Hypoimmunogenic cell mixtures and methods of use thereof

Abstract

Featured are cell mixtures that include a first population of cells containing fibroblasts and a second population of cells containing hypoimmunogenic cells. Also featured are aggregates and engineered tissue constructs that contain the cell mixtures and methods of use thereof, e.g., for administration or implantation into a subject.

Description

[0001] HYPOIMMUNOGENIC CELL MIXTURES AND METHODS OF USE THEREOF

[0002] Background of the Invention

[0003] Many diseases result from damage, malfunction, or loss of a single organ, tissue, or cell type. While certain strategies such as organ transplants can be effective, the demand for replacement organs far exceeds availability, resulting in an average of 18 deaths per day in the United States, alone. Tissue therapeutics, including the development of engineered tissue constructs (e.g., cell-based implants), are among the most promising multidisciplinary approaches to fulfill this demand. However, despite significant advances in the fields of cell biology, microfluidics, and engineering, to date, conventional approaches have failed to re-create functional tissues at a scale necessary to impart therapeutic efficacy. Moreover, cells within the tissue construct may stimulate an immune response by the host, thereby rejecting the transplant. Therefore, an unmet need exists for engineered cells and tissues that can engraft and vascularize in vivo and without generating a deleterious immune response by the host in order for the tissue to perform its desired therapeutic function.

[0004] Summary of the Invention

[0005] The present disclosure provides, inter alia, cell mixtures, engineered tissue constructs, kits, and methods of use thereof.

[0006] In one aspect, featured is a cell mixture that includes a first population of cells containing fibroblasts and a second population of cells containing hypoimmunogenic cells.

[0007] In some embodiments, the fibroblasts include primary fibroblasts, induced pluripotent stem cell (iPSC)-derived fibroblasts, or embryonic stem cell (ESC)-derived fibroblasts.

[0008] In some embodiments, the fibroblasts are expanded and / or matured in vitro.

[0009] In some embodiments, the fibroblasts are expanded in vitro.

[0010] In some embodiments, the fibroblasts are matured in vitro.

[0011] In some embodiments, the fibroblasts are expanded and matured in vitro.

[0012] In some embodiments, the primary fibroblasts include normal human dermal fibroblasts (NHDF) or neonatal foreskin fibroblasts.

[0013] In some embodiments, the fibroblasts are not genetically engineered.

[0014] In some embodiments, the fibroblasts are genetically engineered fibroblasts.

[0015] In some embodiments, the genetically engineered fibroblasts include a kill switch.

[0016] In some embodiments, the kill switch includes a suicide gene or an epitope marker.

[0017] In some embodiments, the suicide gene includes herpes simplex virus-thymidine kinase (HSV- TK) or a caspase 9 suicide gene.

[0018] In some embodiments, the caspase 9 suicide gene is an inducible caspase 9 suicide gene.

[0019] In some embodiments, the epitope marker includes a truncated epidermal growth factor receptor (EGFR) variant (huEGFRt) marker or a cluster of differentiation 20 (CD20) epitope marker.

[0020] In some embodiments, the fibroblasts are immunogenic.

[0021] In some embodiments, the fibroblasts are human fibroblasts.

[0022] In some embodiments, the hypoimmunogenic cells include parenchymal cells. In some embodiments, the parenchymal cells include hepatocytes, hepatocyte precursor cells, pancreatic cells, pancreatic precursor cells, myocytes, neurons, enterocytes, adipocytes, splenic cells, kidney cells, biliary cells, Kupffer cells, stellate cells, cardiac muscle cells, alveolar cells, bronchiolar cells, club cells, urothelial cells, mucous cells, parietal cells, chief cells, G cells, goblet cells, enteroendocrine cells, Paneth cells, M cells, tuft cells, glial cells, gall bladder cells, keratinocytes, melanocytes, Merkel cells, Langerhans cells, osteocytes, osteoclasts, esophageal cells, photoreceptor cells, or corneal epithelial cells.

[0023] In some embodiments, the parenchymal cells include hepatocytes or hepatocyte precursor cells.

[0024] In some embodiments, the hepatocytes include primary human hepatocytes, iPSC-derived hepatocytes, or ESC-derived hepatocytes.

[0025] In some embodiments, the hepatocytes include expanded human hepatocytes or expanded and partially reprogrammed human hepatocytes.

[0026] In some embodiments, the hepatocytes are genetically engineered hepatocytes.

[0027] In some embodiment, genetically engineered hepatocytes contain one or more polynucleotides encoding one or more polypeptides for secretion.

[0028] In some embodiments, the parenchymal cells include pancreatic cells or pancreatic precursor cells.

[0029] In some embodiments, the pancreatic cells include pancreatic endocrine cells or pancreatic endocrine cells.

[0030] In some embodiments, the pancreatic cells include alpha, beta, gamma, delta, or epsilon cells, or a combination thereof.

[0031] In some embodiments, the pancreatic cells include primary human pancreatic cells, iPSC-derived pancreatic cells, or ESC-derived pancreatic cells.

[0032] In some embodiments, the pancreatic cells are genetically engineered pancreatic cells.

[0033] In some embodiments, the hypoimmunogenic cells lack expression of one or more of the following endogenous genes: beta-2-microglobulin (B2M), human leukocyte antigen (HLA)-A, HLA-B, HLA-C, class II major histocompatibility complex transactivator (CIITA), PVR cell adhesion molecule (PVR), or any combination thereof.

[0034] In some embodiments, the hypoimmunogenic cells include one or more alterations that inactivate the endogenous B2M, HLA-A, HLA-B, HLA-C, CIITA, or PVR gene.

[0035] In some embodiments, the hypoimmunogenic cells express one or more of the following genes: cluster of differentiation (CD) 47, HLA-C, HLA-E, HLA-G, programmed death-ligand 1 (PD-L1 ), programmed death-ligand 1 (PD-L2), B7-H2, B7-H3, B7-H4, cytotoxic T-lymphocyte associated protein 4 (CTLA4), CD39, CD73, CD24, CD27, CD35, CD46, CD55, CD59, CD155, CD200, indoleamine 2, 3- dioxygenase 1 (IDO1 ), interleukin (IL)-10, IL-35, Fas ligand (FASL), CC motif chemokine ligand 21 (CCL21 ), milk fat globule-EGF factor 8 protein (MFG-E8), serpin B9 (SERPINB9), double homeobox 4 (DUX4), transforming growth factor beta-1 (TGFB1 ), carcinoembryonic antigen (CEA) cell adhesion molecule 1 (CEACAM1 ), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3; also known as HAVCR2), lymphocyte-activation gene 3 (LAG-3), adenosine A2a receptor (A2AR; also known as ADORA2A), B And T Lymphocyte Associated (BTLA), killer Ig-like receptor (KIR), V-domain Ig suppressor of T cell activation (VISTA), or any combination thereof. In some embodiments, expression of the one or more genes is controlled by one or more inducible promoters or constitutive promoters.

[0036] In some embodiments, the hypoimmunogenic cells overexpress one or more of CD47, HLA-C, HLA-E, HLA-G, PD-L1 , PD-L2, B7-H2, B7-H3, B7-H4, CTLA4, CD39, CD73, CD24, CD27, CD35, CD46, CD55, CD59, CD155, CD200, IDO1 , IL-10, IL-35, FASL, CCL21 , MFG-E8, SERPINB9, DUX4, TGFB1 , CEACAM1 , TIM-3, LAG-3, A2AR, BTLA, KIR, VISTA, or any combination thereof relative to a wild-type cell of the same cell type as the hypoimmunogenic cell.

[0037] In some embodiments, a ratio of the first population of cells to the second population of cells is from 10:1 to 1 :10.

[0038] In some embodiments, the first population of cells is present at a density of 1 x 104cells / mL to 1 x 108cells / mL.

[0039] In some embodiments, the first population of cells is present at a density of 1 x 105cells / mL to 3 x 107cells / mL.

[0040] In some embodiments, the density of the fibroblasts is 6 x 105cells / mL.

[0041] In some embodiments, the second population of cells are present at a density of 1 x 104cells / mL to 1 x 108cells / mL.

[0042] In some embodiments, the second population of cells includes hepatocytes, and the density of the hepatocytes is 1 x 105cells / mL to 3 x 107cells / mL.

[0043] In some embodiments, the density of the hepatocytes is 3 x 105cells / mL.

[0044] In some embodiments, the hypoimmunogenic cells are human cells.

[0045] In some embodiments, the cell mixture further includes one or more additional cell populations.

[0046] In some embodiments, the cell mixture is an aggregate.

[0047] In another aspect, featured is an engineered tissue construct suitable for implantation into a subject, that includes the cell mixture of any of the above embodiments in a biocompatible scaffold.

[0048] In some embodiments, the biocompatible scaffold includes fibrin.

[0049] In some embodiments, the fibrin is human fibrin.

[0050] In some embodiments, the engineered tissue construct further includes a reinforcing agent.

[0051] In some embodiments, the reinforcing agent includes fibrin, surgical mesh, alginate, collagen, polyethylene glycol), polyvinylidene acetate (PVDA), polyvinylidene fluoride (PVDF), poly(lactic-co- glycolic) acid (PLGA), and poly (l-lactic acid) (PLLA).

[0052] In some embodiments, the engineered tissue construct further includes a population of endothelial cells.

[0053] In some embodiments, the population of endothelial cells is arranged as one or more cords.

[0054] In another aspect, featured is a kit that includes the cell mixture or engineered tissue construct of any of the above embodiments and instructions to implant the cell mixture or the engineered tissue construct into a subject in need thereof.

[0055] In another aspect, featured is a method for promoting engraftment and / or vascularization of a population of hypoimmunogenic cells in a subject in need thereof. The method includes administering or implanting the cell mixture or engineered tissue construct of any of the above embodiments into the subject in combination with an immunosuppressive regimen; and maintaining the immunosuppressive regimen for a time sufficient for engraftment and / or vascularization of the hypoimmunogenic cells in the subject.

[0056] In another aspect, featured is a method for promoting engraftment and / or vascularization of a population of hypoimmunogenic cells in a subject in need thereof. The method includes providing a subject who has been administered or implanted the cell mixture or engineered tissue construct of any of the above embodiments into the subject in combination with an immunosuppressive regimen; and maintaining the immunosuppressive regimen for a time sufficient for engraftment and / or vascularization of the hypoimmunogenic cells in the subject.

[0057] In another aspect, featured is the cell mixture or engineered tissue construct of any of the above embodiments for use in a method of promoting engraftment and / or vascularization of a population of hypoimmunogenic cells in a subject in need thereof. The use includes administering or implanting the cell mixture or the engineered tissue construct into the subject in combination with an immunosuppressive regimen; and maintaining the immunosuppressive regimen for a time sufficient for engraftment and / or vascularization of the hypoimmunogenic cells in the subject.

[0058] In some embodiments, the immunosuppressive regimen is maintained for 1 day to 6 months (e.g., 1 day to 7 days, e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, e.g., 1 week to 4 weeks, e.g., 1 week, 2 weeks, 3 weeks, or 4 weeks, e.g., 1 month to 6 months, e.g., 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months).

[0059] In some embodiments, the immunosuppressive regimen is maintained for 7 days to 2 months.

[0060] In some embodiments, the immunosuppressive regimen is maintained for 14 days to 1 month (e.g., 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or 31 days).

[0061] In some embodiments, the method further includes withdrawing the immunosuppressive regimen.

[0062] In some embodiments, the fibroblasts are genetically engineered fibroblasts including a kill switch, and the method further includes activating the kill switch to kill the fibroblasts.

[0063] In some embodiments, the immunosuppressive regimen includes administering one or more immunosuppressive agents to the subject.

[0064] In some embodiments, the one or more immunosuppressive agents include a calcineurin inhibitor, a corticosteroid, an antimetabolite, a mammalian target of rapamycin (mTOR) inhibitor, a T cell costimulation inhibitor, an antibody, or any combination thereof.

[0065] In some embodiments, the calcineurin inhibitor is cyclosporine or tacrolimus; the corticosteroid is prednisone, prednisolone, methylprednisolone, cortisone, or hydrocortisone; the antimetabolite is mycophenolic acid, mycophenolate sodium, mycophenolate mofetil, or azathioprine; the mTOR inhibitor is sirolimus or everolimus; the co-stimulation inhibitor is belatacept; and / or the antibody is alemtuzumab, rabbit antithymocyte globulin (rATG), horse antithymocyte globulin (hATG), basiliximab, or rituximab.

[0066] In another aspect, featured is a method for promoting engraftment and / or vascularization of a population of hypoimmunogenic cells in a subject in need thereof. The method includes administering or implanting the cell mixture or engineered tissue construct of any of the above embodiments into the subject; and activating the kill switch to kill the fibroblasts at a time point following engraftment and / or vascularization of the hypoimmunogenic cells in the subject. In another aspect, featured is the cell mixture or engineered tissue construct of any of the above embodiments for use in a method for promoting engraftment and / or vascularization of a population of hypoimmunogenic cells in a subject in need thereof. The use includes administering or implanting the cell mixture or the engineered tissue construct into the subject and activating the kill switch to kill the fibroblasts at a time point following engraftment and / or vascularization of the hypoimmunogenic cells in the subject.

[0067] In some embodiments, the fibroblasts are allogeneic relative to the subject.

[0068] In some embodiments, the subject is a human.

[0069] Definitions

[0070] As used herein, the term “administering,” “administration,” and the like refer to giving a dosage of a composition (e.g., a cell mixture, an engineered tissue construct, or an immunosuppressive agent) to a subject. The compositions utilized in the methods described herein can be administered, for example, parenterally, intramuscularly, intravenously, intradermally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctivally, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, by inhalation, by injection, by infusion, by continuous infusion, by catheter, or in lipid compositions. The administration may be local or systemic. The route of administration can vary depending on various factors (e.g., the compound or composition being administered and the severity of the condition, disease, or disorder being treated).

[0071] As used herein, the terms “implanting,” “implantation,” and the like refer to directly placing one or more cell mixtures or engineered tissue constructs in any effective implantation site, such as a site that is suitable for neovascularization in a subject (e.g., a human subject). Exemplary implantation sites include an extraperitoneal space, an extrapleural space, a surface of the liver (e.g., on the surface of a renal capsule), a muscle site (e.g., a surface of a muscle, within a muscle sheath, or beneath a muscle, including but not limited to the following muscles: a rectus abdominis, an abdominal oblique, a transversus abdominus, a quadriceps femoris, a gluteus maximus, a semimembranosus, a semitendinosus, a biceps femoris, a deltoid, a biceps, or a latissimus dorsi), a pleural space, an omentum site (e.g., an omentum pedicle flap, an omentum free flap, an omental bursa, or the omentum in situ), a subcutaneous site, a surface of the pancreas, a surface of the spleen, a surface of the kidney, a bone marrow site, a bursa site, a peritoneal cavity site (e.g., a mesentery site), and / or a lesser sac site, among others.

[0072] As used herein, the term “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and nonhuman primates such as monkeys), rabbits, deer, and rodents (e.g., mice and rats). In certain embodiments, the subject is a human.

[0073] As used in the context of the present disclosure, an “engineered tissue construct” refers to a mixture of cultured cells (e.g., fibroblasts and hypoimmunogenic cells) and a biocompatible scaffold (e.g., fibrin). Cells can be from established cell lines, or they can be primary cells, where “primary cells,” “primary cell lines,” and “primary cultures” are used interchangeably herein to refer to cells and cell cultures that have been derived from and allowed to grow in vitro for a limited number of passages, e.g., splitting, of the culture. For example, primary cultures can be cultures that have been passaged 0 times, 1 time, 2 times, 4 times, 5 times, 10 times, or 15 times, but not enough times go through the crisis stage. Primary cell lines can be maintained for fewer than 10 passages in vitro. If the cells are primary cells, such cells can be harvested from an individual by any convenient method. For example, cells from tissues such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach, etc. are most conveniently harvested by biopsy. An appropriate solution can be used for dispersion or suspension of the harvested cells. Such solution will generally be a balanced salt solution, e.g., normal saline, phosphate-buffered saline (PBS), Hank's balanced salt solution, etc., conveniently supplemented with fetal calf serum or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration, generally from 5-25 mM. Convenient buffers include HEPES, phosphate buffers, lactate buffers, etc. The cells can be used immediately, or they can be stored, frozen for long periods of time, being thawed, and capable of being reused. In such cases, the cells will usually be frozen in 10% DMSO, 50% serum, 40% buffered medium, or some other such solution as is commonly used in the art to preserve cells at such freezing temperatures and thawed in a manner as commonly known in the art for thawing frozen cultured cells. For example, hepatocytes may be isolated by conventional methods which can be adapted for human liver biopsy or autopsy material (e.g., to garner primary human hepatocytes). Primary cells may be expanded and / or matured in vitro prior to administration or implantation into a subject.

[0074] As used herein, the term “cell type” refers to a group of cells sharing a phenotype that is statistically separable based on gene expression data. For example, cells of a common cell type may share similar structural and / or functional characteristics, such as similar gene activation patterns and antigen presentation profiles. Cells of a common cell type may include those that are isolated from a common tissue (e.g., epithelial tissue, neural tissue, connective tissue, or muscle tissue) and / or those that are isolated from a common organ, tissue system, blood vessel, or other structure and / or region in an organism.

[0075] As used herein, a scaffold (e.g., a hydrogel scaffold) is considered “biocompatible” when is it does not exhibit toxicity when introduced into a subject (e.g., a human). In the context of the present disclosure, it is preferable that the biocompatible scaffold does not exhibit toxicity towards the cells of the engineered tissue construct or when implanted in vivo in a subject (e.g., a human). For example, with respect to hepatocytes, hepatotoxicity can be measured, for example, by determining hepatocytes apoptotic death rate (e.g., wherein an increase in apoptosis is indicative of hepatotoxicity), transaminase levels (e.g., wherein an increase in transaminase levels is indicative of hepatotoxicity), ballooning of the hepatocytes (e.g., wherein an increase in ballooning is indicative of hepatotoxicity), microvesicular steatosis in the hepatocytes (e.g., wherein an increase in steatosis is indicative of hepatotoxicity), biliary cells death rate (e.g., wherein an increase in biliary cells death rate is indicative of hepatotoxicity), y- glutamyl transpeptidase (GGT) levels (e.g., wherein an increase in GGT levels is indicative of hepatotoxicity). A biocompatible scaffold can include, but is not limited to, fibrin and heparin. The biocompatible scaffold may be a biocompatible hydrogel scaffold. As used herein, the term “hydrogel” refers to a network of polymer chains that are hydrophilic in nature, such that the material absorbs a high volume of water or other aqueous solution. Hydrogels can include, for example, at least 70% volume / volume (v / v) water, at least 80% v / v water, at least 90% v / v water, at least 95%, 96%, 97%, 98% and even 99% or greater v / v water (or other aqueous solution). Hydrogels can include natural or synthetic polymers, the polymeric network often featuring a high degree of crosslinking. Hydrogels also possess a degree of flexibility very similar to natural tissue, due to their significant water content. Hydrogels are particularly useful in tissue engineering applications as scaffolds for culturing cells. In certain embodiments, the hydrogels are made of biocompatible polymers.

