Manufacturing method of recellularized bio-artificial organs and method of using them

Abstract

Methods and compositions relating to at least partially recellularized human organs are provided herein. Various methods for decellularizing non-human animal organs and recellularizing the non-human animal extracellular matrix with cellular compositions. Furthermore, compositions and methods for treating liver disease or other diseases (such as acute liver failure) using an extracorporeal bioartificial liver or other organs are provided herein.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority and benefit under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 511,413, filed on June 30, 2023, the entire content of which is incorporated herein by reference for all purposes and is owned by the common assignee.

Background Art

[0002] Organ transplantation is common throughout the United States and the world. Organs such as the liver, kidney, pancreas, lung, and heart are commonly transplanted to extend the life of the recipient. However, organs are often in short supply, creating a demand for organs and a waiting list. As an example, acute liver failure (ALF) can be a life - threatening serious disease in patients, characterized by the rapid onset of liver biochemical abnormalities, coagulation disorders, and often progression to encephalopathy. With changes in medical management, the clinical outcomes of ALF patients have been steadily improving, but the gold standard for treating ALF remains liver transplantation, with a 1 - year survival rate of 91%. According to data from the ALFSG (Acute Liver Failure Study Group) registry and the SRTR (Scientific Registry of Transplant Recipients), it has been reported that only 64% of the patients listed for transplantation received a life - saving organ. Strategies are needed to alleviate the long waiting lists for organ transplantation and improve the quality of life of patients.

Summary of the Invention

[0003] Recellularized bioartificial organs for use in the treatment of various diseases are provided herein. In some embodiments, the recellularized bioartificial organ includes a liver, or the liver has reduced and / or inactivated microbial particles, can be cultured for a long period (e.g., more than 4 days) before use in transplantation or in an ex vivo blood circuit without systemic anticoagulation, and can maintain liver function (e.g., ammonia clearance and urea production) even after storage at low temperatures for more than 6 hours. Furthermore, methods for producing bioartificial liver tissue and methods for treating patients with an in vitro liver are provided herein.

[0004] A method for producing at least partially recellularized organ compositions, including but not limited to the liver, kidney, lung, and heart, is provided herein, comprising: (a) treating a non-human animal organ with an antiviral treatment; (b) perfusing the non-human animal organ to decellularize it to obtain a decellularized extracellular matrix; and (c) contacting the decellularized extracellular matrix with a cell composition comprising a population of human cells to form an at least partially recellularized organ composition.

[0005] A method for producing a liver composition that is at least partially recellularized is provided herein, comprising: (a) treating a non-human animal liver with an antiviral treatment; (b) perfusing the non-human animal liver to decellularize it to obtain a decellularized extracellular matrix; and (c) contacting the decellularized extracellular matrix with a cell composition comprising a population of human hepatocytes to form a liver composition that is at least partially recellularized.

[0006] A method for producing an organ composition that is at least partially recellularized is provided herein, comprising: (a) treating a non-human animal organ with an antiviral treatment; (b) perfusing the non-human animal organ to decellularize it to obtain a decellularized extracellular matrix; and (c) contacting the decellularized extracellular matrix with a cell composition containing a population of human cells to form an organ composition that is at least partially recellularized.

[0007] A method for producing an organ composition that is at least partially recellularized is provided herein, comprising: (a) treating a non-human animal organ with an antiviral treatment; (b) perfusing the non-human animal organ to decellularize it to obtain a decellularized extracellular matrix; (c) contacting the decellularized extracellular matrix with a first cell composition comprising a population of human vascular endothelial cells; and (d) contacting the decellularized extracellular matrix with a second cell composition to form an organ composition that is at least partially recellularized.

[0008] A method for producing a liver composition that is at least partially recellularized is provided herein, comprising: (a) treating a non-human animal liver with an antiviral treatment; (b) perfusing the non-human animal liver to decellularize it to obtain a decellularized extracellular matrix; (c) contacting the decellularized extracellular matrix with a first cell composition comprising a population of human vascular endothelial cells; and (d) contacting the decellularized extracellular matrix with a second cell composition comprising a population of human hepatocytes to form a liver composition that is at least partially recellularized.

[0009] A composition comprising at least partially recellularized organs produced by any of the methods provided herein is provided herein.

[0010] A composition comprising a liver composition that has been at least partially recellularized by any of the methods provided herein is provided herein.

[0011] A partially recellularized liver is provided herein, comprising (a) a porcine extracellular matrix; and (b) a population of human endothelial cells and human hepatocytes engrafted in the porcine extracellular matrix, wherein the partially recellularized liver exhibits increased ammonia clearance compared to a population of porcine hepatocytes engrafted in the porcine extracellular matrix.

[0012] A partially recellularized liver is provided herein, comprising (a) a porcine extracellular matrix with reduced microbial particles and perfusion-decellularized; and (b) a population of human endothelial cells and human hepatocytes engrafted in the porcine extracellular matrix, wherein the partially recellularized liver exhibits increased ammonia clearance compared to a population of porcine hepatocytes engrafted in the porcine extracellular matrix.

[0013] A composition comprising at least partially recellularized liver and an extracellular matrix protein or solution is provided herein. Furthermore, a composition further comprising a cell culture medium is provided herein.

[0014] An ex vivo method for treating liver disease in a subject, comprising constructing a blood circuit, the blood circuit containing, in fluid communication, blood from the subject and at least partially recellularized liver provided herein, the at least partially recellularized liver filtering the blood from the subject and removing ammonia, thereby providing a method for treating liver disease in the subject.

[0015] A method for treating liver disease in a subject is provided herein, comprising administering to the subject at least partially recellularized liver provided herein, thereby treating liver disease in a subject. Furthermore, a method is provided herein in which the liver disease includes acute hepatic failure (ALF).

[0016] Reference All publications, patents, and patent applications described herein are incorporated herein by reference to the same extent as in the individual publications, patents, or patent applications that are explicitly and individually indicated to be incorporated by reference.

[0017] The novel features of the disclosed embodiments are clearly defined in the appended claims. A deeper understanding of the features and advantages of the disclosed embodiments can be obtained by referring to the following embodiments for carrying out the invention, which describe preferred embodiments in which the principles of the disclosed embodiments are utilized, and to the following appended drawings. [Brief explanation of the drawing]

