Engineered tissue constructs for the treatment of hyperammonemia

Abstract

The present disclosure provides implantable engineered tissue constructs with hepatocytes and stromal cells for treating hyperammonemia.

Description

[Technical field]

[0001] The present disclosure relates to the use of engineered tissue constructs comprising hepatocytes and stromal cells to treat hyperammonemia. [Background technology]

[0002] Ammonia is highly toxic and is produced during metabolism in all organs. Hyperammonemia is caused by impaired ammonia detoxification and / or increased ammonia production. In mammals, the urea cycle detoxifies ammonia by enzymatically converting it to urea, which is then excreted in the urine. For example, impaired ammonia detoxification can be caused by urea cycle disorders (UCDs) in which a urea cycle enzyme is deficient (e.g., ornithine transcarbamylase deficiency). The National Urea Cycle Disorders Foundation estimates the prevalence of UCDs to be 1 in 8,500 births. In addition, non-UCD disorders such as organic acidemia, congenital lactic acidosis, fatty acid oxidation disorders, dibasic amino acid deficiency, transient hyperammonemia, Reye's syndrome, severe perinatal asphyxia, viral-associated hyperammonemia, infections, drug-induced hyperammonemia, liver disease, acute liver failure, acute exacerbation of chronic liver failure, transjugular intrahepatic portosystemic shunt-induced hyperammonemia, hepatic encephalopathy, liver cirrhosis, end-stage liver disease, parenteral hypercaloric nutrition, thyroid disease, Hashimoto's encephalopathy, blood disorders, gastric bypass surgery, gastrointestinal bleeding, malnutrition, generalized seizures, organ transplantation, and renal failure can also cause hyperammonemia. Hyperammonemia can cause vomiting, respiratory alkalosis, hypothermia, or death, in addition to neurological symptoms (e.g., convulsions, ataxia, stroke-like lesions, coma, psychosis, vision loss, acute encephalopathy, cerebral edema).

[0003] Current therapies for hyperammonemia aim to reduce ammonia excess but are widely considered suboptimal. For example, in the case of UCD, most patients require significant dietary changes consisting of protein restriction. However, low-protein diets must be carefully monitored because excessive restriction of protein intake causes the body to break down muscle, resulting in the production of ammonia. Therefore, many patients require supplementation with ammonia-scavenging drugs (e.g., sodium phenylbutyrate, sodium benzoate, and glycerol phenylbutyrate), and one or more of these drugs must be administered three to four times daily. Side effects of these drugs include nausea, vomiting, irritability, loss of appetite, and menstrual disorders in women. For children, delivery of food and medicines may require a gastrostomy tube that is surgically implanted into the stomach, or a nasogastric tube that is manually inserted through the nose into the stomach. If these treatment options fail, liver transplantation may be required. Thus, there is a significant unmet need for effective and reliable long-term treatments for diseases associated with hyperammonemia. Summary of the Invention

[0004] The present disclosure provides compositions and methods that can be used to treat hyperammonemia. The compositions and methods of the present disclosure can be used to administer engineered tissue constructs comprising hepatocyte and fibroblast cell populations to a patient (e.g., a mammalian patient, e.g., a human patient) having a urea cycle disorder, organic acidemia, congenital lactic acidosis, fatty acid oxidation disorder, dibasic amino acid deficiency, transient hyperammonemia, Reye's syndrome, severe perinatal asphyxia, viral-associated hyperammonemia, infectious disease, drug-induced hyperammonemia, liver disease, acute liver failure, acute exacerbation of chronic liver failure, transjugular intrahepatic portosystemic shunt-induced hyperammonemia, hepatic encephalopathy, liver cirrhosis, end-stage liver disease, parenteral hypercaloric nutrition, thyroid disease, Hashimoto's encephalopathy, blood disorder, gastric bypass surgery, gastrointestinal bleeding, malnutrition, generalized seizures, organ transplant, or renal failure in an amount effective to treat hyperammonemia.