[0076] The term “adherence material” is a material incorporated into the cell mixture disclosed herein to which a cell or microorganism has some affinity, such as a binding agent. The material can be incorporated, for example, into a hydrogel. The material and a cell or microorganism interact through any means including, for example, electrostatic or hydrophobic interactions, covalent binding, or ionic attachment. The material may include, but is not limited to, antibodies, proteins, peptides, nucleic acids, peptide aptamers, nucleic acid aptamers, sugars, proteoglycans, or cellular receptors.

[0077] As used herein, the term “hypoimmunogenic cell” or “hypoimmune cell” refers to a cell that gives rise to a reduced immunological rejection response when transferred into an allogeneic host, e.g., compared to a wild-type or immunogenic cell of the same cell type. In some embodiments, hypoimmunogenic cells do not give rise to an immune response. Thus, “hypoimmunogenic” may refer to a significantly reduced or eliminated immune response when compared to the immune response of a cell prior to immuno-engineering. In some embodiments, a hypoimmunogenic cell lacks expression of one or more of beta-2-microglobulin (B2M), human leukocyte antigen (HLA)-A, HLA-B, HLA-C, class II major histocompatibility complex transactivator (CIITA), PVR cell adhesion molecule (PVR), or any combination thereof. In other embodiments, a hypoimmunogenic cell has reduced expression of one or more of B2M, HLA-A, HLA-B, HLA-C, CIITA, PVR, or any combination thereof, e.g., relative to a wild-type cell of the same cell type as the hypoimmunogenic cell. In some embodiments, a hypoimmunogenic cell expresses one or more of cluster of differentiation cluster of differentiation (CD) 47, HLA-C, HLA-E, HLA-G, programmed death-ligand 1 (PD-L1 ), programmed death-ligand 1 (PD-L2), B7-H2, B7-H3, B7-H4, cytotoxic T-lymphocyte associated protein 4 (CTLA4), CD39, CD73, CD24, CD27, CD35, CD46, CD55, CD59, CD155, CD200, indoleamine 2, 3-dioxygenase 1 (IDO1 ), interleukin (IL)-10, IL-35, Fas ligand (FASL), CC motif chemokine ligand 21 (CCL21 ), milk fat globule-EGF factor 8 protein (MFG-E8), serpin B9 (SERPINB9), double homeobox 4 (DUX4), TGFB1 , carcinoembryonic antigen (CEA) cell adhesion molecule 1 (CEACAM1 ), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3; also known as HAVCR2), lymphocyte-activation gene 3 (LAG-3), adenosine A2a receptor (A2AR; also known as ADORA2A), B And T Lymphocyte Associated (BTLA), killer Ig-like receptor (KIR), V-domain Ig suppressor of T cell activation (VISTA), or any combination thereof. In some embodiments, a hypoimmunogenic cell overexpresses one or more of CD47, HLA-C, HLA-E, HLA-G, PD-L1 , PD-L2, B7- H2, B7-H3, B7-H4, CTLA4, CD39, CD73, CD24, CD27, CD35, CD46, CD55, CD59, CD155, CD200, IDO1 , IL-10, IL-35, FASL, CCL21 , MFG-E8, SERPINB9, DUX4, TGFB1 , CEACAM1 , TIM-3, LAG-3, A2AR, BTLA, KIR, VISTA, or any combination thereof, e.g., relative to a wild-type cell of the same cell type as the hypoimmunogenic cell. In some embodiments, a hypoimmunogenic cell lacks expression of one or more of B2M, HLA-A, HLA-B, HLA-C, CIITA, PVR, or any combination thereof, and expresses (e.g., overexpresses) one or more of CD47, HLA-C, HLA-E, HLA-G, PD-L1 , PD-L2, B7-H2, B7-H3, B7- H4, CTLA4, CD39, CD73, CD24, CD27, CD35, CD46, CD55, CD59, CD155, CD200, IDO1 , IL-10, IL-35, FASL, CCL21 , MFG-E8, SERPINB9, DUX4, TGFB1 , CEACAM1 , TIM-3, LAG-3, A2AR, BTLA, KIR, VISTA, or any combination thereof.

[0078] As used herein, the term “level” refers to a level of a protein, as compared to a reference. The reference can be any useful reference, as defined herein. By a “decreased level” and an “increased level” of a protein is meant a decrease or increase in protein level, as compared to a reference (e.g., a decrease or an increase by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, 500%, or more; a decrease or an increase of more than 10%, 15%, 20%, 50%, 75%, 100%, or 200%, as compared to a reference; a decrease or an 15 increase by less than 0.01 -fold, 0.02-fold, 0.1 -fold, 0.3-fold, 0.5-fold, 0.8-fold, or less; or an increase by more than 1 .2-fold, 1 .4-fold, 1 .5-fold, 1 .8-fold, 2.0-fold, 3.0-fold, 3.5-fold, 4.5-fold, 5.0- fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 1000-fold, or more). A level of a protein may be expressed in mass / vol (e.g., g / dL, mg / mL, pg / mL, or ng / mL) or percentage relative to total protein in a sample.

[0079] As used herein, the terms “treat” and “treatment” refer to therapeutic treatment, in which the object is to prevent or slow down (lessen) an undesired physiological change, such as the progression of a disease or disorder, e.g., a liver disease (e.g., acute liver failure, a urea cycle disorder, or hyperbilirubinemia (e.g., in a subject having Crigler-Najjar syndrome)) or diabetes (e.g., type 1 diabetes or type 2 diabetes). For example, for a liver disease, beneficial or desired clinical results may include, but are not limited to, the reduction of ammonia, an improvement in a test of gallbladder ejection fraction, or the alleviation of symptoms of acute liver failure, a urea cycle disorder, or hyperbilirubinemia (e.g., in a subject having Crigler-Najjar syndrome). The concentration of ammonia protein or the gallbladder ejection fraction may be determined using assays known in the art, for example, using a hepatobiliary iminodiacetic acid scan.

[0080] The term “acute liver failure” includes, but is not limited to, the conditions referred to by the terms hyperacute liver failure, acute liver failure, subacute liver failure, and fulminant hepatic failure (FHF). As used herein, fulminant hepatic failure” or “FHF” are used interchangeable and are defined as the severe impairment of hepatic functions in the absence of pre-existing liver disease. For example, FHF may result from exposure of a susceptible individual to an agent capable of producing serious hepatic injury. Examples of such agents include infectious agents, excessive alcohol, hepatotoxic metabolites, and hepatotoxic compounds (e.g., drugs). Other causes of FHF include congenital abnormalities, autoimmune disease, and metabolic disease. In many cases the precise etiology of FHF is unknown (e.g., idiopathic).

[0081] As used herein, the term “urea cycle disorder” refers to any disorder that is caused by a defect or malfunction in the urea cycle. The urea cycle is a cycle of biochemical reactions that produces urea from ammonia, a product of protein catabolism. Specific types of urea cycle disorder include, but are not limited to, phosphate synthetase 1 (CPS1 ) deficiency, ornithine transcarbamylase (OTC) deficiency, argininosuccinate synthetase (ASS1 ) deficiency, argininosuccinate lyase (ASL) deficiency, arginase-1 (ARG1 ) deficiency, N-acetylglutamate synthetase (NAGS) deficiency, ornithine translocase (ORNT1 ) deficiency, and citrin deficiency. A urea cycle disorder may be characterized by an aberrant level of ammonia (e.g., an ammonia level of greater than or equal to 80 pmol / L). As used herein, “Crigler-Najjar syndrome” refers to a condition characterized by high levels of bilirubin in the blood (hyperbilirubinemia). Bilirubin is produced when red blood cells are broken down. This substance is removed from the body only after it undergoes a chemical reaction in the liver, which converts the toxic form of bilirubin (called unconjugated bilirubin) to a nontoxic form called conjugated bilirubin. Subjects with Crigler-Najjar syndrome have a buildup of unconjugated bilirubin in their blood (unconjugated hyperbilirubinemia). Crigler-Najjar syndrome is classified into two subtypes, type I and type II. As used herein, “Crigler-Najjar type I” refers to a subtype of Crigler-Najjar syndrome in which mutations in the B-UGT1 gene cause the resulting expressed enzyme, B-UGT, to be completely inactive. Thus, Crigler-Najjar type I patients exhibit a complete absence of B-UGT activity. As used herein, “Crigler-Najjar type II” refers to a subtype of Crigler-Najjar syndrome in which mutations in the B-UGT1 gene cause B- UGT to be partially inactive. Thus, B-UGT activity is reduced in patients with Crigler-Najjar type II and such patients exhibit a strongly reduced bilirubin conjugation capacity compared to healthy subjects.

[0082] As used herein, the term “suitable for neovascularization” refers to conditions and / or environmental characteristics fit for the formation of new blood vessels. Generally, neovascularization means the formation of new blood vessels in injured tissue or in tissue not normally containing blood vessels or the formation of novel blood vessels (e.g., arterioles, venules, and capillaries) of a higher density than usual in said tissue. For example, a site that is suitable for neovascularization may have an existing microvessel density of greater than 3.6 vessels / mm2(e.g., greater than 3.7 vessels / mm2, 3.8 vessels / mm2, 3.9 vessels / mm2, 4 vessels / mm2, 4.1 vessels / mm2, 4.2 vessels / mm2, 4.3 vessels / mm2, 4.4 vessels / mm2, 4.5 vessels / mm2, 5 vessels / mm2, 6 vessels / mm2, 7 vessels / mm2, 8 vessels / mm2, 9 vessels / mm2, 10 vessels / mm2, 50 vessels / mm2, 100 vessels / mm2, 200 vessels / mm2, 300 vessels / mm2, 400 vessels / mm2, 500 vessels / mm2, 1000 vessels / mm2, 2000 vessels / mm2, 3000 vessels / mm2, 4000 vessels / mm2, or 4500 vessels / mm2).

[0083] As used herein, an “extraperitoneal space” refers to a space outside the peritoneal cavity, which is the cavity containing the organs in the abdomen. An extraperitoneal space may be, for example, a preperitoneal space, a retroperitoneal space, or a subperitoneal space. A preperitoneal space is the space between the peritoneum internally and the transversalis fascia externally. Organs in the preperitoneal space include, for example, the liver, spleen, stomach, superior part of the duodenum, jejunum, ileum, transverse colon, sigmoid colon and superior part of the rectum. A retroperitoneal space is the area in the back of the abdomen behind the peritoneum. Organs in the retroperitoneal space include, for example, kidneys, adrenal glands, pancreas, nerve roots, lymph nodes, abdominal aorta, inferior vena cava, and parts of the duodenum and colon. A subperitoneal space is a continuous interconnecting space beneath the peritoneum containing the extraperitoneal space, the ligaments and mesenteries, and their suspended organs. Organs in the subperitoneal space include, for example, the bladder, the cervix of the uterus, and the last part of the rectum. An engineered tissue construct that is implanted in an extraperitoneal space (e.g., preperitoneal space, a retroperitoneal space, or a subperitoneal space) is implanted on a surface within the extraperitoneal space.

[0084] As used herein, an “extrapleural space” refers to a region between the inner surface of the ribs and intercostal muscles on one side and the parietal pleura on the other. An engineered tissue construct that is implanted in an extrapleural space is implanted on a surface within the extrapleural space. As used herein, a “pleural space” or “pleural cavity” is a cavity that exists between the parietal and visceral pleura. An engineered tissue construct that is implanted in a pleural space is implanted on a surface within the pleural space.

[0085] As used herein, an “omentum” is a large flat adipose tissue layer nestling on the surface of the intra-peritoneal organs. The greater omentum is a large apron-like fold of visceral peritoneum that hangs down from the stomach. The lesser omentum is the double layer of peritoneum that extends from the liver too the lesser curvature of the stomach, and to the first part of the duodenum.

[0086] As used herein, a “lesser sac” or “omental bursa” refers to the cavity in the abdomen that is formed by the lesser and greater omentum.

[0087] As used herein, a “kill switch” refers to a control mechanism that can be used to deplete or kill a cell (e.g., a fibroblast). Exemplary kill switches include, without limitation, suicide genes (e.g., herpes simplex virus-thymidine kinase (HSV-TK) or a caspase 9 suicide gene (e.g., an inducible caspase 9 suicide gene; see, e.g., Straathof et al. Blood 105(11 ):4247-4254, 2005 and Di Stasi et al. N. Engl. J. Med. 365(18):1673-1683, 2011 )) and epitope markers (e.g., a truncated epidermal growth factor receptor (EGFR) variant (huEGFRt) marker (see, e.g., Wang et al. Blood 118(5):1255-1263, 2011 ), a cluster of differentiation 20 (CD20) epitope marker, a CD34 epitope marker, or a multi-epitope marker (e.g., RQR8; see Mosti et al. Gene Therapy 28:602-612, 2021 )). A kill switch may be activated, e.g., by administering a factor to a subject that activates a suicide gene or by administering an antibody or other binding agent that binds to an epitope marker. For example, an inducible caspase 9 suicide gene may include a nucleic acid encoding the intracellular portion of the human caspase 9 protein, fused to a drug-binding domain derived from the human FK506-binding protein. Cells (e.g., fibroblasts) can be genetically engineered to express the suicide gene. Administration to the subject of an otherwise insert small molecule (e.g., AP1903 (rimiducid)) induces dimerization of the inducible caspase 9 protein, leading to activation of the apoptosome and subsequent cell death. Thus, in the example of inducible caspase 9, a kill switch may be activated by administering AP1903 to the subject. In another example, for an epitope marker, activation of the kill switch may comprise administering to the subject an antibody that binds to the epitope marker. For example, with respect to huEGFRt, activation of the kill switch may include administration of cetuximab to the subject, resulting in killing (e.g., via antibody-dependent cellular cytotoxicity (ADCC) or complementdependent cytotoxicity (CDC)) of the cell expressing the epitope marker.

[0088] Detailed Description

[0089] The present invention features cell mixtures containing a population of fibroblasts and a population of hypoimmunogenic cells. Also featured are aggregates and engineered tissue constructs containing the cell mixtures and methods of use thereof. Typically, a cell or tissue implant (e.g., containing allogeneic cells) may elicit an immune response upon implantation, thereby causing rejection of the cells or tissue implant. The present invention solves this problem by including a population of hypoimmunogenic cells that reduces or eliminates an immune response by a host subject, thereby reducing the risk of or eliminating tissue or graft rejection. The mixture also contains a population of fibroblasts, which are advantageous for promoting engraftment and vascularization of a tissue construct into a subject, while the hypoimmunogenic cells perform a therapeutic function without stimulating an immune response by the host subject. Upon engraftment, the fibroblasts may be subsequently removed, if desired, either by allowing an immune response or providing an external stimulus (e.g., activating a kill switch), or both, to kill the fibroblasts. The cell mixtures and engineered tissue constructs described herein can be used for methods of treating a disease or disorder, such as a liver disease or diabetes.

[0090] The sections that follow provide a description of the cell mixtures, engineered tissue constructs, and methods of making and using the same.

[0091] Hypoimmunogenic Cells

[0092] Hypoimmunogenic cells are cells the give rise to a reduced or eliminated immunological rejection response when transferred into an allogeneic host. In some embodiments, hypoimmunogenic cells do not give rise to any immune response. Thus, the hypoimmunogenic cells elicit a significantly reduced or abrogated immune response when compared to the immune response of the same cell prior to immunoengineering.

[0093] Any suitable hypoimmunogenic cells may be used in the cell mixtures, engineered tissue constructs, and methods disclosed herein. In some embodiments, a hypoimmunogenic cell lacks expression of one or more of beta-2-microglobulin (B2M), human leukocyte antigen (HLA)-A, HLA-B, HLA-C, class II major histocompatibility complex transactivator (CIITA), PVR cell adhesion molecule (PVR), or any combination thereof. In some embodiments, a hypoimmunogenic cell expresses (e.g., overexpresses) one or more of cluster of differentiation (CD) 47, HLA-C, HLA-E, HLA-G, programmed death-ligand 1 (PD-L1 ), programmed death-ligand 1 (PD-L2), B7-H2, B7-H3, B7-H4, cytotoxic T- lymphocyte associated protein 4 (CTLA4), CD39, CD73, CD24, CD27, CD35, CD46, CD55, CD59, CD155, CD200, indoleamine 2, 3-dioxygenase 1 (IDO1 ), interleukin (IL)-10, IL-35, Fas ligand (FASL), CC motif chemokine ligand 21 (CCL21 ), milk fat globule-EGF factor 8 protein (MFG-E8), serpin B9 (SERPINB9), double homeobox 4 (DUX4), TGFB1 , carcinoembryonic antigen (CEA) cell adhesion molecule 1 (CEACAM1 ), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3; also known as HAVCR2), lymphocyte-activation gene 3 (LAG-3), adenosine A2a receptor (A2AR; also known as ADORA2A), B And T Lymphocyte Associated (BTLA), killer Ig-like receptor (KIR), V-domain Ig suppressor of T cell activation (VISTA), or any combination thereof. Any of the cell types described herein may be genetically modified to produce hypoimmunogenic cells. For example, hepatocytes may be engineered to be hypoimmunogenic hepatocytes. Similarly, pancreatic cells (e.g., beta cells) may be engineered to be hypoimmunogenic pancreatic cells (e.g., beta cells).

[0094] In some embodiments, any of the hypoimmunogenic cells disclosed herein lacks expression of B2M. In some embodiments, any of the hypoimmunogenic cells disclosed herein lacks expression of HLA-A. In some embodiments, any of the hypoimmunogenic cells disclosed herein lacks expression of HLA-B. In some embodiments, any of the hypoimmunogenic cells disclosed herein lacks expression of HLA-C. In some embodiments, any of the hypoimmunogenic cells disclosed herein lacks expression of CIITA. In some embodiments, any of the hypoimmunogenic cells disclosed herein lacks expression of PVR. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) CD47. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) HLA-E. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) HLA-G. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) PD-L1 . In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) CTLA4. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses CD39. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) CD73. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) CD24. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) CD27. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) CD35. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) CD46. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) CD55. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) CD59. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) CD200. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) HLA-C. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) IDO1 . In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) IL-10. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) IL-35. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) FASL. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) CCL21 . In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) MFG-E8. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) SERPINB9. In some embodiments, any of the hypoimmunogenic cells disclosed herein expresses (e.g., overexpresses) DUX4. In some embodiments, the hypoimmunogenic cells express a CTLA4-immunoglobulin fusion protein (CTLA4-lg). See, e.g., Rong et al. Cell Stem Cell. 14:121 -130, 2014.

[0095] In some embodiments, the hypoimmunogenic cells may have reduced expression or lack expression of one or more HLA class I genes (e.g., via knockout of B2M or knockout of HLA-A, HLA-B, and / or HLA-C, e.g., knockout of HLA-A and HLA-B). In some embodiments, hypoimmunogenic cells lacking one or more HLA class I genes (e.g., via knockout of B2M or knockout of HLA-A, HLA-B, and / or HLA-C, e.g., knockout of HLA-A and HLA-B) also may express (e.g., overexpress) HLA-E (e.g., a singlechain HLA-E). See, e.g., Gornalusse et al. Nat. Biotechnol. 35:765-772, 2017.

