Decellularized tissue scaffolds, implants including same, and methods of producing and using same

Abstract

Provided herein is a method for producing a tissue graft. The graft may include a living cell and an attachment scaffold. The method may include rinsing a tissue sample; incubating the sample in a hypertonic salt solution to produce a hypertonic solution-treated tissue sample; incubating the treated tissue sample with an ionic detergent to produce a detergent-treated tissue sample; incubating the detergent-treated sample with a DNase to produce a DNase-treated scaffold; incubating the DNase-treated scaffold with a virus-inactivating compound to produce a virus- inactivated scaffold; and contacting the virus-inactivated scaffold with a living cell population to produce a tissue graft.

Description

DECELLULARIZED TISSUE SCAFFOLDS, IMPLANTS INCLUDING SAME, AND METHODS OF PRODUCING AND USING SAMEFIELD

[0001] The present disclosure relates to decellularized tissue scaffolds suitable for tissue implantations.BACKGROUND

[0002] In recent years, extracellular matrices (ECM) derived from decellularized tissues as three-dimensional (3D) scaffolds have drawn increasing attention as a tissue engineering strategy for regenerating, repairing, or replacing malfunctioning tissues and organs. 3D scaffolds may serve as an adhesive substrate for implanted cells and provide physical support to guide the formation of the new tissues or organs. Such scaffolds may benefit from the structure and signals of the decellularized ECM to modulate or maintain the differentiated phenotype of seeded cells.

[0003] The primary criterion for establishing sufficient decellularization may be that the resulting tissue matrix has significantly less double-stranded DNA than the original tissue. Preferably, the amount of DNA remaining may be no more than 90% or no more than 95% of the original amount. Removing cellular and immunogenic material may reduce risk of immune reactions, inflammation, and implant rejection by the host.

[0004] Removing the majority of cellular components while retaining sufficient ECM structure and composition to support cell viability poses a challenge. It has proven difficult to consistently achieve production of safe and effective decellularized ECM scaffolds. The challenge may be increased by the requirement to avoid using components or reagents not suitable for human implant production, especially if they cannot be readily and reliably removed during scaffold production.

[0005] There is an unmet need for decellularization processes devoid of potentially harmful surfactants or other reagents used for removal of cellular and nuclear material while retaining an effective ECM architecture. Such decellularization processes may be useful for preparing scaffolds for production of live-cell implants for human clinical use.SUMMARY110BET01PCT 1

[0006] Provided herein is a method for producing a cell-free attachment scaffold from a tissue sample. The method may include rinsing the tissue sample.

[0007] The method may include incubating the rinsed tissue sample in a hypertonic solution. This step may generate a hypertonic solution-treated tissue sample.

[0008] The method may include incubating the hypertonic solution-treated tissue sample with an ionic detergent. This step may generate a detergent-treated sample. The ionic detergent may be an anionic detergent. The ionic detergent may be deoxycholate or a salt thereof.

[0009] The method may include incubating the detergent-treated sample with a nuclease. In some embodiments, the nuclease is a DNase. This step may generate a nuclease-treated or DNase- treated scaffold.

[0010] The method may include incubating the DNase-treated scaffold with a virusinactivating compound. This step may generate a virus-free and / or cell-free attachment scaffold.

[0011] Also provided herein is a method for producing a tissue graft. The graft may include a living cell population and an attachment scaffold. The method may include any or all of the aforementioned steps for producing a cell-free attachment scaffold.

[0012] The method may include the step of contacting the virus-inactivated scaffold with a living cell population. This step may generate a tissue graft.

[0013] Also provided herein is a tissue graft, wherein the tissue graft comprises a living cell population and an attachment scaffold, and wherein the scaffold comprises Collagens III and IV, Fibronectin, heparan sulfate, Laminin, and Elastin; and the scaffold supports attachment of pancreatic islet cells to the scaffold and viability of the pancreatic islet cells.

[0014] Also provided herein is a tissue graft, wherein the tissue graft comprises a living cell population and an attachment scaffold, and wherein the scaffold comprises Collagens I, III, IV, V, and VI; and Fibronectin, heparan sulfate, Laminin, and Elastin; and the scaffold supports attachment of pancreatic islet cells to the scaffold and viability of the pancreatic islet cells.

[0015] Also provided herein is a method of treating diabetes. The method may include any or all of the aforementioned steps for producing a tissue graft.

[0016] The method may include administering the tissue graft to a subject.110BET01PCT 2

[0017] Also provided herein is a method of treating diabetes. The method may include administering a tissue graft to a subject. The tissue graft may include a living cell and an attachment scaffold. The scaffold may include levels of Collagens I, in, IV, V, and VI; Fibronectin, heparan sulfate, Laminin, and / or Elastin that are effective to facilitate attachment of cells to the scaffold and support viability of the cells.

[0018] Also provided herein is a method of treating diseases and disorders, for example diabetes. The method may include administering a tissue graft to a subject. The tissue graft may have been created by any or all of the aforementioned steps for producing a tissue graft.BRIEF DESCRIPTION OF THE FIGURES

[0019] This patent application file contains figures executed in color. Copies of the patent application file with the color drawings can be obtained upon request and payment of the necessary fee.

[0020] Fig. 1 is a diagram of an exemplary decellularization protocol.

[0021] Fig. 2 contain images showing immunofluorescence following staining with fluorescent antibodies, showing removal of DNA (left panels) and SLA (right panels) from decellularized scaffolds (bottom panels), as compared to MOs (top panels).

[0022] Figs. 3A-D contain immunofluorescence images. Fig. 3A shows staining of, from left to right, nuclei, Collagen III, Laminin (left panel); and nuclei, Collagen III, and heparan sulfate (right panel), before treatment (top row), or following NaOH treatment (middle row) or NaOH + H2O2 (bottom row). Fig. 3B shows staining of (L-R) nuclei, Fibronectin, Laminin (top panel); and nuclei, Collagen IV, and heparan sulfate (bottom panel), following decellularization with NaOH (rows 1 and 3) or Tergitol™ (rows 2 and 4). Fig. 3C shows staining of (L-R) nuclei, Fibronectin, Laminin (rows 1 and 3); and nuclei, Collagen IV, and heparan sulfate (rows 2 and 4), following decellularization with NaOH (top panel) or CHAPS (bottom panel). Fig. 3D shows staining of (L-R) nuclei, Collagen IV, and heparan sulfate (top row); nuclei, Collagen III, and Laminin (middle row); and nuclei, Fibronectin, and Elastin (bottom row), following decellularization with NaOH (left panel) or deoxycholate (right panel).

[0023] Fig. 4 contains immunofluorescence images of liver tissue before (left 3 columns) and after (right 3 columns) decellularization with deoxycholate. Stained are nuclei, Elastin, and110BET01PCT 3Collagen III (top row); nuclei, Collagen IV, and Laminin (middle row); and nuclei, Fibronectin, and heparan sulfate (bottom row).

[0024] Figs. 5A-C and E-F contain immunofluorescence images. Fig. 5A shows staining of, from left to right, nuclei, Insulin, MafA (rows 1 and 3); and nuclei, PDX1, and Glucagon (rows 2 and 4), 2 days (top panel) or 16 days (bottom panel) following seeding of beta cells from ChiPSC22-derived beta cells onto deoxycholate-decellularized, lung-derived scaffolds. Fig. 5B shows staining of (L-R) nuclei, Insulin, MafA (top panel); and nuclei, Glucagon, and PDX1 (bottom panel), 14 days following seeding of TC6 cells onto deoxycholate- (rows 1 and 3) or NaOH-decellularized (rows 2 and 4) lung-derived scaffolds. Fig. 5C shows staining of (L-R) nuclei, PDX1, and Glucagon (rows 1 and 3); and nuclei, Insulin, and MafA (rows 2 and 4), following seeding of TC6 cells onto CHAPS-decellularized (top panel) or NaOH-decellularized (bottom panel) scaffolds. Fig. 5D is a chart showing TC6 cell numbers (vertical axis; expressed as percentage of control) on NaOH (control) (left dataset) and deoxycholate (“deoxy”; right dataset) scaffolds. Figs. 5E-F show staining of (L-R) nuclei, Insulin, and MAFA (top panel); and nuclei, Glucagon, and PDX1 (bottom panel); 2 days (rows 1 and 3) or 16 days (rows 2 and 4); following seeding of pancreatic islet-cell spheroids onto deoxycholate-decellularized scaffolds from lung (E) or liver (F) tissue.

[0025] Fig. 6 is a diagram showing structures of exemplary triazole-containing alginate compounds.

[0026] Fig. 7 is a graph showing quantitation of C-peptide levels (vertical axis) in the media as measured by ELISA, following incubation of ESC-derived pancreatic islet cells seeded on scaffolds (left series of bars) or of unattached pancreatic islet-cell spheroids (right series) in medium. The averages and standard deviations (error bars) of several samples are shown. The left and right bars in each series show C-peptide under low- and high-glucose conditions, respectively.

[0027] Fig. 8 contains graphs showing C-peptide levels (vertical axis) in mouse serum while fasting or 45 min. after glucose challenge (first and second bars of each series, respectively). Mice were implanted with empty scaffolds subcutaneously (SC) or intraperitoneally (IP) (first dataset; average of these two groups is shown) or scaffolds seeded with ESC-derived pancreatic islet cell spheroids SC or IP (second and third datasets, respectively), and C-peptide levels were measured on days 28, 44, 77, and 92 (upper left and right graphs and lower left and right graphs,110BET01PCT 4respectively) after implantation in mice given empty scaffolds SC / IP (left data set pair; data combined; zero values); pancreatic islet-cell scaffolds SC (middle data set pair); and pancreatic islet-cell scaffolds IP (right data set pair).