[0005] In one embodiment, the disclosure provides a method for treating a variety of conditions, including urea cycle disorders, organic acidemias, congenital lactic acidosis, fatty acid oxidation disorders, dibasic amino acid deficiencies, transient hyperammonemia, Reye's syndrome, severe perinatal asphyxia, viral-associated hyperammonemia, infections (e.g., urinary tract infections, or infections caused by Proteus mirabilis, E. coli, or Klebsiella), drug-induced hyperammonemia (e.g., caused by the drugs valproic acid, topiramate, carbamazepine, salicylates, sulfadiazine, carbonic anhydrase inhibitors, carbonic anhydrase inhibitors with valproic acid, or chemotherapy), liver disease (e.g., biliary obstruction, alpha-1 antitrypsin deficiency, Wilson's disease, cystic fibrosis, galactosemia, or tyrosinemia), acute liver failure, acute exacerbation of chronic liver failure, transjugular vein purpura, pulmonary edema, and / or pulmonary edema. Provided is a method of treating hyperammonemia in a subject having intrahepatic portosystemic shunt-induced hyperammonemia, hepatic encephalopathy, liver cirrhosis, end-stage liver disease, parenteral hypercaloric nutrition, thyroid disease, Hashimoto's encephalopathy, blood disorders (e.g., multiple myeloma or acute leukemia), gastric bypass surgery, gastrointestinal bleeding, malnutrition, generalized stroke, organ transplant, or renal failure, comprising transplanting one or more engineered tissue constructs comprising a population of hepatic cells and optionally a population of stromal cells (e.g., fibroblasts) in an amount effective to treat hyperammonemia in the subject.

[0006] In another aspect, the disclosure provides a method for the reduction of ammonia levels in a subject in need thereof (e.g., a urea cycle disorder, organic acidemias, congenital lactic acidosis, fatty acid oxidation disorders, dibasic amino acid deficiencies, transient hyperammonemia, Reye's syndrome, severe perinatal asphyxia, viral-associated hyperammonemia, infections (e.g., urinary tract infections, or infections caused by Proteus mirabilis, E. coli, or Klebsiella), drug-induced hyperammonemia (e.g., caused by the drugs valproic acid, topiramate, carbamazepine, salicylates, sulfadiazine, carbonic anhydrase inhibitors, carbonic anhydrase inhibitors with valproic acid, or chemotherapy), liver disease (e.g., biliary obstruction, alpha-1 antitrypsin deficiency, Wilson's disease, cystic fibrosis, galactosemia, or tyrosinemia), acute liver failure, acute increase in chronic liver failure, The present invention provides a method for treating ammonia levels in a subject having hepatic encephalopathy, transjugular intrahepatic portosystemic shunt-induced hyperammonemia, hepatic encephalopathy, cirrhosis, end-stage liver disease, parenteral hypercaloric nutrition, thyroid disease, Hashimoto's encephalopathy, blood disorders (e.g., multiple myeloma or acute leukemia), gastric bypass surgery, gastrointestinal bleeding, malnutrition, generalized stroke, organ transplant, or renal failure, comprising transplanting one or more engineered tissue constructs comprising a population of hepatic cells and optionally a population of stromal cells (e.g., fibroblasts) in an amount effective to reduce ammonia levels in the subject.

[0007] In some embodiments of any of the above aspects, the hepatocyte population comprises an amount of hepatocytes equivalent to 0.5%-30% (e.g., 0.5%-30%, 0.6%-30%, 0.7%-30%, 0.8%-30%, 0.9%-30%, 1%-30%, 2%-30%, 3%-30%, 4%-30%, 5%-30%, 10%-30%, or 20%-30%) of the subject's total liver mass.

[0008] In some embodiments of any of the above aspects, the hepatocyte population comprises an amount of hepatocytes equivalent to 0.5%-20% (e.g., 0.5%-20%, 0.6%-20%, 0.7%-20%, 0.8%-20%, 0.9%-20%, 1%-20%, 2%-20%, 3%-20%, 4%-20%, 5%-20%, or 10%-20%) of the mass of the subject's remaining liver.