[0018] [Figure 1A] Figures 1A to 1C show schematic diagrams of one embodiment of a method for producing a recellular liver and a method for treating subjects with liver disease. Figure 1A shows a schematic diagram including photographs of a natural pig liver being harvested, decellularized, and recellularized with HUVEC and human hepatocytes. Figure 1B shows a schematic diagram of a bioreactor station used to culture the artificial liver. Figure 1C shows a cell culture scheme showing a co-culture of HUVEC and human hepatocytes, including a scaffold equilibration and qualification period, endothelial culture consisting of HUVEC, and a 3-day available treatment window. [Figure 1B] Figures 1A to 1C show schematic diagrams of one embodiment of a method for producing a recellular liver and a method for treating subjects with liver disease. Figure 1A shows a schematic diagram including photographs of a natural pig liver being harvested, decellularized, and recellularized with HUVEC and human hepatocytes. Figure 1B shows a schematic diagram of a bioreactor station used to culture the artificial liver. Figure 1C shows a cell culture scheme showing a co-culture of HUVEC and human hepatocytes, including a scaffold equilibration and qualification period, endothelial culture consisting of HUVEC, and a 3-day available treatment window. [Figure 1C]Figures 1A to 1C show schematic diagrams of one embodiment of a method for producing a recellular liver and a method for treating subjects with liver disease. Figure 1A shows a schematic diagram including photographs of a natural pig liver being harvested, decellularized, and recellularized with HUVEC and human hepatocytes. Figure 1B shows a schematic diagram of a bioreactor station used to culture the artificial liver. Figure 1C shows a cell culture scheme showing a co-culture of HUVEC and human hepatocytes, including a scaffold equilibration and qualification period, endothelial culture consisting of HUVEC, and a 3-day available treatment window. [Figure 2A]Figures 2A-2I show cell identification and histological features of extracorporeal bioartificial livers (BELs). HUVECs and PHLCs were characterized before seeding and within the graft. CD31 and CD105 expression in HUVECs was assessed by flow cytometry before seeding into decellularized grafts. Figure 2A shows a representative histogram of endothelial marker expression in HUVECs. Figure 2B shows a graph from Figure 1A showing the percentage of HUVECs stained as CD31-positive and CD105-positive (n=25). Figure 2C shows a graph of glucose consumption rate during endothelial culture of decellularized scaffolds. Shaded areas indicate two standard deviations from the mean. Figure 2D shows the flow cytometry scatter profile of the PHLC population. Hepatocytes are indicated by gated areas based on size and complexity. Gating is representative. Figure 2E shows ASGR expression in a representative PHLC population. Gating indicates positive expression compared to appropriate isotype controls. The shown population is a representative example of the PHLC population after separation. Figure 2F shows the quantification of hepatocytes within the BEL population determined from the scatter plot gating strategy. (n=14) Figure 2G shows hematoxylin-eosin (H&E) staining of cross-sections of BEL recellularized with HUVEC and PHLC one day after hepatocyte seeding. Figure 2H shows H&E staining of cross-sections of blood vessels lined with HUVEC in BEL recellularized with HUVEC and PHLC. Arrows indicate representative regions of blood vessels lined with HUVEC. Figure 2I shows H&E staining of high-magnification cross-sections of BEL recellularized with HUVEC and PHLC hepatocytes one day after hepatocyte seeding. Arrows indicate HUVEC lining the blood vessels. [Figure 2B]Figures 2A-2I show cell identification and histological features of extracorporeal bioartificial livers (BELs). HUVECs and PHLCs were characterized before seeding and within the graft. CD31 and CD105 expression in HUVECs was assessed by flow cytometry before seeding into decellularized grafts. Figure 2A shows a representative histogram of endothelial marker expression in HUVECs. Figure 2B shows a graph from Figure 1A showing the percentage of HUVECs stained as CD31-positive and CD105-positive (n=25). Figure 2C shows a graph of glucose consumption rate during endothelial culture of decellularized scaffolds. Shaded areas indicate two standard deviations from the mean. Figure 2D shows the flow cytometry scatter profile of the PHLC population. Hepatocytes are indicated by gated areas based on size and complexity. Gating is representative. Figure 2E shows ASGR expression in a representative PHLC population. Gating indicates positive expression compared to appropriate isotype controls. The shown population is a representative example of the PHLC population after separation. Figure 2F shows the quantification of hepatocytes within the BEL population determined from the scatter plot gating strategy. (n=14) Figure 2G shows hematoxylin-eosin (H&E) staining of cross-sections of BEL recellularized with HUVEC and PHLC one day after hepatocyte seeding. Figure 2H shows H&E staining of cross-sections of blood vessels lined with HUVEC in BEL recellularized with HUVEC and PHLC. Arrows indicate representative regions of blood vessels lined with HUVEC. Figure 2I shows H&E staining of high-magnification cross-sections of BEL recellularized with HUVEC and PHLC hepatocytes one day after hepatocyte seeding. Arrows indicate HUVEC lining the blood vessels. [Figure 2C]Figures 2A-2I show cell identification and histological features of extracorporeal bioartificial livers (BELs). HUVECs and PHLCs were characterized before seeding and within the graft. CD31 and CD105 expression in HUVECs was assessed by flow cytometry before seeding into decellularized grafts. Figure 2A shows a representative histogram of endothelial marker expression in HUVECs. Figure 2B shows a graph from Figure 1A showing the percentage of HUVECs stained as CD31-positive and CD105-positive (n=25). Figure 2C shows a graph of glucose consumption rate during endothelial culture of decellularized scaffolds. Shaded areas indicate two standard deviations from the mean. Figure 2D shows the flow cytometry scatter profile of the PHLC population. Hepatocytes are indicated by gated areas based on size and complexity. Gating is representative. Figure 2E shows ASGR expression in a representative PHLC population. Gating indicates positive expression compared to appropriate isotype controls. The shown population is a representative example of the PHLC population after separation. Figure 2F shows the quantification of hepatocytes within the BEL population determined from the scatter plot gating strategy. (n=14) Figure 2G shows hematoxylin-eosin (H&E) staining of cross-sections of BEL recellularized with HUVEC and PHLC one day after hepatocyte seeding. Figure 2H shows H&E staining of cross-sections of blood vessels lined with HUVEC in BEL recellularized with HUVEC and PHLC. Arrows indicate representative regions of blood vessels lined with HUVEC. Figure 2I shows H&E staining of high-magnification cross-sections of BEL recellularized with HUVEC and PHLC hepatocytes one day after hepatocyte seeding. Arrows indicate HUVEC lining the blood vessels. [Figure 2D]Figures 2A-2I show cell identification and histological features of extracorporeal bioartificial livers (BELs). HUVECs and PHLCs were characterized before seeding and within the graft. CD31 and CD105 expression in HUVECs was assessed by flow cytometry before seeding into decellularized grafts. Figure 2A shows a representative histogram of endothelial marker expression in HUVECs. Figure 2B shows a graph from Figure 1A showing the percentage of HUVECs stained as CD31-positive and CD105-positive (n=25). Figure 2C shows a graph of glucose consumption rate during endothelial culture of decellularized scaffolds. Shaded areas indicate two standard deviations from the mean. Figure 2D shows the flow cytometry scatter profile of the PHLC population. Hepatocytes are indicated by gated areas based on size and complexity. Gating is representative. Figure 2E shows ASGR expression in a representative PHLC population. Gating indicates positive expression compared to appropriate isotype controls. The shown population is a representative example of the PHLC population after separation. Figure 2F shows the quantification of hepatocytes within the BEL population determined from the scatter plot gating strategy. (n=14) Figure 2G shows hematoxylin-eosin (H&E) staining of cross-sections of BEL recellularized with HUVEC and PHLC one day after hepatocyte seeding. Figure 2H shows H&E staining of cross-sections of blood vessels lined with HUVEC in BEL recellularized with HUVEC and PHLC. Arrows indicate representative regions of blood vessels lined with HUVEC. Figure 2I shows H&E staining of high-magnification cross-sections of BEL recellularized with HUVEC and PHLC hepatocytes one day after hepatocyte seeding. Arrows indicate HUVEC lining the blood vessels. [Figure 2E]Figures 2A-2I show cell identification and histological features of extracorporeal bioartificial livers (BELs). HUVECs and PHLCs were characterized before seeding and within the graft. CD31 and CD105 expression in HUVECs was assessed by flow cytometry before seeding into decellularized grafts. Figure 2A shows a representative histogram of endothelial marker expression in HUVECs. Figure 2B shows a graph from Figure 1A showing the percentage of HUVECs stained as CD31-positive and CD105-positive (n=25). Figure 2C shows a graph of glucose consumption rate during endothelial culture of decellularized scaffolds. Shaded areas indicate two standard deviations from the mean. Figure 2D shows the flow cytometry scatter profile of the PHLC population. Hepatocytes are indicated by gated areas based on size and complexity. Gating is representative. Figure 2E shows ASGR expression in a representative PHLC population. Gating indicates positive expression compared to appropriate isotype controls. The shown population is a representative example of the PHLC population after separation. Figure 2F shows the quantification of hepatocytes within the BEL population determined from the scatter plot gating strategy. (n=14) Figure 2G shows hematoxylin-eosin (H&E) staining of cross-sections of BEL recellularized with HUVEC and PHLC one day after hepatocyte seeding. Figure 2H shows H&E staining of cross-sections of blood vessels lined with HUVEC in BEL recellularized with HUVEC and PHLC. Arrows indicate representative regions of blood vessels lined with HUVEC. Figure 2I shows H&E staining of high-magnification cross-sections of BEL recellularized with HUVEC and PHLC hepatocytes one day after hepatocyte seeding. Arrows indicate HUVEC lining the blood vessels. [Figure 2F]Figures 2A-2I show cell identification and histological features of extracorporeal bioartificial livers (BELs). HUVECs and PHLCs were characterized before seeding and within the graft. CD31 and CD105 expression in HUVECs was assessed by flow cytometry before seeding into decellularized grafts. Figure 2A shows a representative histogram of endothelial marker expression in HUVECs. Figure 2B shows a graph from Figure 1A showing the percentage of HUVECs stained as CD31-positive and CD105-positive (n=25). Figure 2C shows a graph of glucose consumption rate during endothelial culture of decellularized scaffolds. Shaded areas indicate two standard deviations from the mean. Figure 2D shows the flow cytometry scatter profile of the PHLC population. Hepatocytes are indicated by gated areas based on size and complexity. Gating is representative. Figure 2E shows ASGR expression in a representative PHLC population. Gating indicates positive expression compared to appropriate isotype controls. The shown population is a representative example of the PHLC population after separation. Figure 2F shows the quantification of hepatocytes within the BEL population determined from the scatter plot gating strategy. (n=14) Figure 2G shows hematoxylin-eosin (H&E) staining of cross-sections of BEL recellularized with HUVEC and PHLC one day after hepatocyte seeding. Figure 2H shows H&E staining of cross-sections of blood vessels lined with HUVEC in BEL recellularized with HUVEC and PHLC. Arrows indicate representative regions of blood vessels lined with HUVEC. Figure 2I shows H&E staining of high-magnification cross-sections of BEL recellularized with HUVEC and PHLC hepatocytes one day after hepatocyte seeding. Arrows indicate HUVEC lining the blood vessels. [Figure 2G]Figures 2A-2I show cell identification and histological features of extracorporeal bioartificial livers (BELs). HUVECs and PHLCs were characterized before seeding and within the graft. CD31 and CD105 expression in HUVECs was assessed by flow cytometry before seeding into decellularized grafts. Figure 2A shows a representative histogram of endothelial marker expression in HUVECs. Figure 2B shows a graph from Figure 1A showing the percentage of HUVECs stained as CD31-positive and CD105-positive (n=25). Figure 2C shows a graph of glucose consumption rate during endothelial culture of decellularized scaffolds. Shaded areas indicate two standard deviations from the mean. Figure 2D shows the flow cytometry scatter profile of the PHLC population. Hepatocytes are indicated by gated areas based on size and complexity. Gating is representative. Figure 2E shows ASGR expression in a representative PHLC population. Gating indicates positive expression compared to appropriate isotype controls. The shown population is a representative example of the PHLC population after separation. Figure 2F shows the quantification of hepatocytes within the BEL population determined from the scatter plot gating strategy. (n=14) Figure 2G shows hematoxylin-eosin (H&E) staining of cross-sections of BEL recellularized with HUVEC and PHLC one day after hepatocyte seeding. Figure 2H shows H&E staining of cross-sections of blood vessels lined with HUVEC in BEL recellularized with HUVEC and PHLC. Arrows indicate representative regions of blood vessels lined with HUVEC. Figure 2I shows H&E staining of high-magnification cross-sections of BEL recellularized with HUVEC and PHLC hepatocytes one day after hepatocyte seeding. Arrows indicate HUVEC lining the blood vessels. [Figure 2H]Figures 2A-2I show the cell identification and tissue images of an extracorporeal bioartificial liver (BEL). HUVEC and PHLC were characterized before seeding and within the graft. The expression of CD31 and CD105 in HUVEC was evaluated by flow cytometry before seeding into the decellularized graft. Figure 2A shows a representative histogram of HUVEC expression of endothelial markers. Figure 2B shows a graph of the percentage of HUVEC stained as CD31 positive and CD105 positive from Figure 1A (n = 25). Figure 2C shows a graph of the glucose consumption rate during the endothelial culture period of the decellularized scaffold. The shaded area indicates two standard deviations from the mean. Figure 2D shows the flow cytometry scatter profile of the PHLC population. Hepatocytes are indicated by the gate region based on size and complexity. The gating is representative. Figure 2E shows the ASGR expression within a representative PHLC population. The gate shows the positive expression when compared to the appropriate isotype control. The population shown is a representative example of the PHLC population after isolation. Figure 2F shows the hepatocyte quantification within the BEL population determined from the scatter plot gating strategy (n = 14). Figure 2G shows Hematoxylin and Eosin (H&E) staining of a cross-section of BEL recellularized with HUVEC and PHLC 1 day after hepatocyte seeding. Figure 2H shows H&E staining of a cross-section of a vessel lined with HUVEC in BEL recellularized with HUVEC and PHLC. The arrow indicates a representative area of the vessel lined with HUVEC. Figure 2I shows H&E staining showing a high magnification cross-section of BEL recellularized with HUVEC and PHLC hepatocytes 1 day after hepatocyte seeding. The arrow indicates HUVEC lining the vessel. [Figure 2I]Figures 2A-2I show cell identification and histological features of extracorporeal bioartificial livers (BELs). HUVECs and PHLCs were characterized before seeding and within the graft. CD31 and CD105 expression in HUVECs was assessed by flow cytometry before seeding into decellularized grafts. Figure 2A shows a representative histogram of endothelial marker expression in HUVECs. Figure 2B shows a graph from Figure 1A showing the percentage of HUVECs stained as CD31-positive and CD105-positive (n=25). Figure 2C shows a graph of glucose consumption rate during endothelial culture of decellularized scaffolds. Shaded areas indicate two standard deviations from the mean. Figure 2D shows the flow cytometry scatter profile of the PHLC population. Hepatocytes are indicated by gated areas based on size and complexity. Gating is representative. Figure 2E shows ASGR expression in a representative PHLC population. Gating indicates positive expression compared to appropriate isotype controls. The shown population is a representative example of the PHLC population after separation. Figure 2F shows the quantification of hepatocytes within the BEL population determined from the scatter plot gating strategy. (n=14) Figure 2G shows hematoxylin-eosin (H&E) staining of cross-sections of BEL recellularized with HUVEC and PHLC one day after hepatocyte seeding. Figure 2H shows H&E staining of cross-sections of blood vessels lined with HUVEC in BEL recellularized with HUVEC and PHLC. Arrows indicate representative regions of blood vessels lined with HUVEC. Figure 2I shows H&E staining of high-magnification cross-sections of BEL recellularized with HUVEC and PHLC hepatocytes one day after hepatocyte seeding. Arrows indicate HUVEC lining the blood vessels. [Figure 3A]Figures 3A to 3H show a functional comparison between recellularized liver (BEL) from pigs and humans. Figure 3A shows a graph showing the removal of ammonia from the culture medium 1 hour after a 200 μM bolus administration to pig and human BELs, with data expressed per million cells (n=23 for pigs, n=16 for humans). Figure 3B shows a graph showing the 1-hour ammonia clearance performed daily for 3 days of BEL culture in pig and human BELs, with data expressed per million cells (n=5 for pigs, n=10 for humans). Figure 3C shows a graph showing glucose consumption calculated daily after 24-hour culture for 3 days in pig and human BELs (n=5 for pigs, n=10 for humans). Figure 3D shows a graph showing the urea concentration in the BEL acclimatization medium collected at the end of the 24-hour culture period, with data expressed per million cells (n=14 for pigs, n=8 for humans). Figure 3E shows a graph of fibrinogen production in BEL-conditioned medium collected at the end of a 24-hour culture period, with data expressed per million cells (n=5 for pigs, n=8 for humans). Figure 3F shows a graph of A1AT production in BEL-conditioned medium collected at the end of a 24-hour culture period, with data expressed per million cells (n=5 for pigs, n=8 for humans). Figure 3G shows a schematic diagram of the circuit for the hemoperfusion test. Figure 3H shows a graph of patency of pig and human BEL measured for 180 minutes in the hemoperfusion circuit (n=6 for pigs, n=3 for humans). Statistical significance: **p<0.01, ***p<0.001, ****p<0.0001, and ns = no significant difference. The results of the t-test between the pig and human data are shown, both (Figures 3B and 3C) showing results with false detection correction. [Figure 3B]Figures 3A to 3H show a functional comparison between recellularized liver (BEL) from pigs and humans. Figure 3A shows a graph showing the removal of ammonia from the culture medium 1 hour after a 200 μM bolus administration to pig and human BELs, with data expressed per million cells (n=23 for pigs, n=16 for humans). Figure 3B shows a graph showing the 1-hour ammonia clearance performed daily for 3 days of BEL culture in pig and human BELs, with data expressed per million cells (n=5 for pigs, n=10 for humans). Figure 3C shows a graph showing glucose consumption calculated daily after 24-hour culture for 3 days in pig and human BELs (n=5 for pigs, n=10 for humans). Figure 3D shows a graph showing the urea concentration in the BEL acclimatization medium collected at the end of the 24-hour culture period, with data expressed per million cells (n=14 for pigs, n=8 for humans). Figure 3E shows a graph of fibrinogen production in BEL-conditioned medium collected at the end of a 24-hour culture period, with data expressed per million cells (n=5 for pigs, n=8 for humans). Figure 3F shows a graph of A1AT production in BEL-conditioned medium collected at the end of a 24-hour culture period, with data expressed per million cells (n=5 for pigs, n=8 for humans). Figure 3G shows a schematic diagram of the circuit for the hemoperfusion test. Figure 3H shows a graph of patency of pig and human BEL measured for 180 minutes in the hemoperfusion circuit (n=6 for pigs, n=3 for humans). Statistical significance: **p<0.01, ***p<0.001, ****p<0.0001, and ns = no significant difference. The results of the t-test between the pig and human data are shown, both (Figures 3B and 3C) showing results with false detection correction. [Figure 3C]Figures 3A - 3H show the functional comparison between porcine and human recellularized livers (BELs). Figure 3A shows a graph indicating that ammonia was removed from the medium 1 hour after a 200 μM bolus administration to porcine BEL and human BEL. The data are presented per million cells (porcine, n = 23; human, n = 16). Figure 3B shows a graph indicating the ammonia clearance performed daily for 1 hour during 3 days of BEL culture in porcine BEL and human BEL. The data are presented per million cells (porcine, n = 5; human, n = 10). Figure 3C shows a graph indicating the glucose consumption calculated daily after 24 - hour culture for 3 days in porcine and human BELs (porcine, n = 5; human, n = 10). Figure 3D shows a graph indicating the urea concentration in the BEL - conditioned medium collected at the end of a 24 - hour culture period. The data are presented per million cells (porcine, n = 14; human, n = 8). Figure 3E shows a graph indicating the fibrinogen production in the BEL - conditioned medium collected at the end of a 24 - hour culture period. The data are presented per million cells (porcine, n = 5; human, n = 8). Figure 3F shows a graph indicating the A1AT production in the BEL - conditioned medium collected at the end of a 24 - hour culture period. The data are presented per million cells (porcine, n = 5; human, n = 8). Figure 3G shows a schematic diagram showing the circuit for the blood perfusion test. Figure 3H shows a graph indicating the viability of porcine BEL and human BEL measured for 180 minutes in the blood perfusion circuit (porcine BEL, n = 6; human BEL, 3). Statistical significance: **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns = no significant difference. Represents the results of a t - test between porcine data and human data. For both (Figure 3B, Figure 3C), the results with false discovery correction are shown. [Figure 3D]Figures 3A to 3H show a functional comparison between recellularized liver (BEL) from pigs and humans. Figure 3A shows a graph showing the removal of ammonia from the culture medium 1 hour after a 200 μM bolus administration to pig and human BELs, with data expressed per million cells (n=23 for pigs, n=16 for humans). Figure 3B shows a graph showing the 1-hour ammonia clearance performed daily for 3 days of BEL culture in pig and human BELs, with data expressed per million cells (n=5 for pigs, n=10 for humans). Figure 3C shows a graph showing glucose consumption calculated daily after 24-hour culture for 3 days in pig and human BELs (n=5 for pigs, n=10 for humans). Figure 3D shows a graph showing the urea concentration in the BEL acclimatization medium collected at the end of the 24-hour culture period, with data expressed per million cells (n=14 for pigs, n=8 for humans). Figure 3E shows a graph of fibrinogen production in BEL-conditioned medium collected at the end of a 24-hour culture period, with data expressed per million cells (n=5 for pigs, n=8 for humans). Figure 3F shows a graph of A1AT production in BEL-conditioned medium collected at the end of a 24-hour culture period, with data expressed per million cells (n=5 for pigs, n=8 for humans). Figure 3G shows a schematic diagram of the circuit for the hemoperfusion test. Figure 3H shows a graph of patency of pig and human BEL measured for 180 minutes in the hemoperfusion circuit (n=6 for pigs, n=3 for humans). Statistical significance: **p<0.01, ***p<0.001, ****p<0.0001, and ns = no significant difference. The results of the t-test between the pig and human data are shown, both (Figures 3B and 3C) showing results with false detection correction. [Figure 3E]Figures 3A to 3H show a functional comparison between recellularized liver (BEL) from pigs and humans. Figure 3A shows a graph showing the removal of ammonia from the culture medium 1 hour after a 200 μM bolus administration to pig and human BELs, with data expressed per million cells (n=23 for pigs, n=16 for humans). Figure 3B shows a graph showing the 1-hour ammonia clearance performed daily for 3 days of BEL culture in pig and human BELs, with data expressed per million cells (n=5 for pigs, n=10 for humans). Figure 3C shows a graph showing glucose consumption calculated daily after 24-hour culture for 3 days in pig and human BELs (n=5 for pigs, n=10 for humans). Figure 3D shows a graph showing the urea concentration in the BEL acclimatization medium collected at the end of the 24-hour culture period, with data expressed per million cells (n=14 for pigs, n=8 for humans). Figure 3E shows a graph of fibrinogen production in BEL-conditioned medium collected at the end of a 24-hour culture period, with data expressed per million cells (n=5 for pigs, n=8 for humans). Figure 3F shows a graph of A1AT production in BEL-conditioned medium collected at the end of a 24-hour culture period, with data expressed per million cells (n=5 for pigs, n=8 for humans). Figure 3G shows a schematic diagram of the circuit for the hemoperfusion test. Figure 3H shows a graph of patency of pig and human BEL measured for 180 minutes in the hemoperfusion circuit (n=6 for pigs, n=3 for humans). Statistical significance: **p<0.01, ***p<0.001, ****p<0.0001, and ns = no significant difference. The results of the t-test between the pig and human data are shown, both (Figures 3B and 3C) showing results with false detection correction. [Figure 3F]Figures 3A to 3H show a functional comparison between recellularized liver (BEL) from pigs and humans. Figure 3A shows a graph showing the removal of ammonia from the culture medium 1 hour after a 200 μM bolus administration to pig and human BELs, with data expressed per million cells (n=23 for pigs, n=16 for humans). Figure 3B shows a graph showing the 1-hour ammonia clearance performed daily for 3 days of BEL culture in pig and human BELs, with data expressed per million cells (n=5 for pigs, n=10 for humans). Figure 3C shows a graph showing glucose consumption calculated daily after 24-hour culture for 3 days in pig and human BELs (n=5 for pigs, n=10 for humans). Figure 3D shows a graph showing the urea concentration in the BEL acclimatization medium collected at the end of the 24-hour culture period, with data expressed per million cells (n=14 for pigs, n=8 for humans). Figure 3E shows a graph of fibrinogen production in BEL-conditioned medium collected at the end of a 24-hour culture period, with data expressed per million cells (n=5 for pigs, n=8 for humans). Figure 3F shows a graph of A1AT production in BEL-conditioned medium collected at the end of a 24-hour culture period, with data expressed per million cells (n=5 for pigs, n=8 for humans). Figure 3G shows a schematic diagram of the circuit for the hemoperfusion test. Figure 3H shows a graph of patency of pig and human BEL measured for 180 minutes in the hemoperfusion circuit (n=6 for pigs, n=3 for humans). Statistical significance: **p<0.01, ***p<0.001, ****p<0.0001, and ns = no significant difference. The results of the t-test between the pig and human data are shown, both (Figures 3B and 3C) showing results with false detection correction. [Figure 3G]Figures 3A to 3H show a functional comparison between recellularized liver (BEL) from pigs and humans. Figure 3A shows a graph showing the removal of ammonia from the culture medium 1 hour after a 200 μM bolus administration to pig and human BELs, with data expressed per million cells (n=23 for pigs, n=16 for humans). Figure 3B shows a graph showing the 1-hour ammonia clearance performed daily for 3 days of BEL culture in pig and human BELs, with data expressed per million cells (n=5 for pigs, n=10 for humans). Figure 3C shows a graph showing glucose consumption calculated daily after 24-hour culture for 3 days in pig and human BELs (n=5 for pigs, n=10 for humans). Figure 3D shows a graph showing the urea concentration in the BEL acclimatization medium collected at the end of the 24-hour culture period, with data expressed per million cells (n=14 for pigs, n=8 for humans). Figure 3E shows a graph of fibrinogen production in BEL-conditioned medium collected at the end of a 24-hour culture period, with data expressed per million cells (n=5 for pigs, n=8 for humans). Figure 3F shows a graph of A1AT production in BEL-conditioned medium collected at the end of a 24-hour culture period, with data expressed per million cells (n=5 for pigs, n=8 for humans). Figure 3G shows a schematic diagram of the circuit for the hemoperfusion test. Figure 3H shows a graph of patency of pig and human BEL measured for 180 minutes in the hemoperfusion circuit (n=6 for pigs, n=3 for humans). Statistical significance: **p<0.01, ***p<0.001, ****p<0.0001, and ns = no significant difference. The results of the t-test between the pig and human data are shown, both (Figures 3B and 3C) showing results with false detection correction. [Figure 3H]Figures 3A to 3H show a functional comparison between recellularized liver (BEL) from pigs and humans. Figure 3A shows a graph showing the removal of ammonia from the culture medium 1 hour after a 200 μM bolus administration to pig and human BELs, with data expressed per million cells (n=23 for pigs, n=16 for humans). Figure 3B shows a graph showing the 1-hour ammonia clearance performed daily for 3 days of BEL culture in pig and human BELs, with data expressed per million cells (n=5 for pigs, n=10 for humans). Figure 3C shows a graph showing glucose consumption calculated daily after 24-hour culture for 3 days in pig and human BELs (n=5 for pigs, n=10 for humans). Figure 3D shows a graph showing the urea concentration in the BEL acclimatization medium collected at the end of the 24-hour culture period, with data expressed per million cells (n=14 for pigs, n=8 for humans). Figure 3E shows a graph of fibrinogen production in BEL-conditioned medium collected at the end of a 24-hour culture period, with data expressed per million cells (n=5 for pigs, n=8 for humans). Figure 3F shows a graph of A1AT production in BEL-conditioned medium collected at the end of a 24-hour culture period, with data expressed per million cells (n=5 for pigs, n=8 for humans). Figure 3G shows a schematic diagram of the circuit for the hemoperfusion test. Figure 3H shows a graph of patency of pig and human BEL measured for 180 minutes in the hemoperfusion circuit (n=6 for pigs, n=3 for humans). Statistical significance: **p<0.01, ***p<0.001, ****p<0.0001, and ns = no significant difference. The results of the t-test between the pig and human data are shown, both (Figures 3B and 3C) showing results with false detection correction. [Figure 4A]Figures 4A to 4F show that human BEL exhibits extended function after the cold storage period. Figure 4A shows a schematic diagram of the assay sampling scheme during the culture period and during the 3-day extended retention period between the treatment window after 14-16 hours of cold storage (CS). Figure 4B shows a graph of ammonia removed from the culture medium by human BEL 1 hour after delivery of a 200 μM ammonia bolus, performed daily during the treatment window after the cold storage period (n=5-7). Figure 4C shows a graph of daily glucose consumption measurements (n=4-9). Figure 4D shows a graph of urea production (n=4-7). Figure 4E shows a graph of fibrinogen (n=4-7). Figure 4F shows a graph of A1AT production (n=4-7, n=2 for measurements collected on days 6 and 7) quantified daily in human BEL after 24 hours of culture during the treatment window after cold storage. Statistical significance: *p<0.05, **p<0.01, ****p<0.0001, and ns = no significant difference. [Figure 4B] Figures 4A to 4F show that human BEL exhibits extended function after the cold storage period. Figure 4A shows a schematic diagram of the assay sampling scheme during the culture period and during the 3-day extended retention period between the treatment window after 14-16 hours of cold storage (CS). Figure 4B shows a graph of ammonia removed from the culture medium by human BEL 1 hour after delivery of a 200 μM ammonia bolus, performed daily during the treatment window after the cold storage period (n=5-7). Figure 4C shows a graph of daily glucose consumption measurements (n=4-9). Figure 4D shows a graph of urea production (n=4-7). Figure 4E shows a graph of fibrinogen (n=4-7). Figure 4F shows a graph of A1AT production (n=4-7, n=2 for measurements collected on days 6 and 7) quantified daily in human BEL after 24 hours of culture during the treatment window after cold storage. Statistical significance: *p<0.05, **p<0.01, ****p<0.0001, and ns = no significant difference. [Figure 4C]Figures 4A to 4F show that human BEL exhibits extended function after the cold storage period. Figure 4A shows a schematic diagram of the assay sampling scheme during the culture period and during the 3-day extended retention period between the treatment window after 14-16 hours of cold storage (CS). Figure 4B shows a graph of ammonia removed from the culture medium by human BEL 1 hour after delivery of a 200 μM ammonia bolus, performed daily during the treatment window after the cold storage period (n=5-7). Figure 4C shows a graph of daily glucose consumption measurements (n=4-9). Figure 4D shows a graph of urea production (n=4-7). Figure 4E shows a graph of fibrinogen (n=4-7). Figure 4F shows a graph of A1AT production (n=4-7, n=2 for measurements collected on days 6 and 7) quantified daily in human BEL after 24 hours of culture during the treatment window after cold storage. Statistical significance: *p<0.05, **p<0.01, ****p<0.0001, and ns = no significant difference. [Figure 4D] Figures 4A to 4F show that human BEL exhibits extended function after the cold storage period. Figure 4A shows a schematic diagram of the assay sampling scheme during the culture period and during the 3-day extended retention period between the treatment window after 14-16 hours of cold storage (CS). Figure 4B shows a graph of ammonia removed from the culture medium by human BEL 1 hour after delivery of a 200 μM ammonia bolus, performed daily during the treatment window after the cold storage period (n=5-7). Figure 4C shows a graph of daily glucose consumption measurements (n=4-9). Figure 4D shows a graph of urea production (n=4-7). Figure 4E shows a graph of fibrinogen (n=4-7). Figure 4F shows a graph of A1AT production (n=4-7, n=2 for measurements collected on days 6 and 7) quantified daily in human BEL after 24 hours of culture during the treatment window after cold storage. Statistical significance: *p<0.05, **p<0.01, ****p<0.0001, and ns = no significant difference. [Figure 4E]Figures 4A to 4F show that human BEL exhibits extended function after the cold storage period. Figure 4A shows a schematic diagram of the assay sampling scheme during the culture period and during the 3-day extended retention period between the treatment window after 14-16 hours of cold storage (CS). Figure 4B shows a graph of ammonia removed from the culture medium by human BEL 1 hour after delivery of a 200 μM ammonia bolus, performed daily during the treatment window after the cold storage period (n=5-7). Figure 4C shows a graph of daily glucose consumption measurements (n=4-9). Figure 4D shows a graph of urea production (n=4-7). Figure 4E shows a graph of fibrinogen (n=4-7). Figure 4F shows a graph of A1AT production (n=4-7, n=2 for measurements collected on days 6 and 7) quantified daily in human BEL after 24 hours of culture during the treatment window after cold storage. Statistical significance: *p<0.05, **p<0.01, ****p<0.0001, and ns = no significant difference. [Figure 4F] Figures 4A to 4F show that human BEL exhibits extended function after the cold storage period. Figure 4A shows a schematic diagram of the assay sampling scheme during the culture period and during the 3-day extended retention period between the treatment window after 14-16 hours of cold storage (CS). Figure 4B shows a graph of ammonia removed from the culture medium by human BEL 1 hour after delivery of a 200 μM ammonia bolus, performed daily during the treatment window after the cold storage period (n=5-7). Figure 4C shows a graph of daily glucose consumption measurements (n=4-9). Figure 4D shows a graph of urea production (n=4-7). Figure 4E shows a graph of fibrinogen (n=4-7). Figure 4F shows a graph of A1AT production (n=4-7, n=2 for measurements collected on days 6 and 7) quantified daily in human BEL after 24 hours of culture during the treatment window after cold storage. Statistical significance: *p<0.05, **p<0.01, ****p<0.0001, and ns = no significant difference. [Figure 5A]Figures 5A to 5D show that human recellularized liver exhibits extended function for at least 7 days. Figure 5A shows a graph of ammonia clearance over time. Figure 5B shows a graph of urea concentration over time. Figure 5C shows a graph of fibrinogen concentration over time. Figure 5D shows a graph of α1-antitrypsin (A1AT) concentration over time. [Figure 5B] Figures 5A to 5D show that human recellularized liver exhibits extended function for at least 7 days. Figure 5A shows a graph of ammonia clearance over time. Figure 5B shows a graph of urea concentration over time. Figure 5C shows a graph of fibrinogen concentration over time. Figure 5D shows a graph of α1-antitrypsin (A1AT) concentration over time. [Figure 5C] Figures 5A to 5D show that human recellularized liver exhibits extended function for at least 7 days. Figure 5A shows a graph of ammonia clearance over time. Figure 5B shows a graph of urea concentration over time. Figure 5C shows a graph of fibrinogen concentration over time. Figure 5D shows a graph of α1-antitrypsin (A1AT) concentration over time. [Figure 5D] Figures 5A to 5D show that human recellularized liver exhibits extended function for at least 7 days. Figure 5A shows a graph of ammonia clearance over time. Figure 5B shows a graph of urea concentration over time. Figure 5C shows a graph of fibrinogen concentration over time. Figure 5D shows a graph of α1-antitrypsin (A1AT) concentration over time. [Figure 6A] Figures 6A to 6D show that human recellularized liver retains function for at least 4 days after cryogenic storage. Figure 6A shows a graph of ammonia clearance over time. Figure 6B shows a graph of urea concentration over time. Figure 6C shows a graph of fibrinogen concentration over time. Figure 6D shows a graph of α1-antitrypsin (A1AT) concentration over time. The arrows indicate 24 hours after returning to ambient temperature perfusion in the bioreactor following a 12-hour cryogenic storage transport simulation (PT = post-transport). [Figure 6B]Figures 6A to 6D show that human recellularized liver retains function for at least 4 days after cryogenic storage. Figure 6A shows a graph of ammonia clearance over time. Figure 6B shows a graph of urea concentration over time. Figure 6C shows a graph of fibrinogen concentration over time. Figure 6D shows a graph of α1-antitrypsin (A1AT) concentration over time. The arrows indicate 24 hours after returning to ambient temperature perfusion in the bioreactor following a 12-hour cryogenic storage transport simulation (PT = post-transport). [Figure 6C] Figures 6A to 6D show that human recellularized liver retains function for at least 4 days after cryogenic storage. Figure 6A shows a graph of ammonia clearance over time. Figure 6B shows a graph of urea concentration over time. Figure 6C shows a graph of fibrinogen concentration over time. Figure 6D shows a graph of α1-antitrypsin (A1AT) concentration over time. The arrows indicate 24 hours after returning to ambient temperature perfusion in the bioreactor following a 12-hour cryogenic storage transport simulation (PT = post-transport). [Figure 6D] Figures 6A to 6D show that human recellularized liver retains function for at least 4 days after cryogenic storage. Figure 6A shows a graph of ammonia clearance over time. Figure 6B shows a graph of urea concentration over time. Figure 6C shows a graph of fibrinogen concentration over time. Figure 6D shows a graph of α1-antitrypsin (A1AT) concentration over time. The arrows indicate 24 hours after returning to ambient temperature perfusion in the bioreactor following a 12-hour cryogenic storage transport simulation (PT = post-transport). [Figure 7] Figure 7 shows one embodiment of the ex vivo blood circuit. [Figure 8] Figure 8 shows a photograph illustrating that electron beam treatment of perfused decellularized pig liver after decellularization impairs the desirable shape and orientation of the extracellular matrix necessary for organ recellularization. [Figure 9] Figure 9 shows a photograph of a pig liver that was frozen after collection and is scheduled to be treated with antiviral therapy before decellularization. [Figure 10]Figure 10 shows a photograph of perfused decellularized pig liver that was treated with electron beam before decellularization. [Figure 11] Figure 11 shows a graph of glucose consumption rates in recellularized livers, comparing livers produced using an antiviral pretreatment process with unpretreated controls. [Figure 12] Figure 12 shows a graph of recellularized liver blood loop patency produced using an antiviral pretreatment step, compared to an unpretreated control. [Modes for carrying out the invention]

[0019] Recellularized bioartificial organs, such as livers (also referred to as at least partially recellularized livers), for use in the treatment of liver diseases are provided herein. Methods for producing at least partially recellularized livers are provided herein. Recellularized bioartificial livers have reduced and / or inactivated microbial particles and can be cultured for extended periods (e.g., more than 4 days) before use in transplantation or in an ex vivo blood circuit. The inventors have found that the health and function of recellularized bioartificial livers, including human cells, can be improved by combining antiviral treatment, the use of decellularized porcine extracellular matrix, and cell seeding conditions. The inventors have also found that recellularized bioartificial livers produced by the methods provided herein can be used ex vivo in a target blood circuit to remove ammonia, produce urea, and produce fibrinogen and α1-antitrypsin (A1AT). This function can be maintained for at least 4 days after cryogenic storage and can function for at least 7 days in a bioreactor. Surprisingly and unexpectedly, the claimed method for producing at least partially recellularized liver improved liver function when human cells were seeded onto decellularized porcine extracellular matrix compared to when decellularized porcine extracellular matrix was recellularized with porcine hepatocytes.