[0028] Fig. 9A is a graph showing glucose levels (vertical axis) of mice at various times (horizontal axis) after challenge by Intraperitoneal Glucose Tolerance Test (IPGTT). Green, red, and blue datasets show combined empty scaffolds groups (MOMs SC / IP), SC pancreatic islet-cell seeded scaffolds (EMPs SC), and IP pancreatic islet-cell scaffolds (EMPs IP), respectively. B. is a graph showing the area under the curve (AUC) of the groups depicted in Fig. 9A, in units of milligram minutes per deciliter (mg*min / dL). C. is a graph showing a time course of random (fed) glucose levels (vertical axis) on different days (horizontal axis) after diabetes induction.

[0029] Fig. 10 contains microscopy images following staining of an excised empty scaffold, SC-seeded scaffold, and IP-seeded scaffold (top, middle, and bottom rows), showing staining for (left to right) Ku80, Insulin, and CD31.

[0030] Figs. 11A-B are microscopy images. A. Hematoxylin and eosin (H&E) staining of tissue sections (n=2; representative images are shown) from MO samples (upper panel) and decellularized scaffolds (lower panel). B. Staining for F4 / 80 (left column), a-smooth muscle actin (a-SMA; middle column), and CD31 (left column), of MO samples (top row), decellularized scaffolds (MOM; second row), talc-injected positive controls (third row), and sham controls (bottom row). Endothelial marker CD31 -positive structures are indicated by arrows.DETAILED DESCRIPTION

[0031] Provided herein is a method for producing a cell-free attachment scaffold from a tissue sample. The scaffold may be biocompatible. The scaffold may be considered safe for human implantation. The scaffold may be carcinogen free. The scaffold may be pathogen free. The scaffold may be free of a host antigen such as Swine Leucocyte Antigen (SLA).

[0032] As used herein, scaffold may refer to a physical structure with which cells associate. The cells may associate or attach upon or into the structure.

[0033] The term biocompatible, as used herein, may describe a material that is substantially non-toxic in the in-vivo environment of its intended use. The environment may be a human body. In some embodiments, the environment may be a body of a cat, a dog, a horse, a mouse, a rat, or110BET01PCT 5a pig. The described compositions and implants may be biocompatible and / or hypoimmunogenic. The compositions and implants may be administrable in conjunction with immune tolerance therapy. In some embodiments, biocompatibility may be gauged by the ability of a material to pass the biocompatibility tests set forth in International Standards Organization (ISO) Standard No. 10993 and / or the U.S. Pharmacopeia (USP) 23 and / or the U.S. Food and Drug Administration (FDA) blue book memorandum No. G95-1, entitled “Use of International Standard ISO- 10993, Biological Evaluation of Medical Devices Part-1 : Evaluation and Testing.” Tests may measure a material's toxicity, infectivity, pyrogenicity, irritation potential, reactivity, hemolytic activity, carcinogenicity and / or immunogenicity. A structure or material that does not cause a significantly adverse, long-lived or escalating biological reaction or response in a majority of patients may be considered biocompatible. A mild, transient inflammation that typically accompanies surgery or implantation of foreign objects into a living organism may be disregarded when evaluating biocompatibility.

[0034] As provided herein, methods have been developed to decellularize tissue without using components unsuitable for preparation of implants for use in humans. Furthermore, the resulting scaffolds may contain substantially native levels of Collagens III and IV, Fibronectin, heparan sulfate, Laminin, and Elastin, factors which may support cell attachment, viability and long-term survival. (Fig- 3)

[0035] Tissue sample may refer to a slice, section, or other fragment of tissue removed from a freshly slaughtered or living organism or from a biopsy, which may be either treated or untreated, prior to performing the described methods. In some embodiments, the mentioned tissue sample may be a lung tissue sample. In some embodiments, the tissue sample may be a liver tissue sample. In some embodiments, the tissue sample may be from another tissue.

[0036] In some embodiments, the attachment scaffold may contain less than 5% of the genomic DNA present in the tissue sample. In some embodiments, the scaffold may contain less than 3%; in other embodiments, less than 2%; or, in other embodiments, less than 1% of the genomic DNA present in the tissue sample.

[0037] In some embodiments, the scaffolds mentioned herein contain less than 10 nanograms (ng.) of host double-stranded DNA per scaffold. In some embodiments, the scaffolds contain less than 5 ng., 3 ng., 2 ng., 1 ng., 500 pg., 300 pg., 200 pg., 100 pg., 50 pg., 30 pg., 20 pg., 10 pg., 5110BET01PCT 6pg., 3 pg., or 2 pg.; or, in other embodiments, less than 1 pg., per scaffold. Host DNA in this context refers to DNA from the tissue used to create the scaffold; and does not refer to DNA from the seeded cells.

[0038] In some embodiments, the scaffolds contain less than 10 ng. of host DNA per implant. In some embodiments, the scaffolds contain less than 5 ng., 3 ng., 2 ng., 1 ng., 500 pg., 300 pg., 200 pg., 100 pg., 50 pg., 30 pg., 20 pg., 10 pg., 5 pg., 3 pg., or 2 pg.; or, in other embodiments, less than 1 pg. of host double-stranded DNA, per implant.

[0039] In some implementations, the scaffolds may be substantially free of antigens from the source tissue sample. In some implementations, the scaffolds may contain no detectable source tissue sample antigen. In some implementations, tissue sample antigen may be detected by antibody staining. In some implementations, the antibody staining may show an antigen level substantially equivalent to a control (for example, a control lacking primary antibody or an isotype control). As provided herein, the described scaffolds were shown to be free of SLA, which was abundant on the host tissue (Fig. 2).

[0040] Described herein are scaffolds approximately up to 300 microns thick. In some the scaffolds may be 6 mm in diameter, length, and / or width and 300 microns thick, or about 8.5 cubic millimeters (mm3). Those skilled in the art will appreciate that a variety of sizes of scaffolds may be produced by the described methods. In some implementations, the scaffolds are between 2-10 mm, between 2-8 mm, between 4-10 mm, between 4-8 mm, or between 5-7 mm in diameter, length, and / or width. In some implementations, the scaffolds are between 100-400, between 150-400, between 150-350, or between 200-350 microns thick. In some implementations, the scaffolds have a volume between 5-20 mm3, between 5-18 mm3, between 5-15 mm3, or between 5-10 mm3. In some embodiments, the scaffolds are between 100-350, between 150-350, between 150-300, or between 200-300 microns thick; and independently are between 2-500 mm, between 10-500 mm, between 2-250 mm, or between 10-250 mm in diameter, length, and / or width. The disclosure is not limited to scaffolds having a round or oval cross section.

[0041] In some embodiments, the described scaffolds lack detectable amounts of host viruses. Examples of host viruses that may be tested for include cytomegalovirus (CMV), Epstein-Barr virus (EBV), and BK virus (BKV). Examples of suitable detection kits include Qiagen™ Artus CMV RGQ, Qiagen Artus EBV, and Qiagen BKV.110BET01PCT 7

[0042] In some embodiments, the described scaffolds contain ECM components that enable vascularization of grafts, enabling therapeutic factors produced by the graft to enter the recipient’s bloodstream.

[0043] The mentioned method for producing a cell-free attachment scaffold may include the step of rinsing the tissue sample. This step may generate a rinsed tissue sample.

[0044] The method may include incubating the rinsed tissue sample in a hypertonic solution. This step may generate a hypertonic solution-treated tissue sample. The solution may be a hypertonic salt solution. The solution may include a biocompatible salt. Biocompatibility may be as defined in ISO Standard No. 10993. The salt may be non-hazardous to humans. The salt may be GRAS, which may refer to a substance considered “generally recognized as safe” by the United States Food and Drug Administration (FDA). The salt may be readily washable in aqueous solution. The term “readily washable”, in this context, may refer to a substance that does not leave a detectable residue on a substrate following three washes of the substrate. In more specific embodiments, the substrate may be the described rinsed tissue sample. An example of a suitable detection method for a substance is high-performance liquid chromatography-mass spectrometry (HPLC-MS).

[0045] The mentioned biocompatible salt may be scaffold-preparation process biocompatible. In this context, the term “scaffold-preparation process-biocompatible” may refer to a substance that does not leave a detectable harmful residue after three washing steps. An example of a suitable method for detecting a substance is HPLC-MS. Those skilled in the art will appreciate that a substance considered non-hazardous to humans may be safe to use in scaffold preparation. Additionally, a substance readily washable in aqueous solution may be safe to use in scaffold preparation, provided that at least three washing steps are performed after introduction of the substance. Scaffold-preparation process biocompatible may refer to a substance that fulfils at least one of the following criteria: (a) is considered non-hazardous to humans; and / or (b) is readily washable in aqueous solution.

[0046] The method for producing a cell-free attachment scaffold may include the step of rinsing the hypertonic solution-treated tissue sample. This step may generate a post-hypertonic solution-treatment rinsed tissue sample.110BET01PCT 8

[0047] The method for producing a cell-free attachment scaffold may include the step of incubating the hypertonic solution-treated tissue sample with an anionic detergent. This step may generate a detergent-treated sample.

[0048] The detergent may be a biocompatible detergent. Biocompatibility may be as defined in ISO Standard No. 10993. The detergent may be non-hazardous to humans. The detergent may be GRAS. The detergent may be readily washable in aqueous solution. The term “readily washable”, in this context, may refer to a substance that does not leave a detectable residue on a substrate following three washes of the substrate. An example of a suitable method for detecting a substance is HPLC-MS. In more specific embodiments, the substrate may be the described detergent-treated sample.

[0049] The mentioned biocompatible detergent may be scaffold-preparation process biocompatible. In this context, the term “scaffold-preparation process-biocompatible” may refer to a substance that does not leave a detectable harmful residue after three washing steps. The process-biocompatible detergent may be non-hazardous to humans. The process-biocompatible detergent may be readily washable. In some embodiments, at least three washing steps are performed after introduction of the detergent. Scaffold-preparation process-biocompatible” may refer to a detergent that fulfils at least one of the following criteria: (a) considered non-hazardous to humans; and / or (b) readily washable in aqueous solution.