[0009] In some embodiments of any of the above aspects, the hepatocyte population comprises at least 3×10 5 ~1.8×10 11 (For example, about 4 × 10 5 ~Approx. 1.8×10 11 , about 5×10 5 ~Approx. 1.8×10 11 , about 6×10 5 ~Approx. 1.8×10 11 , about 7×10 5 ~Approx. 1.8×10 11 , about 8×10 5 ~Approx. 1.8×10 11 , about 9×10 5 ~Approx. 1.8×10 11 , about 1×10 6 ~Approx. 1.8×10 11 , about 2×10 6 ~Approx. 1.8×10 11 , about 3×10 6 ~Approx. 1.8×10 11 , about 4×10 6 ~Approx. 1.8×10 11 , about 5×10 6 ~Approx. 1.8×10 11 , about 6×10 6 ~Approx. 1.8×10 11 , about 7×10 6 ~Approx. 1.8×10 11 , about 8×10 6 ~Approx. 1.8×10 11 , about 9×10 6 ~Approx. 1.8×10 11 , about 1×10 7 ~Approx. 1.8×10 11 , about 2×10 7 ~Approx. 1.8×10 11 , about 3×10 7 ~Approx. 1.8×1011 , about 4×10 7 ~Approx. 1.8×10 11 , about 5×10 7 ~Approx. 1.8×10 11 , about 6×10 7 ~Approx. 1.8×10 11 , about 7×10 7 ~Approx. 1.8×10 11 , about 8×10 7 ~Approx. 1.8×10 11 , about 9×10 7 ~Approx. 1.8×10 11 , about 1×10 8 ~Approx. 1.8×10 11 , about 2×10 8 ~Approx. 1.8×10 11 , about 3×10 8 ~Approx. 1.8×10 11 , about 4×10 8 ~Approx. 1.8×10 11 , about 5×10 8 ~Approx. 1.8×10 11 , about 6×10 8 ~Approx. 1.8×10 11 , about 7×10 8 ~Approx. 1.8×10 11 , about 8×10 8 ~Approx. 1.8×10 11 , about 9×10 8 ~Approx. 1.8×10 11 , about 1×10 9 ~Approx. 1.8×10 11 , about 2×10 9 ~Approx. 1.8×10 11 , about 3×10 9 ~Approx. 1.8×10 11 , about 4×10 9 ~Approx. 1.8×10 11 , about 5×10 9 ~Approx. 1.8×10 11 , about 6×10 9 ~Approx. 1.8×10 11 , about 7×10 9 ~Approx. 1.8×10 11 , about 8×10 9 ~Approx. 1.8×10 11 , about 9×10 9 ~Approx. 1.8×10 11 , about 1×10 10 ~Approx. 1.8×1011 , about 2×10 10 ~Approx. 1.8×10 11 , about 3×10 10 ~Approx. 1.8×10 11 , about 4×10 10 ~Approx. 1.8×10 11 , about 5×10 10 ~Approx. 1.8×10 11 , about 6×10 10 ~Approx. 1.8×10 11 , about 7×10 10 ~Approx. 1.8×10 11 , about 8×10 10 ~Approx. 1.8×10 11 , about 9×10 10 ~Approx. 1.8×10 11 , or approximately 1 × 10 11 ~Approx. 1.8×10 11 ) including hepatocytes.

