Example 5 as well as HepG2 cells as a positive control as previously described (Nakai et al., 2000; PMID: 10989417). Briefly, the activity is measured using an assay that monitors NADH production over time. The activity is then normalized against the total protein added to the reaction using the BCA assay (ThermoFisher, BCA Protein Assay Kit). Further, the cells are incubated with cell culture medium (DMEM), and samples of the cell culture medium are taken at 4 hours and 24 hours to measure the depletion of branched-chain amino acids (BCAAs) from the cells generated in Example 5 and HepG2 cells over time. Cell-free DMEM functions as a negative control. The BCAA concentration is then measured using an enzyme assay (Sigma, catalog number: MAK003).

[0217] Adipocytes differentiated with double-layer hydrogel encapsulation are expected to secrete significant levels of leptin and lipoprotein lipase and exhibit significant levels of BCAA catabolism when assayed in vitro.

[0218] Example 7: In Vitro Testing of ASC Encapsulation in Double-Layer Hydrogels This example demonstrates, among other things, the in vitro activity of encapsulated ASCs by measuring BCAA catabolism.

[0219] The encapsulated cells prepared in Example 4 are evaluated for BCAA catabolic activity. The determination of branched-chain α-keto acid dehydrogenase activity is performed on the cell lysates of the cells prepared in Example 4 as well as on HepG2 cells as a positive control, as previously described (Nakai et al., 2000; PMID: 10989417). Briefly, the activity is measured using an assay that monitors NADH production over time. The activity is then normalized against the total protein added to the reaction using a BCA assay (ThermoFisher, BCA Protein Assay Kit). Furthermore, the depletion of branched-chain amino acids (BCAAs) from the cells generated in Example 4 and HepG2 cells over time is measured by incubating the cells with cell culture medium (DMEM) and collecting samples of the cell culture medium at 4 hours and 24 hours. DMEM without cells functions as a negative control. The BCAA concentration is then measured using an enzyme assay (Sigma, catalog number: MAK003).

[0220] Without being bound to a particular theory, it is expected that ASCs in double-layer hydrogel encapsulation exhibit a significant level of BCAA catabolism.

[0221] Example 8: Therapeutic effect of double-layer hydrogel-encapsulated adipocytes in a mouse model of lipodystrophy and leptin deficiency This example demonstrates, among other things, the in vivo activity of the encapsulated cells after differentiation into adipocytes.

[0222] In this example, the encapsulated cells prepared in Example 5 are evaluated for their in vivo activity in a mouse model of lipodystrophy and leptin deficiency (ob / ob mouse, Jax strain number: 000632). The adipocytes differentiated in Example 5 are transplanted subcutaneously into ob / ob mice at a cell dose of 20 million to 140 million cells per mouse. As a control, another group of ob / ob mice is injected with only HBSS without cells in the same manner. Subsequently, body weight, food intake, plasma triglycerides (ThermoFisher, catalog number: TR22421), plasma insulin (Crystal Chem, catalog number: 900080), and non-fasting glucose (glucometer) are measured over time after the administration of cells or HBSS. In addition, a glucose tolerance test (GTT) is also performed on the 38th day after the administration of cells or HBSS. Briefly, the GTT involves fasting the mice for 6 hours, followed by an intraperitoneal injection of dextrose (1 mg / kg body weight), and then measuring the blood glucose level from the tail at 0, 15, 30, 60, 90, and 120 minutes after the glucose injection using a glucometer. The GTT data are analyzed by the area under the curve (AUC) as well described in the literature on GTT (Virtue et al., 2021: PMID AUC: 34117483).

[0223] Example 9: Therapeutic effect of double-layer hydrogel-encapsulated adipocytes or ASCs in a mouse model of maple syrup urine disease (MSUD) This example, among other things, demonstrates the in vivo activity of encapsulated ASCs or adipocytes.

[0224] In this example, the encapsulated cells generated in Example 4 or Example 5 are evaluated for their in vivo activity in a mouse model of maple syrup urine disease (MSUD) (e.g., Jax strain number: 006999). Either the ASCs generated from Example 4 or the adipocytes differentiated in Example 5 are subcutaneously transplanted into MSUD mice at a cell dose of 20 million to 140 million cells per mouse. As a control, another group of MSUD mice is injected with only HBSS without cells in the same manner. Subsequently, plasma BCAA is measured (Sigma, catalog number: MAK003), and the survival rate is tracked over time.