[0096] In some embodiments, the hypoimmunogenic cells may have reduced expression or lack expression of one or more HLA class II genes (e.g., via knockout of CIITA, RFXANK, RFX5, or RFXAP). In some embodiments, the hypoimmunogenic cells may have reduced expression or lack expression of one or more HLA class II genes via knockout of CIITA.

[0097] In some embodiments, the hypoimmunogenic cells may have reduced expression or lack expression of both HLA class I genes and HLA class II genes (e.g., via knockout of both B2M and CIITA, or knockout of HLA-A, HLA-B, and / or HLA-C and CIITA).

[0098] In some embodiments, the hypoimmunogenic cells may have reduced expression or lack expression of both HLA class I genes and HLA class II genes (e.g., via knockout of both B2M and CIITA, or knockout of HLA-A, HLA-B, and / or HLA-C and CIITA) and express (e.g., overexpress) CD47 (see, e.g., Deuse et al. Nat. Biotechnol. 37:252-258, 2019). In some embodiments, the hypoimmunogenic cells may have reduced expression or lack expression of both HLA class I genes and HLA class II genes (e.g., via knockout of both B2M and CIITA) and express (e.g., overexpress) CD47, HLA-G, and PD-L1 (see, e.g., Han et al. Proc. Natl. Acad. Sci USA 116:10441 -10446, 2019).

[0099] In some embodiments, the hypoimmunogenic cells express a mutant B2M-HLA-E (mBE) and B2M-HLA-G (mBG) fusion proteins, e.g., to protect against allogeneic NK cell-mediated lysis (see, e.g., Guo, et al. European Journal of Immunology. 51 : 2513-2521 , 2021 ).

[0100] Additional hypoimmunogenic cells that can be used in the cell mixtures, engineered tissue constructs, and methods disclosed herein include, without limitation, any hypoimmunogenic cell described in Zhao et al. / Sc / ence 23(6):101162, 2020, WO 2018 / 132783, WO 2021 / 041316, WO 2021 / 022223, or WO 2019 / 014351.

[0101] Hypoimmunogenic cells can be generated by any suitable technique known in the art to the skilled artisan. For example, cells (e.g., iPSCs or hepatocytes) can be gene edited (e.g., using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene editing technology or other gene editing technologies known in the art such as zinc-finger nucleases and transcription activator-like effector nucleases (TALENs)) to reduce expression of, knock out, express, or overexpress one or more of the genes described above. For example, one or both alleles of an endogenous gene may be knocked out to reduce or eliminate expression of the gene (e.g., B2M, HLA-A, HLA-B, HLA-C, CIITA, PVR, or any combination thereof). In another example, a cell may be engineered to express or overexpress one or more of CD47, HLA-C, HLA-E, HLA-G, PD-L1 , PD-L2, B7-H2, B7-H3, B7-H4, CTLA4, CD39, CD73, CD24, CD27, CD35, CD46, CD55, CD59, CD155, CD200, IDO1 , IL-10, IL-35, FASL, CCL21 , MFG-E8, SERPINB9, DUX4, TGFB1 , CEACAM1 , TIM-3, LAG-3, A2AR, BTLA, KIR, VISTA, or any combination thereof, via transduction with a vector (e.g., a viral vector (e.g., a retroviral vector (e.g., a lentiviral vector) or an adeno-associated viral (AAV) vector)).

[0102] The expression of or more of any of the genes disclosed herein, e.g., CD47, HLA-C, HLA-E, HLA-G, PD-L1 , PD-L2, B7-H2, B7-H3, B7-H4, CTLA4, CD39, CD73, CD24, CD27, CD35, CD46, CD55, CD59, CD155, CD200, IDO1 , IL-10, IL-35, FASL, CCL21 , MFG-E8, SERPINB9, DUX4, TGFB1 , CEACAM1 , TIM-3, LAG-3, A2AR, BTLA, KIR, VISTA, or any combination thereof, may be controlled by any suitable control element(s), e.g., promoters or enhancers, including constitutively active promoters (e.g., the human p-actin promoter (ACTB), cytomegalovirus (CMV), elongation factor-1 a, (EF1a), phosphoglycerate kinase (PGK) and ubiquitinC (UbC)) and / or inducible promoters (e.g., a tetracycline- controlled promoter or a doxycycline-inducible promoter). Other constitutively active promoters and inducible promoters are known in the art.

[0103] Cell Populations

[0104] Cell populations may be optimized to maintain the appropriate morphology, phenotype, and cellular function conducive to use in the methods of the disclosure. Cell populations of the cell mixtures and engineered tissue constructs described herein include a population of hypoimmunogenic cells and a population of fibroblasts. The population may include primary cells, engineered cells, cell aggregates, induced pluripotent stem cell (iPSC)-derived cells, embryonic stem cell-derived cells, transdifferentiated cells, expanded cells, expanded and partially reprogrammed cells, or a tissue or portion thereof. The cells may be primary cells, e.g., primary cells that are expanded and / or matured in vitro. In some embodiments, the population of cells includes endocrine, exocrine, paracrine, heterocrine, autocrine, or juxtacrine cells.

[0105] In some embodiments, the population of cells includes Leydig cells, adrenal cortical cells, pituitary cells, thyrocytes, granulosa cells, mammary gland epithelial cells, thymocytes, thymic epithelial cells, hypothalamus cells, skeletal muscle cells, smooth muscle cells, enteroendocrine cells (e.g., L cells and / or chromaffin cells), ovarian cells, parathyroid cells, thyroid cells, and / or neuronal cells.

[0106] In some embodiments, the pituitary cells comprise thyrotropic pituitary cells, lactotropic pituitary cells, corticotropic pituitary cells, somatotropic pituitary cells, and / or gonadotropic pituitary cells.

[0107] In some embodiments, the neuronal cells comprise dopaminergic cells.

[0108] In some embodiments, the hypoimmunogenic cells include parenchymal cells.

[0109] In some embodiments, the parenchymal cells include hepatocytes, hepatocyte precursor cells, pancreatic cells, pancreatic precursor cells, myocytes, neurons, enterocytes, adipocytes, splenic cells, kidney cells, biliary cells, Kupffer cells, stellate cells, cardiac muscle cells, alveolar cells, bronchiolar cells, club cells, urothelial cells, mucous cells, parietal cells, chief cells, G cells, goblet cells, enteroendocrine cells, Paneth cells, M cells, tuft cells, glial cells, gall bladder cells, keratinocytes, melanocytes, Merkel cells, Langerhans cells, osteocytes, osteoclasts, esophageal cells, photoreceptor cells, or corneal epithelial cells.

[0110] In some embodiments, the parenchymal cells include hepatocytes or hepatocyte precursor cells.

[0111] In some embodiments, the hepatocytes include primary human hepatocytes, iPSC-derived hepatocytes, or ESC-derived hepatocytes.

[0112] In some embodiments, the hepatocytes include expanded human hepatocytes or expanded and partially reprogrammed human hepatocytes.

[0113] In some embodiments, the hepatocytes are genetically engineered hepatocytes.

[0114] In some embodiments, genetically engineered hepatocytes contain one or more polynucleotides encoding one or more polypeptides for secretion.

[0115] In some embodiments, the parenchymal cells include pancreatic cells or pancreatic precursor cells.

[0116] In some embodiments, the pancreatic cells include pancreatic endocrine cells or pancreatic endocrine cells.

[0117] In some embodiments, the pancreatic cells include alpha, beta, gamma, delta, or epsilon cells, or a combination thereof.

[0118] In some embodiments, the pancreatic cells include primary human pancreatic cells, iPSC-derived pancreatic cells, or ESC-derived pancreatic cells.

[0119] In some embodiments, the pancreatic cells are genetically engineered pancreatic cells.

[0120] In some embodiments, the population of cells are primary cells. In some embodiments the first population of cells are induced pluripotent (iPSC)-derived cells or embryonic stem cells (ESC)-derived cells.

[0121] In some embodiments, the cells are engineered cells, primary cells, or transdifferentiated cells.

[0122] In some embodiments, the cell mixture or engineered tissue construct includes two or more populations of cells (e.g., two, three, four, five, six, seven, eight, nine, ten, or more populations of cells). In some embodiments, the population of cells includes a population of hepatocytes (e.g., hypoimmune hepatocytes) and a population of fibroblasts.

[0123] In some embodiments, the engineered cells are engineered to express or secrete a protein, such as an antibody, a cytokine, an enzyme, a coagulation factor, or a hormone. The protein may be, for example, an endogenous human protein or an engineered protein.

[0124] Hepatocytes

[0125] The cell mixtures and engineered tissue constructs described herein may include hepatocytes (e.g., hypoimmunogenic hepatocytes). In some embodiments, the hepatocytes are primary human hepatocytes (PHH). In some embodiments, the hepatocytes are derived from stem cells (e.g., induced pluripotent stem cells). In some embodiments, the hepatocytes are expanded human hepatocytes or expanded and partially reprogrammed human hepatocytes. In some embodiments, the hepatocytes are genetically engineered hepatocytes. The genetically engineered hepatocytes may contain one or more polynucleotides encoding one or more polypeptides for secretion.

[0126] In some embodiments, the density of hepatocytes is 0.1 M / mL to 150 M / mL (e.g., 0.2 M / mL to 149 M / mL, 0.3 M / mL to 148 M / mL, 0.4 M / mL to 147 M / mL, 0.5 M / mL to 146 M / mL, 1 M / mL to 145 M / mL, 5 M / mL to 140 M / mL, 10 M / mL to 100 M / mL, 20 M / mL to 50 M / mL, or 30 M / mL to 40 M / mL). For example, in some embodiments, the density of hepatocytes is 0.2 M / mL to 149 M / mL. In some embodiments, the density of hepatocytes is 0.3 M / mL to 148 M / mL. In some embodiments, the density of hepatocytes is 0.4 M / mL to 147 M / mL. In some embodiments, the density of hepatocytes is 0.5 M / mL to 146 M / mL. In some embodiments, the density of hepatocytes is 1 M / mL to 145 M / mL. In some embodiments, the density of hepatocytes is 5 M / mL to 140 M / mL. In some embodiments, the density of hepatocytes is 10 M / mL to 100 M / mL. In some embodiments, the density of hepatocytes is 20 M / mL to 50 M / mL. In some embodiments, the density of hepatocytes is 30 M / mL to 40 M / mL.

[0127] In some embodiments, the engineered tissue construct includes a population of hepatocytes in an amount of from 3 x 105to 1 .8 x 1011(e.g., from 4 x 105to 1 .8 x 1011, from 5 x 105to 1 .8 x 1011, from 6 x

[0128] 105to 1 .8 x 1011, from 7 x 105to 1 .8 x 1011, from 8 x 105to 1 .8 x 1011, from 9 x 105to 1 .8 x 1011, from 1 x

[0129] 106to 1 .8 x 1011, from 2 x 106to 1 .8 x 1011, from 3 x 106to 1 .8 x 1011, from 4 x 106to 1 .8 x 1011, from 5 x

[0130] 106to 1 .8 x 1011, from 6 x 106to 1 .8 x 1011, from 7 x 106to 1 .8 x 1011, from 8 x 106to 1 .8 x 1011, from 9 x

[0131] 106to 1 .8 x 1011, from 1 x 107to 1 .8 x 1011, from 2 x 107to 1 .8 x 1011, from 1 .8 x 107to 1 .8 x 1011, from 4 x 107to 1 .8 x 1011, from 5 x 107to 1 .8 x 1011, from 6 x 107to 1 .8 x 1011, from 7 x 107to 1 .8 x 1011, from 8 x 107to 1 .8 x 1011, from 9 x 107to 1 .8 x 1011, from 1 x 108to 1 .8 x 1011, from 2 x 108to 1 .8 x 1011, from 3 x 108to 1 .8 x 1011, from 4 x 108to 1 .8 x 1011, from 5 x 108to 1 .8 x 1011, from 6 x 108to 1 .8 x 1011, from 7 x 108to 1 .8 x 1011, from 8 x 108to 1 .8 x 1011, from 9 x 108to 1 .8 x 1011, from 1 x 109to 1 .8 x 1011, from 2 x 109to 1 .8 x 1011, from 3 x 109to 1 .8 x 1011, from 4 x 109to 1 .8 x 1011, from 5 x 109to 1 .8 x 1011, from 6 x 109to 1 .8 x 1011, from 7 x 109to 1 .8 x 1011, from 8 x 109to 1 .8 x 1011, from 9 x 109to 1 .8 x 1011, from 1 x 1010to 1 .8 x 1011, from 2 x 1010to 1 .8 x 1011, from 3 x 1010to 1 .8 x 1011, from 4 x 1010to 1 .8 x 1011, from 5 x 1010to 1 .8 x 1011, from 6 x 1010to 1 .8 x 1011, from 7 x 1010to 1 .8 x 1011, from 8 x 1010to 1 .8 x 1011, from 9 x 1010to 1 .8 x 1011, or from 1 x 1011to 1 .8 x 1011) hepatocytes. Fibroblasts

[0132] The cell mixtures and engineered tissue constructs described herein include fibroblasts. Any suitable fibroblasts may be used. In some embodiments, the fibroblasts are human fibroblasts (e.g., dermal fibroblasts, normal human dermal fibroblasts, neonatal foreskin fibroblasts, human lung fibroblasts, human ventricular cardiac fibroblasts, human atrial cardiac fibroblasts, human uterine fibroblasts, human bladder fibroblasts, human gingival fibroblasts, human pericardial fibroblasts, human gall bladder fibroblasts, human portal vein fibroblasts, or human vas deferens fibroblasts). In some embodiments, the fibroblasts are human dermal fibroblasts. In some embodiments, the fibroblasts are normal human dermal fibroblasts. In some embodiments, the fibroblasts are neonatal foreskin fibroblasts. In some embodiments, the fibroblasts human lung fibroblasts. In some embodiments, the fibroblasts are human ventricular cardiac fibroblasts. In some embodiments, the fibroblasts are human atrial cardiac fibroblasts. In some embodiments, the fibroblasts are human uterine fibroblasts. In some embodiments, the fibroblasts are human bladder fibroblasts. In some embodiments, the fibroblasts are human gingival fibroblasts. In some embodiments, the fibroblasts are human pericardial fibroblasts. In some embodiments, the fibroblasts are human gall bladder fibroblasts. In some embodiments, the fibroblasts are human portal vein fibroblasts. In some embodiments, the fibroblasts are vas deferens fibroblasts.

[0133] In some embodiments, the fibroblasts are not genetically engineered.

[0134] In some embodiments, the fibroblasts are genetically engineered fibroblasts.

[0135] In some embodiments, the genetically engineered fibroblasts include a kill switch.

[0136] In some embodiments, the kill switch includes a suicide gene or an epitope marker.

[0137] In some embodiments, the suicide gene includes herpes simplex virus-thymidine kinase (HSV- TK) or a caspase 9 suicide gene.

[0138] In some embodiments, the suicide gene is an inducible caspase 9 suicide gene. Cell-permeable synthetic ligands (e.g., rapamycin, FK506, AP1903, AP20187) that bind to FK506 binding protein 12 (FKBP12) may be used to activate the suicide gene. FKBP12 belongs to the immunophilin family of receptors, a physiological function of which is to bind to and inactivate calcineurin. Calcineurin inhibition leads to impaired T-cell receptor signaling and consequent immunosuppression. The FKBP12 may be an engineered variant, such as FKBP12-F36V. Th FKBP ligand has high affinity and selectivity for FKBP12- F36V and interacts minimally with endogenous FKBP. Administration of the ligand may chemically induce dimerization and induce apoptosis of the cells expressing the suicide gene (see, e.g., Zhou, et al. Gene Therapy of Solid Cancers: Methods and Protocols, 87-105, 2015).

[0139] In some embodiments, the epitope marker includes a truncated epidermal growth factor receptor (EGFR) variant (huEGFRt) marker or a cluster of differentiation 20 (CD20) epitope marker.

[0140] In some embodiments, the fibroblasts are immunogenic.

[0141] In some embodiments, the fibroblasts are allogeneic relative to the subject.

[0142] In some embodiments, the population of fibroblasts includes from 6 x 103to 1 .8 x 1012e.g., from 1 to 1 .8 x 1012, from 10 to 1 .8 x 1012, from 100 to 1 .8 x 1012, from 1 x 103to 1 .8 x 1012, from 2 x 103to 1 .8 x 1012, from 3 x 103to 1 .8 x 1012, from 4 x 103to 1 .8 x 1012, from 5 x 103to 1 .8 x 1012, from 6 x 103to 1 .8 x 1012, from 7 x 103to 1 .8 x 1012, from 8 x 103to 1 .8 x 1012, from 9 x 103to 1 .8 x 1012, from 1 x 104to 1 .8 x 1012, from 2 x 104to 1 .8 x 1012, from 3 x 104to 1 .8 x 1012, from 4 x 104to 1 .8 x 1012, from 5 x 104to 1 .8 x 1012, from 6 x 104to 1 .8 x 1012, from 7 x 104to 1 .8 x 1012, from 8 x 104to 1 .8 x 1012, from 9 x 104to 1 .8 x 1012, from 1 x 105to 1 .8 x 1012, from 2 x 105to 1 .8 x 1012, from 3 x 105to 1 .8 x 1012, from 4 x 105to 1 .8 x 1012, from 5 x 105to 1 .8 x 1012, from 6 x 105to 1 .8 x 1012, from 7 x 105to 1 .8 x 1012, from 8 x 105to 1 .8 x 1012, from 9 x 105to 1 .8 x 1012, from 1 x 106to 1 .8 x 1012, from 2 x 106to 1 .8 x 1012, 3 x 106to 1 .8 x

[0143] 1012, 4 x 106to 1 .8 x 1012, 5 x 106to 1 .8 x 1012, 6 x 106to 1 .8 x 1012, 7 x 106to 1 .8 x 1012, 8 x 106to 1 .8 x

[0144] 1012, 9 x 106to 1 .8 x 1012, from 1 x 107to 1 .8 x 1012, from 2 x 107to 1 .8 x 1012, from 18 x 107to 1 .8 x 1012, from 4 x 107to 1 .8 x 1012, from 5 x 107to 1 .8 x 1012, from 6 x 107to 1 .8 x 1012, from 7 x 107to 1 .8 x 1012, from 8 x 107to 1 .8 x 1012, from 9 x 107to 1 .8 x 1012, from 1 x 108to 1 .8 x 1012, from 2 x 108to 1 .8 x 1012, from 3 x 108to 1 .8 x 1012, from 4 x 108to 1 .8 x 1012, from 5 x 108to 1 .8 x 1012, from 6 x 108to 1 .8 x 1012, from 7 x 108to 1 .8 x 1012, from 8 x 108to 1 .8 x 1012, from 9 x 108to 1 .8 x 1012, from 1 x 109to 1 .8 x 1012, from 2 x 109to 1 .8 x 1012, from 3 x 109to 1 .8 x 1012, from 4 x 109to 1 .8 x 1012, from 5 x 109to 1 .8 x 1012, from 6 x 109to 1 .8 x 1012, from 7 x 109to 1 .8 x 1012, from 8 x 109to 1 .8 x 1012, from 9 x 109to 1 .8 x 1012, from 1 x 1010to 1 .8 x 1012, from 2 x 1010to 1 .8 x 1012, from 3 x 1010to 1 .8 x 1012, from 4 x 1010to 1 .8 x 1012, from 5 x 1010to 1 .8 x 1012, from 6 x 1010to 1 .8 x 1012, from 7 x 1010to 1 .8 x 1012, from 8 x 1010to 1 .8 x 1012, from 9 x 1010to 1 .8 x 1012, from 1 x 1011to 1 .8 x 1012, from 2 x 1011to 1 .8 x 1012, from 3 x 1011to

[0145] 1 .8 x 1012, from 4 x 1011to 1 .8 x 1012, from 5 x 1011to 1 .8 x 1012, from 6 x 1011to 1 .8 x 1012, from 7 x 1011to 1 .8 x 1012, from 8 x 1011to 1 .8 x 1012, from 9 x 1011to 1 .8 x 1012, or from 1 x 1012to 1 .8 x 1012) fibroblasts.