[0010] In some embodiments of any of the above aspects, the optional stromal cell (e.g., fibroblast) population comprises up to 1.8×10 12 (For example, about 1 to about 1.8 × 10 12 , about 10 to about 1.8 x 10 12 , about 100 to about 1.8 x 10 12 , about 1×10 3 ~Approx. 1.8×10 12 , about 2×10 3 ~Approx. 1.8×10 12 , about 3×10 3 ~Approx. 1.8×10 12 , about 4×10 3 ~Approx. 1.8×10 12 , about 5×10 3 ~Approx. 1.8×10 12 , about 6×10 3 ~Approx. 1.8×10 12 , about 7×10 3 ~Approx. 1.8×10 12 , about 8×10 3 ~Approx. 1.8×10 12 , about 9×10 3 ~Approx. 1.8×10 12 , about 1×10 4 ~Approx. 1.8×10 12 , about 2×10 4~Approx. 1.8×10 12 , about 3×10 4 ~Approx. 1.8×10 12 , about 4×10 4 ~Approx. 1.8×10 12 , about 5×10 4 ~Approx. 1.8×10 12 , about 6×10 4 ~Approx. 1.8×10 12 , about 7×10 4 ~Approx. 1.8×10 12 , about 8×10 4 ~Approx. 1.8×10 12 , about 9×10 4 ~Approx. 1.8×10 12 , about 1×10 5 ~Approx. 1.8×10 12 , about 2×10 5 ~Approx. 1.8×10 12 , about 3×10 5 ~Approx. 1.8×10 12 , about 4×10 5 ~Approx. 1.8×10 12 , about 5×10 5 ~Approx. 1.8×10 12 , about 6×10 5 ~Approx. 1.8×10 12 , about 7×10 5 ~Approx. 1.8×10 12 , about 8×10 5 ~Approx. 1.8×10 12 , about 9×10 5 ~Approx. 1.8×10 12 , about 1×10 6 ~Approx. 1.8×10 12 , about 2×10 6 ~Approx. 1.8×10 12 , about 3×10 6 ~Approx. 1.8×10 12 , about 4×10 6 ~Approx. 1.8×10 12 , about 5×10 6 ~Approx. 1.8×10 12 , about 6×10 6 ~Approx. 1.8×10 12 , about 7×10 6 ~Approx. 1.8×10 12 , about 8×10 6 ~Approx. 1.8×10 12 , about 9×10 6~Approx. 1.8×10 12 , about 1×10 7 ~Approx. 1.8×10 12 , about 2×10 7 ~Approx. 1.8×10 12 , about 3×10 7 ~Approx. 1.8×10 12 , about 4×10 7 ~Approx. 1.8×10 12 , about 5×10 7 ~Approx. 1.8×10 12 , about 6×10 7 ~Approx. 1.8×10 12 , about 7×10 7 ~Approx. 1.8×10 12 , about 8×10 7 ~Approx. 1.8×10 12 , about 9×10 7 ~Approx. 1.8×10 12 , about 1×10 8 ~Approx. 1.8×10 12 , about 2×10 8 ~Approx. 1.8×10 12 , about 3×10 8 ~Approx. 1.8×10 12 , about 4×10 8 ~Approx. 1.8×10 12 , about 5×10 8 ~Approx. 1.8×10 12 , about 6×10 8 ~Approx. 1.8×10 12 , about 7×10 8 ~Approx. 1.8×10 12 , about 8×10 8 ~Approx. 1.8×10 12 , about 9×10 8 ~Approx. 1.8×10 12 , about 1×10 9 ~Approx. 1.8×10 12 , about 2×10 9 ~Approx. 1.8×10 12 , about 3×10 9 ~Approx. 1.8×10 12 , about 4×10 9 ~Approx. 1.8×10 12 , about 5×10 9 ~Approx. 1.8×10 12 , about 6×10 9 ~Approx. 1.8×10 12 , about 7×10 9~Approx. 1.8×10 12 , about 8×10 9 ~Approx. 1.8×10 12 , about 9×10 9 ~Approx. 1.8×10 12 , about 1×10 10 ~Approx. 1.8×10 12 , about 2×10 10 ~Approx. 1.8×10 12 , about 3×10 10 ~Approx. 1.8×10 12 , about 4×10 10 ~Approx. 1.8×10 12 , about 5×10 10 ~Approx. 1.8×10 12 , about 6×10 10 ~Approx. 1.8×10 12 , about 7×10 10 ~Approx. 1.8×10 12 , about 8×10 10 ~Approx. 1.8×10 12 , about 9×10 10 ~Approx. 1.8×10 12 , about 1×10 11 ~Approx. 1.8×10 12 , about 2×10 11 ~Approx. 1.8×10 12 , about 3×10 11 ~Approx. 1.8×10 12 , about 4×10 11 ~Approx. 1.8×10 12 , about 5×10 11 ~Approx. 1.8×10 12 , about 6×10 11 ~Approx. 1.8×10 12 , about 7×10 11 ~Approx. 1.8×10 12 , about 8×10 11 ~Approx. 1.8×10 12 , about 9×10 11 ~Approx. 1.8×10 12 , or approximately 1 × 10 12 ~Approx. 1.8×10 12 ) stromal cells (e.g., fibroblasts).