[0020] A method for producing a liver composition that is at least partially recellularized is provided herein. Furthermore, a least partially recellularized liver is provided herein, comprising (a) a decellularized non-human animal extracellular matrix; and (b) a population of human endothelial cells and a population of human hepatocytes engrafted in the non-human animal extracellular matrix, wherein the least partially recellularized liver exhibits increased ammonia clearance compared to a comparison population of non-human animal hepatocytes engrafted in a non-human animal extracellular matrix.

[0021] An ex vivo method for treating liver disease in a subject, comprising constructing a blood circuit, the blood circuit containing, in fluid communication, blood from the subject and at least partially recellularized liver provided herein, the at least partially recellularized liver filtering the blood from the subject and removing ammonia, thereby providing a method for treating liver disease in the subject.

[0022] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art.

[0023] The terms used herein are intended solely to describe specific embodiments and are not intended to limit them. Where used herein, the singular forms “a,” “an,” and “the” are intended to include the plural form unless the context explicitly states otherwise. Furthermore, as used herein, the terms “including,” “includes,” “having,” “has,” and “with,” or their variations thereof, mean “comprising.”

[0024] As used herein, the term “about” and its grammatical equivalents in relation to a base number and its grammatical equivalent may include a range of values ​​within ±10% of that value. For example, the quantity “about 10” includes the quantities from 9 to 11. The term “about” in relation to a base number may also include a range of values ​​within ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of that value.

[0025] As used herein, the term “substantially” may refer to a value close to 100% of a given value. In some embodiments, the term may refer to an amount that may be at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.9%, or about 99.99% of the total amount. In some embodiments, the term may refer to an amount that may be about 100% of the total amount.

[0026] As used herein, the terms “decellularized” or “decellularized” may refer to a biological structure (e.g., an isolated organ or part thereof, or tissue) in which cells and tissue contents have been reduced or removed, leaving an intact, acellular underlying structure. Organs such as kidneys may be composed of various specialized tissues. The specialized tissue structure of an organ, i.e., the parenchyma, may provide specific functions related to that organ. The supporting fibrous network of an isolated organ may be the stroma. Most organs have an interstitial skeleton composed of non-specialized connective tissue supporting specialized tissues. The process of decellularization may remove, at least partially, the cellular portion of a tissue, leaving a complex three-dimensional network of the extracellular matrix (ECM). The ECM underlying structure may be composed primarily of collagen, but may also include cytokines, proteoglycans, laminins, fibrillins, and other proteins secreted by cells. Decellularized structures, at least partially, can provide biocompatible substrates into which various cell populations can be injected, or they can provide biocompatible substrates used for implantation as cell-free medical devices, such as wound care matrices, fistula matrices, void fillers, skin fillers, soft tissue reinforcers, or other substrates that enable cell invasion and remodeling after transplantation or application. Decellularized biological structures can be rigid or semi-rigid and have the ability to change their shape. Examples of decellularized isolated organs include, but are not limited to, parenchymal organs such as the heart, kidneys, liver, lungs, pancreas, brain, bones, spleen, gallbladder, bladder, uterus, ureters, and urethra.

[0027] As used herein, the terms “recellularize” or “re-cellatilize” may refer to the engraftment or distribution of any number of cells or cell compositions provided herein onto a decellularized extracellular matrix. Recellularized organs may have the morphology or activity of native non-decellularized organs.

[0028] The terms “effective dose” or “therapeutic dose” may refer to an amount of a composition, such as a cell-containing composition, that may be sufficient to produce the desired activity when introduced into an isolated organ or a portion thereof provided herein.

[0029] As used herein, the term “functional” and its grammatical equivalents may mean the ability to operate, have an intended purpose, or perform such a purpose. “Functional” may include any percentage from the baseline up to 100% of the intended purpose. For example, functional may include 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or up to approximately 100% of the intended purpose, or an approximate figure thereof. In some embodiments, the term "functional" may mean more than 100% or about 100% of normal function, e.g., 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, or up to about 1000% of the intended purpose.

[0030] As used herein, the term “recipient” and its grammatical equivalents may refer to an object. The object may be a human or a non-human animal. The recipient may also need it, for example, treatment for a disease such as cancer. In some embodiments, the recipient may need preventive therapy. In other cases, the recipient may not need it.

[0031] As used herein, the term “subject” and its grammatical equivalents may refer to a human or a non-human. A subject may be a mammal. A subject may be a human mammal of biological sex, male or female. A subject may be of any age. A subject may be an embryo. A subject may be a newborn or up to approximately 100 years old. A subject may be in need of it. A subject may have a disease, such as cancer. A subject may be premenopausal or menopausal, or may have induced menopause.

[0032] The terms “treatment” or “to treat” and their grammatical equivalents may refer to the medical management of a subject intended to cure, improve, stabilize, or prevent a disease, condition, or disorder. Treatment may include active treatment, i.e., treatment specifically directed toward improvement of a disease, condition, or disorder. Treatment may include causal treatment, i.e., treatment directed toward the removal of the cause of the associated disease, condition, or disorder. Furthermore, this treatment may include palliative treatment, i.e., treatment designed to alleviate symptoms rather than cure the disease, condition, or disorder. Treatment may include preventive treatment, i.e., treatment directed toward minimizing, or partially or completely inhibiting, the onset of a disease, condition, or disorder. Treatment may include supportive treatment, i.e., treatment employed to complement another specific therapy directed toward improvement of a disease, condition, or disorder. In some embodiments, the condition may be pathological. In some embodiments, treatment may not completely cure, improve, stabilize, or prevent the disease, condition, or disorder.

[0033] Antimicrobial treatment of organs Provided herein are at least partially recellularized organs or parts thereof (e.g., liver) that are free of or substantially free of microorganisms and / or have reduced or inactivated microorganisms. In some cases, “microorganism” and “microbial particles” are used interchangeably herein. In some embodiments, microorganisms may be eukaryotic cells or eukaryotes, or prokaryotic cells or prokaryotes, viruses, bacteria, fungi, yeasts, proteins such as prions, parasites, endotoxins, or any combination thereof. In some embodiments, at least partially recellularized organs or parts thereof are free of viruses or viral particles. In some embodiments, antimicrobial treatment includes antiviral treatment.

[0034] Prior to decellularization of the non-human animal organs provided herein, the non-human animal organs are excised or isolated from the non-human animal and frozen. In some embodiments, the isolated non-human animal liver is stored at a temperature of about -80°C to about 0°C. In some embodiments, the isolated non-human animal liver is stored at a temperature of about -20°C to about -10°C.

[0035] Frozen non-human animal organs (e.g., livers) are treated with antiviral treatments provided herein. In some embodiments, the antiviral treatment includes treating or irradiating the non-human animal organs with radiation. In some embodiments, the antiviral treatment includes treating or irradiating the non-human animal organs with electron beams (E-beams). In the irradiation treatment, the electron beam dose is the amount of energy absorbed by the target material (e.g., non-human animal liver), in units of gray, where 1 gray is equal to 1 joule / kilogram. In some embodiments, the non-human animal organs are treated with electron beams at electron doses ranging from about 1 kilogray (kGy) to about 100 kGy. In some embodiments, the electron dose is about 2 kGy to about 50 kGy. In some embodiments, the electron dose is about 5 kGy to about 25 kGy. In some embodiments, the electron dose is about 10 kGy to about 20 kGy. In some embodiments, the electron dose is 10 kGy to 20 kGy. In some embodiments, non-human animal organs are irradiated with electron beams at electron doses of at least 5 kGY, at least 6 kGY, at least 7 kGY, at least 8 kGY, at least 9 kGY, at least 10 kGY, at least 11 kGY, at least 12 kGY, at least 13 kGY, at least 14 kGY, at least 15 kGY, at least 16 kGY, at least 17 kGY, at least 18 kGY, at least 19 kGY, at least 20 kGY, at least 21 kGY, at least 22 kGY, at least 23 kGY, at least 24 kGY, at least 25 kGY, or higher.

[0036] In some embodiments, non-human animal organs are irradiated for a period of about 10 seconds to about 200 seconds. In some embodiments, non-human animal organs are irradiated for a period of at least 30 seconds, at least 1 minute (60 seconds), at least 2 minutes, at least 3 minutes, at least 4 minutes, or at least 5 minutes, or longer. In some embodiments, non-human animal organs are irradiated for a period of about 5 minutes to about 1 hour (60 minutes). In some embodiments, non-human animal organs are irradiated for a period of about 1 hour to about 24 hours. In some embodiments, non-human animal organs are irradiated for a period of about 24 hours (1 day) to about 168 hours (7 days).

[0037] In some embodiments, antimicrobial treatment (e.g., antiviral treatment) includes exposing non-human animal organs to ultraviolet light, plasma, gamma rays, or X-rays. The duration for which non-human animal organs are exposed to the antiviral treatment provided herein depends on the type of treatment used to remove viruses and viral particles.

[0038] In some embodiments, the antiviral treatment includes a chemical treatment. In some embodiments, the chemical treatment is performed in addition to radiation treatment, such as electron beam treatment. In some embodiments, the chemical treatment includes contacting the decellularized extracellular matrix of a non-human animal organ or non-human animal liver with a surfactant, enzyme, antimicrobial agent, peroxide, peroxy acid, or any combination thereof.

[0039] Antimicrobial agents can be non-oxidizing organic compounds that derive their antimicrobial activity through chemical or physicochemical interactions with microorganisms. Suitable antimicrobial agents include high molecular weight quaternary ammonium salts, such as poly[(dimethyliminio)-2-butene-1,4-diyl chloride], [4-tris(2-hydroxyethyl)ammonio]-2-butenyl-w-[tris(2-hydroxyethyl)ammonio]dichloride (chemical registry number 75345-27-6), benzalkonium halides, and biguanides such as salts of alexidine, free alexidine bases, salts of chlorhexidine, hexamethylene biguanides, and polymers thereof.

[0040] Proteolytic enzymes may be used in combination with the antiviral treatments provided herein to simultaneously clean and disinfect a liver that has been at least partially recellularized.

[0041] In some embodiments, non-human animal organs are treated with one or more of the following: peracids, hydrogen peroxide, acetic acid, peracetic acid (PAA), physiological saline, SDS, or sodium hydroxide (NaOH). In some embodiments, non-human animal organs are treated with one or more chemicals selected from acids (e.g., peracids), hydrogen peroxide, chemicals containing hydrogen peroxide, chemicals containing peracids, hydrogen peroxide covalently bonded to an organic part, physiological saline, and sodium-containing solutions. In some embodiments, non-human animal organs are treated with physiological saline. In some embodiments, non-human animal organs are treated with a sodium-containing solution, which may include, for example, 0.1% NaCl, 0.2% NaCl, 0.5% NaCl, 0.7% NaCl, 0.8% NaCl, 0.9% NaCl, 1% NaCl, 1.5% NaCl, 2% NaCl, 2.5% NaCl, 3% NaCl, 3.5% NaCl, 4% NaCl, 4.5% NaCl, 5% NaCl, 5.5% NaCl, 6% NaCl, 6.5% NaCl, 7% NaCl, 8% NaCl, 9% NaCl, 10% NaCl, 12% NaCl, 15% NaCl, 20% NaCl, 23% NaCl, or 25% NaCl. In some embodiments, non-human animals are treated with a disinfectant. In some embodiments, the disinfectant may include saline solution and an acid. In some embodiments, the disinfectant may include saline solution and a peracid (e.g., peracetic acid). In some embodiments, the disinfectant may contain 0.9% NaCl and 600 ppm peracetic acid. In some embodiments, the saline solution may be 1× saline, 2× saline, 5× saline, 7× saline, 10× saline, 12× saline, or 15× saline. In some embodiments, the disinfectant may contain saline and a peracid (e.g., peracetic acid). In some embodiments, the saline solution may be 1× saline. In some embodiments, the acid or peracid in the disinfectant may range from about 25 ppm (parts per million) to about 4000 ppm.In some embodiments, the acid or peracid in the disinfectant solution may be in the range of about 500 ppm to about 700 ppm (500 ppm to 700 ppm), 600 ppm to 650 ppm, 250 ppm to 700 ppm, 250 ppm to 800 ppm, 550 ppm to 1000 ppm, 600 ppm to 700 ppm, 550 ppm to 2000 ppm, 550 ppm to 3000 ppm, 550 ppm to 4000 ppm, 1000 ppm to 2000 ppm, 2000 ppm to 3000 ppm, or 3000 ppm to 4000 ppm. In some embodiments, the acid or peracid is about, at least about, or at most about 10 ppm, 25 ppm, 50 ppm, 75 ppm, 90 ppm, 100 ppm, 125 ppm, 150 ppm, 175 ppm, 200 ppm, 225 ppm, 250 ppm, 275 ppm, 300 ppm, 325 ppm, 350 ppm, 375 ppm, 400 ppm, 425 ppm, 450 ppm, 475 ppm, 500 ppm, 525 ppm, 550 ppm It may be 575 ppm, 600 ppm, 625 ppm, 650 ppm, 675 ppm, 700 ppm, 725 ppm, 750 ppm, 775 ppm, 800 ppm, 900 ppm, 1000 ppm, 1200 ppm, 1400 ppm, 1500 ppm, 1700 ppm, 2000 ppm, 2200 ppm, 2500 ppm, 2750 ppm, 3000 ppm, 3200 ppm, 3500 ppm, 3750 ppm, or 4000 ppm. In some embodiments, the acid or peracid (e.g., peracetic acid) may be about 600 ppm. In some embodiments, the acid or peracid (e.g., peracetic acid) may be about 50 ppm. In some embodiments, the disinfectant solution may have a pH of about 4 to about 10 (e.g., pH 6 to 7, 5 to 8, 5 to 10, 6 to 8, and 5.5 to 9). In some embodiments, the disinfectant solution may have a pH of about 5.00 to about 7.50, about 6.00 to about 8.00, about 6.10 to about 7.00, about 4.50 to about 9.00, or about 6.00 to about 6.50.In some embodiments, the disinfectant solution has a pH of about, at least about, or at most about 4.00, 4.50, 5.00, 5.50, 5.80, 5.90, 6.00, 6.05, 6.10, 6.11, 6.12, 6.13, 6.14, 6.15, 6.16, 6.17, 6.18, 6.19, 6.20, 6.30, 6.40, 6.41, 6.42, 6.43, 6.44, 6.45, 6.50, 6.60, 6.90, 7.00, 7.50, 8.00, 8.50, 9.0, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, or 14. In some embodiments, disinfection of an isolated organ, tissue, or part thereof may last from a few minutes to several days, several weeks, several months, or longer. In some embodiments, disinfection may be performed for about, at least about, or at most about 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 12 hours, 15 hours, 18 hours, 20 hours, 22 hours, 24 hours, 27 hours, 30 hours, 34 hours, 40 hours, 44 hours, 48 ​​hours, or longer. In some embodiments, disinfection may be carried out for about, at least about, or at most about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 14 days, 30 days, or longer. In some embodiments, disinfection may be carried out for about at least about, or at most about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 7 weeks, 8 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, or longer. In some embodiments, non-human animal organs are treated with peroxy acid or hydrogen peroxide. In some embodiments, peroxy acid includes peroxyacetic acid, peracetic acid, peroxycarboxylic acid, derivatives, or combinations thereof.

[0042] Decellularization of organs Provided herein are at least partially recellularized organs (e.g., liver) or parts thereof, prepared from a decellularized extracellular matrix, wherein the decellularized extracellular matrix is ​​of non-human animal origin. Decellularization can be carried out using the methods described in U.S. Patent No. 8,470,520, U.S. Patent No. 11,278,643, U.S. Patent Publication No. 17 / 781,843, and U.S. Patent Publication No. 17 / 111,577, which are incorporated herein by reference in their entirety. In some embodiments, decellularization includes perfusion decellularization.

[0043] The first step in decellularizing an organ or tissue, such as the liver, is, if possible, to insert a cannula into the organ or tissue. The blood vessels, tubes, and / or lumens of the organ or tissue can be cannula-inserted using methods and materials known in the art. The next step in decellularizing an organ or tissue is to perfuse the cannula-inserted organ or tissue with a cell-disrupting medium. Perfusion through the organ can be multidirectional (e.g., anterograde and retrograde). Langendorff perfusion of the heart, as well as physiological perfusion (also known as four-chamber working mode perfusion), is common in the art. See, for example, Dehnert, The Isolated Perfused Warm-Blooded Heart According to Langendorff, In Methods in Experimental Physiology and Pharmacology: Biological Measurement Techniques V. Biomesstechnik-Verlag March GmbH, West Germany, 1988. In short, in Langendorff perfusion, a cannula is inserted into the aorta and connected to a reservoir containing a cell-disrupting medium. The cell-disrupting medium can be delivered retrogradely to the aorta at a constant flow rate, for example, by an infusion pump or roller pump, or by a constant hydrostatic pressure. In either case, the aortic valve is forcibly closed, and the perfusion fluid is directed to the coronary artery orifice (thus perfusing the entire ventricular muscle of the heart), and then flows out into the right atrium via the coronary sinus. In working mode perfusion, a second cannula is connected to the left atrium, and the perfusion can be changed from retrograde to antegrade.

[0044] Methods for perfusing other organs or tissues are known in the art. For example, the following references describe perfusion of the lungs, liver, kidneys, brain, and limbs: Van Putte et al., 2002, Ann. Thorac. Surg., 74(3):893-8; den Butter et al., 1995, Transpl. Int., 8:466-71; Firth et al., 1989, Clin. Sci. (Lond.), 77(6):657-61; Mazzetti et al., 2004, Brain Res., 999(1):81-90; Wagner et al., 2003, J. Artif. Organs, 6(3):183-91.

[0045] In some embodiments, one or more cell disruption media may be used to decellularize an organ or tissue. The cell disruption media typically comprises at least one surfactant, such as SDS, PEG, or Triton® X. The cell disruption media may contain water so that the media is osmotically incompatible with cells. Alternatively, the cell disruption media may contain a buffer (e.g., PBS) for osmotic compatibility with cells. The cell disruption media may also contain enzymes, such as, but not limited to, one or more collagenases, one or more dispases, one or more DNases, or proteases such as trypsin. In some cases, the cell disruption media may also, or instead, contain one or more enzyme inhibitors (e.g., protease inhibitors, nuclease inhibitors, and / or collagenase inhibitors).

[0046] In certain embodiments, the cannula-inserted organ or tissue may be sequentially perfused with two different cell disruption media. For example, the first cell disruption media may contain an anionic surfactant such as SDS, and the second cell disruption media may contain an ionic surfactant such as Triton X. Following perfusion with at least one cell disruption media, the cannula-inserted organ or tissue may be perfused with a solution containing one or more enzymes, such as a lavage solution and / or those provided herein. Alternating the direction of perfusion (e.g., antegrade and retrograde) may help to effectively decellularize the entire organ or tissue. The decellularization provided herein essentially decellularizes the organ from the inside out, resulting in minimal damage to the ECM. The organ or tissue may be decellularized at a suitable temperature of 4–40°C. Depending on the size and weight of the organ or tissue, and the specific surfactant and surfactant concentration in the cell disruption medium, the organ or tissue is typically perfused with the cell disruption medium for approximately 0.1 to 12 hours per gram of parenchymal organ or tissue. Including washing, the organ may be perfused for up to approximately 12 to 72 hours per gram of tissue. The perfusion is usually adjusted to physiological conditions, which include pulsatile flow, velocity, and pressure.

[0047] As provided herein, a decellularized organ or tissue essentially consists of extracellular matrix (ECM) components from all or most of the regions of the organ or tissue, including the ECM components of the vascular tree. ECM components may include any or all of fibronectin, fibrillin, laminin, elastin, members of the collagen family (e.g., collagen I, III, and IV), glycosaminoglycans, ground substance, reticular fibers, and thrombospongin, which may remain organized as defined structures such as the basement membrane. Successful decellularization is defined as the absence of detectable myofilaments, endothelial cells, smooth muscle cells, and nuclei in tissue sections using standard histological staining procedures. Removal of any remaining cellular debris from the decellularized organ or tissue is also preferred but not required.

[0048] In some embodiments, it is important that the morphology and structure of the extracellular matrix (ECM) are maintained (i.e., substantially intact) during and after the decellularization process in order to effectively recellularize and generate organs or tissues. As used herein, “morphology” may refer to the overall shape of an organ or tissue, or the ECM. On the other hand, as used herein, “structure” may refer to the outer surface, inner surface, and the ECM between them.

[0049] The morphology and structure of the ECM can be examined visually and / or histologically. For example, the basement membrane on the outer surface of a parenchymal organ, or within the vascular structure of an organ or tissue, should not be removed or significantly damaged by decellularization. In addition, the microfibrils of the ECM should be similar to those of an undecellularized organ or tissue, or not significantly altered from them.

[0050] One or more compounds may be applied in or on a decellularized organ or tissue, for example, to preserve the decellularized organ or to prepare the decellularized organ or tissue for recellularization, and / or to assist or stimulate cells during the recellularization process. Such compounds include, but are not limited to, one or more growth factors (e.g., VEGF, DKK-1, FGF, bFGF, PDGF, HGF, BMP-1, BMP-4, SDF-1, IGF, and HGF), immunomodulators (e.g., cytokines, glucocorticoids, IL2R antagonists, leukotriene antagonists, etc., including but not limited to antibody therapy, immune response regulation using stem cells, bone marrow transplantation, etc.), and / or factors that modify the coagulation cascade (e.g., aspirin, heparin-binding proteins, and heparin). In addition, decellularized organs or tissues may be further treated, for example, by irradiation (e.g., ultraviolet light, gamma rays), to reduce or eliminate the presence of any type of microorganism remaining on or within the decellularized organs or tissues.