[0050] In some embodiments, the mentioned detergent may be an anionic detergent. In some embodiments, the detergent may be an ionic bile acid. In some embodiments, the ionic detergent may include deoxycholate. In some implementations, the deoxycholate may be sodium deoxycholate. Other non-limiting examples of ionic bile acids include cholic acid and chenodeoxycholic acid. In some embodiments, the ionic bile acid includes a carboxy moiety. In some embodiments, the bile acid may be anionic. In some embodiments, the anionic bile acid includes a carboxy moiety

[0051] In some embodiments, the detergent may be zwitterionic detergent. In some embodiments, the detergent may be a taurine-conjugated. In some embodiments, the detergent may be zwitterionic bile acid. In some embodiments, the detergent may be a taurine-conjugated bile acid. In some embodiments, the detergent may possess both positive and negative electrical charges at physiological pH.110BET01PCT 9

[0052] In some embodiments, the detergent may be a neutral detergent. In some embodiments, the detergent may be a neutral bile acid, a non-limiting example of which is ursodeoxycholic acid (UDCA). In some embodiments, the detergent may lack ionizable groups at physiological pH.

[0053] The method for producing a cell-free attachment scaffold may include the step of rinsing the detergent-treated tissue sample. This step may generate a post-detergent treatment- rinsed tissue sample.

[0054] The method may include the step of incubating the detergent-treated sample with a nuclease, for example a DNase. This step may generate a nuclease-treated or DNase-treated scaffold.

[0055] The DNase may be a biocompatible DNase. Biocompatibility may be as defined in ISO Standard No. 10993. The DNase may be non-hazardous to humans. The DNase may be GRAS. The DNase may be readily washable in aqueous solution. The term “readily washable”, in this context, may refer to a DNase that does not leave a detectable residue on a substrate following three washes of the substrate. In more specific embodiments, the substrate may be the described DNase-treated scaffold. The absence of residual DNase may be tested using resDetect™ DNase Activity Assay Kit (Fluorescence), commercially available from Aero Biosystems.

[0056] The mentioned biocompatible DNase may be scaffold-preparation process biocompatible. In this context, the term “scaffold-preparation process-biocompatible” may refer to a substance that does not leave a detectable harmful residue after three washing steps. The process-biocompatible DNase may be non-hazardous to humans. The process-biocompatible DNase may be readily washable. In some embodiments, at least three washing steps are performed after introduction of the DNase. Scaffold-preparation process-biocompatible” may refer to a DNase that fulfils at least one of the following criteria: (a) considered non-hazardous to humans; and / or (b) readily washable in aqueous solution.

[0057] The mentioned DNase may be, in some embodiments, a DNA endonuclease. In some embodiments the DNase may be a non-sequence specific DNA endonuclease. In some embodiments the DNase may be DNase I. Non-limiting examples of DNase I enzymes are DNase I Type IV, DNase I Type II, and DNase I-XT. Non-limiting examples of other non-sequence specific DNA endonucleases are Duplex DNase, and micrococcal nuclease.110BET01PCT 10

[0058] The method for producing a cell-free attachment scaffold may include the step of rinsing the DNase-treated scaffold. This step may generate a post-DNase treatment-rinsed tissue sample.

[0059] The method may include the step of incubating the DNase-treated scaffold with a virusinactivating compound. This step may generate a virus-inactivated attachment scaffold. The virus- inactivated attachment scaffold may be the final product of the mentioned method.

[0060] The virus-inactivating compound may be a biocompatible compound. Biocompatibility may be as defined in ISO Standard No. 10993. The compound may be non-hazardous to humans. The compound may be GRAS. The compound may be readily washable in aqueous solution. The term “readily washable”, in this context, may refer to a substance that does not leave a detectable residue on a substrate following three washes of the substrate. In more specific embodiments, the substrate may be the described virus-inactivated attachment scaffold. An example of a suitable method for detecting a substance is HPLC-MS.

[0061] The mentioned biocompatible virus-inactivating compound may be scaffoldpreparation process biocompatible. In this context, the term “scaffold-preparation processbiocompatible” may refer to a substance that does not leave a detectable harmful residue after three washing steps. The process-biocompatible compound may be non-hazardous to humans. The process-biocompatible compound may be readily washable. In some embodiments, at least three washing steps are performed after introduction of the compound. Scaffold-preparation processbiocompatible” may refer to a compound that fulfils at least one of the following criteria: (a) considered non-hazardous to humans; (b) readily washable in aqueous solution; and / or (c) sufficiently volatile that no trace of the substance is detectable after a one-day incubation at room temperature. Preferably, the virus-inactivating compound should not deplete the described ECM components.

[0062] The mentioned virus-inactivating compound may be, in some embodiments, a reactive oxygen species. In some embodiments, the compound may form a free radical upon reaction with a free proton. In some embodiments, the compound may form an oxygen radical upon reaction with a free proton. In some embodiments, the compound may form an inorganic radical anion upon contact with an extracellular matrix. In some embodiments, the compound may form hydrogen peroxide upon contact with an extracellular matrix. In some embodiments, the110BET01PCT 11compound may be hydrogen peroxide. In some embodiments, virus inactivation is performed by a physical treatment. In some embodiments, the physical treatment is gamma irradiation. In some embodiments, the gamma irradiation may be performed at low temperature, for example between 0-5°C. In some embodiments, the physical treatment is supercritical CO2. In some embodiments, the virus inactivation step is selected from hydrogen peroxide treatment, gamma-irradiation treatment, and supercritical CO2 treatment.

[0063] Treatment with gamma-irradiation is known in the art, and is described, for example, in Hume AJ etal., 2016. Inactivation of RNA viruses by gamma irradiation: a study on mitigating factors. Viruses 8: E204, and the references cited therein. Treatment with supercritical CO2 is known in the art, and is described, for example, in J Fages et al., Viral Inactivation of Human Bone Tissue Using Supercritical Fluid Extraction. ASAIO J 44, 289-293 (1998) and the references cited therein.

[0064] In some embodiments, the virus-inactivating compound is selected from a reactive oxygen species,

[0065] The method for producing a cell-free attachment scaffold may include the step of rinsing the virus-inactivated attachment scaffold. This step may generate a post-virus-inactivation- rinsed attachment scaffold. This scaffold may be the final product of the mentioned method.

[0066] The method for producing a cell-free attachment scaffold may include the step of contacting the virus-inactivated scaffold with a buffer. The method may include the step of suspending the scaffold in a buffer. The method may include the step of submerging the scaffold in a buffer. The buffer may be biocompatible. Biocompatibility may be as defined in ISO Standard No. 10993. This composition, including the scaffold and the buffer, may be the final product of the mentioned method.

[0067] Also provided herein is a method of producing a tissue graft. The tissue graft may include a population of living cells and an attachment scaffold. The tissue graft may be biocompatible. Biocompatibility may be as defined in ISO Standard No. 10993. Biocompatibility may be as defined as lacking a detectable amount of any known substance considered toxic in humans. Toxicity may be evaluated according to ISO Standard No. 10993. The tissue graft may be considered safe for human implantation. The tissue graft may be carcinogen free.110BET01PCT 12

[0068] The method may include the step of rinsing a tissue sample. This rinsing step may be according to any of the embodiments mentioned herein, for example in the context of a method for producing a cell-free attachment scaffold.

[0069] In some embodiments, the mentioned tissue sample may be a lung tissue sample. In some embodiments, the tissue sample may be a liver tissue sample. In some embodiments, the tissue sample may be from another tissue.

[0070] The method for producing a tissue graft may include the step of incubating the rinsed tissue sample in a hypertonic salt solution. This step may generate a hypertonic solution-treated tissue sample. This step may be according to any of the embodiments mentioned herein, for example in the context of a method for producing a cell-free attachment scaffold.

[0071] The method may include the step of rinsing the hypertonic solution-treated tissue sample. This step may generate a post-hypertonic solution-treatment rinsed tissue sample.

[0072] The method for producing a tissue graft may include the step of incubating the hypertonic solution-treated tissue sample with an anionic detergent. This step may generate a detergent-treated tissue sample. This step may be according to any of the embodiments mentioned herein, for example in the context of a method for producing a cell-free attachment scaffold.

[0073] The method may include the step of rinsing the detergent-treated tissue sample. This step may generate a post-detergent treatment-rinsed tissue sample.

[0074] The method for producing a tissue graft may include the step of incubating the detergent-treated tissue sample with a DNase. This step may generate a DNase-treated scaffold. This step may be according to any of the embodiments mentioned herein, for example in the context of a method for producing a cell-free attachment scaffold.

[0075] The method may include the step of rinsing the DNase-treated scaffold. This step may generate a post-DNase treatment-rinsed tissue sample.

[0076] The method for producing a tissue graft may include the step of incubating the DNase- treated scaffold with a virus-inactivating compound. This step may generate a virus-inactivated attachment scaffold. This step may be according to any of the embodiments mentioned herein, for example in the context of a method for producing a cell-free attachment scaffold.110BET01PCT 13

[0077] The method may include the step of rinsing the virus-inactivated attachment scaffold.This step may generate a post-virus-inactivator-rinsed attachment scaffold.

[0078] The method for producing a tissue graft may include the step of contacting the virus- inactivated scaffold with a living cell population. The method may include the step of seeding a living cell population onto the scaffold. The method may include the step of incubating the scaffold with a living cell population. This step may generate the tissue graft. Non-limiting examples of suitable media are media used to support mammalian cell growth, for example adherent cell growth. A non-limiting example of such a medium is DMEM + human serum albumin (HSA) + a serum substitute, for example for pancreatic islet cells.

[0079] In some embodiments, the mentioned living cell population may be of a different tissue type than the tissue sample used as the source of the scaffold. In some embodiments, the living cell population may be from the same tissue type as the scaffold source.

[0080] In some embodiments, the attachment scaffold may contain less than 5% of the genomic DNA present in the source tissue. In some embodiments, the scaffold may contain < 3%; in other embodiments, < 2%; or, in other embodiments, < 1 % of the genomic DNA present in the source tissue.