[0146] In some embodiments, the fibroblasts are expanded in vitro. In some embodiments, the fibroblasts are matured in vitro. In some embodiments, the fibroblasts are expanded and matured in vitro.

[0147] Aggregated Hypoimmunogenic Cells and Fibroblasts

[0148] The cellular compositions disclosed herein can be provided as a suspension in a biocompatible scaffold containing hypoimmunogenic cells and fibroblasts. In some embodiments, the population of hypoimmunogenic cells and the population of fibroblasts are aggregated in spheroids. For example, in some embodiments, the population of hypoimmunogenic cells and the population of fibroblasts are aggregated in spheroids and the spheroids are distributed non-homogenously, in a layer, along the z-axis of the biocompatible scaffold. In some embodiments, the spheroids are distributed homogenously along the x-axis of the biocompatible scaffold. In some embodiments, the spheroids are distributed homogenously along the y-axis of the biocompatible scaffold.

[0149] In some embodiments, the cell mixtures or aggregates are in a biocompatible scaffold, and the population of hypoimmunogenic cells and the population of fibroblasts together account for at least 70% (e.g., at least 71 %, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 95%, or 100%) of the total cells in the engineered tissue construct. For example, in some embodiments, the population of hypoimmunogenic cells and the population of fibroblasts together account for at least 71 % of the total cells in the engineered tissue construct. In some embodiments, the population of hypoimmunogenic cells and the population of fibroblasts together account for at least 72% of the total cells in the engineered tissue construct. In some embodiments, the population of hypoimmunogenic cells and the population of fibroblasts together account for at least 73% of the total cells in the engineered tissue construct. In some embodiments, the population of hypoimmunogenic cells and the population of fibroblasts together account for at least 74% of the total cells in the engineered tissue construct. In some embodiments, the population of hypoimmunogenic cells and the population of fibroblasts together account for at least 75% of the total cells in the engineered tissue construct. In some embodiments, the population of hypoimmunogenic cells and the population of fibroblasts together account for at least 80% of the total cells in the engineered tissue construct. In some embodiments, the population of hypoimmunogenic cells and the population of fibroblasts together account for at least 85% of the total cells in the engineered tissue construct. In some embodiments, the population of hypoimmunogenic cells and the population of fibroblasts together account for at least 90% of the total cells in the engineered tissue construct. In some embodiments, the population of hypoimmunogenic cells and the population of fibroblasts together account for at least 95% of the total cells in the engineered tissue construct. In some embodiments, the population of hypoimmunogenic cells and the population of fibroblasts together account for 100% of the total cells in the engineered tissue construct.

[0150] In some embodiments, the cell mixtures or aggregates are distributed non-homogenously along the z-axis of the biocompatible scaffold. In some embodiments, the cell mixtures or aggregates are distributed homogenously along the x-axis of the biocompatible scaffold. In some embodiments, the cell mixtures or aggregates are distributed homogenously along the y-axis of the biocompatible scaffold.

[0151] In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :10 to 10:1 . In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :10 to 4:1 (e.g., 1 :10 to 4:1 , 1 :10 to 3:1 , 1 :10 to 2:1 , 1 :10 to 1 :1 , 1 :9 to 4:1 , 1 :9 to 3:1 , 1 :9 to 2:1 , 1 :9 to 1 :1 , 1 :8 to 4:1 , 1 :8 to 3:1 , 1 :8 to 2:1 , 1 :8 to 1 :1 , 1 :7 to 4:1 , 1 :7 to 3:1 , 1 :7 to 2:1 , 1 :7 to 1 :1 , 1 :6 to 4:1 , 1 :6 to 3:1 , 1 :6 to 2:1 , 1 :6 to 1 :1 , 1 :5 to 4:1 , 1 :5 to 3:1 , 1 :5 to 2:1 , 1 :5 to 1 :1 , 1 :4 to 4:1 , 1 :4 to 3:1 , 1 :4 to 2:1 , 1 :4 to 1 :1 , 1 :3 to 4:1 , 1 :3 to 3:1 , 1 :3 to 2:1 , 1 :3 to 1 :1 , 1 :2 to 4:1 , 1 :2 to 3:1 , 1 :2 to 2:1 , 1 :2 to 1 :1 , 1 :1 to 4:1 , 1 :1 to 3:1 , 1 :1 to 2:1 , or 1 :0 to 1 :1 ).

[0152] For example, in some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :9 to 4:1 . In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :8 to 4:1 . In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :7 to 4:1 . In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :6 to 4:1 . In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :5 to 4:1 . In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :4 to 4:1 . In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :3 to 4:1 . In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :2 to 4:1 . In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :1 to 4:1 . In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :0 to 4:1 .

[0153] In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :10 to 3:1 . In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :9 to 3:1 . In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :8 to 3:1 . In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :7 to 3:1 . In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :6 and 3:1 . In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :5 to 3:1 . In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :4 to 3:1 . In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :3 to 3:1 . In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :2 to 3:1 . In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :1 to 3:1 . In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :0 to 3:1 .

[0154] In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :10 to 2:1 . In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :9 to 2:1 . In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :8 to 2:1 . In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :7 to 2:1 . In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :6 to 2:1 . In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :5 to 2:1 . In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :4 to 2:1 . In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :3 to 2:1 . In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :2 to 2:1 . In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :1 to 2:1 . In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :0 to 2:1 .

[0155] In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :10 to 1 :1 . In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :9 to 1 :1 . In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :8 to 1 :1 . In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :7 to 1 :1 . In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :6 to 1 :1 . In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :5 to 1 :1 . In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :4 to 1 :1 . In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :3 to 1 :1 . In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :2 to 1 :1 . In some embodiments, the ratio of hypoimmunogenic cells to fibroblasts is from 1 :1 to 1 :0.

[0156] In some embodiments, the layer of cell mixtures or aggregates in the biocompatible scaffold is from 100 pm to 1 mm (e.g., 200 pm to 900 pm, 300 pm to 800 pm, 400 pm to 700 pm, or 500 pm to 600 pm) thick. For example, in some embodiments, the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is from 200 pm to 900 pm thick. In some embodiments, the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is from 300 pm to 800 pm thick. In some embodiments, the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is from 400 pm to 700 pm thick. In some embodiments, the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is from 500 pm to 600 pm thick.

[0157] In some embodiments, the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 100 pm thick. In some embodiments, the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 200 pm thick. In some embodiments, the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 300 pm thick. In some embodiments, the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 400 pm thick. In some embodiments, the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 500 pm thick. In some embodiments, the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 600 pm thick. In some embodiments, the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 700 pm thick. In some embodiments, the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 800 pm thick. In some embodiments, the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 900 pm thick. In some embodiments, the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 1 mm thick. In some embodiments, the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 2 mm thick.

[0158] In some embodiments, the density of hypoimmunogenic cells in the layer of cell mixtures or aggregates in the biocompatible scaffold is from 0.06 M / cm2to 150 M / cm2(e.g., 0.07 M / cm2to 149 M / cm2, 0.08 M / cm2to 148 M / cm2, 0.09 M / cm2to 147 M / cm2, 0.1 M / cm2to 146 M / cm2, 0.2 M / cm2to 145 M / cm2, 0.3 M / cm2to 140 M / cm2, 0.4 M / cm2to 130 M / cm2, 0.5 M / cm2to 120 M / cm2, 1 M / cm2to 1 10 M / cm2, 2 M / cm2to 100 M / cm2, 3 M / cm2to 50 M / cm2, 4 M / cm2to 40 M / cm2, 5 M / cm2to 30 M / cm2, or 10 M / cm2to 20 M / cm2). For example, in some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is from 0.07 M / cm2to 149 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is from 0.08 M / cm2to 148 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is from 0.09 M / cm2to 147 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is from 0.1 M / cm2to 146 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is from 0.2 M / cm2to 145 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is from 0.3 M / cm2to 140 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is from 0.4 M / cm2to 130 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is from 0.5 M / cm2to 120 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is from 1 M / cm2to 1 10 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is from 2 M / cm2to 100 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is from 3 M / cm2to 50 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is from 4 M / cm2to 40 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is from 5 M / cm2to 30 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is from 10 M / cm2to 20 M / cm2.

[0159] In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 0.06 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 0.07 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 0.08 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 0.09 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 0.1 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 0.2 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 0.3 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 0.4 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 0.5 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 1 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 2 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 3 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 4 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 5 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 10 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 20 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 30 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 40 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 50 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 100 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 1 10 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 120 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 130 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 140 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 145 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 146 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 147 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 148 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 149 M / cm2. In some embodiments, the density of hypoimmunogenic cells in the layer of hypoimmunogenic cell and fibroblast aggregates in the biocompatible scaffold is 150 M / cm2.

[0160] In some embodiments, the thickness of the layer is dependent upon the hypoimmunogenic celldensity.

[0161] Other Cell Types

[0162] In some embodiments, the cell mixture or engineered tissue construct includes other cell types. In some embodiments, the cell mixture or engineered tissue construct includes less than 30% (e.g., less than 29%, 28%, 27%, 26%, 25%, 20%, 15%, 10%, or 21%) of other cell types. For example, other cell types may include stellate cells, Kupffer cells, pit cells, cholangiocytes, portal fibroblasts, and liver sinusoidal endothelial cells. In some embodiments, the cell mixture or engineered tissue construct includes less than 30% (e.g., less than 29%, 28%, 27%, 26%, 25%, 20%, 15%, 10%, or 21%) of stellate cells. In some embodiments, the cell mixture or engineered tissue construct includes less than 30% (e.g., less than 29%, 28%, 27%, 26%, 25%, 20%, 15%, 10%, or 21%) of Kupffer cells. In some embodiments, the cell mixture or engineered tissue construct includes less than 30% (e.g., less than 29%, 28%, 27%, 26%, 25%, 20%, 15%, 10%, or 21%) of pit cells. In some embodiments, the cell mixture or engineered tissue construct includes less than 30% (e.g., less than 29%, 28%, 27%, 26%, 25%, 20%, 15%, 10%, or 21%) of bile duct cells.

[0163] In some embodiments, the cell mixture or engineered tissue construct includes endothelial cells. In some embodiments, the cell mixture or engineered tissue construct includes up to 30% (e.g., up to 29%, 28%, 27%, 26%, 25%, 20%, 15%, 10%, or 1%) of endothelial cells. In some embodiments, the population of endothelial cells is arranged as one or more cords.

[0164] Biocompatible Hydrogel Scaffolds

[0165] The cellular compositions disclosed herein can be provided as a suspension in a biocompatible scaffold containing the cells, e.g., hypoimmunogenic cells and fibroblasts. In some embodiments, the population of cells (e.g., the population of hypoimmunogenic cells and the population of fibroblasts) are aggregated in spheroids. In some embodiments, the biocompatible scaffold has an x-axis, a y-axis, and a z-axis. For example, in some embodiments, the population of cells, e.g., fibroblasts and hypoimmunogenic cells, are aggregated in spheroids and the spheroids are distributed non- homogenously, in a layer, along the z-axis of the biocompatible scaffold. In some embodiments, the spheroids are distributed homogenously along the x-axis of the biocompatible scaffold. In some embodiments, the spheroids are distributed homogenously along the y-axis of the biocompatible scaffold.

[0166] The biocompatible scaffold may be liquid, gel, semi-solid, or solid at room temperature (e.g., 25 °C). The biocompatible scaffold may be biodegradable or non-biodegradable. In some embodiments, the scaffold is bioresorbable or bioreplaceable. In some embodiments, the scaffold includes fibrin (e.g., human fibrin or human fibrinogen, e.g., FIBRYGA®). Exemplary biocompatible scaffolds include polymers and hydrogels include collagen, fibrinogen, fibrin, chitosan, MATRIGEL™, dextrans including chemically cross-linkable or photo-cross-linkable dextrans, processed tissue matrix such as submucosal tissue, PEG hydrogels (e.g., heparin-conjugated PEG hydrogels), poly(lactic-co-glycolic acid) (PLGA), hydroxyethyl methacrylate (HEMA), gelatin, alginate, agarose, polysaccharides, hyaluronic acid (HA), peptide-based self-assembling gels, thermo-responsive poly(NIPAAm). A number of biopolymers are known to those skilled in the art (Bryant and Anseth, J. Biomed. Mater. Res. 59(1 ):63-72, 2002; Mann et al., Biomaterials. 22 (22): 3045-3051 , 2001 ; Mann et al., Biomaterials. 22 (5):439-444, 2001 , and Peppas et al., Eur. J. Pharm. Biopharm. 50(1 ), 27-46, 2000; all incorporated by reference). In other embodiments, the biocompatible scaffold may contain a biopolymer having any of a number of growth factors, adhesion molecules, degradation sites or bioactive agents to enhance cell viability or for any number of other reasons. Such molecules are well known to those skilled in the art.

[0167] In some embodiments, the PEG hydrogel may be chemically cross-linkable and / or modified with bifunctional groups.

[0168] In certain embodiments, the biocompatible scaffold includes allogeneic components, autologous components, or both allogeneic components and autologous components. In certain embodiments, the biocompatible scaffold includes synthetic or semi-synthetic materials. In certain embodiments, the biocompatible scaffold includes a framework or support, such as a fibrin-derived scaffold.

[0169] In some embodiments, the biocompatible scaffold is fibrin.

[0170] Biocompatible hydrogel scaffolds suitable for use include any polymer that is gellable in situ, e.g., one that does not require chemicals or conditions (e.g., temperature or pH) that are not cytocompatible. This includes both stable and biodegradable biopolymers.

[0171] Polymers for use herein are preferably crosslinked, for example, ionically crosslinked. In certain embodiments, the methods and constructs described herein use polymers in which polymerization can be promoted photochemically (i.e., photo-crosslinked), by exposure to an appropriate wavelength of light (i.e. , photopolymerizable) or a polymer which is weakened or rendered soluble by light exposure or other stimulus. Although some of the polymers listed above are not inherently light sensitive (e.g., collagen, HA), they may be made light sensitive by the addition of acrylate or other photosensitive groups.

[0172] In certain embodiments, the method utilizes a photoinitiator. A photoinitiator is a molecule that is capable of promoting polymerization of hydrogels upon exposure to an appropriate wavelength of light as defined by the reactive groups on the molecule. In the context of the disclosure, photoinitiators are cytocompatible. A number of photoinitiators are known that can be used with different wavelengths of light. For example, 2,2-dimethoxy-2-phenyl-acetophenone, HPK 1 -hydroxycyclohexyl-phenyl ketone and Irgacure 2959 (hydroxyl-1 -[4-(hydroxyethoxy)phenyl]-2methyl-1 propanone) are all activated with UV light (365 nm). Other crosslinking agents activated by wavelengths of light that are cytocompatible (e.g., blue light) can also be used with the engineered tissue constructs and methods described herein.

[0173] In other embodiments, the method involves the use of polymers bearing non-photochemically polymerizable moieties. In certain embodiments, the non-photochemically polymerizable moieties are Michael acceptors. Non-limiting examples of such Michael acceptor moieties include a,p-unsaturated ketones, esters, amides, sulfones, sulfoxides, and phosphonates. Additional non-limiting examples of Michael acceptors include quinines and vinyl pyridines. In some embodiments, the polymerization of Michael acceptors is promoted by a nucleophile. Suitable nucleophiles include, but are not limited to thiols, amines, alcohols, and molecules possessing thiol, amine, and alcohol moieties. In certain embodiments, the disclosure features the use of thermally crosslinked polymers.

[0174] In some embodiments, the z-axis of the biocompatible scaffold is from 500 pm to 5 mm (e.g., 600 pm to 4 mm, 700 pm to 3 mm, 800 pm to 2 mm, or 900 pm to 1 mm). For example, in some embodiments, the z-axis of the biocompatible scaffold is from 600 pm to 4 mm. In some embodiments, the z-axis of the biocompatible scaffold is from 700 pm to 3 mm. In some embodiments, the z-axis of the biocompatible scaffold is from 800 pm to 2 mm. In some embodiments, the z-axis of the biocompatible scaffold is from 900 pm to 1 mm.

[0175] In some embodiments, the z-axis of the biocompatible scaffold is 500 pm. In some embodiments, the z-axis of the biocompatible scaffold is 600 pm. In some embodiments, the z-axis of the biocompatible scaffold is 700 pm. In some embodiments, the z-axis of the biocompatible scaffold is 800 pm. In some embodiments, the z-axis of the biocompatible scaffold is 900 pm. In some embodiments, the z-axis of the biocompatible scaffold is 1 mm. In some embodiments, the z-axis of the biocompatible scaffold is 2 mm. In some embodiments, the z-axis of the biocompatible scaffold is 3 mm. In some embodiments, the z-axis of the biocompatible scaffold is 4 mm. In some embodiments, the z-axis of the biocompatible scaffold is 5 mm.

[0176] In some embodiments, the ratio of height of the biocompatible scaffold to height of the layer of cell aggregates, e.g., hypoimmunogenic cell and fibroblast aggregates, in the biocompatible scaffold is from 20:1 to 1 :1 (e.g., 19:1 to 1 :1 , 18:1 to 1 :1 , 17:1 to 1 :1 , 16:1 to 1 :1 , 15:1 to 1 :1 , 14:1 to 1 :1 , 13:1 to 1 :1 , 12:1 to 1 :1 , 11 :1 to 1 :1 , 10:1 to 1 :1 , 9:1 to 1 :1 , 8:1 to 1 :1 , 7:1 to 1 :1 , 6:1 to 1 :1 , 5:1 to 1 :1 , 4:1 to 1 :1 , 3:1 to 1 :1 , or 2:1 to 1 :1 ). For example, in some embodiments the ratio of height of the biocompatible scaffold to height of the layer is from 19:1 to 1 :1 . In some embodiments, the ratio of height of the biocompatible scaffold to height of the layer is from 18:1 to 1 :1 . In some embodiments, the ratio of height of the biocompatible scaffold to height of the layer is from 17:1 to 1 :1 . In some embodiments, the ratio of height of the biocompatible scaffold to height of the layer is from 16:1 to 1 :1 . In some embodiments, the ratio of height of the biocompatible scaffold to height of the layer is from 15:1 to 1 :1 . In some embodiments, the ratio of height of the biocompatible scaffold to height of the layer is from 14:1 to 1 :1 . In some embodiments, the ratio of height of the biocompatible scaffold to height of the layer is from 13:1 to 1 :1 . In some embodiments, the ratio of height of the biocompatible scaffold to height of the layer is from 12:1 to 1 :1 . In some embodiments, the ratio of height of the biocompatible scaffold to height of the layer is from 11 :1 to 1 :1 . In some embodiments, the ratio of height of the biocompatible scaffold to height of the layer is from 10:1 to 1 :1 . In some embodiments, the ratio of height of the biocompatible scaffold to height of the layer is from 9:1 to 1 :1 . In some embodiments, the ratio of height of the biocompatible scaffold to height of the layer is from 8:1 to 1 :1 . In some embodiments, the ratio of height of the biocompatible scaffold to height of the layer is from 7:1 to 1 :1 . In some embodiments, the ratio of height of the biocompatible scaffold to height of the layer is from 6:1 to 1 :1 . In some embodiments, the ratio of height of the biocompatible scaffold to height of the layer is from 5:1 to 1 :1 . In some embodiments, the ratio of height of the biocompatible scaffold to height of the layer is from 4:1 to 1 :1 . In some embodiments, the ratio of height of the biocompatible scaffold to height of the layer is from 3:1 to 1 :1 .