[0051] In some embodiments, perfusion decellularization of the extracellular medium (ECM) from an organ or tissue may preserve native microstructures such as intact blood vessels and / or microvascular systems compared to other decellularization techniques, such as immersion-based decellularization. For example, perfusion decellularized ECM from an organ or tissue can preserve collagen content and other binding and signaling factors, as well as vascular structure, thus providing a niche environment with native cues for maintaining the functional differentiation or cellular function of introduced cells. In one embodiment, perfusion decellularized ECM from an organ or tissue may be perfused with cells and / or culture medium under appropriate conditions, including appropriate pressure and flow to mimic conditions normally found in vivo, using the vascular structure of the perfusion decellularized ECM. Normal pressure in a human-sized organ can range from approximately 40 mmHg to approximately 200 mmHg, and the resulting flow rate depends on the diameter of the inflow vessel for perfusion. In a normal human heart, the resulting perfusion flow is approximately 20 mL / min / 100g to 200 mL / min / 100g. Using such a system, seeded cells can achieve seeding concentrations approximately 5 to 1000 times higher than those achieved under two-dimensional cell culture conditions. Unlike two-dimensional culture systems, the ECM environment allows for further functional differentiation of cells, for example, differentiation of progenitor cells into cells exhibiting sustained organ-specific or tissue-specific phenotypes.

[0052] In some embodiments, perfusion decellularization involves cannula insertion into an organ or part thereof. In some embodiments, at least one cannula insertion is introduced into the organ or part thereof. In some embodiments, at least two cannula insertions are introduced into the organ or part thereof. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 cannula insertions are introduced into the organ or part thereof. In some cases, the cannula may be part of a cannula insertion system. The cannula insertion system may include a hollow tube of appropriate size for introduction into a blood vessel, tube, lumen, or any combination thereof in the organ or tissue. Typically, in an organ, at least one blood vessel, tube, and / or lumen is cannula inserted. The perfusion device or cannula insertion system may include a holding container for a solution (e.g., a cell disruption medium) and a mechanism (e.g., a pump, pneumatics, gravity) for circulating the fluid into the organ through one or more cannulas. Sterility of organs or tissues during decellularization and / or recellularization can be maintained by using various techniques known in the art, such as controlling and filtering airflow and / or perfusing with antibiotics, antifungals, or other antimicrobial agents, to prevent the growth of undesirable microorganisms. In some embodiments, the systems provided herein may have the ability to monitor specific perfusion characteristics (e.g., pressure, volume, flow pattern, temperature, gas, pH), mechanical forces (e.g., ventricular wall motion and stress), and electrical stimulation (e.g., pacing). In some embodiments, the vascular bed may change during the course of decellularization and recellularization (e.g., vascular resistance, volume), and a pressure-controlled perfusion device or cannula insertion system may be advantageous to avoid or reduce fluctuations. The effectiveness of perfusion can be evaluated in effluent and tissue sections. Perfusion flow rate, flow pattern, temperature, O2 and CO2 partial pressures, and pH can be monitored using standard methods. In some embodiments, sensors may be used to monitor systems (e.g., bioreactors) and / or organs or tissues.Sonomicrometry, micromanometry, and / or conductance measurements may be used to obtain pressure-volume or preload recruitable stroke work information related to the movement and performance of the myocardial wall. Sensors may be used to monitor, for example, the pressure of the fluid passing through the cannulated organ or tissue; the ambient temperature and / or the temperature of the organ or tissue within the system; the pH and / or flow rate of the fluid passing through the cannulated organ or tissue; and / or the biological activity of the organ or tissue being recellularized. In addition to having sensors to monitor such features, a system for decellularizing and / or recellularizing an organ or tissue may also include means for maintaining or regulating such features. Means for maintaining or regulating such features may include components such as thermometers, thermostats, electrodes, pressure sensors, overflow valves, valves for changing the fluid flow rate, valves for opening and closing fluid connections to a solution used to change the pH of the solution, balloons, external pacemakers, and / or compliance chambers. To help ensure stable conditions (e.g., temperature), the chamber, reservoir, and tubing may be equipped with water jackets.

[0053] In some embodiments, the decellularization method includes preparing an organ or part thereof, inserting a cannula into the organ or part thereof, and perfusing the cannulated organ or part thereof with a solution or medium through the cannula insertion. In some embodiments, cannula insertion is performed in a lumen, blood vessel, tube, or a combination thereof. In some embodiments, about 1 to 3, about 1 to 5, about 2 to 3, about 2 to 5, and about 1 to 8 solutions may be used for organ perfusion. In some embodiments, the solution is perfused at least twice. In some embodiments, the solution is perfused through the organ or part thereof at least 3, 4, 5, 6, 7, 8, 9, or up to 10 times. Various solutions and mediums may be employed during recelluarization. In some embodiments, the solution may be selected from the group consisting of cell disruption solutions, washing solutions, disinfectants, or combinations thereof.

[0054] In some embodiments, a cell disruption solution may contain at least one surfactant (Table 1). A surfactant can be an amphiphilic molecule that may contain both a nonpolar "tail" having aliphatic or aromatic properties and a polar "head." The ionic nature of the polar head group can form the basis for a broad classification of surfactants. Surfactants can be ionic (charged, anionic, or cationic), nonionic (uncharged), or zwitterionic (having both positive and negative charged groups, but with a net charge of zero). In some embodiments, a surfactant can be denaturing or non-denaturing with respect to protein structure. Denaturing surfactants can be anionic, such as sodium dodecyl sulfate (SDS), or cationic, such as ethyltrimethylammonium bromide (ETMAB). These surfactants denature proteins by completely disrupting membranes and disrupting protein-protein interactions. Non-denatured surfactants can be classified into nonionic surfactants such as Triton X-100, NP40, and Tween®, bile salts such as cholates, and zwitterionic surfactants such as CHAPS.

[0055] [Table 1]

[0056] In some embodiments, a washing solution may be used during decellularization. The washing solution may be used to remove residual solutions, such as cell disruption solutions, from organs or parts thereof, and in addition, to remove residual cellular components, enzymes, or combinations thereof. Suitable washing solutions may include water, filtered water, phosphate-buffered saline (PBS), and combinations thereof. PBS can maintain a constant pH and cellular molar osmotic concentration. The pH of most biological materials falls within the range of approximately 7 to 7.6. Any concentration of PBS can be used as a washing solution, such as 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or up to approximately 100% PBS. In some embodiments, chemicals may be added to the washing solution. These chemicals may include antibiotics, DNase I, disinfectants, etc.

[0057] In some embodiments, a disinfectant solution may be used during decellularization. The disinfectant solution may contain any number of agents, such as antibiotics, disinfectants, or combinations thereof. In some embodiments, antibiotics that may be used in the decellularization solution may be selected from the group consisting of actinomycin, ampicillin, carbenicillin, cefotaxime, fosmidomycin, gentamicin, kanamycin, neomycin, amphotericin, penicillin, polymyxin, streptomycin, broad selection antibiotics, and combinations thereof. Any concentration of antibiotic may be introduced into the disinfectant solution. Preferred concentrations of antibiotics may be 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or up to approximately 60%. Suitable concentrations of antibiotics are 0.5 U / ml, 1 U / ml, 5 U / ml, 10 U / ml, 20 U / ml, 30 U / ml, 40 U / ml, 50 U / ml, 60 U / ml, 70 U / ml, 80 U / ml, 90 U / ml, 100 U / ml, 110 U / ml, 120 U / ml, 130 U / ml, 140 U / ml, 150 U / ml, 160 U / ml, 170 U / ml, 180 U / ml, 190 U / ml, 200 U / ml, 300 U / ml, 400 U / ml, 500 U / ml, 600 U / ml, 700 U / ml, 800 U / ml, 900 U / ml, 1000 U / ml, and up to approximately 1500 U / ml. Suitable concentrations of antibiotics are 0.5μg / ml, 1μg / ml, 1.5μg / ml, 2μg / ml, 2.5μg / ml, 3μg / ml, 3.5μg / ml, 4μg / ml, 4.5μg / ml, 5μg / ml, 5.5μg / ml, 6μg / ml, 6.5μg / ml, 7μg / ml, 7.5μ g / ml, 8 μg / ml, 8.5 μg / ml, 9 μg / ml, 9.5 μg / ml, 10 μg / ml, 15 μg / ml, 20 μg / ml, 25 μg / ml, 30 μg / ml, 35 μg / ml, 40 μg / ml, 45 μg / ml, 50 μg / ml, or up to about 60 μg / ml.In some embodiments, the antibiotics may be 1% benzalkonium chloride, 100 U / ml penicillin G, 100 U / ml streptomycin, and 0.25 μg / ml amphotericin B.

[0058] Generally, mild (i.e., nonionic) surfactants at moderate concentrations can promote cell lysis and extraction of soluble proteins (often in their natural form) by impairing the integrity of the cell membrane. Using specific buffering conditions, various surfactants can effectively penetrate between the membrane bilayers at concentrations sufficient to form mixed micelles with isolated phospholipids and membrane proteins. In some embodiments, denaturing surfactants such as SDS can bind to both membrane (hydrophobic) and non-membrane (water-soluble, hydrophilic) proteins at concentrations below CMC (i.e., as monomers). The reaction is driven by equilibrium until saturation. Therefore, the free concentration of monomers determines the surfactant concentration. SDS binding is cooperative (i.e., the binding of one SDS molecule increases the probability that another SDS molecule will bind to the same protein), transforming most proteins into rigid, rod-like structures with lengths proportional to their molecular weight. In some embodiments, non-denaturing surfactants such as Triton X-100 have rigid, bulky, nonpolar heads and do not penetrate water-soluble proteins. As a result, non-denaturing surfactants generally do not disrupt the innate interactions and structure of water-soluble proteins and do not possess cooperative binding properties. The main effect of non-denaturing surfactants is to associate with the hydrophobic portions of membrane proteins, thereby conferring miscibility to them.

[0059] In some embodiments, a system for generating an organ or part thereof or tissue may be controlled by a computer-readable storage medium combined with a programmable processor (for example, the computer-readable storage medium used herein stores instructions for the programmable processor to perform specific steps). For example, such a storage medium may, in combination with the programmable processor, receive and process information from one or more sensors. Such a storage medium may also work with the programmable processor to send information and instructions back to the bioreactor and / or organ or tissue. In some embodiments, the biological activity of the organ or tissue undergoing recellularization may be monitored. This biological activity may be that of the organ or part thereof or tissue itself, for example, in the case of cardiac tissue, such as electrical activity, mechanical activity, mechanical pressure, contractility, and / or wall stress. In addition, the biological activity of cells attached to or engrafted on the organ or part thereof or tissue may be monitored, for example, with respect to ion transport / exchange activity, cell division, and / or cell viability. In some embodiments, it may be useful to simulate active loading on the organ or part thereof during recellularization. In some embodiments, the computer-readable storage medium of the present invention may be used in combination with a programmable processor to coordinately control components necessary for monitoring and maintaining an active load on an organ or tissue. In some cases, the weight of an organ or part thereof or tissue may be input to the computer-readable storage medium provided herein, which, in combination with a programmable processor, can calculate the exposure time and perfusion pressure for that particular organ or tissue. Such a storage medium may record preload and afterload (pressure before and after perfusion, respectively), as well as flow rate. In this embodiment, for example, the computer-readable storage medium, in combination with a programmable processor, may regulate perfusion pressure, direction of perfusion, and / or type of perfusion solution via one or more pump and / or valve controls.

[0060] In some embodiments, perfusion decellularization of an organ or part thereof may be approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or up to approximately 100% more effective than non-perfusion-based decellularization systems. Decellularization of an organ or part thereof can be determined using a variety of means. In some embodiments, decellularization can be determined by histological examination. Histological examination can demonstrate the absence or reduction of cytomaterial, nuclei, and combinations thereof in the decellularized organ or part thereof, as well as the preservation of whole structures such as lobules and central veins. In some embodiments, decellularization can be determined by immunohistochemical staining. Immunohistochemical staining can demonstrate the deficiency of cytofactors such as galactosyl-α(1,3)galactose (α-Gal) after perfusion decellularization. In some embodiments, decellularization can be determined using DNA quantification. DNA quantification may include assays such as Picogreen. DNA quantification assays can determine the amount of DNA loss in an organ or a part thereof.

[0061] Perfusion-based decellularized organs, or parts thereof, retain a native scaffold containing a suitable microenvironment necessary for the introduction of organ-specific cells, along with an intact vascular network for reconnection to the target blood supply and a capsule capable of maintaining physiological pressure. These components are important when perfusion recellularization is subsequently used. Perfusion recellularization also utilizes perfusion to rearrange cells with vascular and organ-specific regenerative capabilities onto the organ. These cells migrate to a suitable microenvironment (via relevant signaling protein markers remaining within the perfusion decellularized scaffold) as the organ grows and matures in a bioreactor under normal physiological conditions. The resulting organ can then be transplanted using the same techniques as current organ transplantation. The scaffold created by perfusion decellularization can accept and incorporate various cells onto the organ scaffold being used.

[0062] Immersion decellularization In some embodiments, immersion-based decellularization of an organ or part thereof may be performed. In some embodiments, an entire organ or part thereof may be decellularized by removing all cells and tissue contents from the organ. In some embodiments, decellularization may include a series of sequential extractions. In some embodiments, the first step may involve the removal of cell debris and the solubilization of cell membranes. This may be followed by the solubilization of nuclear cytoplasmic components and nuclear components. In some embodiments, an organ may be decellularized by removing the cell membranes and cell debris surrounding the organ using a gentle mechanical destruction method. A gentle mechanical destruction method may destroy cell membranes. However, damage to or disturbance of the complex internal basis of the biological structure should be avoided in the decellularization process. A gentle mechanical destruction method may include scraping the surface of the organ, shaking the organ, or agitating the organ in an appropriate volume of fluid, such as distilled water. In some embodiments, a gentle mechanical disruption method may involve magnetically agitating the organ or part thereof in an appropriate volume of distilled water (e.g., using a magnetic stirring bar and magnetic plates) until the cell membrane is disrupted and the cell fragments are removed from the organ or part thereof. After the cell membrane has been removed, the nuclear and cytoplasmic components of the biological structure are removed. This may be done by solubilizing the cellular and nuclear components without disrupting the underlying structure. Nonionic surfactants or surfactants may be used to solubilize the nuclear components.Examples of nonionic surfactants (detergents or surfactants) include the Triton series, available from Rohm & Haas, Philadelphia, Pennsylvania (including Triton X-100, Triton N-101, Triton X-114, Triton X-405, Triton X-705, and Triton DF-16, which are commercially available from many vendors); and the Tween series, e.g., monolaurate (Tween 20), monopalmitate (Tween 40), monooleate (Tween 40) Examples include, but are not limited to, sodium cholate, deoxycholate, CHAPS, saponins, n-decyl β-D-glucopyranoside, n-heptyl β-D-glucopyranoside, n-octyl α-D-glucopyranoside, and nonidet P-40. 80)

[0063] Physical processing In some cases, physical treatment of an organ or part thereof may be performed to achieve decellularization. Physical treatment may be used to lyse, kill, and remove cells from the ECM or part thereof. Physical treatment may utilize temperature, force, pressure, and electrical disruption. In some cases, the thermal method may use rapid freeze-thaw as a mechanism. For example, by freezing the tissue, fine ice crystals may form around the cell membrane, and the cells may be lysed. After the cells have been lysed, the tissue may be further exposed to a liquid form of chemical that can break down and wash away any residual or undesirable components. In some cases, the thermal method may preserve the physical structure of the ECM scaffold. Organs or parts thereof, and tissues, may be decellularized at appropriate temperatures. Appropriate temperatures may be around 4°C, 8°C, 10°C, 12°C, 14°C, 16°C, 18°C, 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, 32°C, 34°C, 36°C, 38°C, 40°C, 45°C, 50°C, 55°C, 60°C, or up to approximately 70°C. Physical treatment may also include the use of pressure. Pressurized decellularization may involve the controlled use of hydrostatic pressure applied to tissues, organs, or parts thereof. Pressurized decellularization may be carried out at high temperatures in some cases to avoid unsupervised ice crystal formation. In some cases, electrical disruption of organs or parts thereof may be performed. Electrical disruption may be performed to dissolve cells present inside the tissue or organ. By exposing tissues, organs, or parts thereof to electrical pulses, micropores may be formed in the cell membrane. Cells may die after their homeostatic electrical equilibrium is disrupted by the applied stimulus. This electrical process is described as non-thermal irreversible electroporation (NTIRE).

[0064] Chemical treatment and enzymatic treatment In some cases, chemical treatment of an organ or part thereof may be performed to achieve decellularization. Chemicals and / or salts thereof for use in chemical treatment may be selected for decellularization depending on the thickness of the tissue or organ, the extracellular matrix composition, and the intended use. For example, enzymes would not be used in collagenous tissue because they disrupt connective tissue fibers. However, if collagen is not present in high concentrations or is not required in the tissue, enzymes may be a viable option for decellularization. Chemicals and / or salts thereof may be used to kill or remove cells and may be, but are not limited to, acids, alkalis, ionic surfactants, nonionic surfactants, and zwitterionic surfactants. In some cases, one or more chemicals may constitute a cell disruption medium. A cell disruption medium may include at least one surfactant such as sodium dodecyl sulfate (SDS), polyethylene glycol (PEG), or Triton X. Surfactants can effectively act to dissolve cell membranes and expose their contents to further degradation. For example, after SDS lyses the cell membrane, endonucleases and / or exonucleases may degrade the genetic contents, while other cellular components may be solubilized and washed away from the matrix. In some cases, surfactants can be mixed with alkaline and / or acidic treatments because they have the ability to degrade nucleic acids and solubilize cytoplasmic inclusions.

[0065] One or more cell disruption media may be used to decellularize an organ or tissue. The cell disruption media may contain at least one surfactant, such as SDS, PEG, or Triton X. The cell disruption media may contain water so that the media is osmotically incompatible with cells. Alternatively, the cell disruption media may contain a buffer (e.g., PBS) for osmotic compatibility with cells. The cell disruption media may also contain enzymes, for example, one or more collagenases, one or more dispases, one or more DNases, one or more proteases, and any combination thereof. In some cases, the cell disruption media may also, or instead, contain one or more enzyme inhibitors (e.g., protease inhibitors, nuclease inhibitors, and / or collagenase inhibitors). The cell disruption media may contain water so that the media is osmotically incompatible with cells. Alternatively, the cell disruption medium may contain a buffer (e.g., PBS) for osmotic compatibility with cells. The cell disruption medium may also contain enzymes, for example, one or more collagenases, one or more dispases, one or more DNases, or proteases such as trypsin. In some cases, the cell disruption medium may also, or instead, contain one or more enzyme inhibitors (e.g., protease inhibitors, nuclease inhibitors, and / or collagenase inhibitors). In some cases, nonionic surfactants such as Triton X-100 may be used. Triton X-100 can disrupt interactions between lipids and between lipids and proteins. In some cases, Triton X-100 may not disrupt protein-protein interactions, which may be beneficial in preserving the ECM. In some cases, EDTA may be used. EDTA can be a chelating agent that binds to calcium, which can be a component for proteins to interact with each other. By rendering calcium unavailable, EDTA can prevent endogenous proteins between cells from binding to each other.EDTA is sometimes used in combination with trypsin, an enzyme that acts as a protease, cleaving existing bonds between endogenous proteins in adjacent cells within a tissue.

[0066] Surfactants are effective for approximately 10 minutes, 30 minutes, 60 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, and 32 hours. It can be administered for 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 ​​hours, 49 hours, 50 hours, 51 hours, 52 hours, 53 hours, 54 hours, 55 hours, 56 hours, 57 hours, 58 hours, 59 hours, 60 hours, 70 hours, 80 hours, 90 hours, or up to approximately 100 hours.

[0067] Depending on the size and / or weight of the organ or part thereof, chemical treatments such as surfactants may be applied to the organ or part thereof using a cell disruption medium for approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 hours, and up to approximately 20 hours per gram of parenchymal organ or tissue.

[0068] Including rinsing, organs can be treated for up to approximately 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, and more. Perfusion may be performed for 38 hours, up to approximately 39 hours, up to approximately 40 hours, up to approximately 41 hours, up to approximately 42 hours, up to approximately 43 hours, up to approximately 44 hours, up to approximately 45 hours, up to approximately 46 hours, up to approximately 47 hours, up to approximately 48 hours, up to approximately 49 hours, up to approximately 50 hours, up to approximately 51 hours, up to approximately 52 hours, up to approximately 53 hours, up to approximately 54 hours, up to approximately 55 hours, up to approximately 56 hours, up to approximately 57 hours, up to approximately 58 hours, up to approximately 59 hours, up to approximately 60 hours, up to approximately 70 hours, up to approximately 80 hours, up to approximately 90 hours, or up to approximately 100 hours. In some cases, an organ or part thereof may be perfused for approximately 12 to approximately 72 hours per gram of tissue. In some embodiments, perfusion may be adjusted to physiological conditions, which include pulsatile flow, velocity, pressure, and any combination thereof.

[0069] In some cases, an organ, part thereof, or tissue may be sequentially brought into contact with at least two different cell disruption media. For example, the first cell disruption media may contain an anionic surfactant such as SDS, and the second cell disruption media may contain an ionic surfactant such as Triton X. Following contact, such as perfusion with at least one cell disruption media, the cannula-inserted organ or tissue may be perfused with a solution containing one or more enzymes, such as a lavage solution and / or those provided herein. In some cases, alternating the direction of perfusion (e.g., anterograde and retrograde) may help to effectively decellularize the organ, part thereof, or tissue. The decellularization provided herein may decellularize the organ or part thereof from the inside out, resulting in minimal damage to the ECM.

[0070] In some cases, a sequential decellularization method may include contacting an organ or part thereof with a cell disruption medium such as an SDS surfactant, followed by a washing step, followed by the addition of one or more chemical substances, followed by contact with a surfactant, and finally, at least one washing step. A sequential decellularization method may include at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelfth, thirteen, thirteen, thirteen, thirteen, fifteen, or up to fifteen, contact steps with any medium or solution provided herein.

[0071] The buffers provided herein may be in concentrations of approximately 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or up to approximately 100%.