[0225] Without being bound by a particular theory, it is expected that the transplanted cells will reduce plasma BCAA levels and extend survival.

[0226] References Gramignoli, R.; Green, M. L.; Tahan, V.; Dorko, K.; Skvorak, K. J.; Marongiu, F.; Zao, W.; Venkataramanan, R.; Ellis, E. C.; Geller, D.; Breite, A. G.; Dwulet, F. E.; McCarthy, R. C.; Strom, S. C. Development and application of purified tissue dissociation enzyme mixtures for human hepatocyte isolation. Cell Transplant. 21(6):1245 - 1260; 2012. Gramignoli, R.; Dorko, K.; Tahan, V.; Skvorak, K. J.; Ellis, E.; Jorns, C.; Ericzon, B. G.; Fox, I. J.; Strom, S. C. Hypothermic storage of human hepatocytes for transplantation. Cell Transplant. 23(9):1143 - 1151; 2014. Mossanen, J.C.; Tacke, F.; Acetaminophen-induced acute liver injury in mice. Laboratory Animals 49(1):30-36; 2015. Viswanathan, P.; Sharma, Y.; Jaber, F.-L.; Tchaikovskaya, T.; Gupta, S. Transplanted hepatocytes rescue mice in acetaminophen-induced acute liver failure through paracrine signals of hepatic ATM and STAT3 pathways. FASEB J. 35(4):e21471; 2021.

[0227] Equivalents One of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by the following claims.

[0228] As used herein, all headings are for organizational purposes only and are in no way intended to limit the disclosure. The contents of any individual section may be equally applicable to all sections.

[0229] Incorporation by reference All patents and publications referred to herein are hereby incorporated by reference in their entirety.

Claims

1. A bilayer semipermeable fibrosis-resistant hydrogel polymer enclosure, (a) An outer hydrogel layer comprising the enclosure, which optionally contains one or more rejection inhibitors that reduce rejection reactions when implanted in a mammalian subject; (b) An inner hydrogel layer disposed within the outer hydrogel layer, which optionally contains one or more active substances that promote cell viability, cell differentiation, cell health, cell function, and / or the reduction and / or prevention and / or inhibition of apoptosis, and / or the prevention of rejection and / or fibrosis, and further optionally the outer hydrogel layer and the inner hydrogel layer are separated by a liquid layer, such as a culture medium; and (c) An internal aqueous chamber disposed within the internal hydrogel layer, suitable for accommodating at least 10 living mammalian cells, and optionally comprising an internal aqueous chamber whose volume allows for the growth of the at least 10 cells within the chamber, A hydrogel polymer enclosure suitable for administration to human subjects.

2. The hydrogel polymer enclosure according to claim 1, wherein the internal and external hydrogel layers independently comprise alginate, methacrylate, or a combination thereof, and optionally, the alginate or methacrylate comprises one or more zwitterionic groups selected from phosphorbetaine, sulfobetaine, carboxybetaine, cysteine, sulfopyridinium betaine, phosphorylcholine, and sulfobetainesiloxane.

3. The hydrogel polymer enclosure according to claim 1, wherein the internal aqueous chamber contains cells, and optionally the cells are mammalian cells, and optionally the mammalian cells include cells derived from human induced pluripotent stem cells (iPSCs), adipose-derived stem cells (ASCs), or embryonic stem cells (ESCs).