[0177] In some embodiments, the biocompatible scaffold includes a synthetic heparin mimetic. In particular, the synthetic polymer of the invention may include an amount of negative charge that, in some embodiments, is similar to the amount of negative charge present in heparin. Accordingly, the synthetic polymer of the disclosure can mimic the functional properties of heparin. For example, the synthetic polymer of the disclosure has the potential to bind various bioactive agents, e.g., growth factors, that naturally bind to heparin. Therefore, the synthetic polymer of the disclosure, as well as the hydrogel comprising the synthetic polymer described herein can bind various bioactive agents, e.g., growth factors, thereby preventing the bioactive agents from diffusing away and maintaining the bioactive agents at a high concentration locally, so that they can act on cells and promote various cell functions.

[0178] Engineered Tissue Constructs

[0179] Engineered tissue constructs described herein include a population of cells or a cell mixture (e.g., hypoimmunogenic cells and fibroblasts) and a biocompatible scaffold.

[0180] In some embodiments, the engineered tissue construct includes two or more populations of cells (e.g., two, three, four, five, six, seven, eight, nine, ten, or more populations of cells).

[0181] The shape of the engineered tissue construct may depend on the site of implantation and / or the disease or disorder to be treated. In some embodiments, the engineered tissue construct is triangular, rectangular (e.g., square), or circular. In some embodiments, the engineered tissue construct has a thickness than is substantially less than its length and width. For example, the engineered tissue construct may be substantially flat, e.g., a flat rectangle, triangle, or disc shape.

[0182] The engineered tissue construct may have a length, width, and thickness that are each, independently, from 0.1 mm to 100 cm, e.g., from 0.1 mm to 1 mm (e.g., 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, or 1 mm), from 1 mm to 1 cm (e.g., 1 mm, 2 mm, 3 mm,

[0183] 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, or 1 cm), from 1 cm to 10 cm (e.g., 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, or 10 cm), or from 10 cm to 100 cm (e.g., 20 cm, 30 cm, 40 cm, 50 cm, 60 cm, 70 cm, 80 cm, 90 cm, or 100 cm). In some embodiments, the thickness of the engineered tissue construct is from 0.1 mm to 1 cm, e.g., from 0.1 mm to 1 mm (e.g., 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, or 1 mm) or from 1 mm to 1 cm (e.g., 1 mm, 2 mm, 3 mm, 4 mm,

[0184] 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, or 1 cm), and the length and / or width are each, independently, from 1 cm to 10 cm (e.g., 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, or 10 cm) or from 10 cm to 100 cm (e.g., 20 cm, 30 cm, 40 cm, 50 cm, 60 cm, 70 cm, 80 cm, 90 cm, or 100 cm). In some embodiments, the length and width are each, independently, at least 5-fold (e.g., at least 5-fold, at least 6-fold, 7-fold, 8- fold, 9-fold, 10-folder, 20-fold, 30-fold, 40-fold, 50-fold, or more) greater than the thickness of the engineered tissue construct.

[0185] In some embodiments, the engineered tissue construct has a surface area of 10 cm2to 2,000 cm2, e.g., from 10 cm2to 100 cm2(e.g., 10 cm2, 20 cm2, 30 cm2, 40 cm2, 50 cm2, 60 cm2, 70 cm2, 80 cm2, 90 cm2, or 100 cm2), 100 cm2to 1 ,000 cm2(e.g., 100 cm2, 200 cm2, 300 cm2, 400 cm2, 500 cm2, 600 cm2, 700 cm2, 800 cm2, 900 cm2, or 1 ,000 cm2), or 1 ,000 cm2to 2,000 cm2(e.g., 1 ,000 cm2, 1 ,100 cm2, 1 ,200 cm2, 1 ,300 cm2, 1 ,400 cm2, 1 ,500 cm2, 1 ,600 cm2, 1 ,700 cm2, 1 ,800 cm2, 1 ,900 cm2, or 2,000 cm2).

[0186] In some embodiments, the cells are present (e.g., encapsulated) on a first face of the engineered tissue construct. In some embodiment, the cells are present (e.g., encapsulated) on a first face and a second face of the engineered tissue construct. In some embodiments, the first face of the engineered tissue construct containing the encapsulated cells contacts a site of implantation. In some embodiments, the method includes implanting a plurality of engineered tissue constructs. For example, in some embodiments, the engineered tissue construct has a thickness that is substantially less than its length and width (e.g., a substantially flat rectangle, triangle, or disc shape) and a stack of engineered tissue constructs is implanted in a site of implantation. In other examples, the members of the plurality may be implanted in different implantation sites.

[0187] In another aspect, the cells are provided in the form of an aggregate (e.g., a spheroidal aggregate). In some embodiments, the cell populations are admixed under conditions which cause the two cell populations to form aggregates. In some embodiments, the cell populations are admixed using tissue fabrication techniques. In some embodiments, two or more cell populations are co-cultured. In some embodiments, the two or more cell populations are cocultured by hanging drop, microwell molding, non-adhesive surfaces, spheroid suspension culture using a spinner flask, vertical wheel bioreactor, horizontal wheel bioreactor, or a microfluidic spheroid system. Additional methods include those using acoustical waves and using positively charged surfaces on a plate.

[0188] In other aspects, the compositions provided herein can contain additional components, including but not limited to, growth factors, ligands, cytokines, drugs, and the like. In some embodiments, the cell mixtures can include molecules which elicit additional microenvironmental cues such as small molecules or growth factors which stimulate or enhance proliferation and expansion of a cell population.

[0189] The properties of the cell aggregates of the present disclosure can be varied to suit a particular application. In certain embodiments, the density of the cell aggregates can be changed. In certain embodiments, cell aggregates of different diameters can be fabricated. In certain embodiments, the overall network organization of the one or more cell aggregates can be defined, for example, by the number, three-dimensional organization, alignment, diameters, density, and the like.

[0190] In certain embodiments, the engineered tissue construct can contain one or more bioactive substances. Examples of bioactive substance(s) include, but are not limited to, hormones, neurotransmitters, growth factors, hormone, neurotransmitter or growth factor receptors, interferons, interleukins, chemokines, cytokines, colony stimulating factors, chemotactic factors, extracellular matrix components, and adhesion molecules, ligands and peptides; such as growth hormone, parathyroid hormone bone morphogenetic protein, transforming growth factor-alpha, TGF-beta1 , TGF-beta2, stromal cell growth factor, granulocyte / macrophage colony stimulating factor, epidermal growth factor, platelet derived growth factor, insulin-like growth factor, scatter factor / hepatocyte growth factor, fibrin, dextran, matrix metalloproteinases, collagen, fibronectin, vitronectin, hyaluronic acid, an RGD-containing peptide or polypeptide, an angiopoietin and vascular endothelial cell growth factor.

[0191] In certain embodiments, the engineered tissue constructs disclosed herein include one or more adherence materials to facilitate maintenance of the desired phenotype of the grafted cells in vivo. The material may include, but is not limited to, antibodies, proteins, peptides, nucleic acids, peptide aptamers, nucleic acid aptamers, sugars, proteoglycans, or cellular receptors.

[0192] The type of adherence materials (e.g., extracellular matrix (ECM) materials, sugars, proteoglycans, etc.) will be determined, in part, by the cell type or types (e.g., fibroblasts and hypoimmunogenic cells) to be cultured. ECM molecules found in a cell's native microenvironment are useful in maintaining the function of both primary cells, precursor cells, and / or cell lines. In some embodiments, the engineered tissue construct further includes a biocompatible scaffold (e.g., biocompatible hydrogel scaffold). For example, in some embodiments, the biocompatible scaffold is fibrin (e.g., human fibrin or human fibrinogen, e.g., FIBRYGA®). In some environments, the biocompatible scaffold is resorbable. In some embodiments, the fibrin includes polymerized fibrinogen. In some embodiments, the fibrinogen is reconstituted in a hypertonic ionic strength solution prior to polymerization (e.g., a high glucose solution, e.g., Dulbecco's Modified Eagle Medium (DMEM)). In some embodiments, the biocompatible scaffold is bioresorbable. In some embodiments, the biocompatible scaffold includes a synthetic heparin mimetic. In particular, the synthetic polymer of the disclosure may include an amount of negative charge that, in some embodiments, is similar to the amount of negative charge present in heparin. Accordingly, the synthetic polymer of the disclosure can mimic the functional properties of heparin. For example, the synthetic polymer of the disclosure has the potential to bind various bioactive agents, e.g., growth factors, that naturally bind to heparin. Therefore, the synthetic polymer of the disclosure, as well as the hydrogel comprising the synthetic polymer described herein can bind various bioactive agents, e.g., growth factors, thereby preventing the bioactive agents from diffusing away and maintaining the bioactive agents at a high concentration locally, so that they can act on cells and promote various cell functions.

[0193] In some embodiments, the engineered tissue construct includes from 1 x 106cells / mL to 1 x 108cells / mL, e.g., from 1 x 106cells / mL to 10 x 106cells / mL (e.g., 1 x 106cells / mL, 2 x 106cells / mL, 3 x 106cells / mL, 4 x 106cells / mL, 5 x 106cells / mL, 6 x 106cells / mL, 7 x 106cells / mL, 8 x 106cells / mL, 9 x 106cells / mL, or 1 x 107cells / mL) or from 1 x 107cells / mL to 1 x 108cells / mL (e.g., 2 x 107cells / mL, 3 x 107cells / mL, 4 x 107cells / mL, 5 x 107cells / mL, 6 x 107cells / mL, 7 x 107cells / mL, 8 x 107cells / mL, 9 x 107cells / mL, or 1 x 108cells / mL).

[0194] In some embodiments, the engineered tissue construct is from 0.1 mL to 5 L (e.g., 0.2 mL to 5 L, 0.3 mL to 5 L, 0.4 mL to 5 L, 0.5 mL to 5 L, 1 mL to 5 L, 5 mL to 5 L, 10 mL to 5 L, 100 mL to 5 L, 1 L to 5 L, 2 L to 5 L, 3 L to 5 L, or 4 L to 5 L) in volume. In some embodiments, the engineered tissue construct has a volume of from 20 mL and 1 .2 L, e.g., from 20 mL to 100 mL (e.g., 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, or 100 mL), from 100 mL to 500 mL (e.g., 100 mL, 150 mL, 200 mL, 250 mL, 300 mL, 350 mL, 400 mL, 450 mL, or 500 mL), or from 500 mL to 1 .2 L (e.g., 500 mL, 600 mL, 700 mL, 800 mL, 900 mL, 1 L, 1 .1 L, or 1 .2 L). For example, in some embodiments, the engineered tissue construct is from 0.2 mL to 5 L in volume. In some embodiments, the engineered tissue construct is from 0.3 mL to 5 L in volume. In some embodiments, the engineered tissue construct is from 0.4 mL to 5 L in volume. In some embodiments, the engineered tissue construct is from 0.5 mL to 5 L in volume. In some embodiments, the engineered tissue construct is from 1 mL to 5 L in volume. In some embodiments, the engineered tissue construct is from 5 mL to 5 L in volume. In some embodiments, the engineered tissue construct is from 10 mL to 5 L in volume. In some embodiments, the engineered tissue construct is from 100 mL to 5 L in volume. In some embodiments, the engineered tissue construct is from 1 mL to 5 L in volume. In some embodiments, the engineered tissue construct is from 2 mL to 5 L in volume. In some embodiments, the engineered tissue construct is from 3 mL to 5 L in volume. In some embodiments, the engineered tissue construct is from 4 mL to 5 L in volume.

[0195] In some embodiments, the engineered tissue construct further includes a reinforcing agent. In some embodiments, the reinforcing agent is selected from the list of fibrin, surgical mesh, alginate, collagen, polyethylene glycol), polyvinylidene acetate (PVDA), polyvinylidene fluoride (PVDF), poly(lactic-co-glycolic) acid (PLGA), and poly (l-lactic acid) (PLLA). In some embodiments, the reinforcing agent is fibrin. In some embodiments, the reinforcing agent is surgical mesh. In some embodiments the reinforcing agent is alginate. In some embodiments, the reinforcing agent is collagen. In some embodiments, the reinforcing agent is polyethylene glycol). In some embodiments, the reinforcing agent is PVDA. In some embodiments, the reinforcing agent is PVDF. In some embodiments, the reinforcing agent is PLGA. In some embodiments, the reinforcing agent is PLLA. In some embodiments, the reinforcing agent is any suitable agent.

[0196] In some embodiments, the engineered tissue construct may be any shape (e.g., cylindrical, square, or square with rounded corners).

[0197] In some embodiments, the engineered tissue construct has a serpentine topography (e.g., to increase surface area).

[0198] Methods of Administration and Implantation

[0199] Described herein are methods for administering or implanting a cell mixture or an engineered tissue construct that includes a population of hypoimmunogenic cells and a population of fibroblasts. The methods may include administering or implanting the engineered tissue construct in a subject (e.g., a human subject).

[0200] Also featured herein is a method of promoting engraftment and / or vascularization of a population of hypoimmunogenic cells in a subject by administering or implanting the cell mixture or engineered tissue construct into the subject in combination with an immunosuppressive regimen; and maintaining the immunosuppressive regimen for a time sufficient for engraftment and / or vascularization of the hypoimmunogenic cells in the subject (e.g., a human subject).

[0201] Also featured herein is a method for promoting engraftment and / or vascularization of a population of hypoimmunogenic cells in a subject in need thereof. The method includes providing a subject who has previously been administered or implanted the cell mixture or engineered tissue construct into the subject in combination with an immunosuppressive regimen; and maintaining the immunosuppressive regimen for a time sufficient for engraftment and / or vascularization of the hypoimmunogenic cells in the subject.

[0202] Also featured herein is a method for promoting engraftment and / or vascularization of a population of hypoimmunogenic cells in a subject in need thereof. The method includes administering or implanting the cell mixture or engineered tissue construct into the subject and activating a kill switch to kill the fibroblasts at a time point following engraftment and / or vascularization of the hypoimmunogenic cells in the subject.

[0203] Any of the cell mixtures, aggregates thereof, or engineered tissue constructs may be used in any of the methods described herein.

[0204] The cell mixture or engineered tissue construct may be administered by any suitable route or implanted to any suitable site. In some embodiments, the cell mixture or engineered tissue construct is implanted in in an extraperitoneal space, in an extrapleural space, or on a liver surface. In some embodiments, upon implantation, the population of cells are engrafted and vascularized in the subject.

[0205] In some embodiments, the method includes implanting the cell mixture or engineered tissue construct in a human subject in an extraperitoneal space, in an extrapleural space, on a surface of the liver, in a muscle site (e.g., on a surface of a muscle, within a muscle sheath, or beneath a muscle), in a pleural space, in an omentum site, in a subcutaneous site, on a surface of the pancreas, on a surface of the spleen, on a surface of the kidney, in a bone marrow site, in a bursa site, in a peritoneal site (e.g., mesentery), or in a lesser sac site.

[0206] The cell mixtures or engineered tissue constructs described herein can be administered or implanted in a subject. Non-limiting examples of non-human subjects include non-human primates, dogs, cats, mice, rats, guinea pigs, rabbits, fowl, pigs, horses, cows, goats, or sheep. In certain embodiments, the subject is any animal. In certain embodiments, the subject can be any mammal. In certain embodiments, the subject is a human.

[0207] In some embodiments, the subject has an age of from 1 day to 120 years (e.g., from 1 day to 7 days, from 1 day to 1 month, from 1 day to 6 months, from 1 day to 1 year, from 1 day to 2 years, from 1 month and 12 months, from 1 month and 6 months, from 6 months and 2 years, from 1 year and 2 years, from 1 year and 5 years, from 1 year and 10 years, from 10 years and 20 years, from 10 years and 50 years, from 50 years and 80 years, or from 60 years to 90 years). In some embodiments, the subject has an age of from 1 day to 1 year. In some embodiments, the subject is a newborn or an infant.

[0208] In some embodiments, the cell mixture or engineered tissue construct is administered or implanted into a subject in an extraperitoneal space, in an extrapleural space, on a surface of the liver, in a muscle site (e.g., a surface of a muscle, within a muscle sheath, or beneath a muscle), in a pleural space, in an omentum site, in a subcutaneous site, on a surface of the pancreas, on a surface of the spleen, on a surface of the kidney, in a bone marrow site, in a bursa site, in a peritoneal site, or in a lesser sac site.

[0209] In some embodiments, the cell mixture or engineered tissue construct is administered or implanted into the subject in an extraperitoneal space, in an extrapleural space, or on a liver surface. In some embodiments, the cell mixture or engineered tissue construct is implanted in the extraperitoneal space. In some embodiments, the cell mixture or engineered tissue construct is implanted in a pre- peritoneal space, a retroperitoneal space, or a subperitoneal space.

[0210] In some embodiments, the cell mixture or engineered tissue construct is administered or implanted on the surface of the liver.

[0211] In some embodiments, the cell mixture or engineered tissue construct is layered on the dome of the liver and / or covered with omentum.

[0212] In some embodiments, the muscle site is a surface of a muscle. In some embodiments, the muscle site is within a muscle sheath. In some embodiments, the muscle site is beneath a muscle.

[0213] In some embodiments, the omentum site includes an omentum pedicle flap, an omentum free flap, an omental bursa, or the omentum in situ.

[0214] In some embodiments, the cell mixture or engineered tissue construct is implanted subcutaneously with an omental flap, subcutaneously with an adjuvant, or subcutaneously with an arteriovenous fistula.

[0215] In some embodiments, the cell mixture or engineered tissue construct is administered or implanted into the subject in an implantation site selected from the group consisting of the peritoneum, an extraperitoneal site (e.g., retroperitoneum), pre-peritoneal space, or a subperitoneal space), peritoneal cavity (e.g., omentum or mesentery), rectus abdominis muscle, abdominal oblique muscle, quadriceps femoris muscle, gluteus maximus, a hamstring muscle (e.g., a semimembranosus, a semitendinosus, or a biceps femoris), deltoid, biceps, latissimus dorsi, extraperitoneal fat, and renal capsule; an extraperitoneal site, a site on the surface of the liver, or an extrapleural site; or a site that is suitable for neovascularization. For example, in some embodiments, the peritoneum is the retroperitoneum. In some embodiments, the peritoneal cavity is the omentum. In some embodiments, the peritoneal cavity is the mesentery. In some embodiments, the omentum is the greater omentum or the omental bursa. In some embodiments, the mesentery is the small intestinal mesentery. In some embodiments, the cell mixture or engineered tissue construct is implanted into the subject as a pedicled omental wrap or an omental wrap.