[0072] Organ recellularization Decellularized organs and parts thereof provided herein can be recellularized. Organs or tissues can be produced by contacting a decellularized organ or tissue provided herein, such as a decellularized non-human animal liver, with a cell population. In some embodiments, the decellularized extracellular matrix provided herein is contacted with a cell composition comprising one or more cell populations or cell types. In some embodiments, the decellularized extracellular matrix provided herein is contacted with a first cell composition and a second cell composition. In some embodiments, the first cell composition comprises an endothelial cell population. In some embodiments, the second cell composition comprises a hepatocyte population. In some embodiments, the cell composition provided herein is a mixture of different cell types. For example, the cell composition may comprise a hepatocyte population and an endothelial cell population.

[0073] A partially recellularized liver comprising a first cell composition comprising human endothelial cells and a second cell composition comprising human hepatocytes is provided herein. In some embodiments, the decellularized extracellular matrix is ​​brought into contact with the second cell composition when the first cell composition is characterized to have a glucose consumption rate of at least about 10 mg / hour, at least about 20 mg / hour, at least about 30 mg / hour, at least about 40 mg / hour, at least about 50 mg / hour, at least about 60 mg / hour, at least about 70 mg / hour, at least about 80 mg / hour, at least about 90 mg / hour, or at least about 100 mg / hour.

[0074] In some embodiments, the cell composition may include a population of regenerative cells. As used herein, regenerative cells are any cells used to re-cell a decellularized organ or tissue. Regenerative cells may be totipotent, pluripotent, or multipotent cells, and may have an undetermined or determined differentiation fate. Regenerative cells may also be a single lineage of cells. In addition, regenerative cells may be undifferentiated, partially differentiated, or fully differentiated cells. Regenerative cells as used herein include embryonic stem cells (as defined by the National Institutes of Health (NIH); see, for example, the glossary at stemcells.nih.gov on the World Wide Web). Regenerative cells also include progenitor cells, precursor cells, and “adult”-derived stem cells, including umbilical cord cells and fetal stem cells. Examples of regenerative cells that may be used to re-cell an organ or part thereof, as provided herein, may include, but are not limited to, embryonic stem cells, umbilical cord blood cells, tissue-derived stem cells or progenitor cells, bone marrow-derived stem cells or progenitor cells, blood-derived stem cells or progenitor cells, adipose tissue-derived stem cells or progenitor cells, mesenchymal stem cells (MSCs), skeletal muscle-derived cells, induced pluripotent stem cells (iPSCs), genetically modified cells from which immunogenic factors, including but not limited to HLA, have been removed, or multipotent adult progenitor cells (MAPCs). Additional regenerative cells that may be used include tissue-specific stem cells, including cardiac stem cells (CSCs), multipotent adult cardiac-derived stem cells, cardiac fibroblasts, cardiac microvascular endothelial cells, or aortic endothelial cells. Bone marrow-derived stem cells such as bone marrow mononuclear cells (BM-MNCs), endothelial or vascular stem cells or progenitor cells, and peripheral blood-derived stem cells such as endothelial progenitor cells (EPCs) may also be used as regenerative cells. In some embodiments, the number of regenerative cells that can be introduced into a decellularized organ or part thereof to generate an organ or tissue may depend on both the organ (e.g., which organ, its size and weight) or tissue and the type and developmental stage of the regenerative cells.Different types of cells may have different tendencies regarding the population density they reach. Similarly, different organs or tissues may be recellularized at different densities. For example, a decellularized organ or tissue may be “seeded” with at least about 1,000 (e.g., at least 10,000, 100,000, 1,000,000, 10,000,000, or 100,000,000) regenerative cells; or about 1,000 cells / mg of tissue (wet weight, i.e., before decellularization) to about 10,000,000 cells / mg of tissue (wet weight) may be attached thereto. In some embodiments, regenerative cells may be introduced (“seeded”) into a decellularized organ or tissue by injection into one or more sites.

[0075] The methods for recellularizing tissue or organ matrix provided herein also include reendothelialization of the tissue or organ matrix with endothelial cells or endothelial progenitor cells. In one embodiment, endothelial cells and endothelial progenitor cells are obtained by culturing embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) under appropriate conditions for inducing stem cells into endothelial lineages. Endothelial progenitor cells are cells that are beginning to differentiate into endothelial cells or have the potential to differentiate into endothelial cells (e.g., multipotent cells; e.g., lineage-restricted cells; e.g., cells destined to become endothelial cells) but are not considered fully differentiated endothelial cells. For example, endothelial cells typically express platelet endothelial cell adhesion molecule-1 (PECAM1; also known as CD31) and may also express one or more of the following markers: VEGFR-1 (also known as Flt-1), VEGFR-2 (also known as Flk-1), guanylate-binding protein-1 (GBP-1), thrombomodulin (also known as CD141), VE-cadherin (also known as CD144), von Willebrand factor (vWF), and intercellular adhesion molecule 2 (ICAM-2). In general, endothelial progenitor cells are also capable of taking up acetylated LDL and may migrate toward VEGF and / or form tubes on Matrigel.

[0076] ESCs or iPSCs can be further cultured under conditions that result in fully differentiated endothelial cells. In addition, endothelial cells can be obtained from any number of sources, such as blood, skin, liver, heart, lung, retina, and any other tissue or organ that contains endothelial cells. Representative endothelial cells include, but are not limited to, vascular endothelial cells, bone marrow endothelial cells, circulating blood endothelial cells, human aortic endothelial cells, human brain microvascular endothelial cells, human dermal microvascular endothelial cells, human intestinal microvascular endothelial cells, human pulmonary microvascular endothelial cells, human microvascular endothelial cells, hepatic sinusoidal endothelial cells, human saphenous vein endothelial cells, human umbilical vein endothelial cells, lymphatic endothelial cells, microvessel endothelial cells, microvascular endothelial cells, pulmonary artery endothelial cells, retinal capillary endothelial cells, retinal microvascular endothelial cells, vascular endothelial cells, umbilical cord blood endothelial cells, and combinations thereof. As those skilled in the art will understand, this is not intended to be a comprehensive enumeration of endothelial cells.

[0077] Endothelial cells can be obtained, for example, from one of the many biological material depositaries worldwide. See, for example, the United States Cell Culture Lineage Preservation Center (ATCC.org on the World Wide Web) or the International Depositary Authority of Canada (IDAC; nml-lnm.gc.ca on the World Wide Web). Endothelial cells or endothelial progenitor cells can also be obtained from the recipient individual of the transplanted tissue or organ matrix. These cells are considered autologous to the recipient. In addition, under certain circumstances, the relationship between the tissue or organ matrix and the endothelial cells or endothelial progenitor cells may be homogeneous (i.e., different individuals of the same species); in other cases, the relationship between the tissue or organ matrix and the endothelial cells or endothelial progenitor cells may be heterogeneous (i.e., individuals of different species).

[0078] Compositions comprising endothelial cells or endothelial progenitor cells are typically delivered to a tissue or organ matrix under physiological conditions (e.g., 37°C) and non-physiological conditions (e.g., 4–35°C) in a cell-compatible solution (e.g., in a physiological composition). Physiological compositions as used herein may include, but are not limited to, buffers, nutrients (e.g., sugars, carbohydrates), enzymes, growth and / or differentiation media, cytokines, antibodies, inhibitors, growth factors, salt solutions, or serum-derived proteins. As used herein, a composition "essentially consisting of" endothelial cells or endothelial progenitor cells is a composition that substantially does not contain cells other than endothelial cells or endothelial progenitor cells, but may still contain any components that may be present in a physiological composition (e.g., buffers, nutrients, etc.).

[0079] To optimize reendothelialization, endothelial cells or endothelial progenitor cells are typically introduced into the organ or tissue matrix by perfusion. Similar to pre-cellular perfusion, and as described in International Publication No. 2007 / 025233, perfusion is carried out via vascular or vascular-like structures of the organ or tissue matrix. Perfusion for reendothelialization of the organ or tissue matrix should be carried out at a flow rate sufficient to circulate the physiological cellular composition through the vascular structures. Perfusion with endothelial cells or endothelial progenitor cells may be multidirectional (e.g., anterograde and retrograde) to further optimize reendothelialization. Following cell perfusion, a resting period may be followed to enhance engraftment before reperfusion of the organ or tissue matrix.

[0080] In some embodiments, at least one type of cell may be introduced into a decellularized organ or a portion thereof. For example, a cell population may be injected at multiple locations within the decellularized organ or tissue, or different cell types may be injected into different parts of the decellularized organ or a portion thereof. Alternatively, in addition to injection, regenerative cells, cell populations, or cell cocktails may be introduced by perfusion into a cannula-inserted decellularized organ or a portion thereof. For example, regenerative cells may be perfused into a decellularized organ using a perfusion medium, which may then be converted into a proliferation and / or differentiation medium to induce growth and / or differentiation of the regenerative cells. During recellularization, the organ or tissue may be maintained under conditions that allow at least a portion of the regenerative cells to proliferate, multiply, differentiate, and any combination thereof in the decellularized organ or a portion thereof. In some embodiments, these conditions may include, but are not limited to, appropriate temperature, pressure, electrical activity, mechanical activity, force, appropriate amounts of O2 and / or CO2, appropriate amounts of humidity, sterile or near-sterile conditions, and any combination thereof. During recellularization, the decellularized organ or tissue, and the regenerative cells attached thereto, may be maintained in a suitable environment. For example, regenerative cells may require nutritional supplements (e.g., nutrients and / or carbon sources such as glucose), exogenous hormones or growth factors, and / or a specific pH.

[0081] In some embodiments, the regenerative cells provided herein may be homogeneous with respect to a decellularized organ or part thereof (e.g., a human decellularized organ or tissue seeded with human regenerative cells), or the regenerative cells may be heterogeneous with respect to a decellularized organ or part thereof (e.g., a pig decellularized organ or tissue seeded with human regenerative cells). As used herein, “homogeneous” means cells obtained from the same species from which the organ or tissue originates (e.g., self, or from related or unrelated individuals), while “heterogeneous” means cells obtained from a different species from which the organ or tissue originates.

[0082] In some embodiments, endothelial cells can be perfused into a decellularized liver. The endothelial cell population can engraft in the decellularized liver matrix as described above. In some embodiments, the recellularized organ may include fenestrated endothelium that was not present in the decellularized liver and / or in the seeded or introduced cell population, but may be present after engraftment and / or migration into the decellularized liver matrix.

[0083] A liver that is at least partially recellularized, including human hepatocytes, is provided herein. In some embodiments, the human hepatocytes are primary human hepatocytes. In some embodiments, the human hepatocytes are derived from or differentiated from human stem cells. In some embodiments, the human stem cells are embryonic stem cells, induced pluripotent stem cells, or adult stem cells. In some embodiments, the human hepatocytes are derived from or differentiated from progenitor cells. In some embodiments, the human hepatocytes are human hepatocytes differentiated in vitro.

[0084] In some embodiments, the decellularized extracellular matrix is ​​coated with additional extracellular matrix proteins or solutions. For example, the additional extracellular matrix proteins or solutions may include, but are not limited to, fibronectin, fibrillin, laminin, elastin, members of the collagen family (e.g., collagen I, III, and IV), and solubilized basement membrane matrix (MATRIGEL®) secreted by Engelblesse-Holm-Swam mouse sarcoma cells.

[0085] Characterization of recellularized organs Functional liver tissue and at least partially recellularized liver that remove ammonia, produce urea, and express both endothelial and hepatocyte surface markers are provided herein.

[0086] The recellularized organs and compositions provided herein may be assayed for a range of cell surface markers and / or genes associated with the corresponding primary organ (e.g., liver). The recellularized organs and compositions provided herein may be assayed in comparison to a population of cells that have not engrafted in a decellularized extracellular matrix.

[0087] The transformation or plasticity of at least partially decellularized livers provided herein can be monitored by determining the expression levels of specific genes or proteins. For example, genes in the parenchymal niche or sinusoidal niche of a recellularized organ can be used to assess the formation and structure of the recellularized organ. Genetic markers that can be used to determine sinusoidal marker expression in some embodiments may be, but are not limited to, VEGFR-3, D2-40, STAB2, CD31, RPL19, or LYVE-1. In some cases, a whole biopsy of a seeded liver graft may be taken to examine for the presence or absence of expression of sinusoidal markers, including, but not limited to, VEGFR-3, D2-40, STAB2, CD31, RPL19, or LYVE-1. In some cases, direct detection of increased sinusoidal gene expression and / or fenestration, measured after engraftment into parenchymal space and / or migration and / or proliferation.

[0088] In some embodiments, the at least partially recellularized liver provided herein includes one or more surface markers selected from the group consisting of: CD31, CD105, and asialoglycoprotein receptor 1 (ASGR1). In some embodiments, the at least partially recellularized liver provided herein includes two or more surface markers selected from the group consisting of: CD31, CD105, and asialoglycoprotein receptor 1 (ASGR1). In some embodiments, the at least partially recellularized liver provided herein includes surface markers selected from the group consisting of: CD31, CD105, albumin, van Willebrand factor (vWF), lymphatic endothelial hyaluronan receptor 1 (LYVE-1), TEK receptor tyrosine kinase (Tie-2), and asialoglycoprotein receptor 1 (ASGR1). Methods for measuring gene expression include, for example, polymerase chain reaction (PCR), microarrays, sequencing, and Northern or Southern blotting. Methods for measuring or tracking protein expression include, for example, Western blotting, immunoadsorption assays (e.g., enzyme-linked immunosorbent assay or ELISA), flow cytometry, and microscopy.

[0089] The recellularized artificial liver provided herein has a functional urea cycle in which waste products (ammonia) are removed from the blood by metabolism to urea. Ammonia is produced from protein catabolism, whether due to a high-protein diet, deamination reactions, or prolonged periods of starvation. Ammonia is also naturally produced by the gut microbiota. In muscle and peripheral tissues, glutamate is the amino acid that accepts free ammonia, and this acceptance leads to the formation of glutamine. Glutamine is then transported out of muscle and peripheral tissues and utilized by the liver. Glutaminase breaks down glutamine into glutamate and ammonia. Glutamate also produces additional urea via the enzyme glutamate dehydrogenase. From here, ammonia is first taken up into the mitochondria of hepatocytes, and finally urea is formed. Urea then leaves the cytoplasm of hepatocytes and is finally excreted in the urine.

[0090] Provided herein are at least partially recellularized livers characterized by increased ammonia clearance levels compared to a population of hepatocytes that have not engrafted in the extracellular matrix of pigs. In some embodiments, the at least partially recellularized livers are characterized by having ammonia clearance after cryogenic storage. In some embodiments, the at least partially recellularized livers are cryogenically stored for 6 hours or more and have ammonia clearance. Methods for measuring ammonia clearance include ammonia clearance assays described in the examples.

[0091] A partially recellularized liver characterized by having urea production is provided herein. Furthermore, a partially recellularized liver characterized by having increased urea production compared to a population of hepatocytes that have not engrafted in the extracellular matrix of pigs is provided herein. In some embodiments, the partially recellularized liver is characterized by having urea production after cryogenic storage. In some embodiments, the partially recellularized liver is cryogenically stored for 6 hours or more and has urea production.

[0092] Provided herein are at least partially recellularized livers characterized by increased levels of α1-antitrypsin (A1AT) compared to hepatocyte populations that have not engrafted in the extracellular matrix of pigs. A1AT is a protease inhibitor produced by the liver that protects tissues in the body from inflammation and improves lung elasticity. Loss of A1AT may be associated with chronic and uncontrolled tissue breakdown.

[0093] Provided herein are at least partially recellularized livers characterized by increased levels of fibrinogen compared to hepatocyte populations that have not engrafted in the extracellular matrix of pigs. Fibrinogen is produced by the liver to regulate blood coagulation (clotting).

[0094] The following are provided herein: at least partially recellularized livers characterized by reduced levels of microbial proteins or microbial activity compared to at least partially recellularized livers that have not been treated with antiviral treatment (e.g., electron beam and PAA). The at least partially recellularized livers provided herein can be maintained in culture for extended periods (e.g., more than 4 days) due to improved overall health and safety of the recellularized organ.

[0095] Methods for measuring urea production, microbial proteins, microbial activity, fibrinogen, and A1AT include, for example, Western blot assays, colorimetric assays, chemical assays, liquid chromatography, or immunoadsorption assays (e.g., ELISA). Additional methods are further provided in the following examples.

[0096] The patency of the recellularized organs provided herein can be evaluated over time. In some cases, patency can be assayed for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 hours. In some cases, patency can be assayed for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days.

[0097] In some embodiments, the functionality of the recellularized organs provided herein is evaluated by determining the consumption of specific metabolites (i.e., glucose, lactate, glutamine, glutamic acid, and ammonia). Such consumption can be determined by continuously perfusing the metabolites and measuring the rate of consumption of the metabolites over time, for example, using changes in electrochemical potential. Methods for detecting these metabolites are readily apparent to those skilled in the art, and sensors for determining these metabolites are readily available.

[0098] In some embodiments, the rate of metabolite consumption can be used to determine the success of endothelial cell engraftment onto a decellularized matrix. For example, the glucose consumption rate may correlate with the success of endothelialization in recellularized liver. Furthermore, since the glucose consumption rate may correlate with in vivo graft patency, it can be used as a surrogate for in vivo patency. The consumption of other metabolites such as lactate, glutamine, glutamic acid, and ammonia is also expected to predict in vivo patency.

[0099] Uses of organs and parts thereof The decellularized and recellularized organs or parts thereof provided herein can be used in a variety of applications. For example, the organs or parts thereof can be transplanted into a subject or used ex vivo in a blood circuit for hepatic clearance of blood from ammonia and other toxins.

[0100] Ex vivo blood circuit Methods for treating a subject using an ex vivo blood circuit are provided herein. In some embodiments, the method includes constructing a blood circuit, which is in fluid communication with blood from a subject and at least partially recellularized liver provided herein, the at least partially recellularized liver filtering the blood from the subject and removing ammonia, thereby treating liver disease in the subject. An ex vivo hemoperfusion circuit may be constructed to filter the blood of a subject and remove ammonia from the blood. An exemplary ex vivo hemoperfusion circuit is shown in Figure 7. An ex vivo blood circuit may be used, for example, to assist organ transplantation while a subject is awaiting organ transplantation, or to prolong the survival of a subject with a fatal disease.

[0101] In some embodiments, the ex vivo circuit includes a bioreactor. The bioreactor may be used as part of the system or ex vivo blood circuit provided herein to deliver physical, electrical, chemical, or a combination thereof to an organ or a part thereof (e.g., a liver that has been at least partially recellularized). In some embodiments, the bioreactor may include means for increasing the oxygen level in the culture medium. In some embodiments, the elevated oxygen levels may range from approximately 22% to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, and 95% to 100%. In some embodiments, the increased oxygen levels may fluctuate over culture time. In some embodiments, an increase in oxygen levels in the culture medium in which cells are cultured may result in decreased glucose consumption, delayed metabolic conversion to glycosylation, decreased lactate production, decreased ammonia concentration, increased ammonia clearance rate, stability of the maturation phenotype, proliferation, or any combination thereof. In some embodiments, an increase in oxygen levels in the culture medium in which cells such as hepatocytes are cultured may result in decreased glucose consumption, phenotypic stability as measured by metabolic activity, decreased or delayed glucose production, secretion of coagulation factors, decreased lactate production, decreased ammonia concentration, increased ammonia clearance, or any combination thereof. In some embodiments, the presence of elevated oxygen levels in the culture medium in which one or more cells, such as hepatocytes, are cultured may extend the ability to remove ammonia. In some embodiments, elevated oxygen levels may be generated by direct oxygenation, in-line oxygenation, gas-permeable materials, or any combination thereof. In some cases, direct oxygenation may include the use of a membrane oxygenation chamber. In some embodiments, direct oxygenation may include the use of a bubbler.In some embodiments, inline oxygenation may involve the use of an inline oxygen supply. In some embodiments, the culture medium may be delivered by a peristaltic pump. In some embodiments, the culture medium may be passed through a gas-permeable material, allowing for gas exchange through the material. In some embodiments, gas exchange may include oxygen exchange, nitrogen exchange, carbon dioxide exchange, or any combination thereof. In some embodiments, the at least partially permeable tubing may include silicone tubing. In some embodiments, the silicone tubing may enable oxygen exchange and create an elevated oxygen level in the culture medium. In some embodiments, the oxygen level in the culture medium may be elevated by direct injection of a mixture of oxygen and one or more other gases. In some embodiments, one or more other gases may include nitrogen, carbon dioxide, or a combination of the two. In some embodiments, the oxygen level in the culture medium can be increased by injecting gases containing approximately 40% oxygen, 45% oxygen, 50% oxygen, 55% oxygen, 60% oxygen, 65% oxygen, 70% oxygen, 75% oxygen, 80% oxygen, 85% oxygen, 90% oxygen, 95% oxygen, or 100% oxygen. In some embodiments, the oxygen level in the culture medium can be increased by injecting approximately 100% pure oxygen. In some embodiments, the increased oxygen level in the culture medium, facilitated by oxygen-carrying molecules, can increase accessibility to cells within an isolated organ or part thereof. In some cases, the cells may include seeded hepatocytes. In some cases, the isolated organ or part thereof may include an extracellular matrix (ECM) graft. In some cases, the culture medium can be hyperoxygenated before seeding cells into the isolated organ or part thereof. In some cases, the culture medium may be hyperoxygenated before seeding hepatocytes onto the ECM graft. In some cases, the oxygen level may be adjusted based on metrics. In some cases, the metrics may be evaluated or adjusted and may include glucose levels, lactate levels, pCO2, pH, ammonia levels, pyruvate levels, other measurable parameters, and any combination thereof in the culture medium.In some embodiments, a system comprising any of the compositions disclosed herein may be disclosed herein. In some embodiments, the system may comprise at least one of a bioreactor, a pump, a housing, tubing, oxygen-permeable tubing, an incubator, a motor, a computer, a storage medium, a biological safety cabinet, an incubator, or any combination thereof. In some embodiments, cells are stored in an incubator. In some embodiments, the incubator can control temperature, gas concentration, humidity, and any combination thereof.

[0102] Organ transplant In some embodiments, compositions provided herein, such as partially recellularized organs (e.g., liver), may be transplanted into subjects with disease. Related diseases that may require organ transplantation include, but are not limited to, organ failure, cardiomyopathy, cirrhosis, chronic obstructive pulmonary disease, pulmonary edema, biliary atresia, emphysema and pulmonary hypertension, coronary heart disease, valvular heart disease, congenital heart disease, coronary artery disease, pancreatitis, cystic fibrosis, diabetes mellitus, hepatitis, hypertension, idiopathic pulmonary fibrosis, polycystic kidney disease, short bowel syndrome, injury, congenital defects, genetic disorders, autoimmune diseases, and any combination thereof. Implants according to the present invention may be used to replace or enhance existing tissue. For example, to treat a subject with renal impairment by replacing a dysfunctional kidney with an exogenous or artificial kidney. The subject may be monitored for improvement in renal impairment after transplantation of an exogenous kidney. Any decellularized organ or part thereof provided herein may be used for transplantation into a subject.