[0081] In some embodiments, the living cells of the tissue graft may be pancreatic cells. In some embodiments, the living cells may be pancreatic islet cells. In some embodiments, the living cells may be pancreatic progenitor cells. In some embodiments, the living cells may be a non- pancreatic cell type.

[0082] In some embodiments, the living cells may be human cells. In some embodiments, the living cells may be from a non-human, mammalian species. In some embodiments, the living cells may be non-mammalian cells.

[0083] In some embodiments, the living cells may be embryonic stem cell (ESC)-derived. In some embodiments, the living cells may be induced pluripotent stem cell (iPSC)-derived. In some embodiments, the living cells may be primary cells.

[0084] The term pancreatic islet cells, as used herein, may refer to cells having a pancreatic phenotype. The term may encompass (Insulin-secreting) beta cells, (Glucagon-secreting) alpha cells, (Somatostatin-secreting) delta cells, (Pancreatic polypeptide-secreting) PP cells (F cells),110BET01PCT 14and (Ghrelin-secreting) Epsilon cells. In some embodiments, at least 40% of the pancreatic islet cells are beta cells. In some embodiments, at least 50% of the islet cells are beta cells. In some embodiments, at least 10%, or in other embodiments, at least 15%, of the islet cells are alpha cells.

[0085] In some embodiments, the pancreatic islet cells are ESC derived. In some embodiments, the pancreatic islet cells are iPSC-derived. In some embodiments, the pancreatic islet cells are primary pancreatic cells.

[0086] In some embodiments, the term pancreatic islet cells may encompass ESC-derived pancreatic cells, iPSC-derived pancreatic cells, and primary pancreatic cells

[0087] In some embodiments, the term pancreatic islet cells may encompass fully differentiated pancreatic islet cells and pancreatic progenitor cells. In some implementations, one of these cell types is present in a described living cell population. In other embodiments, both cell types are present in the population.

[0088] Incubation of the scaffold with a living cell population may be performed in a growth medium or culture medium. Those skilled in the art will appreciate that the particular growth medium utilized is not critical for carrying out the described methods and procedures. In some embodiments, the medium is suitable for supporting viability of mammalian cells. In some embodiments, the medium supports viability of adherent mammalian cells. Non-limiting examples of basal mammalian culture media are Dulbecco’s Modified Eagle Medium (DMEM), DMEM Fl 2, Minimum Essential Medium (MEM), Roswell Park Memorial Institute (RPMI), Media 199 (M199 media), Ham’s F-12 Nutrient Mixture, and Ham’s F-12 Nutrient Mixture.

[0089] The basal medium may be supplemented with a serum, non-limiting examples of which are fetal bovine serum (FBS) and fetal calf serum; or a serum replacement, non-limiting examples of which are KnockOut™ Serum Replacement (commercially available from Gibco™ and reported to contain amino acids, vitamins, transferrin or substitutes, insulin or insulin substitutes, trace elements, collagen precursors, and albumin preloaded with lipid), human Platelet Lysate (HPL) (commercially available from PL BioScience), and Serum Replacements 1, 2, and 3 (commercially available from Sigma- Aldrich™).

[0090] As provided herein, different types of insulin-secreting cells were seeded onto the described decellularized scaffolds and incubated in growth medium, to test whether the scaffolds110BET01PCT 15had the necessary ECM components physically and functionally support the cells. The pancreatic cells securely attached to the scaffold and expressed Insulin, Glucagon, and the pancreatic beta cell markers PDX-1 and MAF bZIP transcription factor A (MafA) (Figs. 5A-E).

[0091] Those skilled in the art will appreciate that the protein encoded by the PDX-1 gene is a transcriptional activator of several genes, including insulin, somatostatin, glucokinase, islet amyloid polypeptide, and glucose transporter type 2 (NCBI entry for Gene ID: 3651).

[0092] Those skilled in the art will appreciate that MafA is specifically expressed in 0 cells and is a key regulatory factor for maintaining adult 0-cell function” (Jiani Liang el al., MafA Regulation in 0-Cells: From Transcriptional to Post-Translational Mechanisms. Biomolecules. 2022 Apr; 12(4): 535).

[0093] Also provided herein is a tissue graft. The tissue graft may include a population of living cells (also referred to as a “living cell population”) and an attachment scaffold. The living cells may be attached to the scaffold. The living cells may be adhered to the scaffold. The tissue graft may be biocompatible. Biocompatibility may be as defined in ISO Standard No. 10993. Biocompatibility may be as defined as lacking a detectable amount of any known substance considered toxic in humans. Toxicity may be evaluated according to ISO Standard No. 10993. The tissue graft may be considered safe for human implantation. The tissue graft may be carcinogen free.

[0094] In some embodiments, the attachment scaffold may have been generated from a source tissue. In some embodiments, the source tissue may be lung tissue. In some embodiments, the source tissue may be liver tissue. In some embodiments, the scaffold may have been generated from any of the herein-described protocols for generating an attachment scaffold.

[0095] In some embodiments, the attachment scaffold may contain < 5% of the genomic DNA present in the source tissue. In some embodiments, the scaffold may contain, in some embodiments, < 3%, < 2%; or, < 1% of the genomic DNA present in the source tissue.

[0096] In some embodiments, the attachment scaffold may contain less than 5% of source genomic DNA present in the living cell population.

[0097] In some embodiments, the attachment scaffold may include the factors Collagens III and IV, Fibronectin, heparan sulfate, Laminin, and Elastin. In some embodiments, the attachment110BET01PCT 16scaffold may also include Collagen V. In some embodiments, the attachment scaffold may also include Collagen VI. In some embodiments, the attachment scaffold may include Collagens I, III, IV, V, and VI, Fibronectin, heparan sulfate, Laminin, and Elastin. In some embodiments, the attachment scaffold may also include Collagen V. The mentioned factors may each be present at a level that is detectably above background by immunofluorescence (for example, a control lacking primary antibody or an isotype control). In some embodiments, the factors may each be present at a level that is stained by immunofluorescence comparably to the source tissue before the decellularization process. In some embodiments, the level of immunofluorescence staining may be judged by a person skilled in the art of immunofluorescence to reflect an abundance at least 30% of the amount in the source tissue before decellularization. In some embodiments, the staining level may be judged by the skilled person to reflect an abundance at least 50% or the amount in the tissue before decellularization.

[0098] In some embodiments, the mentioned factors may collectively be present at a level effective to facilitate attachment of pancreatic islet cells to the scaffold and viability of the islet cells. In some embodiments, the factors may be present at a level that supports attachment of islet cells to the scaffold and viability of the islet cells. In some embodiments, the factors may support attachment and viability of the cells to the scaffold for 6 weeks, in other embodiments, for 8 weeks; in other embodiments, for 10 weeks; or, in other embodiments, for 12 weeks. In some embodiments, the factors may support proliferation of an Insulin-secreting cell line (for example, a beta cell-derived cell line; a non-limiting example of which is the Beta-TC-6 cell line) for at least 4 days, when incubated under suitable conditions (a non-limiting example of which is TC6 medium (DMEM 15% FCS, 0.05 mM P-mercaptoethanol, 1% Glutamine, 1% PS, 1% Antibiotic / Antimycotic) for TC-6 cells). In some implementations, the cells proliferate to reach at least 1.25-fold, at least 1.5 fold, or at least 1.7 fold of the original cell number at 7 days in culture. As provided herein (Fig. 5D), Insulin-secreting cell lines exhibit significantly enhanced proliferation on the herein-described scaffolds vs. scaffolds prepared by other methods.

[0099] In some embodiments, the mentioned factors may collectively be present at a level effective to facilitate attachment of pancreatic islet cells to the scaffold and secretion of Insulin (e.g., measured using C-peptide levels) by the islet cells. In some embodiments, two days after seeding the cells on the scaffold, the cells secrete at least 100 picomoles per liter (pmol / L) of C- peptide per 100,000 cells, during a one-hour incubation in high-glucose (e.g., 20 mM) Krebs 110BET01PCT 17solution, according to a standard Glucose-stimulated Insulin Secretion (GSIS) protocol. (For example, 250,000 cells may be incubated in 1 milliliter (ml.) high-glucose Krebs solution. In some embodiments, the cells secrete at least 200, in other embodiments, at least 300, in other embodiments, at least 500, in other embodiments, at least 1000, in other embodiments, at least 1500, in other embodiments, at least 2000 pmol / L of C-peptide per 100,000 cells, under the aforementioned conditions two days after seeding. In some embodiments, the cells secrete at least 200, in other embodiments, at least 300, in other embodiments, at least 500, in other embodiments, at least 1000, in other embodiments, at least 1500, in other embodiments, at least 2000 pmol / L of C-peptide per 100,000 cells, under the aforementioned conditions 7 days after seeding. In some embodiments, the factors may support attachment of the cells to the scaffold and Insulin secretion for at least 1 week, in other embodiments, at least 2 weeks; in other embodiments, at least 4 weeks; or, in other embodiments, at least 6 weeks. Those skilled in the art will appreciate that C-peptide levels are a surrogate for insulin levels.

[0100] In some embodiments, the living cells of the tissue graft are of a tissue type different from the source tissue used to produce the attachment scaffold. In some embodiments, the living cells are from the same tissue type as the source tissue of the attachment scaffold.

[0101] Also provided herein is a method of treating diabetes in a subject in need thereof. Also provided herein is a method of improving glucose responsiveness in a subject in need thereof. Also provided herein is a method of improving glucose control in a subject in need thereof. Also provided herein is a method of restoring glucose tolerance in a subject in need thereof.

[0102] Also provided herein is a method of increasing Insulin levels in a subject in need thereof. The subject may have an Insulin deficiency. The administered compositions may enable Insulin secretion into the bloodstream of the subject. The Insulin secretion may be responsive to elevated blood glucose levels. Elevated blood glucose levels may refer to levels over 100 mg / dL (milligrams per deciliter); in other embodiments, 125 mg / dL; or in other embodiments, 140 mg / dL.