[0216] In some embodiments, one or more cell mixtures or engineered tissue constructs is implanted in one or more implantation sites selected from the group consisting of the peritoneum, retroperitoneum, peritoneal cavity (e.g., omentum or mesentery), rectus abdominis muscle, abdominal oblique muscle (including an internal oblique muscle or an external oblique muscle), transversus abdominis muscle, quadriceps femoris muscle, gluteus maximus, a hamstring muscle (e.g., a semimembranosus muscle, a semitendinosus muscle, or a biceps femoris muscle), deltoid , biceps, latissimus dorsi, extraperitoneal fat, and renal capsule; an extraperitoneal site, a surface of the liver, or an extrapleural site; or a site that is suitable for neovascularization. For example, in some embodiments, the cell mixture or engineered tissue construct is implanted in the retroperitoneum. For example, in some embodiments, two engineered tissue constructs are implanted bilaterally on an omentum site.

[0217] The cell mixture or engineered tissue construct can be implanted in any suitable manner, often with pharmaceutically acceptable carriers. In some embodiments, the cell mixture or engineered tissue construct is implanted on a surface of a tissue or organ. In some embodiments, the cell mixture or engineered tissue construct is implanted on an orthotopic site. In other embodiments, the cell mixture or engineered tissue construct is implanted on an ectopic site.

[0218] Any suitable approach may be used to perform the implantation. For example, the engineered tissue construct may be implanted using an open surgical procedure or a minimally invasive surgery. In some embodiments, the cell mixture or engineered tissue construct may be implanted using an open surgical procedure. In other embodiments, the engineered tissue construct may be implanted using a minimally invasive surgery.

[0219] In some embodiments, the methods of implantation include dissecting the site of implantation. In some embodiments, the methods of implantation include forming a pocket for the implant. For example, the site of implantation may be dissected down through skin, fat, connective tissue and / or muscle, e.g., to the peritoneum. Dissection (e.g., blunt dissection) of connective tissue, e.g., between the peritoneum and muscle, may be performed to form an implant pocket. One or more sutures may be added to mechanically stabilize the graft at the desired site of implantation, e.g., in the pocket.

[0220] The engineered tissue construct may be affixed to the subject using any suitable approach. For example, the engineered tissue construct may be affixed using sutures, staples, or by welding (e.g., laser tissue welding). In some embodiments, the engineered tissue construct is affixed by one or more sutures or one or more staples. In some embodiments, the engineered tissue construct is affixed by suturing adjoining tissue to restrain migration of the engineered tissue construct. For example, in some embodiments, the engineered tissue construct is implanted at an extraperitoneal site, and the engineered tissue construct is affixed by suturing the muscle fascia to the peritoneum at one or more positions surrounding the engineered tissue construct. In some embodiments, the engineered tissue construct is not directly sutured or stapled (in other words, the sutures or staples do not penetrate the engineered tissue construct itself). In some embodiments, the engineered tissue construct is implanted in a site that is suitable for neovascularization. In some embodiments, a site that it suitable for neovascularization is one having a microvessel density of from 3.6 vessels / mm2to 4500 vessels / mm2(e.g., 3.7 vessels / mm2to 4000 vessels / mm2, 3.8 vessels / mm2to 3500 vessels / mm2, 3.9 vessels / mm2to 3000 vessels / mm2, 4 vessels / mm2to 2500 vessels / mm2, 5 vessels / mm2to 2000 vessels / mm2, 10 vessels / mm2to 1000 vessels / mm2, or 100 vessels / mm2).

[0221] In some embodiments, a site that is suitable for neovascularization may have an existing microvessel density of greater than 3.7 vessels / mm2. In some embodiments, a site that is suitable for neovascularization may have an existing microvessel density of greater than 4 vessels / mm2. In some embodiments, a site that is suitable for neovascularization may have an existing microvessel density of greater than 4.5 vessels / mm2. In some embodiments, a site that is suitable for neovascularization may have an existing microvessel density of greater than 5 vessels / mm2. In some embodiments, a site that is suitable for neovascularization may have an existing microvessel density of greater than 10 vessels / mm2. In some embodiments, a site that is suitable for neovascularization may have an existing microvessel density of greater than 50 vessels / mm2. In some embodiments, a site that is suitable for neovascularization may have an existing microvessel density of greater than 100 vessels / mm2. In some embodiments, a site that is suitable for neovascularization may have an existing microvessel density of greater than 1000 vessels / mm2. In some embodiments, a site that is suitable for neovascularization may have an existing microvessel density of greater than 4500 vessels / mm2. Autologous, allogenic, or xenogenic cells may be used. The cells may be implanted in any physiologically acceptable medium. In one embodiment, the cells are cryopreserved in 5-20% DMSO, 5% dextrose and autologous serum. As is familiar to those of skill in the art, dosage of the cells of the present invention to be implanted in vivo is determined with reference to various parameters, including the species of the host, the age, weight, and disease status. Dosage also depends upon the location to be targeted within the subject. For example, implantation of the engineered tissue construct into the omentum may require different dosages than implantation to the mesentery. The dosage is preferably chosen so that implantation causes an effective result, which can be measured by molecular assays (e.g., a liver function test) or by monitoring a suitable symptom in the subject (e.g., symptoms of Crigler-Najjar).

[0222] In some embodiments, the cell mixture or engineered tissue construct is administered to the subject.

[0223] In other embodiments, the cell mixture or engineered tissue construct is implanted into the subject.

[0224] In some embodiments, following administration or implantation of the cell mixture or engineered tissue construct, the cell mixture or engineered tissue construct persists for at least two weeks (e.g., at least three weeks, one month, two months, three months, four months, five months, six months, seven months, ten months, eleven months, one year, five years, ten years, or the lifetime of a subject in whom the cell mixture or engineered tissue construct has been administered or implanted). In some embodiments, following administration or implantation of the cell mixture engineered tissue construct, the cell mixture or engineered tissue construct persists until an organ transplant occurs (e.g., until the patient receives a liver transplant).

[0225] Immunosuppressive Regimens

[0226] In some embodiments, the methods described herein administering an immunosuppressive regimen including one or more immunosuppressive or immunomodulatory drugs to a subject to modulate an immune response in conjunction with administration or implantation of a cell mixture or engineered tissue construct described herein. In some embodiments, the immune response is a humoral response or antibody-mediated response.

[0227] In some embodiments, the immunosuppressive regimen is administered or has previously been administered to prevent graft rejection or promote graft survival.

[0228] The cell mixtures or engineered tissue constructs disclosed herein can be administered in combination with one or more additional immunosuppressive therapies including, but not limited to, drugs which inhibit T-cell activation (e.g.; calcineurin inhibitors (CNI), e.g., tacrolimus (PROGRAF® or; ASTAFRAF®)); systemic immunosuppressants for universal transplant immunotolerance (corticosteroids such as methylprednisolone (MEDROL® or; SOLU-MEDROL®); prednisone or prednisolone); cyclosporine (NEORAL®; SANDIMMUNE® or; GENGRAF®); co-stimulation blockade therapy such as abatacept (ORENCIA®) and belatacept (NULOJIX®); anti-metabolites much as mycophenolate motefil (CELLCEPT® or; MYFORTIC®), mycophenolic acid, mycophenolate sodium, or azathioprine (IMURAN®); mammalian target of rapamycin (mTOR) inhibitors such as sirolimus (RAPAMUNE®) and everolimus (AFINITOR®); T-cell depleting monoclonal antibodies such as muromonab-CD3 (OKT3); alemtuzumab (Campath® or LEMTRADA®); ATG (THYMOBLOBULIN® or; ATGAM®); B-cell depleting monoclonal antibodies such as rituximab (RITUXAN®); proteasome inhibitors such as bortezomib (VELCADE®); IL-2-Ra monoclonal antibodies such as daclizumab (ZENAPAX®); basiliximab (SIMULECT®); , rituximab; lymphocyte integrin blockade monoclonal antibodies such as natalizumab (TYSABRI®); N-Acetyl Cysteine (NAC); hepatitis B vaccine (HEPLISAV-B®); glecaprevir and pibrentasvir (MAVYRET®); sofosbuvir (VOSEVI®); obeticholic acid (OCALIVA®); elbasvir and grazoprevir (ZEPATIER®); cholic acid (CHOLBAM®); daclatasvir (DAKLINZA®); ombitasvir, paritaprevir, and ritonavir (TECHNIVIE™); simeprevir (OLYSIO™); sofosbuvir (SOVALDI®); telaprevir (INCIVEK™); boceprevir (VICTRELIS™); tenofovir disoproxil fumarate (VIREAD®); telbivudine (TYZEKA™); entecavir (BARACLUDE™); adefovir (HEPSERA®); peginterferon alfa-2a (PEGASYS®); peginterferon alfa-2b (PEGINTRON®); or ribavirin and twinrix. Additional agents include glitazones and vitamin E.

[0229] In some embodiments, the cell mixture or engineered tissue constructs disclosed herein can be administered in combination with one or more additional immunosuppressive therapies including but not limited to a PEGylated anti-CD28 monovalent monoclonal antibody fragment (e.g., anti-human CD28 FR104) or domain antibody such as lulizumab (BMS-931699), an IL-2Ra specific antibody for Treg expansion (e.g., a Fc IL-2 mutein (e.g., AMG-592)), a PEGylated IL-2 antibody, a humanized lgG1 anti- CD40L antagonist (e.g., AT-1501 ), a bivalent anti-CD40L domain antibody such as letolizumab (BMS- 986004), an Fc silent human IgG 1 anti-CD40 antibody such as VIB4920 or iscalimab (CFZ533), imlifidase, or a human anti-IL6 monoclonal antibody such as clazakizumab (CSL300). In some embodiments, the one or more immunosuppressive agents comprise a calcineurin inhibitor, a corticosteroid, an antimetabolite, an mTOR inhibitor, a T cell costimulation inhibitor, an antibody, or any combination thereof.

[0230] In some embodiments, (i) the calcineurin inhibitor comprises cyclosporine or tacrolimus; (ii) the corticosteroid comprises prednisone, prednisolone, methylprednisolone, cortisone, or hydrocortisone; (iii) the antimetabolite comprises mycophenolic acid, mycophenolate sodium, mycophenolate mofetil, or azathioprine; (iv) the mTOR inhibitor comprises sirolimus or everolimus; (v) the co-stimulation inhibitor comprises belatacept; and / or (vi) the antibody comprises alemtuzumab, rabbit antithymocyte globulin (rATG), horse antithymocyte globulin (hATG), basiliximab, or rituximab.

[0231] In some embodiments, the immunosuppressive regimen is maintained for 1 day to 6 months (e.g., 1 day to 7 days, e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, e.g., 1 week to 4 weeks, e.g., 1 week, 2 weeks, 3 weeks, or 4 weeks, e.g., 1 month to 6 months, e.g., 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months). In some embodiments, the immunosuppressive regimen is maintained for 7 days to 2 months. In some embodiments, the immunosuppressive regimen is maintained for 14 days to 1 month (e.g., 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or 31 days).

[0232] In some embodiments, the immunosuppressive regimen is withdrawn following engraftment and / or vascularization of the cell mixture or the engineered tissue construct in the subject.

[0233] Methods for Making Engineered Tissue Constructs

[0234] Provided herein are methods for making engineered tissue constructs. In general, the methods for making an engineered tissue construct described herein involve mammalian (e.g., human) cells. The methods for making an engineered tissue construct described herein may involve one or more cell types. Any of the cell mixtures disclosed herein, or aggregates thereof, can be used to produce an engineered tissue construct. In some embodiments, the engineered tissue construct includes hypoimmunogenic cells (e.g., hepatocytes, e.g., primary human hepatocytes (PHH)) and fibroblasts (e.g., human dermal fibroblasts, e.g., normal human dermal fibroblasts, neonatal foreskin fibroblasts, human lung fibroblasts, human ventricular cardiac fibroblasts, human atrial cardiac fibroblasts, human uterine fibroblasts, human bladder fibroblasts, human gingival fibroblasts, human pericardial fibroblasts, human gall bladder fibroblasts, human portal vein fibroblasts, or human vas deferens fibroblasts).

[0235] Frozen master cell banks (MCB) may be sourced through external suppliers and may be received as cryopreserved cells. In some examples, all cell types are terminally differentiated cells isolated from primary donors obtained with appropriate donor consent for therapeutic use. For example, hepatocytes (e.g., PHH) may be obtained from cadaveric donors via collagenase perfusion, Percoll density gradient purification, and subsequent cryopreservation to create an MCB. Hepatocytes may be stored cryopreserved until initiation of a manufacturing build. Prior to accepting the lot as a released MCB, release testing is conducted on hepatocyte (e.g., PHH) candidate MCBs to establish that their performance characteristics meet acceptance criteria for characterization, release, and stability. Fibroblasts e.g., human dermal fibroblasts, e.g., normal human dermal fibroblasts, neonatal foreskin fibroblasts, human lung fibroblasts, human ventricular cardiac fibroblasts, human atrial cardiac fibroblasts, human uterine fibroblasts, human bladder fibroblasts, human gingival fibroblasts, human pericardial fibroblasts, human gall bladder fibroblasts, human portal vein fibroblasts, or human vas deferens fibroblasts) may be, for example, isolated from a single donor of neonatal foreskin by physical separation of dermal and epidermal layers and sequential digestion with dispase and collagenase. After isolation, fibroblasts may be minimally expanded and cryopreserved to create a frozen MCB. Frozen MCBs may be shipped to the manufacturing site and fibroblasts are expanded to create working cell banks (WCB), which may be then cryopreserved until initiation of a manufacturing build. These WCB may be released based on specific acceptance criteria prior to use in the manufacturing process.

[0236] Upon initiation of a manufacturing build, fibroblasts (e.g., human dermal fibroblasts, e.g., normal human dermal fibroblasts, neonatal foreskin fibroblasts, human lung fibroblasts, human ventricular cardiac fibroblasts, human atrial cardiac fibroblasts, human uterine fibroblasts, human bladder fibroblasts, human gingival fibroblasts, human pericardial fibroblasts, human gall bladder fibroblasts, human portal vein fibroblasts, or human vas deferens fibroblasts) may be thawed from their respective WCB expanded, and tested to measure viability and cell count. Hepatocytes (e.g., PHH) may be thawed from the hepatocyte MCB and tested to measure viability and cell count prior to optionally being combined at a ratio (e.g., 1 :2) with fibroblasts, centrifuged into arrays of microwells (e.g., pyramidal microwells), and incubated for 2-3 days to promote self-assembly of the cells into multicellular hepatic aggregates (e.g., spheroidal aggregates). Hepatic aggregates (e.g., spheroidal aggregates) may be deemed acceptable for encapsulation after microscopic confirmation of compaction.

[0237] The cell mixtures or aggregates may then be encapsulated with a solution (e.g., a fibrinogen solution) that is polymerized (e.g., with thrombin). These encapsulation steps may occur within a mold (e.g., a cylindrical mold) that controls the overall dimensions of the engineered tissue construct to be 500 pm to 5 mm in thickness and with an outer diameter of 6 mm to 100 cm (e.g., 7 mm to 999 mm, 8 mm to 998 mm, 9 mm to 997 mm, 10 mm to 996 mm, 20 mm to 995 mm, 30 mm to 990 mm, 40 mm to 980 mm, 60 mm to 960 mm, 90 mm to 930 mm, 100 mm to 900 mm, 200 mm to 800 mm, 300 mm to 700 mm, 400 mm to 600 mm, or 500 mm). The thickness may be controlled by the volume of cell-hydrogel suspension and targeted to be 2 mm in thickness.

[0238] In some embodiments, the mold may be any shape (e.g., cylindrical, square, or square with rounded corners).

[0239] Within the solution (e.g., a fibrinogen solution that is polymerized e.g., with thrombin), the hypoimmunogenic cell / fibroblast aggregates are allowed to non-homogenously distribute (e.g., by gravity) along the z-axis of the biocompatible scaffold into a layer (e.g., to settle), thereby forming a one-sided engineered tissue construct.

[0240] In some embodiments, two or more of the one-sided engineered tissue constructs are assembled with each of the layers facing outwardly, respectively, thereby forming a two-sided engineered tissue construct.

[0241] The engineered tissue constructs of the present disclosure can be formed by a process described herein. In some embodiments, engineered tissue constructs with defined cellular configurations in a biocompatible hydrogel scaffold may be prepared by photopatterning PEG hydrogels containing the hypoimmunogenic cell and fibroblast populations, resulting in a hydrogel network consisting of 3D cell hypoimmunogenic cells and fibroblasts. Further control of cell orientation within these patterned domains may be achieved utilizing dielectrophoretic patterning techniques. Dielectrophoresis (DEP) can be used alone for patterning of cells in relatively homogeneous slabs of hydrogel or in conjunction with the photopolymerization method.

[0242] In some embodiments, organizing cells and material into spatial arrangements, such as engineered tissue constructs, can be accomplished by physically constraining the placement of cells / material by the use of wells or grooves, or injecting cells into microfluidic channels or oriented void spaces / pores. In certain embodiments, the cells can be organized by physically positioning cells with electric fields, magnetic tweezers, optical tweezers, ultrasound waves, pressure waves, or micromanipulators. In some embodiments, the population of hypoimmunogenic cells and the population of fibroblasts are aggregated in spheroids and the spheroids are allowed (e.g., by gravity) to non- homogenously distribute along the z-axis of the biocompatible scaffold into a layer.

[0243] In certain embodiments, the method for fabricating engineered tissue constructs and embedding the constructs in extracellular matrix includes (1 ) generating 3D templates that have been defined with channels or trenches, (2) suspending the population of cells and the population of cells in liquid collagen and centrifuging these cells into the channels of the template, (3) removing excess cell / collagen suspension to allow aggregates to form, and (4) removing aggregates from templates via encapsulation in an extracellular matrix scaffold.

[0244] In some embodiments, the method for fabricating the engineered tissue constructs includes (1 ) suspending the population of cells in a naturally derived and / or synthetic scaffolding, (2) placing the suspended cells into the channels of a 3D template, and (3) allowing the cells to form one or more aggregates at least partially embedded in the naturally derived and / or synthetic scaffolding. In some embodiments, the 3D template can be generated by molding, templating, photolithography, printing, deposition, sacrificial molding, stereolithography, or a combination thereof.

[0245] In some embodiments, an engineered tissue construct can be fabricated through the use a custom 3D printer technology to extrude lattices of carbohydrate glass filaments with predefined diameters, spacings and orientations. For example, in some embodiments, soluble (clinical-grade, sterile) fibrinogen and thrombin are combined and poured over the lattice. After the solution has polymerized into insoluble fibrin, the carbohydrate filaments are dissolved, leaving behind channels within the fibrin. The channels can then be filled with a suspension of cells in a naturally derived or synthetic scaffolding (e.g., soluble type I collagen) that subsequently is polymerized to trap the cells within the channels.