[0103] In some embodiments, compositions provided herein, such as parenchymal organs or parts thereof, may have about 1% to about 100% of their original function after decellularization. In some embodiments, compositions provided herein, such as parenchymal organs or parts thereof, may have about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or up to about 100% of their original function after decellularization.

[0104] In some embodiments, certain organs or parts thereof may be suitable for transplantation even if they function less than their counterparts. For example, the liver and kidneys may require approximately 20% of their total organ function to provide the organ function necessary to save a person from liver failure or to wean a person off dialysis. In some embodiments, the liver and kidneys may require approximately 20-30%, 30-40%, 20-50%, 20-60%, and 40-60% of their total organ function to be suitable for transplantation. In some embodiments, organs may function at the same level as their counterparts. For example, the heart is more complex in that it may require approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or up to approximately 100% of its function at the time of transplantation.

[0105] Furthermore, compositions and methods for producing artificial organs or parts thereof containing cell populations are also provided herein. In some embodiments, at least two cell populations can be introduced into a decellularized organ or part thereof. Organs that can be produced include, but are not limited to, the heart, kidney, liver, pancreas, spleen, bladder, ureter, urethra, skeletal muscle, small and large intestines, esophagus, stomach, brain, spinal cord, and bone.

[0106] In some cases, a recellularized liver can be transplanted into a recipient. The recellularized liver provided herein is transplanted as a functional organ. In some cases, function can be determined through the patency of the organ's vascular structure over a long period. Patency can be measured, for example, using the methods described in the following examples. For example, the graft can be connected to a peristaltic pump and subjected to physiologically achievable venous pressure. In some cases, a pressure of 5–50 mmHg may be used to determine patency through the venous vascular structure, or a pressure of 40–120 mmHg may be used to determine patency through the arterial vascular structure.

[0107] In some embodiments, the lifespan of a subject may be extended after transplantation of a composition such as an organ or part thereof provided herein. For example, the lifespan of a subject may be extended by approximately 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 15 years, 20 years, 30 years, 40 years, 50 years, 60 years, 70 years, 80 years, 90 years, or up to approximately 100 years after transplantation. In some embodiments, transplantation of a composition such as an organ or part thereof provided herein may reduce the need for secondary treatment in the subject. Secondary treatment may include dialysis, pacemakers, ventilators, and combinations thereof.

[0108] In vitro screening assay and filtration The decellularized and recellularized organs or parts thereof provided herein may also be used in vitro to screen a wide variety of compounds for the efficacy and cytotoxicity of pharmaceuticals, chemicals, and growth / regulatory factors. Cultures may be maintained in vitro and exposed to the compounds under test. The activity of cytotoxic compounds may be measured by their ability to damage or kill cells in culture. This can be readily assessed by vital staining methods. The effects of growth / regulatory factors may be assessed by analyzing the cellular contents of the matrix, for example, by total cell count and differential cell count. This can be achieved using standard cytological and / or histological methods, including the use of immunocytochemical methods with antibodies that define type-specific cellular antigens. The effects of various drugs on normal cells cultured in reconstructed artificial organs may be evaluated.

[0109] The decellularized and recellularized organs or parts thereof provided herein may be used in vitro to filter aqueous solutions; for example, a reconstructed artificial kidney may be used to filter blood. By using a reconstructed kidney, a system is provided having morphological features similar to in vivo kidney products. This system may be suitable for hemodialysis. In some embodiments, this system may also be useful for hemofiltration to remove water and low molecular weight solutes from blood. The artificial kidney may be maintained in vitro and may be exposed to blood that can be injected into the luminal side of the artificial kidney. The treated aqueous solution may be recovered from the outside of the lumen of the artificial kidney. Filtration efficiency may be evaluated by measuring the ion or metabolic waste content of the filtered and unfiltered blood.

[0110] Delivery vehicle The decellularized and recellularized organs or parts thereof provided herein may be used as vehicles for introducing genes and gene products in vivo to assist or improve transplant outcomes, and / or for use in gene therapy. For example, cultured cells such as endothelial cells may be engineered to express gene products. Cells may be engineered to express gene products transiently and / or under inductive control, or to express gene products as chimeric fusion proteins anchored to endothelial cells, for example, as chimeric molecules consisting of an intracellular domain and / or transmembrane domain of a receptor or receptor-like molecule and a gene product as an extracellular domain fused thereto. In another embodiment, endothelial cells may be genetically engineered to express a gene that is deficient in a patient, or a gene that would exert a therapeutic effect. The gene of interest introduced into the endothelial or parenchymal cells must be related to the disease being treated. For example, in the case of renal impairment, endothelial or cultured kidney cells may be engineered to express a gene product that would improve renal impairment.

[0111] Furthermore, the decellularized and recellularized organs or parts thereof provided herein may be used to produce recombinant proteins, such as α1-antitrypsin (A1AT) or fibrinogen. The recombinant proteins provided herein may be isolated for use in the treatment of diseases.

[0112] Combination therapy In some cases, recellularized livers may be transplanted or used in an ex vivo cycle, along with systemic administration of immunosuppressants. In some embodiments, administration of immunosuppressants prolongs the patency of the transplanted organ. In some cases, the immunosuppressants may be corticosteroids, Janus kinase inhibitors, calcinulin inhibitors, mTOR inhibitors, IMDH inhibitors, biological agents, monoclonal antibodies, or any combination thereof. Examples of corticosteroids may include prednisone, budesonide, prednisolone, and methylprednisolone. Examples of Janus kinase inhibitors may include tofacitinib. Examples of calcinulin inhibitors may include cyclosporine and tacrolimus. Examples of mTOR inhibitors may include sirolimus and everolimus. Examples of IMDH inhibitors may include azathioprine, leflunomide, and mycophenolic acid. Examples of immunosuppressive biological agents may include abatacept, adalimumab, anakinra, certolizumab, etanercept, golimumab, infliximab, ixekizumab, natalizumab, rituximab, secukinumab, tocilizumab, ustekinumab, and vedolizumab. Examples of immunosuppressive monoclonal antibodies may include basiliximab and daclizumab. Such immunosuppressants may be administered to recipients of recellularized livers via enteral routes (including oral, gastric or duodenal feeding tube, rectal suppositories and rectal enema), parenteral routes (including intra-arterial, intracardiac, intraventricular, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intrathecal, intravascular, intravenous, intravitreous, epidural, and subcutaneous injections or infusions), inhalation, percutaneous, transmucosal, sublingual, buccal, or topical (including supercutaneous, dermal, enema, eye drops, ear drops, intranasal, and vaginal) administration.Immunosuppressants are available in doses of approximately 1mg to 1000mg, 5mg to 1000mg, 10mg to 1000mg, 15mg to 1000mg, 20mg to 1000mg, 25mg to 1000mg, 30mg to 1000mg, 35mg to 1000mg, 40mg to 1000mg, 45mg to 1000mg, 50mg to 1000mg, 55mg to 1000mg, 60mg to 1000mg, 65mg to 1000mg, 70mg to 1000mg, 75mg to 1000mg, 80mg to 1000mg, 85mg to 1000mg, 90mg to 1000mg, 95mg to 1000mg, and 1 The drug can be administered to the recipient in doses of approximately 00mg to 1000mg, approximately 150mg to 1000mg, approximately 200mg to 1000mg, approximately 250mg to 1000mg, approximately 300mg to 1000mg, approximately 350mg to 1000mg, approximately 400mg to 1000mg, approximately 450mg to 1000mg, approximately 500mg to 1000mg, approximately 550mg to 1000mg, approximately 600mg to 1000mg, approximately 650mg to 1000mg, approximately 700mg to 1000mg, approximately 750mg to 1000mg, approximately 800mg to 1000mg, approximately 850mg to 1000mg, approximately 900mg to 1000mg, or approximately 950mg to 1000mg.

[0113] Other embodiments and uses of the present invention will become apparent to those skilled in the art by considering this specification and the practice of the invention provided herein. For any reason whatsoever, all U.S. patents and other references referred herein are incorporated by specific reference. The specification and examples should be considered merely illustrative, and the true scope and spirit of the invention are shown by the following claims.

[0114] Preferred embodiment: A method for producing an organ composition that is at least partially recellularized, including but not limited to the liver, kidney, lung, and heart, is provided herein, comprising: (a) treating a non-human animal organ with an antiviral treatment; (b) perfusing and decellularizing the non-human animal organ to obtain a decellularized extracellular matrix; and (c) contacting the decellularized extracellular matrix with a cell composition containing a population of human cells to form an organ composition that is at least partially recellularized. A method for producing an organ composition that is at least partially recellularized is also provided herein, comprising: (a) treating a non-human animal liver with an antiviral treatment; (b) perfusing and decellularizing the non-human animal liver to obtain a decellularized extracellular matrix; and (c) contacting the decellularized extracellular matrix with a cell composition containing a population of human hepatocytes to form an organ composition that is at least partially recellularized. Furthermore, a method is provided herein in which the antiviral treatment comprises irradiating the non-human animal liver with an electron beam (E beam). Furthermore, methods are provided herein that include irradiation, which involves exposing a non-human animal liver to an electron dose of about 2 kGy to about 50 kGy. Furthermore, methods are provided herein that include irradiation, which involves exposing a non-human animal liver to an electron dose of about 5 kGy to about 25 kGy. Furthermore, methods are provided herein that include irradiation, which involves exposing a non-human animal liver to an electron dose of about 10 kGy to 20 kGy. Furthermore, methods are provided herein that further include contacting a decellularized extracellular matrix with a peroxy acid or hydrogen peroxide. Furthermore, methods are provided herein that include antiviral treatment, which involves irradiating a non-human animal liver with an electron beam (E-beam), and further include contacting a decellularized extracellular matrix with a peroxy acid or hydrogen peroxide. Furthermore, methods are provided herein in which the peroxy acid includes peroxyacetic acid, peracetic acid, peroxycarboxylic acid, derivatives thereof, or combinations thereof. Furthermore, the method provided herein further comprises contacting the decellularized extracellular matrix with an additional cell composition comprising a population of human vascular endothelial cells (HUVECs). The method provided herein further comprises a cell composition comprising both a population of human hepatocytes and a population of HUVECs.Furthermore, a method is provided herein in which the human hepatocyte population is primary human hepatocytes. Furthermore, a method is provided herein in which the human hepatocyte population is in vitro differentiated human hepatocytes. Furthermore, a method is provided herein in which the in vitro differentiated human hepatocytes are derived from or differentiated from an embryonic stem cell population, an induced pluripotent stem cell (iPSC) population, or an adult stem cell population. Furthermore, a method is provided herein in which the human hepatocyte population is contacted with a protease before contacting the decellularized extracellular matrix with the cell composition in step (c). Furthermore, a method is provided herein characterized in that the at least partially recellularized liver has an increased ammonia clearance level compared to a hepatocyte population that has not engrafted in the decellularized extracellular matrix. Furthermore, a method is provided herein in which the non-human animal is a non-human mammal. Furthermore, a method is provided herein in which the non-human mammal is an ungulate. Furthermore, a method is provided herein in which the ungulate is a pig. Furthermore, a method is provided herein in which the non-human animal liver is frozen before antiviral treatment.

[0115] A method for producing an organ composition that is at least partially recellularized is provided herein, comprising: (a) treating a non-human animal organ with an antiviral treatment; (b) perfusing the non-human animal organ to decellularize it to obtain a decellularized extracellular matrix; and (c) contacting the decellularized extracellular matrix with a cell composition comprising a population of human cells to form an organ composition that is at least partially recellularized. Furthermore, a method is provided herein in which the antiviral treatment comprises irradiating the non-human animal organ with an electron beam (E beam). Furthermore, a method is provided herein in which the irradiation comprises exposing the non-human animal organ to an electron dose of about 2 kGy to about 50 kGy. Furthermore, a method is provided herein in which the irradiation comprises exposing the non-human animal organ to an electron dose of about 5 kGy to about 25 kGy. Furthermore, a method is provided herein in which the irradiation comprises exposing the non-human animal organ to an electron dose of about 10 kGy to 20 kGy. Furthermore, methods are provided herein that further include contacting a decellularized extracellular matrix with at least one of a peroxy acid or hydrogen peroxide. Furthermore, methods are provided herein that further include contacting a decellularized extracellular matrix with at least one of a peroxy acid or hydrogen peroxide, wherein the antiviral treatment comprises irradiating a non-human animal organ with an electron beam (E-beam). Furthermore, methods are provided herein that include peroxy acid comprising peroxyacetic acid, peracetic acid, peroxycarboxylic acid, derivatives thereof, or combinations thereof. Furthermore, methods are provided herein that further include contacting a decellularized extracellular matrix with an additional cell composition comprising a population of human vascular endothelial cells (HUVECs). Furthermore, methods are provided herein that include both a population of human organ cells and a population of HUVECs. Furthermore, methods are provided herein that include a population of human hepatocytes, human kidney cells, human lung cells, human cardiac cells, or cells of any other human parenchymal organ. Furthermore, methods are provided herein that include a population of human cells being in vitro differentiated human cells. Furthermore, the Specified Method provides a method in which in vitro differentiated human cells are differentiated from an embryonic stem cell population, an induced pluripotent stem cell (iPSC) population, or an adult stem cell population.Furthermore, a method is provided herein in which a population of human cells is brought into contact with a protease before the decellularized extracellular matrix is ​​brought into contact with the cell composition in step (c). Furthermore, a method is provided herein characterized in that at least partially recellularized organs have increased cellular activity compared to the cellular activity of human cell populations that have not engrafted in the decellularized extracellular matrix. Furthermore, a method is provided herein in which the non-human animal is a non-human mammal. Furthermore, a method is provided herein in which the non-human mammal is an ungulate. Furthermore, a method is provided herein in which the ungulate is a pig. Furthermore, a method is provided herein in which the non-human animal organ is frozen before antiviral treatment.

[0116] A method for producing an organ composition that is at least partially recellularized is provided herein, comprising: (a) treating a non-human animal organ with an antiviral treatment; (b) perfusing the non-human animal organ to decellularize it to obtain a decellularized extracellular matrix; (c) contacting the decellularized extracellular matrix with a first cell composition comprising a population of human vascular endothelial cells; and (d) contacting the decellularized extracellular matrix with a second cell composition to form an organ composition that is at least partially recellularized.

[0117] A method for producing a liver composition that is at least partially recellularized is provided herein, comprising: (a) treating a non-human animal liver with an antiviral treatment; (b) perfusing the non-human animal liver to decellularize it to obtain a decellularized extracellular matrix; (c) contacting the decellularized extracellular matrix with a first cell composition comprising a population of human vascular endothelial cells; and (d) contacting the decellularized extracellular matrix with a second cell composition comprising a population of human hepatocytes to form a liver composition that is at least partially recellularized. Furthermore, a method for contacting a decellularized extracellular matrix with a second cell composition is provided herein, wherein the first cell composition is characterized by having a glucose consumption rate of at least about 10 mg / hour. Furthermore, a method for contacting a decellularized extracellular matrix with a second cell composition is provided herein, wherein the first cell composition is characterized by having a glucose consumption rate of at least about 20 mg / hour. Furthermore, a method for contacting a decellularized extracellular matrix with a second cell composition is provided herein, wherein the first cell composition is characterized by having a glucose consumption rate of at least about 30 mg / hour. Furthermore, a method for contacting a decellularized extracellular matrix with a second cell composition is provided herein, wherein the first cell composition is characterized by having a glucose consumption rate of at least about 40 mg / hour. Furthermore, a method for contacting a decellularized extracellular matrix with a second cell composition is provided herein, wherein the first cell composition is characterized by having a glucose consumption rate of at least about 50 mg / hour. Furthermore, a method for contacting a decellularized extracellular matrix with a second cell composition is provided herein, wherein the first cell composition is characterized by having a glucose consumption rate of at least about 60 mg / hour. Furthermore, a method for contacting a decellularized extracellular matrix with a second cell composition is provided herein, wherein the first cell composition is characterized by having a glucose consumption rate of at least about 70 mg / hour.Furthermore, a method is provided herein for contacting a decellularized extracellular matrix with a second cell composition, wherein the first cell composition is characterized by having a glucose consumption rate of at least about 80 mg / hour. Furthermore, a method is provided herein for contacting a decellularized extracellular matrix with a second cell composition, wherein the first cell composition is characterized by having a glucose consumption rate of at least about 90 mg / hour. Furthermore, a method is provided herein for contacting a decellularized extracellular matrix with a second cell composition, wherein the first cell composition is characterized by having a glucose consumption rate of at least about 100 mg / hour. Furthermore, a method is provided herein for contacting a decellularized extracellular matrix with a second cell composition at least 10 days after contacting the decellularized extracellular matrix with the first cell composition. Furthermore, a method is provided herein for contacting a decellularized extracellular matrix with a second cell composition at least 12 days after contacting the decellularized extracellular matrix with the first cell composition. Furthermore, a method is provided herein in which the decellularized extracellular matrix is ​​brought into contact with a second cell composition 10 to 20 days after contacting the decellularized extracellular matrix with a first cell composition. Furthermore, a method is provided herein characterized in that the at least partially recellularized liver composition has an increased ammonia clearance level compared to a hepatocyte population that is not in the form of the at least partially recellularized liver composition. Furthermore, a method is provided herein in which antiviral treatment comprises irradiating the decellularized extracellular matrix with an electron beam (E beam). Furthermore, a method is provided herein in which irradiation comprises exposing a non-human animal liver to an electron dose of about 2 kGy to about 50 kGy. Furthermore, a method is provided herein in which irradiation comprises exposing a non-human animal liver to an electron dose of about 5 kGy to about 25 kGy. Furthermore, a method is provided herein in which irradiation comprises exposing a non-human animal liver to an electron dose of about 10 kGy to 20 kGy. Furthermore, a method is provided herein that further comprises contacting the decellularized extracellular matrix with a peroxy acid or hydrogen peroxide.Furthermore, methods are provided herein in which the peroxy acid comprises peroxyacetic acid, peracetic acid, peroxycarboxylic acid, derivatives thereof, or combinations thereof. Furthermore, methods are provided herein in which the human hepatocyte population is primary human hepatocytes. Furthermore, methods are provided herein in which the human hepatocyte population is in vitro differentiated human hepatocytes. Furthermore, methods are provided herein in which the in vitro differentiated human hepatocytes are derived from or differentiated from an embryonic stem cell population, an induced pluripotent stem cell (iPSC) population, or adult stem cells. Furthermore, methods are provided herein in which the human hepatocyte population is contacted with a protease before contacting the decellularized extracellular matrix with the second cell composition in step (d). Furthermore, methods are provided herein in which the at least partially recellularized liver composition exhibits increased ammonia clearance levels compared to a hepatocyte population that is not in the form of the at least partially recellularized liver composition. Furthermore, methods are provided herein in which the non-human animal is a non-human mammal. Furthermore, methods are provided herein in which the non-human mammal is an ungulate. Furthermore, a method is provided herein in which the ungulate is a pig. Furthermore, a method is provided herein in which the liver of a non-human animal is frozen before antiviral treatment.

[0118] A composition comprising at least partially recellularized organs produced by any of the methods provided herein is provided herein.

[0119] A composition comprising a liver composition at least partially recelled, prepared by any of the methods provided herein, is provided herein. A liver at least partially recelled, comprising (a) a porcine extracellular matrix; (b) a population of human endothelial cells and human hepatocytes engrafted in the porcine extracellular matrix, wherein the liver at least partially recelled has increased ammonia clearance compared to a population of porcine hepatocytes engrafted in the porcine extracellular matrix. A liver at least partially recelled, comprising (a) a porcine extracellular matrix with reduced microbial particles and perfusion-decellated; (b) a population of human endothelial cells and human hepatocytes engrafted in the porcine extracellular matrix, wherein the liver at least partially recelled has increased ammonia clearance compared to a population of porcine hepatocytes engrafted in the porcine extracellular matrix, is provided herein. Furthermore, at least partially recellularized livers are provided herein, wherein the recellularized livers contain one or more surface markers selected from the group consisting of differentiation antigen group (CD) 31, CD105, and asialoglycoprotein receptor 1 (ASGR1). Furthermore, at least partially recellularized livers are provided herein, characterized in that the recellularized livers have increased α1-antitrypsin (A1AT) levels compared to hepatocyte populations that have not engrafted in the porcine extracellular matrix. Furthermore, at least partially recellularized livers are provided herein, characterized in that the recellularized livers have increased fibrinogen levels compared to hepatocyte populations that have not engrafted in the porcine extracellular matrix. Furthermore, at least partially recellularized livers are provided herein, characterized in that the recellularized livers have increased fibrinogen levels compared to porcine hepatocyte populations that have engrafted in the porcine extracellular matrix.Furthermore, the following are provided herein: a partially recellularized liver characterized in that the partially recellularized liver exhibits increased ammonia clearance levels compared to a human hepatocyte population that has not engrafted in the porcine extracellular matrix. Furthermore, the following are provided herein: a partially recellularized liver characterized in that the partially recellularized liver has ammonia clearance after cryogenic storage, wherein the cryogenic storage period is at least 6 hours. Furthermore, the following are provided herein: a partially recellularized liver characterized in that the partially recellularized liver has urea production after cryogenic storage, wherein the cryogenic storage period is at least 6 hours. Furthermore, the following are provided herein: a partially recellularized liver further comprising an antimicrobial agent. Furthermore, the following are provided herein: a partially recellularized liver in which the antimicrobial agent is an antiviral agent. Furthermore, the following are provided herein: a partially recellularized liver in which the antiviral agent comprises peroxyacetic acid. Furthermore, the following are provided herein: a partially recellularized liver in which the human hepatocyte population is primary human hepatocytes. Furthermore, the Specified Provisions also provide a liver in which the human hepatocyte population is in vitro differentiated human hepatocytes, at least partially recellularized. Furthermore, the Specified Provisions also provide a liver in which the in vitro differentiated human hepatocytes are derived from or differentiated from an embryonic stem cell population, an induced pluripotent stem cell (iPSC) population, or adult stem cells.