[0103] The method of treating diabetes, improving glucose responsiveness, improving glucose control, restoring glucose tolerance, or increasing Insulin levels may include the step of rinsing a tissue sample. This rinsing step may be according to any of the embodiments mentioned herein, for example in the context of a method for producing a cell-free attachment scaffold.110BET01PCT 18

[0104] In some embodiments, the mentioned tissue sample may be a non-pancreatic tissue sample. In some embodiments, the tissue sample may be a lung tissue sample. In some embodiments, the tissue sample may be a liver tissue sample. In some embodiments, the tissue sample may be from another non-pancreatic tissue.

[0105] The method of treating diabetes, improving glucose responsiveness, improving glucose control, restoring glucose tolerance, or increasing Insulin levels may include the step of incubating the rinsed tissue sample in a hypertonic salt solution. This step may produce a hypertonic solution- treated tissue sample. This step may be according to any of the embodiments mentioned herein, for example in the context of a method for producing a cell-free attachment scaffold.

[0106] The method may include the step of rinsing the hypertonic solution-treated tissue sample. This step may generate a post-hypertonic solution-treatment rinsed tissue sample.

[0107] The method of treating diabetes, improving glucose responsiveness, improving glucose control, restoring glucose tolerance, or increasing Insulin levels may include the step of incubating the hypertonic solution-treated tissue sample with an anionic detergent. This step may produce a detergent-treated tissue sample. This step may be according to any of the embodiments mentioned herein, for example in the context of a method for producing a cell-free attachment scaffold.

[0108] The method may include the step of rinsing the detergent-treated tissue sample. This step may generate a post-detergent treatment-rinsed tissue sample.

[0109] The method of treating diabetes, improving glucose responsiveness, improving glucose control, restoring glucose tolerance, or increasing Insulin levels may include the step of incubating the detergent-treated tissue sample with a DNase. This step may produce a DNase-treated scaffold. This step may be according to any of the embodiments mentioned herein, for example in the context of a method for producing a cell-free attachment scaffold.

[0110] The method may include the step of rinsing the DNase-treated scaffold. This step may generate a post-DNase treatment-rinsed tissue sample.

[0111] The method of treating diabetes, improving glucose responsiveness, improving glucose control, restoring glucose tolerance, or increasing Insulin levels may include the step of incubating the DNase-treated scaffold with a virus-inactivating compound. This step may produce a virus-110BET01PCT 19inactivated attachment scaffold. This step may be according to any of the embodiments mentioned herein, for example in the context of a method for producing a cell-free attachment scaffold.

[0112] The method may include the step of rinsing the virus-inactivated attachment scaffold. This step may generate a post-virus-inactivator-rinsed attachment scaffold.

[0113] The method of treating diabetes, improving glucose responsiveness, improving glucose control, restoring glucose tolerance, or increasing Insulin levels may include the step of contacting the virus-inactivated scaffold with a population of living pancreatic islet cells. The method may include the step of seeding a living cell population onto the scaffold. The method may include the step of incubating the scaffold with a living cell population. In some embodiments, the pancreatic islet cells are stem cell-derived. In some embodiments, the stem cells may be ESC or iPSC. In some embodiments, the islet cells are primary cells.

[0114] The above step may generate a tissue graft. The graft may contain pancreatic islet cells. The graft may be biocompatible. Biocompatibility may be as defined in ISO Standard No. 10993. Biocompatibility may be as defined as lacking a detectable amount of any known substance considered toxic in humans. Toxicity may be evaluated according to ISO Standard No. 10993.

[0115] Also provided herein is a method of treating diabetes in a subject in need thereof. Also provided herein is a method of improving glucose responsiveness in a subject in need thereof. Also provided herein is a method of improving glucose control in a subject in need thereof. Also provided herein is a method of restoring glucose tolerance in a subject in need thereof. The method of treating diabetes, improving glucose responsiveness, improving glucose control, restoring glucose tolerance, or increasing Insulin levels may include the step of administering to the subject a tissue graft. In some embodiments, the tissue graft may include a population of pancreatic islet cells and an attachment scaffold. In some embodiments, the tissue graft may be capable of vascularization after implantation. In some embodiments, the tissue graft may be vascularized after implantation.

[0116] In some embodiments, the scaffold may be derived from a non-pancreatic tissue sample. In some embodiments, the tissue sample may be a lung tissue sample. In some embodiments, the tissue sample may be a liver tissue sample. In some embodiments, the tissue sample may be from another non-pancreatic tissue.110BET01PCT 20

[0117] In some embodiments, the pancreatic islet cells are stem cell-derived. In some embodiments, the stem cells may be ESC or iPSC. In some embodiments, the islet cells are primary cells.

[0118] The graft used in the described method may be biocompatible. Biocompatibility may be as defined in ISO Standard No. 10993. Biocompatibility may be as defined as lacking a detectable amount of any known substance considered toxic in humans. Toxicity may be evaluated according to ISO Standard No. 10993.

[0119] In some embodiments, the mentioned scaffold may contain levels of Collagens in and IV, Fibronectin, heparan sulfate, Laminin, and Elastin that are effective to facilitate attachment of pancreatic islet cells to the scaffold and support viability of the islet cells. In some embodiments, the mentioned scaffold may contain levels of Collagens I, III, IV, V and VI, Fibronectin, heparan sulfate, Laminin, and Elastin that are effective to facilitate attachment of pancreatic islet cells to the scaffold and support viability of the islet cells. In some embodiments, the factors may support attachment and viability of the cells to the scaffold for 1 week, in other embodiments, for 2 weeks; in other embodiments, for 4 weeks; or, in other embodiments, for 6 weeks.

[0120] In some embodiments, the factors may collectively be present at a level effective to facilitate attachment of pancreatic islet cells to the scaffold and secretion of Insulin by the islet cells. In some embodiments, the cells secrete at least 100 pmol / L, 200 pmol / L, 300 pmol / L, or 500 pmol / L of C-peptide during one-hour incubation in Krebs solution. In some embodiments, the factors may support attachment of the cells to the scaffold and Insulin secretion for 1 week, for 2 weeks, for 4 weeks; or, in other embodiments, for 6 weeks.

[0121] Also provided herein is a method of treating diabetes in a subject in need thereof. Also provided herein is a method of improving glucose responsiveness in a subject in need thereof. Also provided herein is a method of improving glucose control in a subject in need thereof. Also provided herein is a method of restoring glucose tolerance in a subject in need thereof. Also provided herein is a method of treating a disease or disorder resulting from inadequate glucose metabolism in a subject in need thereof. The method of treating diabetes, improving glucose responsiveness, improving glucose control, restoring glucose tolerance, or treating a disease or disorder resulting from inadequate glucose metabolism may include the step of administering to the subject a tissue graft. The graft may include a living cell and an attachment scaffold. The tissue110BET01PCT 21graft may have been created by a process described herein. In some embodiments, the graft may be capable of inducing vascularization after implantation. In some embodiments, the tissue graft may be vascularized after implantation.

[0122] The process for creating the graft may include the step of rinsing a tissue sample. This rinsing step may be according to any of the embodiments mentioned herein, for example in the context of a method for producing a cell-free attachment scaffold.

[0123] In some embodiments, the mentioned tissue sample may be a lung tissue sample. In some embodiments, the tissue sample may be a liver tissue sample. In some embodiments, the tissue sample may be from another tissue.

[0124] The process for creating the graft may include the step of incubating the rinsed tissue sample in a hypertonic salt solution. This step may generate a hypertonic solution-treated tissue sample. This step may be according to any of the embodiments mentioned herein, for example in the context of a method for producing a cell-free attachment scaffold.

[0125] The process for creating the graft may include the step of rinsing the hypertonic solution-treated tissue sample. This step may generate a post-hypertonic solution-treatment rinsed tissue sample.

[0126] The process for creating the graft may include the step of incubating the hypertonic solution-treated tissue sample with an anionic detergent. This step may generate a detergent-treated tissue sample. This step may be according to any of the embodiments mentioned herein, for example in the context of a method for producing a cell-free attachment scaffold.

[0127] The process for creating the graft may include the step of rinsing the detergent-treated tissue sample. This step may generate a post-detergent treatment-rinsed tissue sample.

[0128] The process for creating the graft may include the step of incubating the detergent- treated tissue sample with a DNase. This step may generate a DNase-treated scaffold. This step may be according to any of the embodiments mentioned herein, for example in the context of a method for producing a cell-free attachment scaffold.

[0129] The process for creating the graft may include the step of rinsing the DNase-treated scaffold. This step may generate a post-DNase treatment-rinsed tissue sample.110BET01PCT 22

[0130] The process for creating the graft may include the step of incubating the DNase-treated scaffold with a virus-inactivating compound. This step may generate a virus-inactivated attachment scaffold. This step may be according to any of the embodiments mentioned herein, for example in the context of a method for producing a cell-free attachment scaffold.

[0131] The process for creating the graft may include the step of rinsing the virus-inactivated attachment scaffold. This step may generate a post-virus-inactivation-rinsed attachment scaffold.

[0132] The process for creating the graft may include the step of contacting the virus- inactivated scaffold with a living cell population. The method may include the step of seeding a living population of pancreatic cells onto the scaffold. The method may include the step of incubating the scaffold with a living cell population. This step may generate the tissue graft.

[0133] Also provided herein is a method of manufacturing a medicament or pharmaceutical composition for treating diabetes. Also provided herein is a method of manufacturing a medicament or pharmaceutical composition for improving glucose responsiveness. Also provided herein is a method of manufacturing a medicament or pharmaceutical composition for improving glucose control. Also provided herein is a method of manufacturing a medicament or pharmaceutical composition for restoring glucose tolerance. Also provided herein is a method of manufacturing a medicament or pharmaceutical composition for treating a disease or disorder resulting from inadequate glucose metabolism. The method of manufacturing a medicament or pharmaceutical composition may include the step of manufacturing a tissue graft. The graft may include a living cell and an attachment scaffold. In some embodiments, the graft may be capable of inducing vascularization after implantation. In some embodiments, the tissue graft may be vascularized after implantation.