[0246] The methods allow for the formation of three-dimensional scaffolds from hundreds of micrometers to tens of centimeters in length and width, and tens of micrometers to hundreds of micrometers in height. A resolution of up to 100 micrometers in the photopolymerization method and possible single cell resolution (10 pm) in the DEP method is achievable. Photopolymerization apparatus, DEP apparatus, and other methods to produce 3-dimensional co-cultures are described in U.S. Pat. No. 8,906,684, which is incorporated herein by reference.

[0247] The cells can be cultured in vitro under various culture conditions. The cells (e.g., primary cells) can be expanded in culture, e.g., grown under conditions that promote their proliferation. Culture medium can be liquid or semi-solid, e.g., containing agar, methylcellulose, and the like. The cell population can be suspended in an appropriate nutrient medium, such as Iscove's modified DMEM or RPMI 1640, normally supplemented with fetal calf serum (5-10%), L-glutamine, a thiol, particularly 2-mercaptoethanol, and antibiotics, e.g., penicillin and streptomycin. The culture can contain growth factors to which the regulatory T cells are responsive. Growth factors, as defined herein, can be molecules capable of promoting survival, growth and / or differentiation of cells, either in culture or in the intact tissue, through specific effects on a transmembrane receptor. Growth factors include polypeptides and non-polypeptide factors.

[0248] The cells produced by the methods described herein can be used immediately in the making of an engineered tissue construct. Alternatively, the cells can be frozen at liquid nitrogen temperatures and stored for long periods of time, being thawed and capable of being reused. For example, the cells can be frozen in 10% dimethylsulfoxide (DMSO), 50% serum, 40% buffered medium, or some other such solution as is commonly used in the art to preserve cells at such freezing temperatures and thawed in a manner as commonly known in the art for thawing frozen cultured cells.

[0249] Methods of Treatment

[0250] The cell mixtures and engineered tissue constructs described herein may be used to treat a disease or disorder in a subject, e.g., a human subject in need thereof. An engineered tissue construct containing a population of mammalian (e.g., human) cells that is implanted into a human subject may be used to treat, for example, a liver disease, diabetes (e.g., type 2 diabetes or type 2 diabetes), an endocrine disorder, a hormonal deficiency, a protein deficiency, impaired biotransformation, or a disease of impaired protein synthesis.

[0251] A cell mixture or an engineered tissue construct may be administered or implanted into a human subject to treat a disease or disorder associated with the liver. The liver disease may be, for example, acute liver failure, acute-on-chronic liver failure, congenital bile acid synthesis defect, Crigler-Najjar syndrome, end stage liver disease, familial hypercholesterolemia, familial hypobetalipoproteinemia, glycogen storage disorder type 1 a, glycogen storage disorder type 4 (Andersen), hepatic encephalopathy, Hunter syndrome (MPS II), infantile refsum disease, lysosomal acid lipase deficiency (LAL-D) (cholesteryl ester storage disease), maple syrup urine disease, Maroteaux-Lamy (MPS VI), methylmalonic acidemia, ornithine transcarbamylase (OTC) deficiency, propionic acidemia, a urea cycle disorder, alpha-1 antitrypsin deficiency, Niemann-Pick type A / B, Niemann-Pick type C, primary hyperoxaluria type 1 , primary hyperoxaluria type 2, primary hyperoxaluria type 3, pyruvate kinase deficiency, liver type, Wilson’s disease, D-bifunctional protein deficiency, Gaucher’s disease, Hurler syndrome (MPS I), hypophosphatasia, morquio A (MPS 4A), morquio B (MPS 4B), multiple acyl-coa dehydrogenase deficiency, Sanfilippo (MPS III), acute hepatic porphyrias (AHP), glycogen storage disorder type 3 (Cori), hereditary angioedema (HAE), hereditary hemochromatosis, homocystinuria cystathionine B-synthase deficiency, isovaleric acidemia, N-acetyglutamate synthetase deficiency (NAGS), pseudoxanthoma elasticum, tyrosinemia, 3-methylcrotonyl-CoA carboxylase deficiency (3-MCC), acute fatty liver of pregnancy, congenital factor V deficiency, congenital factor XI deficiency, congenital fibrinogen disorder, corticosteroid-binding globulin deficiency, cutaneous hepatic porphyrias, Fabry disease, factor VII deficiency, factor X deficiency, familial dysbetalipoproteinemia, familial hypertriglyceridemia, familial lipoprotein lipase deficiency, galactosemia, glutaric acidemia type 1 , glycogen storage disorder type 1 b, glycogen storage disorder type 6 (Hers), hemophilia type A (factor VIII deficiency), hemophilia type B (factor IX), hereditary fructose intolerance, long-chain L-3 hydroxyacyl-CoA dehydrogenase deficiency (LCHAD) deficiency, very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency, 3-hydroxy-3- methylgl utaryl-CoA synthase deficiency, abetalipoproteinemia, acetyl-CoA acetyltransferase-2 deficiency, adenosine kinase deficiency, adult polyglucosan body disease, delta-aminolevulinic acid (ALA) dehydratase (ALAD)-deficiency porphyria, alpha-2-plasmin inhibitor deficiency, aminolaevulinic acid dehydratase deficiency porphyria, atransferrinemia, beta-ketothiolase deficiency, bile acid CoA ligase deficiency and defective amidation, carboxypeptidase N deficiency, cerebral creatine deficiency syndrome 1 , cerebral creatine deficiency syndrome 2, cerebral creatine deficiency syndrome 3, Chanarin- Dorfman syndrome, cirrhosis - dystonia - polycythemia - hypermanganesemia syndrome, combined oxidative phosphorylation deficiency 1 , congenital disorder of deglycosylation, carnitine palmitoyltransferase (CPT) Deficiency, hepatic, type la, deoxyguanosine kinase deficiency, formiminoglutamic aciduria, gamma-glutamylcysteine synthetase deficiency, hepatic lipase deficiency, hepatic tuberculosis, Indian childhood cirrhosis, infantile liver failure syndrome, Lucey-Driscoll syndrome, mitochondrial DNA depletion syndrome, tangier disease, trifunctional protein deficiency, 3-hydroxyacyl- coenzyme A dehydrogenase deficiency, acyl-CoA oxidase deficiency, 3-hydroxy-3-methylglutaric aciduria, 2-methylbutyryl-coa dehydrogenase deficiency, acatalasemia, acquired fructose intolerance, cerebrotendinous xanthomatosis, conjugated hyperbilirubinemia (rotor syndrome), cystic echinococcosis, drug-induced hepatitis, Dubin-Johnson syndrome, focal fatty liver, Gilbert syndrome, glycine n- methyltransferase deficiency, hepatitis A, hepatitis E, liver abscess, liver fibrosis, nodular regenerative hyperplasia, nonalcoholic fatty liver disease (NAFLD), peliosis hepatis, phenylketonuria, short-chain acyl- CoA dehydrogenase deficiency (SCAD), trimethylaminuria, visceral steatosis, vitamin k-dependent clotting factors, combined deficiency of, type 1 and type 2, acute cholangitis I biliary tract infection, alagille syndrome, alphavirus infection, alveolar hydatid disease, benign postoperative, cholestasis, benign recurrent intrahepatic cholestasis, bile acid malabsorption, primary, bile duct cysts, Budd-Chiari syndrome, Caroli disease, cholestasis, clonorchiasis, congenital disorders of glycosylation, congenital hepatic fibrosis, erythropoietic protoporphyrias, familial amyloidosis, familial hypercholanemia, flavivirus infection, hepatic infarction, hepatic veno-occlusive disease, hepatolithiasis, hepatoportal sclerosis, hereditary hemorrhagic telangiectasia, lgG4-related sclerosing cholangitis, intrahepatic cholestasis, isolated neonatal sclerosing cholangitis, non-cirrhotic portal fibrosis, opisthorchiasis, polycystic liver disease, portal hypertension, portal vein thrombosis, primary biliary cholangitis, primary sclerosing cholangitis, progressive familial intrahepatic cholestasis, Reye syndrome, Reynolds syndrome, spontaneous bacterial peritonitis, von Willebrand disease type 3, biliary atresia, biliary dyskinesia, biliary reflux, cholecystitis, cholelithiasis, alcoholic hepatitis, alcoholic liver disease, autoimmune hepatitis, cystic fibrosis liver disease, hepatitis D, hepatotoxicity, lgG4-related hepatopathy, liver cirrhosis, nonalcoholic steatohepatitis (NASH), hyperammonemia, TIPS-induced hyperammonemia, or small for size syndrome.

[0252] In some embodiments, the liver disease is acute liver failure, acute-on-chronic liver failure, congenital bile acid synthesis defect, Crigler-Najjar syndrome, end stage liver disease, familial hypercholesterolemia, familial hypobetalipoproteinemia, glycogen storage disorder type 1 a, glycogen storage disorder type 4 (Andersen), hepatic encephalopathy, Hunter syndrome (MPS II), infantile refsum disease, lysosomal acid lipase deficiency (LAL-D) (cholesteryl ester storage disease), maple syrup urine disease, Maroteaux-Lamy (MPS VI), methylmalonic acidemia, ornithine transcarbamylase (OTC) deficiency, propionic acidemia, or a urea cycle disorder. In some embodiments, the liver disease is acute liver failure, a urea cycle disorder, or Crigler- Najjar syndrome.

[0253] Other diseases and disorders may also be treated. For example, in some embodiments, any disease or disorder that may be treated by implantation of one or more cell types as disclosed herein may be treated.

[0254] For example, in some embodiments, the engineered tissue construct includes pancreatic cells, and the disease is a pancreas-associated disease. For example, in some embodiments, the disease is diabetes (e.g., type 1 diabetes or type 2 diabetes) or pancreatitis. In some examples, the disease is type 1 diabetes. For example, for treatment of type 1 diabetes or pancreatitis, the method may include implanting one or more engineered tissue constructs that include pancreatic cells (e.g., alpha, beta, gamma, delta, epsilon cells, or any combination thereof) at any of the implantation sites described herein. In some embodiments, the pancreatic cells comprise beta cells.

[0255] In another example, the engineered tissue construct comprises adrenal cells, and the disease is an adrenal-associated disease (e.g., Addison’s syndrome, hypercortisolism, or Cushing’s syndrome).

[0256] In another example, the engineered tissue construct comprises parathyroid cells, and the disease is a parathyroid-associated disease (e.g., hypoparathyroidism).

[0257] In another example, the engineered tissue construct comprises thyroid cells, and the disease is a thyroid-associated disease (e.g., hypothyroidism or hyperthyroidism).

[0258] In another example, the engineered tissue construct comprises ovarian cells, and the disease is an ovarian-associated disease.

[0259] In another example, the engineered tissue construct comprises enteroendocrine cells (e.g., L cells and / or chromaffin cells), and the disease is an enteroendocrine cell-associated disease.

[0260] In some embodiments, one or more cell types (e.g., hepatocytes, pancreatic cells, and / or fibroblasts) for an engineered tissue construct are obtained from a subject that has been diagnosed with a disease or disorder (e.g., a liver disease or disorder, a pancreas-associated disease, an adrenal- associated disease, such as one or more of the specific diseases and disorders described herein). In some embodiments, the subject is an adult or a pediatric patient (e.g., a human patient that is younger than 18 years old). The obtained cells may be isolated and then expanded ex vivo under conditions that preserve or restore proliferative and functional capacity. At a time point before or after expansion, the obtained cells may be genetically modified. As an example, the cells obtained from the subject may be hepatocytes. In some embodiments, the hepatocytes obtained from the subject are reprogrammed at a time point prior to or after expansion to enhance hepatocyte function. In some embodiments, at a time point before or after expansion, the hepatocytes are genetically modified (e.g., via a method described herein) to correct a pathogenic mutation associated with a liver disease or disorder (e.g., a liver disease or disorder of the subject, e.g., a liver disease or disorder described herein). In some embodiments, genetic modification is performed using a viral vector (e.g., a lentiviral vector or adeno-associated virus (AAV) vector) or a non-viral vector. In some embodiments, genetic modification is performed using an mRNA-mediated approach. Genetic correction strategies include cDNA insertion, gene replacement, exon skipping, and gene editing approaches, such as CRISPR / Cas9, base editing, or prime editing. In some embodiments, gene editing targets include loci that are known to be associated with a liver disorder, such as ornithine transcarbamylase (OTC), fumarylacetoacetate hydrolase (FAH), carbamoyl phosphate synthetase 1 (CPS1 ), argininosuccinate lyase (ASL), or other loci described herein or known in the art. In some embodiments, the hepatocytes obtained from the subject are reprogrammed at a time point prior to or after expansion to enhance hepatocyte function. In some embodiments, the hepatocytes obtained from the subject are expanded and / or matured (e.g., expanded and / or matured ex vivo) but are not genetically modified. The obtained cells (e.g., expanded, reprogrammed, engineered, and / or matured cells; e.g., hepatocytes, pancreatic cells, and / or fibroblasts) may be encapsulated in an engineered tissue construct (e.g., via a method described herein) and then administered to the same subject from which the one or more cells were obtained (i.e., an autologous cell therapy) via any suitable route of administration, e.g., a route of administration described herein. The engineered tissue construct may be administered to the same subject as an autologous cell therapy to treat a disease or disorder (e.g., a disease or disorder described herein) in the subject.

[0261] Kits

[0262] The compositions described herein can be provided in a kit. The kit can include a package insert that instructs a user of the kit, such as a physician, to implant the cell mixture or engineered tissue construct. The kit may optionally include surgical equipment or another device for implanting the engineered tissue construct. In some embodiments, the kit may include one or more additional therapeutic agents.

[0263] Examples

[0264] The following examples are put forth so as to provide those of ordinary skill in the art with a description of how the compositions and methods described herein may be used and evaluated and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.

[0265] Example 1 : Cell Mixtures and Engineered Tissue Constructs Containing Fibroblasts and Hepatocytes

[0266] Cell mixtures that include fibroblasts and hypoimmunogenic hepatocytes are cultured together to form aggregates, e.g., as described in U.S. Provisional Patent Application No. 63 / 346,582, U.S. Provisional Patent Application No. 63 / 346,575, U.S. Provisional Patent Application No. 63 / 419,260, or International Patent Application No. PCT / US2022 / 047760, each of which is incorporated by reference herein in its entirety. In some examples, the hypoimmunogenic hepatocytes are genetically engineered to lack expression of one or more of the following endogenous genes: beta-2-microglobulin (B2M), human leukocyte antigen (HLA)-A, HLA-B, HLA-C, class II major histocompatibility complex transactivator (CIITA), PVR cell adhesion molecule (PVR), or any combination thereof, and / or to express one or more of the following genes: cluster of differentiation (CD) 47, HLA-C, HLA-E, HLA-G, programmed death-ligand 1 (PD-L1 ), programmed death-ligand 1 (PD-L2), B7-H2, B7-H3, B7-H4, cytotoxic T-lymphocyte associated protein 4 (CTLA4), CD39, CD73, CD24, CD27, CD35, CD46, CD55, CD59, CD155, CD200, indoleamine 2, 3-dioxygenase 1 (IDO1 ), interleukin (IL)-10, IL-35, Fas ligand (FASL), CC motif chemokine ligand 21 (CCL21 ), milk fat globule-EGF factor 8 protein (MFG-E8), serpin B9 (SERPINB9), double homeobox 4 (DUX4), TGFB1 , carcinoembryonic antigen (CEA) cell adhesion molecule 1 (CEACAM1 ), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3; also known as HAVCR2), lymphocyte-activation gene 3 (LAG-3), adenosine A2a receptor (A2AR; also known as ADORA2A), B And T Lymphocyte Associated (BTLA), killer Ig-like receptor (KIR), V-domain Ig suppressor of T cell activation (VISTA), or any combination thereof.

[0267] In some examples, engineered tissue constructs are formed using the aggregates, e.g., as described in U.S. Provisional Patent Application No. 63 / 346,529, US Provisional Patent Application No. 63 / 419,254, or International Patent Application No. PCT / US2022 / 047730, each of which is incorporated by reference herein in its entirety.

[0268] The aggregate or engineered tissue construct is administered or implanted into a human subject, e.g., as described in U.S. Patent Application No. 17 / 827,128 or International Patent Application No. PCT / US2022 / 047764, each of which is incorporated by reference herein in its entirety. The aggregate or engineered tissue construct is administered or implanted into the human subject in combination with an immunosuppressive regimen, e.g., including tacrolimus, cyclosporine, mycophenolate mofetil, azathioprine, everolimus, sirolimus, a corticosteroid, or a combination thereof. The immunosuppressive regimen is maintained while the aggregate or engineered tissue construct engrafts and / or vascularizes in the subject, e.g., for 14 days to 1 month. Following engraftment and / or vascularization, the immunosuppressive regimen is withdrawn, allowing for removal of the fibroblasts from the aggregate or engineered tissue construct via the immune system, leaving behind the hypoimmunogenic hepatocytes, which can be used to treat a liver disease as described herein.

[0269] It is expected that the methods described herein will retain the increased viability and vascularization conferred by the presence of the fibroblasts in the aggregate or engineered tissue construct while allowing their removal following successful engraftment and / or vascularization. This approach allows for using non-genetically modified allogeneic fibroblasts in the aggregates or engineered tissue constructs while reducing the need for long-term immunosuppression.

[0270] Example 2: Cell Mixtures and Engineered Tissue Constructs Containing Fibroblasts and Pancreatic Cells

[0271] Cell mixtures that include fibroblasts and hypoimmunogenic pancreatic cells (e.g., beta cells) are cultured together to form aggregates, e.g., as described in U.S. Provisional Patent Application No. 63 / 346,582, U.S. Provisional Patent Application No. 63 / 346,575, U.S. Provisional Patent Application No. 63 / 419,260, or International Patent Application No. PCT / US2022 / 047760. In some examples, the hypoimmunogenic pancreatic cells (e.g., beta cells) are genetically engineered to lack expression of one or more of the following endogenous genes: B2M, HLA-A, HLA-B, HLA-C, CIITA, PVR, or any combination thereof, and / or to express one or more of the following genes: CD47, HLA-C, HLA-E, HLA-G, PD-L1 , PD-L2, B7-H2, B7-H3, B7-H4, CTLA4, CD39, CD73, CD24, CD27, CD35, CD46, CD55, CD59, CD155, CD200, ID01 , IL-10, IL-35, FASL, CCL21 , MFG-E8, SERPINB9, DUX4, TGFB1 , CEACAM1 , TIM-3, LAG-3, A2AR, BTLA, KIR, VISTA, or any combination thereof.

[0272] In some examples, engineered tissue constructs are formed using the aggregates, e.g., as described in U.S. Provisional Patent Application No. 63 / 346,529, US Provisional Patent Application No. 63 / 419,254, or International Patent Application No. PCT / US2022 / 047730.