[0011] In some embodiments, the hepatocytes are primary human hepatocytes. In some embodiments, the hepatocytes are derived from stem cells (e.g., induced pluripotent stem cells). In some embodiments, the stromal cells are fibroblasts. In some embodiments, the fibroblasts are selected from the group consisting of normal human dermal fibroblasts and neonatal foreskin fibroblasts. For example, in some embodiments, the fibroblasts are neonatal foreskin fibroblasts. In some embodiments, the fibroblasts are normal human dermal fibroblasts.

[0012] In some embodiments of any of the above aspects, the ratio of hepatocytes:stromal cells (e.g., fibroblasts) is 1:10 to 4:1 (e.g., 1:10 to 4:1, 1:10 to 3:1, 1:10 to 2:1, 1:10 to 1:1, 1:9 to 4:1, 1:9 to 3:1, 1:9 to 2:1, 1:9 to 1:1, 1:8 to 4:1, 1:8 to 3:1, 1:8 to 2:1, 1:8 to 1:1, 1:7 to 4:1, 1:7 to 3:1, 1:7 to 2:1, 1:7 to 1:1 , 1:6-4:1, 1:6-3:1, 1:6-2:1, 1:6-1:1, 1:5-4:1, 1:5-3:1, 1:5-2:1, 1:5-1:1, 1:4-4:1, 1:4-3:1, 1:4-2:1, 1:4-1:1, 1:3-4:1, 1:3-3:1, 1:3-2:1, 1:3-1:1, 1:2-4:1, 1:2-3:1, 1:2-2:1, 1:2-1:1, 1:1-4:1, 1:1-3:1, 1:1-2:1, and 1:0-1:1).

[0013] In some embodiments of any of the above aspects, the volume of the implant is 0.1 mL to 5 L (e.g., 0.2 mL to 5 L, 0.3 mL to 5 L, 0.4 mL to 5 L, 0.5 mL to 5 L, 1 mL to 5 L, 5 mL to 5 L, 10 mL to 5 L, 100 mL to 5 L, 1 L to 5 L, 2 L to 5 L, 3 L to 5 L, or 4 L to 5 L).

[0014] In some embodiments, the density of the hepatocytes is 0.1 M / mL to 150 M / mL (e.g., 0.2 M / mL to 149 M / mL, 0.3 M / mL to 148 M / mL, 0.4 M / mL to 147 M / mL, 0.5 M / mL to 146 M / mL, 1 M / mL to 145 M / mL, 5 M / mL to 140 M / mL, 10 M / mL to 100 M / mL, 20 M / mL to 50 M / mL, or 30 M / mL to 40 M / mL). In some embodiments, the density of the hepatocytes is 3 M / mL to 12 M / mL.

[0015] In some embodiments of any of the above aspects, the engineered tissue construct further comprises a biocompatible hydrogel scaffold. For example, in some embodiments, the biocompatible scaffold comprises fibrin. In some embodiments, the biocompatible scaffold comprises heparin. In some embodiments, the heparin is a synthetic heparin mimetic.