[0134] The process for creating the graft may include the step of rinsing a tissue sample. This rinsing step may be according to any of the embodiments mentioned herein, for example in the context of a method for producing a cell-free attachment scaffold.

[0135] In some embodiments, the mentioned tissue sample may be a lung tissue sample. In some embodiments, the tissue sample may be a liver tissue sample. In some embodiments, the tissue sample may be from another tissue.110BET01PCT 23

[0136] The process for creating the graft may include the step of incubating the rinsed tissue sample in a hypertonic salt solution. This step may generate a hypertonic solution-treated tissue sample. This step may be according to any of the embodiments mentioned herein, for example in the context of a method for producing a cell-free attachment scaffold.

[0137] The process for creating the graft may include the step of rinsing the hypertonic solution-treated tissue sample. This step may generate a post-hypertonic solution-treatment rinsed tissue sample.

[0138] The process for creating the graft may include the step of incubating the hypertonic solution-treated tissue sample with an anionic detergent. This step may generate a detergent-treated tissue sample. This step may be according to any of the embodiments mentioned herein, for example in the context of a method for producing a cell-free attachment scaffold.

[0139] The process for creating the graft may include the step of rinsing the detergent-treated tissue sample. This step may generate a post-detergent treatment-rinsed tissue sample.

[0140] The process for creating the graft may include the step of incubating the detergent- treated tissue sample with a DNase. This step may generate a DNase-treated scaffold. This step may be according to any of the embodiments mentioned herein, for example in the context of a method for producing a cell-free attachment scaffold.

[0141] The process for creating the graft may include the step of rinsing the DNase-treated scaffold. This step may generate a post-DNase treatment-rinsed tissue sample.

[0142] The process for creating the graft may include the step of incubating the DNase-treated scaffold with a virus-inactivating compound. This step may generate a virus-inactivated attachment scaffold. This step may be according to any of the embodiments mentioned herein, for example in the context of a method for producing a cell-free attachment scaffold.

[0143] The process for creating the graft may include the step of rinsing the virus-inactivated attachment scaffold. This step may generate a post-virus-inactivation-rinsed attachment scaffold.

[0144] The process for creating the graft may include the step of contacting the virus- inactivated scaffold with a living cell population. The method may include the step of seeding a living population of pancreatic cells onto the scaffold. The method may include the step of incubating the scaffold with a living cell population. This step may generate the tissue graft.110BET01PCT 24Exemplary cell populations

[0145] In some embodiments, the living cells used in the described tissue grafts and methods may be from a different species from the source tissue of the attachment scaffold. In some embodiments, the living cells are from the same species type, but a different species individual, as the source tissue of the attachment scaffold. In some embodiments, the living cells are from the same individual as the source tissue of the attachment scaffold.

[0146] In some embodiments, the living cells used in the described tissue grafts and methods may be from a different species from the recipient, which may be referred to as the living cells being xenogeneic relative to the recipient. In some embodiments, the living cells are from the same species type, but a different species individual, as the recipient, which may be referred to as the living cells being allogeneic relative to the recipient. In some embodiments, the living cells are from the same individual as the recipient, which may be referred to as the living cells being autologous relative to the recipient.

[0147] Reference herein to a “living” cell population, “living” pancreatic islet cell population, and similar terms does not require, in some embodiments, that every cell in the population is alive. In some embodiments, the term means that at least 40% of the cells in the population are alive. In some embodiments, the term means that at least 50%; in other embodiments, at least 60%; in other embodiments, at least 70%; in other embodiments, at least 80%; in other embodiments, at least 90%; or, in other embodiments, at least 95% of the cells in the population are alive.

[0148] In some embodiments, the living cells may be pancreatic cells. In some embodiments, the living cells may be pancreatic islet cells. In some embodiments, at least 50% of the islet cells are beta cells. In some embodiments, at least 10%; in other embodiments, at least 15% of the islet cells are alpha cells. In some embodiments, the living cells may be pancreatic progenitor cells. In some embodiments, the living cells may be a cell type that produces a missing factor for a subject in need thereof. In some embodiments, the living cells may be another cell type.

[0149] In some embodiments, the living cells may be human cells. In some embodiments, the living cells may be from another mammalian species. In some embodiments, the living cells may be non-mammalian cells.110BET01PCT 25

[0150] In some embodiments, the living cells may be iPSC-derived. In some embodiments, the living cells may be primary cells.

[0151] In some embodiments, the attachment scaffold may contain less than 5% of the amount genomic DNA present in the source tissue. In some embodiments, the scaffold may contain less than 3%, less than 2%, less or than 1% of the amount of genomic DNA present in the source tissue.

[0152] The described methods of treating diabetes, improving glucose responsiveness, improving glucose control, restoring glucose tolerance, or treating other diseases and disorders resulting from inadequate glucose metabolism may include the step of administering the tissue graft to the subject. In some embodiments, the graft is implanted into the subject. In some embodiments, an immunosuppressant is administered together with the graft. In some embodiments, tolerization therapy may be administered to the subject prior to introducing the graft. In some embodiments, the graft may be capable of inducing vascularization after implantation.

[0153] In some embodiments, the graft may be administered subcutaneously or by insertion into the omentum. Subcutaneous administration may be performed by any method known in the art, a non-limiting example of which is the Peel-Away® Introducer Sheath by Cook Medical. Omental administration may be performed by any method known in the art, a non-limiting examples of which are described in Damyar, K et al. 2021 (An overview of current advancements in pancreatic islet transplantation into the omentum. Islets, 13(5-6), 115-120) and Yu, M et al. (Islet transplantation in the subcutaneous space achieves long-term euglycaemia in preclinical models of type 1 diabetes. Nat Metab 2, 1013-1020 (2020).

[0154] In some embodiments, the described graft may be hypoimmunogenic. In some embodiments, the scaffold of the graft may not appreciably stimulate fibrotic capsule formation. In some embodiments, the fibrotic capsule formation induced by the scaffold may be not substantially greater than an extruded poly(lactic-co-glycolic acid) (PLGA) implant. In some embodiments, the scaffold may not appreciably stimulate cellular infiltration. In some embodiments, the cellular infiltration induced by the scaffold may be not substantially greater than an extruded PLGA implant.

[0155] In some embodiments, the graft may include a hypoimmunogenic scaffold. In some embodiments, the graft may include hypoimmunogenic cells. In some embodiments, the graft may include a hypoimmunogenic scaffold and hypoimmunogenic cells. In some embodiments, the cells110BET01PCT 26may be autologous cells. In some embodiments, the cells may be substantially Human Leukocyte Antigen (HLA)-matched to the graft recipient. In some embodiments, the cells may be fully HLA- matched to the graft recipient.

[0156] In some embodiments, the graft may be treated to reduce its immunogenicity. In some embodiments, the graft may be encapsulated in a selectively permeable coating (e.g., a coating that permits passage of most nutrients, oxygen, and hormones between the graft and its surroundings while preventing passage of immune cells and large immunoglobulins). Non-limiting examples of coatings to reduce immunogenicity are cellulose, agarose, collagen, chitosan, poly (ethylene glycol) (PEG), gelatin, poly (2-hydroxyethyl methacrylate), and alginate. In some embodiments, alginate capsules may be additionally coated with a thin polycation layer, such as poly-L- ornithine (PLO), polyallylamine, or polyvinylamine.

[0157] In some embodiments, the graft immunogenicity may be reduced by using living cells from HLA superdonors of common HLA types. Such cell lines are commercially available, for example from Cellular Dynamics International, Inc.

[0158] In some embodiments, the cells of the graft may be subject to genome editing to reduce the immunogenicity of the graft. Non-limiting examples of such methods are described in Deuse T et al., (Hypoimmunogenic derivatives of induced pluripotent stem cells evade immune rejection in fully immunocompetent allogeneic recipients. Nat Biotechnol. 2019 Mar;37(3):252-258) and the references cited therein.

[0159] Non-limiting examples of immunomodulatory cells are regulatory T cells (Tregs), mesenchymal stem cells (MSCs), dendritic cells, amniotic epithelial cells, and Sertoli cells. In some embodiments, the graft may be implanted together with immunomodulatory biomaterials. In some embodiments, the biomaterial may be conjugated to an immunomodulatory moiety. In some embodiments, the biomaterial may release an immunomodulatory moiety. Non-limiting examples of immunomodulatory moieties are Fas Ligand (FasL), programmed death ligand- 1 (PD-L1), tacrolimus (FK506), transforming growth factor-beta 1 (TGF-pi), Interleukin 33, and triazole- containing compounds. Non-limiting examples of immunomodulatory moieties are the triazole- containing compounds depicted in Fig. 5 and other triazole-containing analogs described in Katie Lu K, et al. (Combinatorial islet protective therapeutic approaches in P-cell transplantation: Rationally designed solutions using a target product profile. Review FASEB Bioadv. 2023 Jun110BET01PCT 272;5(7):287-304. doi: 10.1096 / fba.2023-00029. eCollection 2023 Jul), and the references cited therein; and other moieties described in David A Alagpulinsa et al. (Alginate-microencapsulation of human stem cell-derived 0 cells with CXCL12 prolongs their survival and function in immunocompetent mice without systemic immunosuppression. Am J Transplant. 2019 Jul;19(7):1930-1940. doi: 10.1111 / ajt.15308) and the references cited therein. Other methods for reducing immunogenicity of grafts are described in Grimus S et al. (Immunoprotection Strategies in 0-Cell Replacement Therapy: A Closer Look at Porcine Islet Xenotransplantation. Adv Sci (Weinh). 2024 Aug;l l(31):e2401385. doi: 10.1002 / advs.202401385) and the references cited therein.EXAMPLESExample 1: Tissue biopsy preparation.