[0273] The aggregate or engineered tissue construct is administered or implanted into a human subject, e.g., as described in U.S. Patent Application No. 17 / 827,128 or International Patent Application No. PCT / US2022 / 047764. The aggregate or engineered tissue construct is administered or implanted into the human subject in combination with an immunosuppressive regimen, e.g., including tacrolimus, cyclosporine, mycophenolate mofetil, azathioprine, everolimus, sirolimus, a corticosteroid, or a combination thereof. The immunosuppressive regimen is maintained while the aggregate or engineered tissue construct engrafts and / or vascularizes in the subject, e.g., for 14 days to 1 month. Following engraftment and / or vascularization, the immunosuppressive regimen is withdrawn, allowing for removal of the fibroblasts from the aggregate or engineered tissue construct via the immune system, leaving behind the hypoimmunogenic pancreatic cells (e.g., beta cells). For example, in the case of hypoimmunogenic beta cells, the beta cells can secrete insulin and treat diabetes (e.g., type 1 diabetes or type 2 diabetes) in the human subject.

[0274] It is expected that the methods described herein will retain the increased viability and vascularization conferred by the presence of the fibroblasts in the aggregate or engineered tissue construct while allowing their removal following successful engraftment and / or vascularization. This approach allows for using non-genetically modified allogeneic fibroblasts in the aggregates or engineered tissue constructs while reducing the need for long-term immunosuppression.

[0275] Example 3: Cell Mixtures and Engineered Tissue Constructs Containing Fibroblasts Having Kill Switches and Pancreatic Cells

[0276] Cell mixtures that include fibroblasts and hypoimmunogenic pancreatic cells (e.g., beta cells) are cultured together to form aggregates, e.g., as described in U.S. Provisional Patent Application No. 63 / 346,582, U.S. Provisional Patent Application No. 63 / 346,575, U.S. Provisional Patent Application No. 63 / 419,260, or International Patent Application No. PCT / US2022 / 047760. In some examples, the hypoimmunogenic pancreatic cells (e.g., beta cells) are genetically engineered to lack expression of one or more of the following endogenous genes: B2M, HLA-A, HLA-B, HLA-C, CIITA, PVR, or any combination thereof, and / or to express one or more of the following genes: CD47, HLA-C, HLA-E, HLA-G, PD-L1 , PD-L2, B7-H2, B7-H3, B7-H4, CTLA4, CD39, CD73, CD24, CD27, CD35, CD46, CD55, CD59, CD155, CD200, IDO1 , IL-10, IL-35, FASL, CCL21 , MFG-E8, SERPINB9, DUX4, TGFB1 , CEACAM1 , TIM-3, LAG-3, A2AR, BTLA, KIR, VISTA, or any combination thereof. In this example, the hypoimmunogenic pancreatic cells (e.g., beta cells) also are genetically engineered to contain a kill switch such as inducible caspase 9 suicide gene.

[0277] In some examples, engineered tissue constructs are formed using the aggregates, e.g., as described in U.S. Provisional Patent Application No. 63 / 346,529, US Provisional Patent Application No. 63 / 419,254, or International Patent Application No. PCT / US2022 / 047730.

[0278] The aggregate or engineered tissue construct is administered or implanted into a human subject, e.g., as described in U.S. Patent Application No. 17 / 827,128 or International Patent Application No. PCT / US2022 / 047764. The aggregate or engineered tissue construct is administered or implanted into the human subject in combination with an immunosuppressive regimen, e.g., including tacrolimus, cyclosporine, mycophenolate mofetil, azathioprine, everolimus, sirolimus, a corticosteroid, or a combination thereof. The immunosuppressive regimen is maintained while the aggregate or engineered tissue construct engrafts and / or vascularizes in the subject, e.g., for 14 days to 1 month. Following engraftment and / or vascularization, the immunosuppressive regimen is withdrawn, allowing for removal of the fibroblasts from the aggregate or engineered tissue construct via the immune system, leaving behind the hypoimmunogenic pancreatic cells (e.g., beta cells). Concomitant with withdrawal of the immunosuppression, the subject may be administered a cell-permeable synthetic ligand (e.g., rapamycin, FK506, AP1903, or AP20187) that binds to FKBP12 to activate the inducible caspase 9.

[0279] It is expected that the methods described herein will retain the increased viability and vascularization conferred by the presence of the fibroblasts in the aggregate or engineered tissue construct while allowing their removal following successful engraftment and / or vascularization. This approach allows for using non-genetically modified allogeneic fibroblasts in the aggregates or engineered tissue constructs while reducing the need for long-term immunosuppression.

[0280] Example 4: Cell Mixtures and Engineered Tissue Constructs Containing Hypoimmunogenic Fibroblasts and Pancreatic Cells containing Kill Switches without Immunosuppression

[0281] Cell mixtures that include hypoimmunogenic fibroblasts and hypoimmunogenic pancreatic cells (e.g., beta cells) are cultured together to form aggregates, e.g., as described in U.S. Provisional Patent Application No. 63 / 346,582, U.S. Provisional Patent Application No. 63 / 346,575, U.S. Provisional Patent Application No. 63 / 419,260, or International Patent Application No. PCT / US2022 / 047760. In some examples, the hypoimmunogenic fibroblasts and the hypoimmunogenic pancreatic cells (e.g., beta cells) are genetically engineered to lack expression of one or more of the following endogenous genes: B2M, HLA-A, HLA-B, HLA-C, CIITA, PVR, or any combination thereof, and / or to express one or more of the following genes: CD47, HLA-C, HLA-E, HLA-G, PD-L1 , PD-L2, B7-H2, B7-H3, B7-H4, CTLA4, CD39, CD73, CD24, CD27, CD35, CD46, CD55, CD59, CD155, CD200, IDO1 , IL-10, IL-35, FASL, CCL21 , MFG-E8, SERPINB9, DUX4, TGFB1 , CEACAM1 , TIM-3, LAG-3, A2AR, BTLA, KIR, VISTA, or any combination thereof. In this example, the hypoimmunogenic pancreatic cells (e.g., beta cells) also are genetically engineered to contain a kill switch such as inducible caspase 9 suicide gene.

[0282] In some examples, engineered tissue constructs are formed using the aggregates, e.g., as described in U.S. Provisional Patent Application No. 63 / 346,529, US Provisional Patent Application No. 63 / 419,254, or International Patent Application No. PCT / US2022 / 047730.

[0283] The aggregate or engineered tissue construct is administered or implanted into a human subject, e.g., as described in U.S. Patent Application No. 17 / 827,128 or International Patent Application No. PCT / US2022 / 047764. Following engraftment and / or vascularization of the hypoimmunogenic pancreatic cells (e.g., beta cells), the subject may be administered a cell-permeable synthetic ligand (e.g., rapamycin, FK506, AP1903, or AP20187) that binds to FKBP12 to activate the inducible caspase 9.

[0284] It is expected that the methods described herein will retain the increased viability and vascularization conferred by the presence of the fibroblasts in the aggregate or engineered tissue construct while allowing their removal following successful engraftment and / or vascularization.

[0285] Other Embodiments

[0286] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.

[0287] Other embodiments are within the claims.

Claims

CLAIMS1 . A cell mixture comprising:(i) a first population of cells comprising fibroblasts; and(ii) a second population of cells comprising hypoimmunogenic cells.

2. The cell mixture of claim 1 , wherein the fibroblasts comprise primary fibroblasts, induced pluripotent stem cell (iPSC)-derived fibroblasts, or embryonic stem cell (ESC)-derived fibroblasts.

3. The cell mixture of claim 2, wherein the primary fibroblasts comprise normal human dermal fibroblasts (NHDF) or neonatal foreskin fibroblasts.

4. The cell mixture of any one of claims 1 -3, wherein the fibroblasts are not genetically engineered.

5. The cell mixture of any one of claims 1 -3, wherein (i) the fibroblasts are genetically engineered fibroblasts; and / or (ii) the fibroblasts are expanded and / or matured in vitro.

6. The cell mixture of claim 5, wherein the genetically engineered fibroblasts comprise a kill switch.

7. The cell mixture of claim 6, wherein the kill switch comprises a suicide gene or an epitope marker.

8. The cell mixture of claim 7, wherein the suicide gene comprises herpes simplex virus-thymidine kinase (HSV-TK) or a caspase 9 suicide gene.

9. The cell mixture of claim 8, wherein the caspase 9 suicide gene is an inducible caspase 9 suicide gene.

10. The cell mixture of claim 7, wherein the epitope marker comprises a truncated epidermal growth factor receptor (EGFR) variant (huEGFRt) marker or a cluster of differentiation 20 (CD20) epitope marker.11 . The cell mixture of any one of claims 1 -10, wherein the fibroblasts are immunogenic.

12. The cell mixture of any one of claims 1 -11 , wherein the fibroblasts are human fibroblasts.

13. The cell mixture of any one of claims 1 -12, wherein the hypoimmunogenic cells comprise parenchymal cells.

14. The cell mixture of claim 13, wherein the parenchymal cells comprise hepatocytes, hepatocyte precursor cells, pancreatic cells, pancreatic precursor cells, myocytes, neurons, enterocytes, adipocytes, splenic cells, kidney cells, biliary cells, Kupffer cells, stellate cells, cardiac muscle cells, alveolar cells, bronchiolar cells, club cells, urothelial cells, mucous cells, parietal cells, chief cells, G cells, goblet cells, enteroendocrine cells, Paneth cells, M cells, tuft cells, glial cells, gall bladder cells, keratinocytes,melanocytes, Merkel cells, Langerhans cells, osteocytes, osteoclasts, esophageal cells, photoreceptor cells, or corneal epithelial cells.

15. The cell mixture of claim 13 or 14, wherein the parenchymal cells comprise hepatocytes or hepatocyte precursor cells.

16. The cell mixture of claim 14 or 15, wherein the hepatocytes comprise primary human hepatocytes, iPSC-derived hepatocytes, or ESC-derived hepatocytes, expanded hepatocytes, or expanded and partially reprogrammed hepatocytes.

17. The cell mixture of any one of claims 14-16, wherein the hepatocytes are genetically engineered hepatocytes.

18. The cell mixture of claim 14, wherein the parenchymal cells comprise pancreatic cells or pancreatic precursor cells.

19. The cell mixture of claim 18, wherein the pancreatic cells comprise pancreatic endocrine cells or pancreatic endocrine cells.

20. The cell mixture of claim 18, wherein the pancreatic cells comprise alpha, beta, gamma, delta, or epsilon cells, or a combination thereof.21 . The cell mixture of any one of claims 18-20, wherein the pancreatic cells comprise primary human pancreatic cells, iPSC-derived pancreatic cells, or ESC-derived pancreatic cells.

22. The cell mixture of any one of claims 18-21 , wherein the pancreatic cells are genetically engineered pancreatic cells.

23. The cell mixture of any one of claims 1 -22, wherein the hypoimmunogenic cells lack expression of one or more of the following endogenous genes: beta-2-microglobulin (B2M), human leukocyte antigen (HLA)-A, HLA-B, HLA-C, class II major histocompatibility complex transactivator (CIITA), PVR cell adhesion molecule (PVR), or any combination thereof.

24. The cell mixture of claim 23, wherein the hypoimmunogenic cells comprise one or more alterations that inactivate the endogenous B2M, HLA-A, HLA-B, HLA-C, CIITA, or PVR gene.

25. The cell mixture of any one of claims 1 -24, wherein the hypoimmunogenic cells express one or more of the following genes: cluster of differentiation (CD) 47, HLA-C, HLA-E, HLA-G, programmed deathligand 1 (PD-L1 ), programmed death-ligand 1 (PD-L2), B7-H2, B7-H3, B7-H4, cytotoxic T-lymphocyte associated protein 4 (CTLA4), CD39, CD73, CD24, CD27, CD35, CD46, CD55, CD59, CD155, CD200, indoleamine 2, 3-dioxygenase 1 (IDO1 ), interleukin (IL)-10, IL-35, Fas ligand (FASL), CC motif chemokineligand 21 (CCL21 ), milk fat globule-EGF factor 8 protein (MFG-E8), serpin B9 (SERPINB9), double homeobox 4 (DUX4), TGFB1 , carcinoembryonic antigen (CEA) cell adhesion molecule 1 (CEACAM1 ), T- cell immunoglobulin and mucin-domain containing-3 (TIM-3), lymphocyte-activation gene 3 (LAG-3), adenosine A2a receptor (A2AR), B And T Lymphocyte Associated (BTLA), killer Ig-like receptor (KIR), V- domain Ig suppressor of T cell activation (VISTA), or any combination thereof.

26. The cell mixture of claim 25, wherein the expression of the one or more genes is controlled by one or more inducible promoters or constitutive promoters.

27. The cell mixture of claim 25 or 26, wherein the hypoimmunogenic cells overexpress one or more of CD47, HLA-C, HLA-E, HLA-G, PD-L1 , PD-L2, B7-H2, B7-H3, B7-H4, CTLA4, CD39, CD73, CD24, CD27, CD35, CD46, CD55, CD59, CD155, CD200, IDO1 , IL-10, IL-35, FASL, CCL21 , MFG-E8, SERPINB9, DUX4, TGFB1 , CEACAM1 , TIM-3, LAG-3, A2AR, BTLA, KIR, VISTA, or any combination thereof relative to a wild-type cell of the same cell type as the hypoimmunogenic cell.

28. The cell mixture of any one of claims 1 -27, wherein a ratio of the first population of cells to the second population of cells is from 10:1 to 1 :10.

29. The cell mixture of any one of claims 1 -28, wherein the first population of cells is present at a density of 1 x 104cells / mL to 1 x 108cells / mL.

30. The cell mixture of claim 29, wherein the first population of cells is present at a density of 1 x 105cells / mL to 3 x 107cells / mL.31 . The cell mixture of claim 30, wherein the density of the fibroblasts is 6 x 105cells / mL.

32. The cell mixture of any one of claims 1 -31 , wherein the second population of cells are present at a density of 1 x 104cells / mL to 1 x 108cells / mL.

33. The cell mixture of claim 32, wherein the second population of cells comprises hepatocytes and the density of the hepatocytes is 1 x 105cells / mL to 3 x 107cells / mL.

34. The cell mixture of claim 33, wherein the density of the hepatocytes is 3 x 105cells / mL.

35. The cell mixture of any one of claims 1 -34, wherein the hypoimmunogenic cells are human cells.

36. The cell mixture of any one of claims 1 -35, wherein the cell mixture further comprises one or more additional cell populations.

37. The cell mixture of any one of claims 1 -36, which is an aggregate.

38. An engineered tissue construct suitable for implantation into a subject, comprising the cell mixture of any one of claims 1 -37 in a biocompatible scaffold.

39. The engineered tissue construct of claim 38, wherein the biocompatible scaffold comprises fibrin.

40. The engineered tissue construct of claim 39, wherein the fibrin is human fibrin.41 . The engineered tissue construct of any one of claims 38-40, further comprising a reinforcing agent.

42. The engineered tissue construct of claim 41 , wherein the reinforcing agent comprises fibrin, surgical mesh, alginate, collagen, polyethylene glycol), polyvinylidene acetate (PVDA), polyvinylidene fluoride (PVDF), poly(lactic-co-glycolic) acid (PLGA), and poly (l-lactic acid) (PLLA).

43. The engineered tissue construct of any one of claims 38-42, further comprising a population of endothelial cells.

44. The engineered tissue construct of claim 43, wherein the population of endothelial cells is arranged as one or more cords.

45. A kit comprising the cell mixture of any one of claims 1 -37 or the engineered tissue construct of any one of claims 38-44 and instructions to implant the cell mixture or the engineered tissue construct into a subject in need thereof.

46. A method for promoting engraftment and / or vascularization of a population of hypoimmunogenic cells in a subject in need thereof, the method comprising:(a) administering or implanting the cell mixture of any one of claims 1 -37 or the engineered tissue construct of any one of claims 38-44 into the subject in combination with an immunosuppressive regimen; and(b) maintaining the immunosuppressive regimen for a time sufficient for engraftment and / or vascularization of the hypoimmunogenic cells in the subject.

47. A method for promoting engraftment and / or vascularization of a population of hypoimmunogenic cells in a subject in need thereof, the method comprising:(a) providing a subject who has been administered or implanted the cell mixture of any one of claims 1 -37 or the engineered tissue construct of any one of claims 38-44 into the subject in combination with an immunosuppressive regimen; and(b) maintaining the immunosuppressive regimen for a time sufficient for engraftment and / or vascularization of the hypoimmunogenic cells in the subject.

48. The method of claim 46 or 47, wherein the immunosuppressive regimen is maintained for 1 day to 6 months.

49. The method of claim 48, wherein the immunosuppressive regimen is maintained for 7 days to 2 months.

50. The method of claim 49, wherein the immunosuppressive regimen is maintained for 14 days to 1 month.51 . The method of any one of claims 46-50, further comprising (c) withdrawing the immunosuppressive regimen following step (b).

52. The method of any one of claims 46-51 , wherein the fibroblasts are genetically engineered fibroblasts comprising a kill switch, and the method further comprises activating the kill switch to kill the fibroblasts.

53. The method of any one of claims 46-52, wherein the immunosuppressive regimen comprises administering one or more immunosuppressive agents to the subject.

54. The method of claim 53, wherein the one or more immunosuppressive agents comprise a calcineurin inhibitor, a corticosteroid, an antimetabolite, a mammalian target of rapamycin (mTOR) inhibitor, a T cell costimulation inhibitor, an antibody, or any combination thereof.

55. The method of claim 54, wherein:(i) the calcineurin inhibitor comprises cyclosporine or tacrolimus;(ii) the corticosteroid comprises prednisone, prednisolone, methylprednisolone, cortisone, or hydrocortisone;(iii) the antimetabolite comprises mycophenolic acid, mycophenolate sodium, mycophenolate mofetil, or azathioprine;(iv) the mTOR inhibitor comprises sirolimus or everolimus;(v) the co-stimulation inhibitor comprises belatacept; and / or(vi) the antibody comprises alemtuzumab, rabbit antithymocyte globulin (rATG), horse antithymocyte globulin (hATG), basiliximab, or rituximab.

56. A method for promoting engraftment and / or vascularization of a population of hypoimmunogenic cells in a subject in need thereof, the method comprising:(a) administering or implanting the cell mixture of any one of claims 6-37 or an engineered tissue construct comprising the cell mixture into the subject; and(b) activating the kill switch to kill the fibroblasts at a time point following engraftment and / or vascularization of the hypoimmunogenic cells in the subject.

57. The method of any one of claims 46-56, wherein the fibroblasts are allogeneic relative to the subject.

58. The method of any one of claims 46-57, wherein the subject is a human.

59. The cell mixture of any one of claims 1 -37 or the engineered tissue construct of any one of claims 38- 44 for use in a method of promoting engraftment and / or vascularization of a population of hypoimmunogenic cells in a subject in need thereof, the method comprising:(a) administering or implanting the cell mixture of any one of claims 1 -37 or the engineered tissue construct of any one of claims 38-44 into the subject in combination with an immunosuppressive regimen; and(b) maintaining the immunosuppressive regimen for a time sufficient for engraftment and / or vascularization of the hypoimmunogenic cells in the subject.

60. The cell mixture of any one of claims 6-37 or an engineered tissue construct comprising the cell mixture for use in a method for promoting engraftment and / or vascularization of a population of hypoimmunogenic cells in a subject in need thereof, the method comprising:(a) administering or implanting the cell mixture or the engineered tissue construct into the subject; and(b) activating the kill switch to kill the fibroblasts at a time point following engraftment and / or vascularization of the hypoimmunogenic cells in the subject.