[0120] A composition comprising at least partially recellularized liver and an extracellular matrix protein or solution is provided herein. Furthermore, a composition further comprising a cell culture medium is provided herein.

[0121] An ex vivo method for treating liver disease in a subject, comprising constructing a blood circuit, the blood circuit containing, in fluid communication, blood from the subject and at least partially recellularized liver provided herein, the at least partially recellularized liver filtering the blood from the subject and removing ammonia, thereby providing a method for treating liver disease in the subject.

[0122] A method for treating liver disease in a subject is provided herein, comprising administering to the subject at least partially recellularized liver provided herein, thereby treating liver disease in a subject. Furthermore, a method is provided herein in which the liver disease includes acute hepatic failure (ALF). [Examples]

[0123] Example 1. Procurement of pig organs and decellularization of the entire liver. Whole pig livers (500-700 grams) were extracted from pig carcasses, rinsed with PBS, flushed with saline, and frozen. The frozen livers were treated with electron beam (E-beam) before decellularization (E-Beam Services, Cranberry, New Jersey). In preparation for decellularization, the pig livers were thawed at room temperature and cannulas were inserted into the portal vein (PV), suprahepatic inferior vena cava (sIVC), and inferior vena cava (IVC). Perfusion decellularization was performed on the cannulated livers through the PV and sIVC at a target pressure of 12-17 mmHg using 1% Triton X-100 (Amresco, M143) and 0.6% sodium dodecyl sulfate (Amresco, 0227). The decellularized livers were then disinfected with peracetic acid (PAA; USWater, BI0032-6). Decellularized grafts were washed with phosphate-buffered saline (PBS; Corning 21-040-CMX12) and stored. The decellularization process was completed in an ISO Class 7 cleanroom.

[0124] Example 2: Residue Inspection Punch biopsy samples were collected from fully decellularized liver grafts, weighed, and digested prior to the quantification of SDS, Triton, and DNA residues. 80 mg samples for SDS and Triton residues were digested in 15 μL of 20 mg / mL proteinase K (Qiagen), vortexed, and heated at 60°C for 18–24 hours. The samples were then assayed for detectable SDS and Triton via Pace Analytical (Minneapolis, Minnesota). Punch biopsy samples and known standards (Thermo Fisher Scientific) for DNA testing were digested in 40 μL of 20 mg / mL proteinase K (Thermo Fisher Scientific) and then measured using a Qubit3.0 fluorometer (Thermo Fisher Scientific).

[0125] Example 3: Evaluation of the electron beam irradiation process for virus inactivation The evaluation of the effectiveness of electron beams as a method of virus inactivation was conducted through Charles River Laboratories Services, Inc. (Wayne, Pennsylvania) in accordance with the U.S. Food and Drug Administration's Good Manufacturing Practices (GMP) regulations as set forth in 21 Parts 210 and 211 of the Code of Federal Regulations, and there were no deviations from the test method that would affect the quality of integrity of the test results. In short, liver tissue was divided into 1-inch (approximately 2.54 cm) x 2-inch (approximately 5.08 cm) patches, rinsed with 100 mL of 0.9% physiological saline, and incubated with 1 mL of one of the following viruses for 15 minutes: mouse leukemia virus (MuLV), pseudorabies virus (PRV), reoviridae virus (Reo3), or porcine parvovirus (PPV). After virus addition, the material was treated with electron beam irradiation at 10 kGy or 20 kGy (2 aliquots / virus / irradiation run), then sampled and analyzed for viral titer. A reference run was treated similarly, but without electron beam irradiation.

[0126] Example 4: HUVEC cell culture and seeding onto decellularized liver scaffold Human umbilical vein endothelial cells (HUVECs) (Lonza, catalog number C2519A) were cultured at 37°C and 5% CO2 in antibiotic-free endothelial cell growth medium (R&D Systems, CCM027) supplemented with 2% fetal bovine serum (Corning), 50 mg / L ascorbic acid (Sigma-Agentur), 1 mg / L hydrocortisone (Sigma-Agentur), 20 μg / L FGF (R&D Systems, Minnesota), 5 μg / L VEGF (R&D Systems), 15 μg / L R3IGF (Sigma-Agentur), 1000 U / L heparin (Sigma-Agentur), and 1.5 μM acetate (Sigma-Agentur). Cells were harvested at 90-100% confluence using 0.25% trypsin-EDTA (Thermo Fisher). Decellularized pig liver was placed in a bioreactor and perfused with antibiotic-free medium (37°C, 5% CO2) for 72 hours to ensure that there was no microbial contamination. HUVECs collected at passages 5-9 were added to 180 mL of culture medium using a syringe (1.60 × 10⁶). 8 Cells were used and injected via sIVC. Subsequently, the extracorporeal bioartificial liver (BEL) graft was subjected to a 1-hour static culture to allow cell adhesion within the scaffold. During this 1-hour static period, the temperature and dissolved gas concentration in the bioreactor were maintained by perfusing the system with culture medium while bypassing the organ. After the 1-hour static period, 40 × 10 cells were added to 90 mL of culture medium. 6 Each HUVEC was injected into the sIVC using a syringe under a continuous flow rate of 300 mL / min. After overnight incubation, the organs were manually inverted to perfuse into the portal vein. The HUVECs were harvested and seeded into the sIVC via the portal vein in the same manner as described above. After seeding, the culture medium was changed daily, and the volume was continuously adjusted to maintain a glucose level of over 0.2 g / L within a 24-hour period. The culture medium perfusion to the scaffold was maintained at a maximum flow rate of 300 mL / min, with a maximum pressure of 30 mmHg.

[0127] Example 5: Isolation of porcine liver cells As previously described (Anderson BA et. al., 2021), fresh whole livers (450–1000 g) and primary porcine hepatocytes were isolated from pigs. This study was reviewed and approved (No. 040420) by the Institutional Animal Care and Use Committee (IACUC) of MRS / Collagen Solutions. Cell viability and yield were quantified by trypan blue exclusion method on a hemocytometer. The final cell pellet was resuspended in 1 L of University of Wisconsin (UW) Belzer solution (Bridge to Life, Northbrook, Illinois).

[0128] Example 6: Isolation of human liver cells The organs used for research to advance medicine were obtained by Donor Network West, NDRI, and Southwest Transplant Alliance, and were provided free of charge with the consent of the donors or their immediate relatives. Liver tissue was flushed with lactated Ringer's solution (LRS; Hutchins & Hutchins, Virginia) via the inferior vena cava, hepatic artery, and portal vein. The right and left lobes were resected, the exposed vessels were flushed with cold LRS, and cannulas were inserted for perfusion. Liver tissue was perfused with liver perfusion solution I (Vitroprep, North Carolina) for 12–15 minutes (10–50 mL / min), followed by liver perfusion solution II (Vitroprep, North Carolina) for 20–40 minutes. To initiate digestion, both collagenase MA (3 mg / L; Vitasite) and protease BP (2.5 mg / L; Vitasite) were added to liver perfusion fluid II. The solution was not recirculated. The digested tissue was diluted in human hepatocyte isolation medium (HHIM; DMEM containing 10% FBS) and passed through a sieve set of the following sizes (1 mm (arbitrary), 500, 250, 90 μm). The hepatocytes were concentrated by centrifugation (110 × g, room temperature, 10 minutes) and washed once with HHIM. Cell viability and yield were quantified by trypan blue exclusion method on a hemocytometer. The final cell pellet was resuspended in 1 L of UW solution at a concentration of 5 million cells / mL or less.

[0129] Example 7: Flow cytometry HUVECs were collected from the final cell suspension before seeding onto grafts. Cells were fixed with 2% paraformaldehyde (Electron Microscopy Sciences) and stained for CD31 (Bio-Rad, #MCA1738; 1:100 dilution) and CD105 (Bio-Rad, #MCA1557; 1:100 dilution). Expression was assessed by flow cytometry using a BD Accuri C6 flow cytometer (BD Biosciences). Data analysis was performed using FlowJo software.

[0130] Primary human hepatocyte (PHLC) samples were collected from the final cell suspension of newly isolated PHLCs. Cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences), permeabilized with Triton X-100 (Sigma-Ace), and stored in FACS buffer (1×PBS (Corning) containing 10% BSA (Sigma-Ace) and 0.5% NaN3 (Ricca)). Cells were stained for ASGR1 expression (antibody from R&D Systems, #FAB43941R; 1:20 dilution) and analyzed by flow cytometry using an Attune NxT flow cytometer (Thermo Fisher Scientific). Hepatocytes were identified by size using forward and side scattering, and by ASGR1 expression compared to an isotype control (R&D Systems; IC002R; 1:20 dilution). Data analysis was performed using FlowJo software.

[0131] Example 8: Hepatocyte seeding onto an extracorporeal bio-artificial liver (BEL) scaffold Hepatocytes from either pigs or humans were pelletized overnight at 4°C. After overnight storage, the supernatant was removed from the pellet and 10 × 10⁻⁶ cells were collected. 9 1 individual pig liver cells or 5 × 10⁶ 9One of several human hepatocytes was treated with 2% fetal bovine serum (Corning), 50 mg / L ascorbic acid (Sigma-Ace), 20 μg / L FGF (R&D Systems), 5 μg / L VEGF (R&D Systems), 5 μg / L EGF (R&D Systems), 1000 U / L heparin (Sigma-Ace), 3 g / L human albumin (CSL Behring), 150 μg / L linoleic acid (Sigma-Ace), 0.1 μM dexamethasone (Sigma-Ace), 40 μg / L human glucagon (Novalis), 6 mg / L human holotransferrin (Sigma-Ace), 20 μg / L Gly-His-Lys (Sigma-Ace), 5 ug / L sodium selenite, 1 g / L The cells were diluted in Williams E medium (Gibco) supplemented with L-carnitine (Sigma-A), 0.2 g / L-arginine (Sigma-A), and 10 mg / L glycine (Sigma-A). Porcine hepatocytes were diluted in the above medium supplemented with 50 μg / L LONG® Arginine 3 insulin-like growth factor (IGF), and human hepatocytes were diluted in the above medium supplemented with 8.4 U / L human insulin (Novolin). Both human and porcine hepatocytes were injected via sIVCs (typically 12-16 days after initial HUVEC seeding) through a re-endothelialized BEL scaffold perfused at 350 mL / min, by repeated syringe injections at a flow rate of 94 mL / min. The BEL seeded with hepatocytes was then returned to continuous medium perfusion through a PV with co-culture medium at a maximum flow rate of 300 mL / min and a maximum pressure of 30 mmHg.

[0132] Example 9: Analysis of metabolites Culture medium samples were collected daily from the bioreactor and immediately assayed using a CEDEX Bio HT bioanalyzer (Roche) to determine the levels of glucose, ammonia, and lactate dehydrogenase activity in the medium. The measured glucose concentrations were used to calculate the daily consumption during the 24-hour period before a complete medium change and to determine the level of reendothelialization and the amount of medium required for the next medium change. Additional samples were collected daily for the quantification of urea, A1AT, and fibrinogen and stored at -80°C for analysis. Urea levels were analyzed using a commercially available kit (Bioassay Systems, DIUR-100) with minor modifications to the manufacturer's guidance. A1AT quantification was performed using a commercially available kit (Abcam, ab189579) with minor modifications. Fibrinogen was evaluated using a commercially available kit (Abcam, ab241383) with minor modifications.

[0133] Example 10: Sterility Test Due to the short time between the preparation of HUVEC and PHLC and their introduction into the manufacturing process, microbiological testing focused on detecting any contaminants introduced by these starting materials after they were seeded into the decellularized matrix. After HUVEC seeding and after transfer for transport, USP <71> Official sterility tests were conducted, and the interim assessments from days 5-7 were evaluated prior to the results at day 14. A rapid microbial method (RMM) using Gram staining was also performed to aid in real-time detection of microbial contamination. Rapid mycoplasma testing by nucleic acid amplification was performed after HUVEC seeding and before cryogenic storage simulation. Qualification of this method ensured that the detection limit conformed to European Pharmacopoeia 2.6.7, and that the culture method was used for USP certification. <63> The test method demonstrated appropriate comparability.

[0134] Example 11: Ammonia clearance and urea production assay 16–20 hours after seeding hepatocytes, the bioreactor containing two cultured BEL cells was replaced with fresh medium. Ammonium chloride was administered to the system at a final concentration of 200 μM while the medium was being perfused into the system, bypassing the organs. After circulating the ammonium chloride-containing medium for 10 minutes, a sample was taken at hour 0, the organs were exposed to the ammonium chloride-containing medium, and normal medium perfusion was resumed. A sample was taken at 1 hour, and ammonia levels were quantified using a CEDEX Bio HT bioanalyzer.

[0135] Example 12: Data Analysis Ammonia clearance data were collected in real time. Fibrinogen, α1-antitrypsin, and urea production data were collected and analyzed from frozen samples, and statistical significance was examined retrospectively. AC rate was calculated by measuring the difference in ammonia levels between 0 hours and 1 hour after bolus administration. All data comparisons were applied using one-way analysis of variance (ANOVA) (α<0.05 considered significant) between days 1–7, 1–3, and 5–7. A1AT data generated on days 6 and 7 were not included due to the small "N" (sample size). Data were reported as mean and one standard deviation where applicable.

[0136] Example 13: Low-temperature storage simulation For the cryogenic storage test, BEL grafts were removed from the bioreactor perfusion after 72 hours of culture and transferred to a static transport container. The grafts were flushed with 2 L of cold Belzer UW solution at 300 mL / min. The transport containers were stored in an ice-filled cooler for 14–16 hours. After cryogenic storage, the BELs were flushed with 2 L of room temperature PBS at 300 mL / min and returned to the bioreactor system, where perfusion was resumed with 4 L of fresh medium. Ammonia clearance was assessed approximately 10 minutes after the start of medium perfusion. Samples were taken approximately 24 hours after the start of medium perfusion for A1AT and fibrinogen quantification. Ammonia clearance was also assessed at this point.

[0137] Example 14: Blood Perfusion Test For the in vitro hemoperfusion test, each blepharoplast (BEL) was connected to a circuit consisting of silicone tubing, a pressure transducer (Deltran, DPT-100), a peristaltic pump (Cohl-Palmer, 07522-20), and an oxygenator (Livanova, 050703) heated to 37°C in a recirculating water bath and receiving a mixed gas of 20% O2, 5% CO2, and 75% N2. Freshly collected heparinized porcine blood was heated to 37°C and the active clotting time (ACT) was measured (ITC, Hemochron Response). Protamine sulfate solution was gradually added to the blood to neutralize the heparin until the ACT reached 400-600. Two liters of blood were introduced into the circuit and perfused through the portal vein of the BEL at an initial flow rate of 350 mL / min. Pressure was recorded over 180 minutes of hemoperfusion.

[0138] Example 15: Electron beam irradiation inactivates heterotropic viruses in natural pig liver before decellularization. Whole pig livers were collected from pig carcasses, decellularized, and recellularized using HUVEC and PHLC as described above (Figure 1A). To eliminate the risk of heterotropic virus transmission from the decellularized liver scaffolds to human patients, all whole pig livers were treated with electron beam and PAA as a method of virus inactivation prior to the decellularization process. Viral inoculation with mouse leukemia virus (MuLV), pseudorabies virus (PRV), reoviridae virus (Reo3), and porcine parvovirus (PPV), carried out through Charles River Laboratories, Inc. (Wayne, Pennsylvania), was performed on pig livers, followed by treatment with electron beam doses of 10 kGy or 20 kGy. Both 10 kGy and 20 kGy doses showed a log reduction greater than 1, indicating viral inactivation. However, the 20 kGy treatment was more effective in reducing the virus and was selected as the standard treatment for all BELs (Table 2). To ensure that no harmful residues remained as byproducts of the decellularization process, representative lots of decellularized porcine liver scaffolds were tested for the presence of Triton, SDS, and DNA. All assayed grafts passed the desired acceptance criteria, demonstrating that the decellularization process successfully removes harmful residues before seeding live cells (Table 3).

[0139] [Table 2]

[0140] [Table 3]

[0141] Following decellularization, the scaffolds were placed in a bioreactor culture station (Figure 1B) and perfused with culture medium for a 3-day eligibility period before endothelialization by HUVEC via the superior vena cava (superior IVC) and portal vein (Figure 3C). These seeded scaffolds were cultured for 8–16 days to ensure that revascularization by HUVEC was completed before seeding of primary hepatocytes isolated from native human or porcine liver (Figure 1C). The BELs were cultured for 3 days and then analyzed for function during the therapeutic window.

[0142] To confirm the phenotype of HUVECs before seeding onto decellularized pig scaffolds, the expression of endothelium-specific markers CD31 and CD105 was evaluated by flow cytometry (Figure 2A). Both CD31 and CD105 were expressed at high levels in these cells. The proliferation of HUVECs after seeding onto decellularized pig scaffolds was indicated by an increase in glucose consumption over time (Figure 2B), and further indicated by histological examination of co-cultured BEL (Figure 2C), in which HUVECs were observed lining the vascular structure.

[0143] Furthermore, the isolated PHLC population was evaluated using flow cytometry of PHLCs. The phenotype of hepatocytes was confirmed by both the scattering profile (Figure 2D) and the expression of the hepatocyte-specific marker ASGR (Figure 2E). After seeding, this hepatocyte population engrafted into the parenchyma of a revascularized liver scaffold (Figures 2F-2I).

[0144] To confirm the absence of adventitious agents and contaminants, tests were conducted after HUVEC seeding and again at 14-16 days. All tested lots were deemed suitable for use in this manufacturing process and were free from microbial contamination, endotoxins, and mycoplasma (data not shown). Obtaining human hepatocytes presents logistical challenges due to the unpredictable availability of donor livers, in addition to several ethical considerations. Therefore, in a previously published study, primary porcine hepatocytes were used (Anderson BA et. al. 2021). However, to eliminate the immunological risks to patients commonly associated with xenotransplantation, the inventors hereby constructed BELs entirely from human cell types. The function of these humanized BELs was evaluated compared to porcine grafts produced under the same process. Pig and human BELs were subjected to an ammonia clearance assay the day after hepatocyte seeding. One hour after administration of a 200 μM bolus, BELs seeded with human hepatocytes removed a larger amount of ammonia (96 + 31.5 μM) compared to BELs seeded with porcine hepatocytes (Figure 3A). Since some porcine BELs were able to remove more ammonia than human hepatocytes, we decided to compare the persistence of function to evaluate therapeutic potential. Some porcine BELs did indeed remove the same amount of ammonia on day 1 of culture, but did not retain this function by day 3 compared to humanized BELs seeded with PHLCs (Figure 3B). Statistically significant increases in the levels of urea production, fibrinogen, and A1AT, by-products of ammonia metabolism, observed by humanized BELs at the end of the first 24-hour culture period on day 1 (Figures 3C, 3D, and 3E, respectively), suggest that human hepatocytes are a more suitable therapeutic model.

[0145] Patency is a crucial factor in providing a therapeutic window of sufficient length to support patients in a state of acute liver failure. To determine patency in both human and porcine pulmonary artery (BEL), we developed a homemade blood loop that allowed oxygenated and temperature-controlled blood to flow through the BEL while simultaneously monitoring inline pressure as an indicator of blood flow within the organ (Figure 3B). Compared to porcine BEL, human BEL exhibited lower pressures over a 180-minute assay, remaining within the range of normal physiological parameters (Figure 3C).

[0146] BELs seeded with PHLC showed higher urea, fibrinogen, and A1AT production on day 1 post-seeding than BELs seeded with PPLC (Figures 3D-3F). All BELs tested maintained patency and supported continuous flow of oxygenated and temperature-controlled porcine whole blood throughout a 3-hour blood loop assay (Figures 3G-3H). Compared to BELs seeded with PPLC, BELs seeded with PHLC showed lower pressures throughout the 180-minute assay.

[0147] Example 16: Antiviral treated artificial liver exhibits long-term stability. Treating ALF patients in a clinical setting requires overcoming several logistical challenges to provide effective treatment. Bioartificial livers (BELs) must be functionally sustainable to accommodate specific patients and maintain their function after transport. To determine the long-term functional stability of BELs, BELs were cultured for 72 hours after PHLC seeding, with daily sampling and ammonia clearance assessments. The BELs were then subjected to a 14-16 hour low-temperature storage period, reinstalled in a bioreactor, cultured for another 72 hours, and again subjected to daily sampling and ammonia clearance assessments (Figure 4A). The BELs demonstrated the ability to metabolize ammonia throughout the entire 72-hour culture period. Clearance rates decreased immediately after low-temperature storage (within 10 minutes), but stabilized and remained constant after 24 hours of culture in the bioreactor (Figure 4B). Further evidence of ammonia metabolism is supported by urea accumulation in the bioreactor medium at each measured time point (Figure 4C). BEL also demonstrated protein synthesis ability through the production of both fibrinogen and A1AT. The concentrations of both proteins increased throughout the 72-hour culture period after PHLC seeding. Although the measured levels of both decreased after the low-temperature storage period, they were consistent with the concentrations recorded 1–2 days after PHLC seeding (Figure 4D, Figure 4E). The measured levels of these proteins decreased after the low-temperature storage period, but were consistent with the concentrations recorded 1–2 days after PHLC seeding (Figure 4E, Figure 4F).

[0148] To determine the long-term functional stability of BEL, BEL was cultured for up to 5–7 days after PHLC seeding, with daily sampling and ammonia clearance evaluation performed. Subsequently, BEL was subjected to a 14–16 hour low-temperature storage period, then reinstalled in a bioreactor and cultured for a further 5 hours, with daily sampling and ammonia clearance evaluation performed again. BEL demonstrated the ability to metabolize ammonia for at least 7 days of culture (Figures 5A–5D). The clearance rate decreased immediately after low-temperature storage, but stabilized after 24 hours of culture in the bioreactor and remained constant (Figures 6A–6D).

[0149] Daily glucose consumption, measured throughout the culture period, steadily increased before the culture period and stabilized after low-temperature storage, indicating that BEL cells maintained viability and metabolic activity. Further evidence of ammonia metabolism is supported by the presence of urea in the bioreactor medium at each time point measured. BEL also performed protein synthesis, demonstrated by the production of both fibrinogen and A1AT. As shown in Table 4 below, the concentrations of both proteins increased throughout the culture period after PHLC seeding.