[0160] Healthy porcine lungs were harvested from freshly sacrificed animals. Alveoli-rich areas were selected that lacked visible necrotic tissue and did not display an abundance of large arteries, veins, bronchial cartilage, or adipose tissue. Tissue was cut into pieces and stored at -80°C within 10 minutes of lung removal from the animal carcass.

[0161] Sectioning was performed after lung tissue was removed from -80°C storage and kept at -20°C for one day. Tissue was sliced to 600 micron thickness using a Leica CM-1950 cryostat, and then 6-mm (millimeter) sections were obtained using a circular punch biopsy device. Sections were stored at -80°C.Example 2: Methods of scaffold decellularization

[0162] NaCl / deoxycholate / H2O2 procedure

[0163] All decellularization steps were performed at room temperature under gentle shaking at 50-100 rpm. Micro-organs (MOs) appearing whole (not fragmented), which did not form aggregates with other MOs, were selected and decellularized as follows:

[0164] MOs were rinsed three times with sterilized, deionized water, each time for 30-60 minutes on a rotary shaker.

[0165] Samples were next rinsed two times with sterile hypertonic (IM) sodium chloride (NaCl) aqueous solution on the rotary shaker, by incubating under gentle shaking for 30-60110BET01PCT 28minutes, then incubated for 14 hours in the NaCl solution. MOs were then rinsed twice with deionized water as described above.

[0166] Samples were then incubated in 2% deoxycholate solution in deionized water for 90 minutes, then rinsed three times in deionized water.

[0167] Samples were then, in some experiments, incubated in 13.5 Kunitz units / ml DNase I solution for 60 minutes, followed by two washes in deionized water.

[0168] Samples were then incubated in 0.15% H2O2 solution for 60 minutes, followed by two washes in deionized water. Samples were then incubated for 45 minutes in sterile phosphate- buffered saline (PBS) solution devoid of calcium and magnesium and stored at 4°C until testing or cell seeding, as described in subsequent Examples. The products of these decellularization procedures are referred to as “scaffolds” in the remainder of the Examples.

[0169] An exemplary protocol is illustrated in Fig. 1.

[0170] Alternative detergent procedures

[0171] Alternative protocols were tested, wherein alternative detergents, namely 0.5% Tergitol™ (15-S-9) or 8 millimolar (mM) CHAPS detergent (3-((3-cholamidopropyl) dimethylammonio)-l -propanesulfonate), were substituted for deoxy cholate.

[0172] NaCl / NaOH / H2O2 procedure

[0173] In this procedure, two 1.5-hour incubations in 0.1M sodium hydroxide (NaOH) solution were performed instead of the deoxycholate and DNase steps.Example 3: Determination of decellularization of micro-organs

[0174] The DNA content of the MOs and scaffolds was analyzed to evaluate the reduction of intact nuclei and viable cells, using the Qiagen® QIAamp® DNA Micro Kit, Cat. 56304. MOs were lysed at elevated temperatures in the presence of Proteinase K and Buffer ATL. Buffer AL and ethanol were then added to the lysate, which was subsequently transferred onto a QIAamp® MinElute® Column, to allow adsorption of DNA onto the silica-gel membrane. The column membrane was washed using Buffer AW1 followed by Buffer AW2. The adsorbed DNA was then eluted from the column using Buffer Ae or distilled water. The collected DNA was concentrated110BET01PCT 29and weighed to determine the average mass of DNA per 1 -milligram (mg) native tissue in the samples. Table 1 shows DNA removal efficiency after various decellularization protocols.

[0175] Table 1. DNA removal from MOs.* (average of 2 experiments)

[0176] The above results show that the scaffolds were substantially devoid of intact nuclei and viable cells. The scaffolds were also substantially devoid of SLA (Fig. 2).Example 4: Marker staining to evaluate extracellular matrix integrity of micro-organs

[0177] Histology was used to characterize the extracellular matrix (ECM) integrity of decellularized porcine lung scaffolds obtained by the described decellularization protocols. Analysis included immunofluorescent staining of the tissues, followed by image evaluation.

[0178] Samples were incubated in 4% paraformaldehyde (PF A). For immunostaining, the following first antibodies were used in Figs. 3-5: rabbit anti-laminin, rabbit anti-collagen III, rabbit anti collagen type IV, rat anti heparan sulfate, rabbit or mouse anti fibronectin, mouse anti-elastin, mouse or rat anti-insulin, mouse anti-glucagon and rabbit anti-PDXl, followed by speciesmatching fluorescently-labelled secondary antibodies. DAPI or Hoechst 33342 dyes were used for nuclear staining. Fluorescent images were obtained by confocal microscopy.

[0179] The NaOH decellularization protocol resulted in partial degradation of Collagen III and extensive degradation of Laminin and heparan sulfate (Fig. 3A). The Tergitol™ protocol preserved110BET01PCT 30Laminin and Collagen IV, reduced heparan sulfate, and more extensively degraded Fibronectin (Fig. 3B). The CHAPS protocol resulted in degradation of Fibronectin (Fig. 3C). By contrast, the deoxy cholate protocol substantially preserved native levels of Collagens in and IV, Fibronectin, heparan sulfate, Laminin, and Elastin (Fig. 3D).Example 5: Testing alternative tissue sources.

[0180] Porcine liver and pancreas were examined as alternative tissue sources. Pancreatic tissue completely disintegrated after detergent-based decellularization. Liver tissue was successfully used in deoxycholate-based decellularization, particularly following thinner slicing (reduced 50%) than that described in Example 1. 99.9% of porcine DNA was removed by the completion of the deoxycholate and DNase steps. Fig. 4 shows preservation of all examined ECM components after the deoxycholate decellularization process.Example 6: Seeding of pancreatic islets and pancreatic cells on decellularized scaffolds

[0181] Three different types of insulin-secreting cells were seeded onto decellularized scaffolds to test the scaffolds’ ability to physically and functionally support the cells.

[0182] Beta cells derived from Cellartis® hiPS Cells (from ChiPSC22) were obtained from Takara Bio and seeded onto deoxycholate-decellularized, lung-derived scaffolds and incubated at 37°C in Takara’s Cellartis Beta Cell Basal medium under gentle rolling. After seeding, the scaffolds were transferred into a 48 well plate, each in a well. After 2 and 16 days, nuclei staining revealed attachment of the beta cells to the scaffold. Staining with other antibodies demonstrated the expression of Insulin, Glucagon and the pancreatic beta cell markers PDX-1 and MafA (Fig. 5A).

[0183] TC6 cells (murine insulin-secreting [beta] cells) were seeded under gentle rolling; then grown on deoxycholate-, CHAPS-, or NaOH-decellularized, lung-derived scaffolds for 2-28 days in RPMI with 10% FBS under static conditions. Deoxycholate and CHAPS scaffolds supported higher levels of insulin secretion than NaOH scaffolds (Figs. 5B-C). Quantitative evaluation of cellular viability using the XTT assay (Roche; Sigma: Cat. No. 11465015001) 14 days after seeding demonstrated that TC-6 cells on deoxycholate scaffolds showed a 222% higher metabolic activity, a measure of the number of living cells, vs. NaOH scaffolds (p<0.05, Figure 5D).110BET01PCT 31

[0184] Pancreatic islet cell-spheroids, containing alpha, beta, gamma, delta, and epsilon cells, and pancreatic polypeptide were seeded onto deoxycholate-decellularized scaffolds derived from lung or liver tissue and incubated for 18 hours at 37°C in a medium designed for culture of differentiated pancreatic islet cells. After 2 days, the pancreatic islet cells exhibited attachment to the lung and liver scaffolds and expressed Insulin, Glucagon, PDX-1, and MafA (Figs. 5E-F, respectively). Expression was stable throughout the 16-day duration of the experiment.Example 7: Seeding of pancreatic islet cells on decellularized scaffolds confers glucose responsiveness of insulin secretion.

[0185] Analytical Methods

[0186] After the cells were adhered to the scaffold, the cells were stained for various markers. C-peptide secretion was measured after a one-hour incubation in low-glucose (2 mM) or high- glucose (20 mM) Krebs solution, according to a standard GSIS protocol. (Huang M el al., Assessment of the Metabolic Pathways Associated with Glucose-Stimulated Biphasic Insulin Secretion. Mei Endocrinology, Volume 155, Issue 5, 1 May 2014, Pages 1653-1666). Insulin staining was performed after overnight recovery culture medium. C-peptide was detected using Ultrasensitive C-peptide ELISA (Mercodia), cat. No. MRC-10-1141-01.

[0187] Scaffolds were created from porcine lungs according to the deoxycholate procedure described in Examples 1 -2; and seeded, as described in Example 6, with spheroids containing ESC- derived pancreatic islet cells, and incubated for 16 days in growth medium; or ESC-derived pancreatic islet cells were incubated unattached to a scaffold in the same medium. On day 9 of in- vitro culture after seeding, the cells were incubated in low-glucose (2 mM) or high-glucose (20 mM) solution, and C-peptide secretion into the media was measured by ELISA. Scaffold-attached cells secreted C-peptide in a glucose dependent manner (Fig. 7; left dataset), while non-attached spheroids secreted C-peptide in an unregulated fashion (Fig. 7; right dataset), showing that the described decellularized scaffolds conferred glucose responsive insulin secretion by the pancreatic islet cells.Example 8: Insulin expression and vascularization in pancreatic islet-cell seeded scaffolds in mice110BET01PCT 32

[0188] Scaffolds were created from porcine lungs according to the deoxycholate procedure described in Examples 1-2; and seeded, as described in Example 6, with spheroids containing 2 million ESC-derived pancreatic islet cells. The pancreatic islet-cell scaffolds or empty scaffolds were implanted intraperitoneally (IP); or inserted into a small incision pocket on the upper back, after which the incision was closed with a surgical staple (subcutaneous; SC); into healthy, NOD- SCID mice. Diabetes was induced by injecting streptozocin (STZ) on day 33 after scaffold implantation, and insulin secretion (measured by C-peptide levels) was measured in the mice on. Fig. 8 shows that on days 28, 44, 77, and 92 following implantation, SC- and IP-pancreatic isletcell scaffolds consistently secreted insulin in a glucose-regulated fashion (third and fourth datasets, respectively); while no insulin was secreted from empty scaffolds (first and second datasets).