4. (a) The inner hydrogel layer comprises one or more active substances that promote cell viability and / or function, optionally, the one or more active substances that promote cell viability and / or function are polypeptides having a biological function that promotes genetic stability and / or differentiation, optionally, the one or more active substances are selected from one or more of the following: collagen VA1, collagen VA2, collagen VI1, collagen VI2, collagen VI3, fibronectin, laminin, fibrin, fibrinogen alpha, fibrinogen beta, fibrinogen gamma, factor XIII a chain, factor XIII b chain, basement membrane-specific heparan sulfate proteoglycan core protein, and elastin, and / or (b) The outer hydrogel layer comprises one or more rejection inhibitors, optionally the one or more rejection inhibitors being zwitterionic groups, optionally the zwitterionic groups preventing undesirable protein adsorption, optionally the zwitterionic groups being selected from other anti-fouling polymers including sulfobetaine, carboxybetaine, phosphocholine, or a combination thereof, optionally the outer hydrogel comprises a polyethylene glycol (PEG) linker, optionally the outer hydrogel layer reduces, eliminates, or mitigates undesirable foreign body reactions, including macrophage adhesion and lymphocyte activation. The hydrogel polymer enclosure according to claim 1.

5. The hydrogel polymer enclosure according to claim 1, comprising an extracellular matrix (ECM) component, wherein the extracellular matrix (ECM) component is optionally selected from a reconstituted basement membrane, collagen, fibronectin, laminin, fibrin, nestin, perlecan, or a signaling domain (including combinations thereof).

6. (a) substantially spherical, optionally wherein the substantially spherical enclosure has a diameter of about 200 to 1000 μm, 300 to 800 μm, 400 to 600 μm, or 450 to 550 μm, and / or (b) The internal aqueous chamber contains at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, and 140 00, 15000, 16000, 17000, 18000, 19000, 20000 or more mammalian cells having a volume suitable for mammalian cells, which are optionally selected from iPSCs, ASCs, ESCs, or cells differentiated from any of the above, and which optionally have been genetically transformed, wherein the genetic transformation optionally involves the expression of an introduced gene, a growth factor, a ligand, an antibody, a structural protein, or a therapeutic nucleic acid. The hydrogel polymer enclosure according to claim 1.

7. The following: (a) The cells maintain a viability of at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% over a doubling of at least 10, 12, 15, 20, 25, 30 or more times, without maintaining copy number fluctuations during culture. (b) At least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more of the cells are negative for cell death signals over at least 10, 12, 15, 20, 25, 30, or more doublings during culture, or for at least 5, 10, 20, 30 days or more at the time of administration to the subject, and (c) The cells maintain at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% genetic stability during growth over at least 10, 12, 15, 20, 25, 30 or more doublings in culture. A hydrogel polymer enclosure according to claim 6, wherein one or more of the following are applied.

8. A hydrogel polymer enclosure according to claim 1, which, when implanted in a mammalian subject, induces a reduction in one or more levels of: macrophage adhesion, cell-derived innate immune response, or lymphocyte activation, compared to a control of an enclosure that is not resistant to encapsulated cells or fibrosis, and the enclosure does not induce an immune response.

9. (a) The outer hydrogel layer has an average thickness of about 5, 10, 15, 20, 5, 30, 35, 40, 45, 50, 55, or 60 μm. (b) The internal hydrogel layer has an average thickness of approximately 5, 10, 15, 20, 5, 30, 35, 40, 45, 50, 55, or 60 μm. (c) The inner hydrogel layer and / or the outer hydrogel layer have an average pore size of about 0.1, 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μm, and / or (d) The inner hydrogel layer and / or the outer hydrogel layer have an average pore size that allows molecules with a magnitude of 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900 or less than 1000 kDa to pass through, but does not allow molecules with a magnitude greater than 1000 kDa to pass through. The hydrogel polymer enclosure according to claim 1.

10. The hydrogel polymer enclosure according to claim 1, wherein the cell differentiation includes differentiation from stem cells to primary cells.

11. A composition comprising a plurality of hydrogel polymer enclosures as described in claim 1, wherein optionally: (a) The coefficient of variation of the hydrogel polymer enclosure is less than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%. (b) At least about 60, 70, 80, 85, 90, 95% or more of the hydrogel polymer enclosure contains at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 100, 200, 250, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000 or more mammalian cells, and (c) Sterile, pyrogen-free, substantially free of debris, or any or all of the above, A composition to which one or more of the following are applied.