[0150] [Table 4]

[0151] Example 17: Production of an organ with an intact outer surface using a freezing and irradiation process One of the challenges in sterilizing tissues (whole tissue or decellularized tissue) with electron beams or gamma rays is maintaining the shape of the organ throughout the process. Cross-linking can cause organs to retain their shape from when they were treated, resulting in shape and perfusion problems during recellularization. The inventors have developed a method for freezing organs before electron beam treatment. This makes it possible to orient and maintain the organ in a specific shape throughout the entire process.

[0152] Ideally, decellularized organs should be electron beam sterilized prior to recellularization to ensure viral inactivation during the process. Early attempts at electron beam sterilization at this stage presented problems with orientation and shape. Decellularized organs could not maintain their shape while suspended in solution because they deflated during transport (Figure 8). Since the organs did not fully re-expand, this led to problems with reperfusion during recellularization. Organs that were fully expanded immediately before electron beam treatment could fully re-expand, but this method was not feasible from a logistical standpoint.

[0153] To maintain the shape of the organs, the inventors froze the entire pig organs and subjected them to electron beam treatment while frozen. This made it possible to maintain the complete shape and orientation of the organs during both transport and treatment. Once decellularized, the organs could be fully re-expanded for recellularization and functioned similarly to untreated electron beam matrices.

[0154] Cryotherapy can be used to maintain the shape of various organs and tissues during electron beam and gamma ray treatment. This process can also be applied to both natural / whole tissues or previously decellularized tissues.

[0155] A method for producing organs with intact outer surfaces using freezing and irradiation processes is further provided below.

[0156] The entire pig liver is removed and recovered. These non-sterile organs are placed in vacuum-sealed bags, arranged in the same orientation as the original organs. In addition, all organ blood vessels are also arranged in their original orientation. The vacuum-sealed bags are then placed inside a vacuum sealer. The vacuum sealer is then activated to create a vacuum inside the bags containing the organs, and then sealed to maintain that vacuum. The vacuum inside the bags helps to maintain the original organ structure and promotes a uniform distribution of the irradiation treatment. The vacuum-sealed bags containing the organs are then placed in a -20°C freezer. These packages, frozen whole organs, can be stored for a period of time before being transported for electron beam or gamma ray treatment. When transporting for sterilization, the organs are frozen-packaged, and this frozen packaging may include, but is limited to, placing the whole organs in a cushioned, insulated box and transporting them overnight on dry ice to the electron beam irradiation facility. The organs are then irradiated and returned to the end user.

[0157] Frozen livers were transported for electron beam treatment, and the reliability of electron beam-treated livers for recellularization was demonstrated by two independent recellularization experiments. Figure 9 shows a photograph of a frozen liver transported for electron beam treatment, which was laid flat for more effective treatment and for the orientation of both organs and blood vessels. Figure 10 shows a perfused decellularized pig liver.

[0158] Initial results from electron-beam-treated decellularized liver grafts showed frequent deformation. These areas generally appeared as depressions that could not be fully re-expanded, leading to perfusion blockage in certain regions. Figure 8 shows electron-beam-treated grafts with damage to lobes 3 and 4.

[0159] The main advantage of the method provided herein is that, in addition to inactivating viruses, it is possible to stabilize the tissue in a desired shape and orientation during transport and processing. Fresh, unfrozen pig organs can be stabilized, but processing them is logistically difficult because the tissue cannot be stored for as long as frozen tissue. Decellularized organs are easier to store, but much more difficult to stabilize. Exogenous factors are one of the biggest obstacles in biomedical engineering. The process must be able to demonstrate the ability to inactivate or remove bacterial, fungal, and viral contamination of organs. Here, the inventors have developed a method for electron beam treatment of the entire organ before perfusion decellularization and recellularization without adversely affecting downstream processes. The electron beam treatment step makes it possible to inactivate viruses and reduce fungal and bacterial loads before initiating perfusion decellularization and recellularization.

[0160] Example 18: Method for decellularizing organs by perfusion Perfusion decellularization is a process that requires a balance between strong disinfection and gentle decellularization to preserve the integrity of the extracellular matrix and enable effective downstream recellularization. The disinfection process may prove detrimental to the matrix in a way that makes recellularization impossible, and insufficient disinfection can lead to contamination that hinders recellularization and ultimately poses a risk to the patient. Furthermore, viral infections pose a threat to patients in prosthetic organs resulting from perfusion decellularization. Electron beam treatment provides a viral inactivation process essential for the safety of the final organ.

[0161] Pre-decellularization electron beam treatment also provides a means of reducing contamination on the organ as it begins the decellularization process. This is a significant advantage because sourcing sterile organs is difficult to perform in slaughterhouses and often requires sourcing in an operating room environment. Electron beam treatment also provides a method for controlling endotoxins in the final product. While contaminated organs can be disinfected during the decellularization process, endotoxin accumulation can cause significant problems in the time between sourcing and disinfection. Reducing contamination from the electron beam treatment process lowers endotoxin levels that pose a threat to patients (if sterilization occurs after the decellularization process).

[0162] Whole pig organs, such as the liver or kidneys, are collected from donor animals. These non-sterile organs are placed in vacuum-sealed bags, positioned within the range of their original orientation. In addition, all organ blood vessels are also positioned in their original orientation. The vacuum-sealed bags are then placed inside a vacuum sealer. The vacuum sealer is then activated to create a vacuum inside the bags containing the organs, and then sealed to maintain that vacuum. The vacuum inside the bags helps to maintain the original organ structure and promotes a uniform distribution of the irradiation treatment. The vacuum-sealed bags containing the organs are then placed in a -20°C freezer. Freezing the tissues is said to further maintain the orientation of the liver and to temporarily halt and prevent the degradation of the original tissues. These packaged, frozen whole organs can be stored for a period of time before being transported for electron beam irradiation. When transporting for sterilization, the inventors package the frozen whole organs in a cushioned, insulated box for overnight transport on dry ice to the electron beam irradiation facility. Subsequently, the irradiation facility receives the organs and irradiates each organ individually with a predetermined dose according to the inventor's specifications. During process development, electron beam irradiation dose mapping was completed for several livers to ensure appropriate irradiation exposure throughout the liver graft. The entire organ is then returned to the inventors on dry ice and received under controlled conditions in a frozen state. These sterilized organs are then placed in a freezer for a period of time and can be thawed for decellularization.

[0163] Intra-process whole-organ sterilization was performed to avoid adverse effects on downstream decellularization / recellularization. Table 5 shows the assay conditions for each treatment with low-dose or high-dose electron beam compared to the control group.

[0164] [Table 5]

[0165] Recellularized liver showed favorable results in electron beam treatment. Five grafts (three treated with high-dose electron beams and two with low-dose electron beams) showed higher peaks than the control graft. Furthermore, there was little difference between the high-dose (20 kGy) and low-dose (10 kGy) groups (Figure 11).

[0166] Patency results were also favorable, with all grafts passing the assay and being considered patent. No significant trends were observed in pressure during the test. In all groups, at least one graft ended above 4 mmHg, and in all groups, one or more grafts ended at negative pressure, including two grafts from the high-dose group (Figure 12).

[0167] Overall, the data from Example 18 demonstrate that electron beam treatment is feasible for whole-organ sterilization and virus inactivation in decellularized liver.

[0168] Example 19: Consistent production of functionally recellularized liver The data presented herein establish a defined and consistent manufacturing process for producing functional BELs composed entirely of human cell types as bridge therapy for acute liver failure. The decellularization process provided herein generates an extracellular matrix free from heterotropic virus transmission and residual Triton, SDS, or porcine DNA that could cause potential complications in ALF patients. The seeding method provided herein allows for vascular structure coverage by HUVECs and infiltration of PHLCs into the parenchyma. The bioreactor maintains BEL culture parameters to promote consistent cell proliferation, resulting in a positive functional output over a 6-day period.

[0169] One of the fundamental concerns regarding heterologous materials for patient treatment is the transmission of foreign bodies, potentially those that could trigger an immune response in patients already in a critical condition. Since the porcine-based material is a decellularized liver scaffold, this was addressed by employing a viral inactivation strategy utilizing both electron beam irradiation and PAA treatment (orthogonal modalities working together to successfully remove viruses). The model viruses used in this study were selected based on their unique characteristics, derived from their genome, envelope, and morphology. The ability of this treatment to inactivate or remove these viruses builds confidence in the approach as an effective manufacturing method for removing several viruses that could potentially adversely affect patients. Furthermore, the removal of toxic residues as byproducts of the decellularization process was verified, and the sterility of the recellularization process was confirmed, meaning that the BEL described herein is safe for patient treatment.

[0170] Conventional assays on porcine hepatocytes for the development of BELs have shown a risk of xenotropic virus transmission, and the potential immunological response of human patients to porcine cells limits their usefulness as a strategy for providing treatment for ALF. PHLCs not only avoid these concerns but also show increased function throughout the culture period compared to porcine hepatocytes. While some BELs seeded with porcine hepatocytes can remove ammonia at similar levels on day 1, the inability of porcine hepatocytes to maintain function by day 3 of culture makes providing treatment to patients impractical. This is because several logistical hurdles must be considered in transporting organs to patients in need. Furthermore, the low production of fibrinogen and A1AT suggests that PHLCs are inferior material lacking metabolic capacity.

[0171] Following cryogenic storage to mimic the patient transport process, a short-term decrease in ammonia clearance, as well as fibrinogen, A1AT, and urea production, was observed. This was not unexpected, as it is well known that cold ischemia time adversely affects tissue metabolism. However, compared to the first day after cryogenic storage, BEL function remained largely stable throughout the duration of the culture period. The ammonia clearance rates, urea, and fibrinogen production collected after cryogenic storage remained stable for 70 hours beyond the transport period, paving the way for BEL as a promising treatment for ALF patients. Future studies will investigate the ability of BEL produced through a defined, consistent manufacturing process to provide treatment to patients in a clinical setting.

[0172] Acute liver failure is a rapidly devastating disease. The high demand for liver transplants and long waiting times put patients at risk. Extracorporeal bioartificial livers can be used to support patients in the recovery phase or to support patients as a bridge to transplantation. The manufacturing processes provided herein can produce bioartificial liver grafts that can comply with the safety standards required for patient treatment. The ability of bioartificial livers to function over long periods, remain active even after cryogenic storage and transport, and provide sustained function in long treatment windows indicates the potential to provide effective treatment. The assay results in Examples 1-18 above demonstrate that extracorporeal bioartificial livers are products that can be translated into clinical applications for the treatment of ALF.

Claims

1. A method for producing a liver composition that is at least partially recellularized, (a) Treating non-human animal livers with antiviral agents; (b) Perfusing the non-human animal liver to decellularize it and obtain a decellularized extracellular matrix; (c) To form a liver composition that is at least partially recellularized by contacting the decellularized extracellular matrix with a cell composition containing a population of human hepatocytes, Methods that include...

2. The method according to claim 1, wherein the antiviral treatment comprises irradiating the non-human animal liver with an electron beam (E-beam).

3. The method according to claim 2, wherein the irradiation includes exposing the non-human animal liver to an electron dose of about 2 kGy to about 50 kGy.

4. The method according to claim 2, wherein the irradiation includes exposing the non-human animal liver to an electron dose of about 5 kGy to about 25 kGy.

5. The method according to claim 2, wherein the irradiation includes exposing the non-human animal liver to an electron dose of about 10 kGy to 20 kGy.

6. The method according to claim 1, further comprising contacting the decellularized extracellular matrix with at least one of peroxy acid or hydrogen peroxide.

7. The method according to claim 1, wherein the antiviral treatment comprises irradiating the non-human animal liver with an electron beam (E-beam), and the method further comprises contacting the decellularized extracellular matrix with at least one of peroxy acid or hydrogen peroxide.

8. The method according to claim 6 or 7, wherein the peroxy acid includes peroxyacetic acid, peracetic acid, peroxycarboxylic acid, derivatives thereof, or combinations thereof.

9. The method according to claim 1, further comprising contacting the decellularized extracellular matrix with an additional cell composition comprising a population of human vascular endothelial cells (HUVECs).

10. The method according to claim 1, wherein the cell composition comprises both a human hepatocyte population and a HUVEC population.

11. The method according to claim 1, wherein the population of human hepatocytes is primary human hepatocytes.

12. The method according to claim 1, wherein the population of human hepatocytes is in vitro differentiated human hepatocytes.

13. The method according to claim 12, wherein the in vitro differentiated human hepatocytes are differentiated from an embryonic stem cell population, an induced pluripotent stem cell (iPSC) population, or an adult stem cell population.

14. The method according to claim 1, wherein the human hepatocyte population is brought into contact with the protease before the decellularized extracellular matrix is ​​brought into contact with the cell composition in step (c).

15. The method according to claim 1, characterized in that the at least partially recellularized liver has an increased ammonia clearance level compared to a population of hepatocytes that have not engrafted in the decellularized extracellular matrix.

16. The method according to claim 1, wherein the non-human animal liver is derived from a non-human mammal.

17. The method according to claim 16, wherein the non-human mammal is an ungulate.

18. The method according to claim 17, wherein the ungulate is a pig.

19. The method according to claim 1, wherein the non-human animal liver is frozen before the antiviral treatment.

20. A method for producing a liver composition that is at least partially recellularized, (a) Treating non-human animal livers with antiviral agents; (b) Perfusing the non-human animal liver to decellularize it and obtain a decellularized extracellular matrix; (c) Contacting the decellularized extracellular matrix with a first cell composition containing a population of human vascular endothelial cells; and (d) To form a liver composition that is at least partially recellularized by contacting the decellularized extracellular matrix with a second cell composition containing a population of human hepatocytes, Methods that include...

21. The method according to claim 20, wherein the first cell composition is characterized by having a glucose consumption rate of at least about 30 mg / hour, and the decellularized extracellular matrix is ​​brought into contact with the second cell composition.

22. The method according to claim 20, wherein the decellularized extracellular matrix is ​​brought into contact with the second cell composition at least 10 days after the first cell composition has been brought into contact with the decellularized extracellular matrix.

23. The method according to claim 20, wherein the decellularized extracellular matrix is ​​brought into contact with the second cell composition at least 12 days after the first cell composition has been brought into contact with the decellularized extracellular matrix.

24. The method according to claim 20, wherein the decellularized extracellular matrix is ​​brought into contact with the second cell composition 10 to 20 days after the first cell composition has been brought into contact with the decellularized extracellular matrix.

25. The method according to claim 20, characterized in that the at least partially recellularized liver composition has an increased ammonia clearance level compared to a hepatocyte population that is not in the form of the at least partially recellularized liver composition.

26. The method according to claim 20, wherein the antiviral treatment includes irradiating the decellularized extracellular matrix with an electron beam (E-beam).

27. The method according to claim 26, wherein the irradiation includes exposing the non-human animal liver to an electron dose of about 2 kGy to about 50 kGy.

28. The method according to claim 26, wherein the irradiation includes exposing the non-human animal liver to an electron dose of about 5 kGy to about 25 kGy.

29. The method according to claim 26, wherein the irradiation includes exposing the non-human animal liver to an electron dose of about 10 kGy to 20 kGy.

30. The method according to claim 20, further comprising contacting the decellularized extracellular matrix with a peroxy acid or hydrogen peroxide.

31. The method according to claim 30, wherein the peroxy acid includes peroxyacetic acid, peracetic acid, peroxycarboxylic acid, derivatives thereof, or combinations thereof.

32. The method according to claim 31, wherein the population of human hepatocytes is primary human hepatocytes.

33. The method according to claim 20, wherein the population of human hepatocytes is in vitro differentiated human hepatocytes.

34. The method according to claim 33, wherein the in vitro differentiated human hepatocytes are differentiated from an embryonic stem cell population, an induced pluripotent stem cell (iPSC) population, or adult stem cells.

35. The method according to claim 20, wherein the human hepatocyte population is brought into contact with the protease before the decellularized extracellular matrix is ​​brought into contact with the second cell composition in step (d).

36. The method according to claim 20, characterized in that the at least partially recellularized liver composition has an increased ammonia clearance level compared to a hepatocyte population that is not in the form of the at least partially recellularized liver composition.

37. The method according to claim 20, wherein the non-human animal is a non-human mammal.

38. The method according to claim 37, wherein the non-human mammal is an ungulate.

39. The method according to claim 38, wherein the ungulate is a pig.

40. The method according to claim 20, wherein the non-human animal liver is frozen before the antiviral treatment.

41. A composition comprising a liver composition at least partially recellularized, prepared by the method described in any one of claims 1 to 40.

42. A liver that has been recellularized at least partially: (a) Porcine extracellular matrix with reduced microbial particles and perfusion decellularization; (b) Human endothelial cell populations and human hepatocyte populations engrafted in the porcine extracellular matrix, Includes, The aforementioned at least partially recellularized liver exhibits increased ammonia clearance compared to a population of porcine hepatocytes engrafted in the porcine extracellular matrix. A liver that has been recellularized at least partially.

43. The at least partially recellularized liver according to claim 42, wherein the at least partially recellularized liver contains one or more surface markers selected from the group consisting of differentiation antigen group (CD) 31, CD105, and asialoglycoprotein receptor 1 (ASGR1).

44. The at least partially recellularized liver according to claim 42, characterized in that the at least partially recellularized liver has increased α1-antitrypsin (A1AT) levels compared to a population of hepatocytes that have not engrafted in the extracellular matrix of pigs.

45. The at least partially recellularized liver according to claim 42, characterized in that the at least partially recellularized liver has increased fibrinogen levels compared to a population of hepatocytes that have not engrafted in the extracellular matrix of pigs.

46. The at least partially recellularized liver according to claim 42, characterized in that the at least partially recellularized liver has increased fibrinogen levels compared to a population of porcine hepatocytes engrafted in the porcine extracellular matrix.

47. The at least partially recellularized liver according to claim 42, characterized in that the at least partially recellularized liver has an increased ammonia clearance level compared to a population of human hepatocytes that have not engrafted in the extracellular matrix of pigs.

48. The at least partially recellularized liver according to claim 42, characterized in that the at least partially recellularized liver has ammonia clearance after cryogenic storage, wherein the cryogenic storage is for a period of at least 6 hours.

49. The at least partially recellularized liver according to claim 42, characterized in that the at least partially recellularized liver has urea production after cryopreservation, and the cryopreservation is for a period of at least 6 hours.

50. The at least partially recellularized liver according to claim 42, further comprising an antibacterial agent.

51. The at least partially recellularized liver according to claim 50, wherein the antibacterial agent is an antiviral agent.

52. The at least partially recellularized liver according to claim 51, wherein the antiviral agent comprises peroxyacetic acid or peracetic acid.

53. The at least partially recellularized liver according to claim 42, wherein the population of human hepatocytes is primary human hepatocytes.

54. The at least partially recellularized liver according to claim 42, wherein the population of human hepatocytes is in vitro differentiated human hepatocytes.

55. The at least partially recellularized liver according to claim 54, wherein the in vitro differentiated human hepatocytes are differentiated from an embryonic stem cell population, an induced pluripotent stem cell (iPSC) population, or adult stem cells.

56. A composition comprising at least partially recellularized liver according to any one of claims 42 to 55, and an extracellular matrix protein or solution.

57. The composition according to claim 56, further comprising a cell culture medium.

58. An ex vivo method for treating liver disease in the subject, The aforementioned method, This includes creating a blood circuit. The blood circuit contains, in fluid communication with, the blood from the subject and at least partially recellularized liver as described in any one of claims 42 to 55. The at least partially recellularized liver filters the blood from the subject, removes ammonia, A method for treating the liver disease in the subject.

59. A method for treating liver disease in the subject, The aforementioned method, The administration includes targeting at least partially recellularized liver as described in any one of claims 42 to 55, A method for treating the liver disease in the subject.

60. The method according to claim 58 or 59, wherein the liver disease includes acute liver failure (ALF).

61. A method for producing an organ composition that has been at least partially recellularized, (a) Treating non-human animal organs with antiviral agents; (b) Perfusing the non-human animal organs to decellularize them and obtain decellularized extracellular matrix; (c) To form an organ composition that is at least partially recellularized by contacting the decellularized extracellular matrix with a cell composition containing a population of human cells. Methods that include...

62. The method according to claim 61, wherein the antiviral treatment includes irradiating the non-human animal organ with an electron beam (E-beam).

63. The method according to claim 62, wherein the irradiation includes exposing the non-human animal organ to an electron dose of about 2 kGy to about 50 kGy.

64. The method according to claim 62, wherein the irradiation includes exposing the non-human animal organ to an electron dose of about 5 kGy to about 25 kGy.

65. The method according to claim 62, wherein the irradiation includes exposing the non-human animal organ to an electron dose of about 10 kGy to 20 kGy.

66. The method according to claim 61, further comprising contacting the decellularized extracellular matrix with at least one of peroxy acid or hydrogen peroxide.

67. The method according to claim 61, wherein the antiviral treatment comprises irradiating the non-human animal organ with an electron beam (E-beam), and the method further comprises contacting the decellularized extracellular matrix with at least one of peroxy acid or hydrogen peroxide.

68. The method according to claim 66 or 67, wherein the peroxy acid comprises peroxyacetic acid, peracetic acid, peroxycarboxylic acid, derivatives thereof, or combinations thereof.

69. The method according to claim 61, further comprising contacting the decellularized extracellular matrix with an additional cell composition comprising a population of human vascular endothelial cells (HUVECs).

70. The method according to claim 61, wherein the cell composition comprises both a human organ cell population and a HUVEC population.

71. The method according to claim 61, wherein the human cell population includes human liver cells, human kidney cells, human lung cells, human heart cells, or cells of any other human parenchymal organ.

72. The method according to claim 71, wherein the population of human cells is in vitro differentiated human cells.

73. The method according to claim 72, wherein the in vitro differentiated human cells are differentiated from an embryonic stem cell population, an induced pluripotent stem cell (iPSC) population, or an adult stem cell population.

74. The method according to claim 61, wherein the human cell population is brought into contact with a protease before the decellularized extracellular matrix is ​​brought into contact with the cell composition in step (c).

75. The method according to claim 61, characterized in that the at least partially recellularized organ exhibits increased cellular activity compared to the cellular activity of a population of human cells that have not engrafted in a decellularized extracellular matrix.

76. The method according to claim 61, wherein the non-human animal is a non-human mammal.

77. The method according to claim 76, wherein the non-human mammal is an ungulate.

78. The method according to claim 77, wherein the ungulate is a pig.

79. The method according to claim 61, wherein the non-human animal organ is frozen before the antiviral treatment.

80. Any composition, at least partially recellularized organ, at least partially recellularized liver, method, or use thereof provided herein.

Images