[0189] On day 88 following implantation, mice were subjected to Intraperitoneal Glucose Tolerance Test (IPGTT) to measure glucose homeostasis. After overnight fasting, mice were administered 2g / kg of body mass of 20% glucose solution. The seeded scaffold-implanted mice achieved greater glucose control than the empty scaffold-implanted mice (Fig. 9A; red, blue, and green datasets, respectively). The SC and IP seeded scaffold-treated mice each had lower levels than the empty scaffold-implanted mice. A time course of random (freely fed) glucose levels (Fig. 9B) showed that the EMP-implanted mice by SC or IP route (red and blue datasets, respectively) had consistently lower glucose levels than mice with empty scaffolds (green dataset).

[0190] On day 93 following implantation, the implant area was excised from each mouse. The cells were stained with antibodies to detect Ku80 (human cell nuclei) and detect expression of Insulin and CD31 (vascularization) (left to right). Images of an excised empty scaffold, SC-seeded scaffold, and IP-seeded scaffold shown in Fig. 10 (top, middle, and bottom rows) show that the seeded scaffolds expressed Insulin and underwent vascularization.

[0191] To further characterize the immunogenic properties of the decellularized scaffolds, four experimental groups were employed: non-decellularized MOs, decellularized scaffolds, talc injections (positive control for inflammatory response), and sham (negative control). Samples were implanted subcutaneously in immunocompetent BALB / c mice and harvested on day 28 postimplantation for histological and immunofluorescence analyses.110BET01PCT 33

[0192] H&E staining of tissue sections indicated differences in the immunogenicity of the experimental groups. Results (n=2; representative images are shown) showed reduced fibrotic capsule thickness and decreased cellular infiltration in decellularized scaffolds compared to MO samples (Fig. 11 A), indicating a reduction of the foreign body reaction, and thus lower immunogenicity, in the decellularized scaffolds vs. the original tissue samples (MO).

[0193] Immunofluorescence staining (n=3; representative images are shown) using markers for key inflammatory cell types provided additional insights (Fig. 11B). F4 / 80 staining revealed lower macrophage infiltration in decellularized scaffolds compared to MO implants and talc- injected positive controls, corroborating the hypoimmunogenicity of the decellularized scaffolds. Assessment of a-smooth muscle actin (a-SMA) expression showed reduced diffuse (unorganized) a-SMA-positive cells in decellularized scaffolds vs. MO samples, with decellularized scaffolds levels similar to those observed in sham controls, indicating low activation of fibroblasts associated with fibrotic encapsulation. Endothelial marker CD31 -positive structures (indicated by arrows) were observed in the decellularized scaffolds, and dual staining revealed colocalization of organized a-SMA with CD31 staining.

[0194] While the present invention has been particularly described, persons skilled in the art will appreciate that many variations and modifications can be made. Therefore, the invention is not to be construed as restricted to the particularly described embodiments, and the scope and concept of the invention will be more readily understood by reference to the claims, which follow.110BET01PCT 34

Claims

CLAIMS1. A method for producing a cell-free attachment scaffold from a tissue sample, the method comprising: rinsing the tissue sample; incubating the rinsed tissue sample in a hypertonic salt solution to produce a hypertonic solution-treated tissue sample; incubating the hypertonic solution-treated tissue sample with an ionic detergent to produce a detergent-treated sample; incubating the detergent-treated sample with a DNase to produce a DNase-treated scaffold; and incubating the DNase-treated scaffold with a virus-inactivating compound to produce the cell-free attachment scaffold.

2. The method of claim 1, further comprising rinsing the hypertonic solution-treated tissue sample.

3. The method of claim 1, further comprising rinsing the detergent-treated tissue sample.

4. The method of claim 1, wherein the anionic detergent is an ionic bile acid.

5. The method of claim 1, wherein the ionic detergent comprises deoxycholate.

6. The method of claim 1, further comprising contacting the virus-inactivated scaffold with a buffer.

7. The method of claim 1, wherein the tissue sample is a lung tissue sample.

8. The method of claim 1, wherein the tissue sample is a liver tissue sample.

9. A method for producing a tissue graft, wherein the tissue graft comprises a living cell population and an attachment scaffold, the method comprising: rinsing a tissue sample; incubating the rinsed tissue sample in a hypertonic salt solution to produce a hypertonic solution-treated tissue sample;110BET01PCT 35incubating the hypertonic solution-treated tissue sample with an ionic detergent to produce a detergent-treated tissue sample; incubating the detergent-treated tissue sample with a DNase to produce a DNase-treated scaffold; incubating the DNase-treated scaffold with a virus-inactivating compound to produce a virus-inactivated attachment scaffold; and contacting the virus-inactivated scaffold with the living cell population to produce a tissue graft.

10. The method of claim 9, wherein the living cell population is of a different tissue type than the tissue sample.

11. The method of claim 9, wherein the tissue sample is a lung tissue sample.

12. The method of claim 9, wherein the tissue sample is a liver tissue sample.

13. A tissue graft, wherein the tissue graft comprises a living cell population and an attachment scaffold, wherein the living cell population is attached to the attachment scaffold, and wherein the scaffold comprises Collagens in and IV, Fibronectin, heparan sulfate, Laminin, and Elastin; wherein the scaffold supports attachment of pancreatic islet cells to the scaffold and viability of the pancreatic islet cells.

14. The tissue graft of claim 13, wherein the scaffold was produced from a source tissue, and the living cell population is of a tissue type different from the source tissue.

15. The tissue graft of claim 13, wherein the attachment scaffold was generated from lung tissue or liver tissue.

16. The tissue graft of claim 13, wherein the cells are pancreatic islet cells.

17. The tissue graft of claim 13, wherein the scaffold was produced from a source tissue, and the attachment scaffold contains less than 5% of source genomic DNA present in the source tissue.

18. The tissue graft of claim 13, wherein the scaffold is a hypoimmunogenic scaffold.110BET01PCT 3619. A tissue graft for use in treating diabetes in a subject in need thereof, wherein the tissue graft was manufactured by a method comprising: rinsing a tissue sample; incubating the rinsed tissue sample in a hypertonic salt solution to produce a hypertonic solution-treated tissue sample; incubating the hypertonic solution-treated tissue sample with an anionic detergent to produce a detergent-treated tissue sample; incubating the detergent-treated tissue sample with a DNase to produce a DNase-treated scaffold; incubating the DNase-treated scaffold with a virus-inactivating compound to produce a virus-inactivated scaffold; contacting the virus-inactivated scaffold with a population of pancreatic islet cells to produce a tissue graft; and administering the tissue graft to the subject.

20. The tissue graft of claim 19, wherein the tissue sample is from a non-pancreatic tissue type.

21. The tissue graft of claim 20, wherein the tissue type is lung tissue.

22. The tissue graft of claim 20, wherein the tissue type is liver tissue.

23. The tissue graft of claim 19, wherein the pancreatic islet cells are stem cell-derived.

24. The tissue graft of claim 19, wherein the pancreatic islet cells are primary cells.

25. The tissue graft of claim 19, wherein the pancreatic islet cells are autologous cells.

26. The tissue graft of claim 19, wherein the scaffold is a hypoimmunogenic scaffold.

27. A biocompatible tissue graft for use in treating diabetes in a subject in need thereof, wherein the biocompatible tissue graft comprises pancreatic islet cells and an attachment scaffold, and wherein the scaffold comprises levels of Collagens III and IV, Fibronectin, heparan sulfate, Laminin, and Elastin that are effective to facilitate attachment of the pancreatic islet cells to the scaffold and support viability of the pancreatic islet cells.

28. The biocompatible tissue graft of claim 27, wherein the attachment scaffold is from a non- pancreatic tissue type.110BET01PCT 3729. The biocompatible tissue graft of claim 28, wherein the tissue type is lung tissue.

30. The biocompatible tissue graft of claim 28, wherein the tissue type is liver tissue.

31. The biocompatible tissue graft of claim 27, wherein the pancreatic islet cells are stem cell- derived.

32. The biocompatible tissue graft of claim 27, wherein the pancreatic islet cells are primary cells.

33. The biocompatible tissue graft of claim 27, wherein the pancreatic islet cells are autologous pancreatic islet cells.

34. The biocompatible tissue graft of claim 27, wherein the scaffold is a hypoimmunogenic scaffold.

35. A tissue graft for use in treating diabetes in a subject in need thereof, wherein the tissue graft comprises a population of pancreatic islet cells and an attachment scaffold, and wherein the tissue graft was created by a process comprising: rinsing a tissue sample; incubating the rinsed tissue sample in a hypertonic salt solution to produce a hypertonic solution-treated tissue sample; incubating the hypertonic solution-treated tissue sample with an ionic detergent to produce a detergent-treated tissue sample; incubating the detergent-treated tissue sample with a DNase to produce a DNase-treated scaffold; incubating the DNase-treated scaffold with a virus-inactivating compound to produce a virus-inactivated attachment scaffold; and contacting the virus-inactivated scaffold with the population of pancreatic islet cells to produce a tissue graft.

36. The tissue graft of claim 35, wherein the tissue sample is from a non-pancreatic tissue type.

37. The tissue graft of claim 36, wherein the tissue type is lung tissue.

38. The tissue graft of claim 36, wherein the tissue type is liver tissue.110BET01PCT 3839. The tissue graft of claim 35, wherein the pancreatic islet cells are stem cell-derived.

40. The tissue graft of claim 35, wherein the pancreatic islet cells are primary cells.

41. The tissue graft of claim 35, wherein the pancreatic islet cells are autologous cells.

42. The tissue graft of claim 35, wherein the scaffold is a hypoimmunogenic scaffold.110BET01PCT 39

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