[0016] In some embodiments, the engineered tissue construct is implanted into a subject at an implantation site selected from the group consisting of the peritoneum (e.g., retroperitoneum), the peritoneal cavity (e.g., omentum or mesentery), the rectus abdominis muscle, the oblique abdominal muscle, the quadriceps muscle, extraperitoneal fat, and the renal capsule, an extraperitoneal site, a site on the surface of the liver, or an extrapleural site, or a site suitable for vascularization. For example, in some embodiments, the peritoneum is the retroperitoneum. In some embodiments, the peritoneal cavity is the omentum. In some embodiments, the peritoneum is the mesentery. In some embodiments, the omentum is the greater omentum or omental pouch. In some embodiments, the omentum is a pedicled omentum. In some embodiments, the mesentery is the small intestine mesentery. In some embodiments, the implant is implanted into a subject as a pedicled omental wrap or an omental wrap. In some embodiments, the implantation site is a site suitable for vascularization.

[0017] In some embodiments, the engineered tissue construct has about 3.6 vessels / mm 2 Larger (e.g., about 3.7 vessels / mm 2 , 3.8 vessels / mm 2 , 3.9 vessels / mm 2, 4 vessels / mm 2 , 4.1 vessels / mm 2 , 4.2 vessels / mm 2 , 4.3 vessels / mm 2 , 4.4 vessels / mm 2 , 4.5 vessels / mm 2 , 5 vessels / mm 2 , 6 vessels / mm 2 , 7 vessels / mm 2 , 8 vessels / mm 2 , 9 vessels / mm 2 , 10 vessels / mm 2 , 50 vessels / mm 2 , 100 vessels / mm 2 , 200 vessels / mm 2 , 300 vessels / mm 2 , 400 vessels / mm 2 , 500 vessels / mm 2 , 1000 vessels / mm 2 , 2000 vessels / mm 2 , 3000 vessels / mm 2 , 4000 vessels / mm 2 , or 4500 vessels / mm 2 The implant is then implanted into a subject at a transplant site having a microvascular density greater than 100 nm.

[0018] In some embodiments, the engineered tissue construct provides a microenvironment that promotes sustained hepatocyte survival in a subject for at least 3 months (e.g., at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 1 year, at least 5 years, or at least 10 years).

[0019] In some embodiments, the subject is a human. In some embodiments, after implantation of an engineered tissue construct, the subject has a blood glucose level of about 90 μmol / L or less (e.g., about 89 μmol / L, 88 μmol / L, 87 μmol / L, 86 μmol / L, 85 μmol / L, 84 μmol / L, 83 μmol / L, 82 μmol / L, 81 μmol / L, 80 μmol / L, 79 μmol / L, 78 μmol / L, 77 μmol / L, 76 μmol / L, 75 μmol / L, 74 μmol / L, 73 μmol / L, 75 μmol / L, 76 μmol / L, 77 μmol / L, 78 ... μmol / L, 72 μmol / L, 71 μmol / L, 70 μmol / L, 69 μmol / L, 68 μmol / L, 67 μmol / L, 66 μmol / L, 65 μmol / L, 64 μmol / L, 63 μmol / L, 62 μmol / L, 61 μmol / L, 60 μmol / L, 50 μmol / L, 40 μmol / L, 30 μmol / L, 20 μmol / L, 25 μmol / L, or less than 10 μmol / L).

[0020] In some embodiments, after transplantation of the engineered tissue construct, the subject shows a change in one or more parameters in a blood test relative to baseline levels. In some embodiments, the blood test is a liver function test. For example, in some embodiments, the one or more parameters include gamma-glutamyltransferase, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, or albumin levels.

[0021] In some embodiments, after implantation of the engineered tissue construct, the subject shows improvement in a test of gallbladder ejection fraction, e.g., in some embodiments, the test is a hepatobiliary iminodiacetic acid scan.

[0022] In another aspect, the present disclosure provides a kit comprising an engineered tissue construct, wherein the kit further comprises a package insert instructing a user of the kit to implant the engineered tissue construct into a subject according to the method of any one of the above embodiments. [Brief description of the drawings]