12. A hydrogel polymer enclosure according to any one of claims 1 to 10 or a composition according to claim 11 for use in a method of delivering one or more cells, wherein the method comprises bringing a biological tissue into contact with the hydrogel polymer enclosure or the composition, wherein the hydrogel polymer enclosure or the composition comprises one or more cells, optionally mammalian cells, and optionally the biological tissue is located within a mammalian object.

13. A hydrogel polymer enclosure according to any one of claims 1 to 10 or a composition according to claim 11 for use in a method of treating a disease or disorder, the method comprising providing a therapeutically effective amount of the hydrogel polymer enclosure or the composition to a mammalian target tissue in need thereof, wherein the hydrogel polymer enclosure or composition comprises one or more cells, optionally mammalian cells, optionally: (a) The tissue is liver tissue, and one or more cells are hepatocytes, which optionally include hepatocytes derived from iPSCs, ASCs, or ESCs. (b) The mammalian subjects have or are suspected of having a liver disease, and the liver disease is selected from acute liver failure (ALF), ALF caused by or associated with hereditary metabolic disorders (IMD), chronic liver failure, and acute liver injury (ACLF), and (c) said mammalian subject: (i) Diseases resulting in a deficiency in protein synthesis by the liver or a deficiency in multiple proteins, (ii) A disease that results in insufficient metabolic function of the liver, or (iii) A disease that results in the loss of multiple liver functions. Individuals who have or are suspected of having a disease that causes a liver function deficiency selected from the following: A hydrogel polymer enclosure or composition to which one or more of the following are applied.

14. A method for culturing cells, comprising culturing a hydrogel polymer enclosure according to any one of claims 1 to 10 or a composition according to claim 11 in a bioreactor, wherein the hydrogel polymer enclosure or composition comprises one or more cells, and optionally the cells are (a) Distributed on microcarriers; (b) Pre-culturing cells inside a semipermeable enclosure, optionally comprising, before separating the semipermeable enclosure, harvesting the cells and re-encapsulating them in a hydrogel polymer enclosure according to any one of claims 1 to 10; (c) recovered from cryopreservation; or (d) The above combination, method.

15. The hydrogel polymer enclosure or the composition according to any one of claims 1 to 10, or the composition according to claim 11, wherein the hydrogel polymer enclosure or the composition further optionally comprises one or more of the following: DMSO, glycerol, anti-apoptotic compounds, iron chelating agents, serum albumin, or combinations thereof.

16. A bilayer semipermeable fibrosis-resistant hydrogel polymer enclosure, (a) An outer hydrogel layer comprising the enclosure, which optionally contains one or more rejection inhibitors that reduce rejection reactions when implanted in a mammalian subject; (b) An inner hydrogel layer disposed within the outer hydrogel layer, comprising an alginate and containing one or more active substances that promote cell viability, cell differentiation, cell health, cell function, and / or reduction and / or prevention and / or inhibition of apoptosis, and / or prevention of rejection and / or fibrosis, and further optionally, the outer hydrogel layer and the inner hydrogel layer are separated by a liquid layer, such as a culture medium; and (c) An internal aqueous chamber disposed within the internal hydrogel layer, comprising an internal aqueous chamber containing at least 10 living induced pluripotent stem cells (iPSCs), at least 10 living embryonic hepatocytes (ESCs), or at least 10 living adipose-derived hepatocytes (ASCs), A hydrogel polymer enclosure suitable for administration to human subjects.

17. The volume allows at least 10 cells to grow within the chamber. The one or more active substances are extracellular matrix (ECM) proteins, or contain them, and / or The aforementioned cells can stably differentiate into mesoderm, endoderm, or ectoderm lineages. The hydrogel polymer enclosure according to claim 16.

18. A therapeutically effective amount of a hydrogel polymer enclosure according to any one of claims 1 to 10 and 16 to 17, or a dosage form of the composition according to claim 11.

19. A container, optionally a syringe, and / or The hydrogel polymer enclosure or the composition further comprises a pharmaceutically acceptable carrier, diluent, excipient, or vehicle. The dosage form according to claim 18.

20. A hydrogel polymer enclosure according to any one of claims 1 to 10 and 16 to 17, wherein the hydrogel polymer enclosure is manufactured by microfluidic coaxial injection, or the composition according to claim 11.

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