[0023] [Figure 1] Schematic diagram showing the experimental outline of the validation study of ammonia exposure in mice implanted with engineered tissue constructs. Starting on day 6, wild-type (B6EiC3SnF1 / J) and transgenic B6EiC3Sn a / A-Otcspf-ash / J (spfash) mice were subjected to immunosuppression every 2 days or left untreated. Group 1 (healthy control) and group 2 (unhealthy control) were not operated on. Pre-implantation blood sampling was performed on day 1, and then two engineered tissue constructs per mouse in group 3 were implanted into the fat pad near the uterus in the intraperitoneal space. Each construct contained 1.41×106 primary human hepatocytes and 2.82×106 normal human dermal fibroblasts. After implantation, mice were exposed to 7.5 mmol / kg NH4Cl (intraperitoneal administration) on days 4, 11, 18, 25, and 32. Urine was collected on days 7, 14, 21, and 28 followed by sacrifice on day 32. [Diagram 2] FIG. 2 is a graph showing serum human albumin levels in spfash mice implanted with two engineered tissue constructs as described in FIG. [Diagram 3] Clinical observations of wild-type group 1 healthy unoperated control mice (B6EiC3SnF1 / J), transgenic spfash group 2 unhealthy unoperated control mice, and transgenic spfash group 3 mice exposed to 7.5 mmol / kg NH4Cl and implanted with the two engineered tissue constructs, as described in Figure 1, are shown. [Figure 4] FIG. 2 is a graph showing serum ammonia levels in wild-type healthy unoperated control mice (C57BL / 6), spfash unhealthy unoperated control mice, and spfash mice exposed to 7.5 mmol / kg NH4Cl and implanted with two engineered tissue constructs, as described in FIG. [Diagram 5] FIG. 2 is a graph showing serum ammonia levels in spfash unhealthy unoperated control mice and spfash mice implanted with two engineered tissue constructs exposed to 7.5 mmol / kg NH4Cl, as described in FIG. [Figure 6]FIG. 1 is a schematic showing the steps of the manufacturing build of engineered tissue constructs. In step 1, stromal cells (e.g., fibroblasts) (e.g., neonatal human dermal fibroblasts) are thawed from their respective working cell banks (WCBs), expanded, and tested to measure viability and cell number. Hepatocytes (e.g., primary human hepatocytes (PHH)) are thawed from a hepatocyte master cell bank (MCB), tested to measure viability and cell number (step 2), and then combined with stromal cells (e.g., fibroblasts) (e.g., neonatal human fibroblasts) in a ratio (e.g., 1:2), centrifuged, placed into an array of microwells (e.g., pyramidal microwells), and incubated for 2-3 days to encourage the cells to self-assemble into multicellular liver aggregates. Liver aggregates are deemed amenable to encapsulation after microscopic confirmation of compaction. In step 6, the hepatocyte / fibroblast aggregates are encapsulated in a solution (e.g., containing fibrinogen) that has been polymerized (e.g., with thrombin). These encapsulation steps are performed within a mold (e.g., a cylindrical mold) that controls the overall dimensions of the graft to be approximately 2 mm in thickness and 6 mm to 100 cm in outer diameter (e.g., 7 mm to 999 mm, 8 mm to 998 mm, 9 mm to 997 mm, 10 mm to 996 mm, 20 mm to 995 mm, 30 mm to 990 mm, 40 mm to 980 mm, 60 mm to 960 mm, 90 mm to 930 mm, 100 mm to 900 mm, 200 mm to 800 mm, 300 mm to 700 mm, 400 mm to 600 mm, or 500 mm). The thickness is controlled by the volume of the cell-hydrogel suspension. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] definition As used herein, the terms "implanting", "transplantation", and the like refer to the application of one or more engineered tissue constructs directly to any effective transplantation site (e.g., a site suitable for vascularization) of a subject (e.g., a human subject). Exemplary transplantation sites include, among others, the peritoneum (e.g., retroperitoneum), the peritoneal cavity (e.g., omentum or mesentery), rectus abdominis muscle, oblique abdominal muscle, quadriceps muscle, extraperitoneal fat, kidney capsule, extraperitoneal sites, sites on the surface of the liver, and extrapleural sites.

[0025] As used herein, the term "subject" refers to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates (e.g., monkeys)), rabbits, deer, and rodents (e.g., mice and rats). In certain embodiments, the subject is a human.

[0026] As used herein, the terms "comprise," "comprising," "comprises," and "comprised of" are synonymous with "include," "(including)," "includes," or "contain," "containing," "contains," and are inclusive or open-ended terms specifying the presence of what follows, for example, and do not exclude or preclude the presence of additional, unrecited components, features, elements, members, or steps that are known in the art or disclosed herein.

[0027] The term "one or more" or "at least one" (e.g., one or more or at least one member(s) of a group of members) is clear in itself, but by way of further illustration, the term specifically encompasses reference to any one of the members, or any two or more of the members, such as any three or more, four or more, five or more, six or more, or seven or more of the members, up to and including all of the members.

[0028] As used herein, the terms "effective amount," "effective amount," "therapeutically effective amount," and the like, when used in reference to an engineered tissue construct described herein, refer to an amount of hepatocytes and stromal cells (e.g., fibroblasts) in the engineered tissue construct sufficient to effect a beneficial or desired outcome (e.g., a clinical outcome) when implanted into a subject (e.g., a mammal, e.g., a human). For example, in the context of treating hyperammonemia (e.g., in a patient with a urea cycle disorder), these terms refer to an amount of hepatocytes and stromal cells (e.g., fibroblasts) sufficient to effect a therapeutic response compared to the response obtained without implantation of the engineered tissue construct of interest. An "effective amount," "therapeutically effective amount," and the like, of an engineered tissue construct of the present disclosure also includes an amount that effects a beneficial or desired outcome in a subject compared to a control.

[0029] As used herein, the terms "treat" and "treatment" refer to therapeutic treatments aimed at preventing or slowing (alleviating) undesirable physiological changes (e.g., progression of hyperammonemia). Beneficial or desired clinical outcomes include, but are not limited to, reduction of ammonia, improvement of gallbladder ejection fraction tests, or alleviation of symptoms of hyperammonemia. Ammonia protein concentrations or gallbladder ejection fraction can be quantified using assays known in the art, for example, using a hepatobiliary iminodiacetic acid scan.

[0030] As used herein, the term "hyperammonemia" refers to a metabolic disorder characterized by an excess of ammonia in the blood. As used herein, "hyperammonemia" includes primary and secondary hyperammonemia, as well as acquired and congenital hyperammonemia.

[0031] As used herein, the term "urea cycle disorder" refers to any disorder caused by defects or malfunctions in the urea cycle. The urea cycle is a cycle of biochemical reactions that produces urea from ammonia, a product of protein catabolism. Specific types of urea cycle disorders include, but are not limited to, phosphate synthase 1 (CPS1) deficiency, ornithine transcarbamylase (OTC) deficiency, argininosuccinate synthase (ASS1) deficiency, argininosuccinate lyase (ASL) deficiency, arginase-1 (ARG1) deficiency, N-acetylglutamate synthase (NAGS) deficiency, ornithine translocase (ORNT1) deficiency, and citrin deficiency. Urea cycle disorders may be characterized by abnormal ammonia levels (e.g., ammonia levels of about 90 μmol / L or higher).

[0032] As used in the context of this disclosure, "engineered tissue construct" refers to a mixture of cultured hepatocytes (e.g., primary human hepatocytes) and stromal cells (e.g., fibroblasts) (e.g., neonatal foreskin stromal cells (e.g., fibroblasts)). The relative volume of the engineered tissue construct can be from 0.5 mL to 5 L. In some embodiments, the engineered tissue construct further comprises a biocompatible hydrogel scaffold (e.g., fibrin).

[0033] The cells may be from an established cell line or may be primary cells, where "primary cells", "primary cell lines", and "primary cultures" are used interchangeably herein to refer to cells and cell cultures that are derived from culture and grown in vitro (e.g., split) for a limited number of passages of the culture. For example, a primary culture may be a culture that has been passaged 0, 1, 2, 4, 5, 10, or 15 times (but not enough times to go through a crisis stage). A primary cell line may be maintained in vitro for less than 10 passages. If the cells are primary cells, such cells may be harvested from an individual by any convenient method. For example, cells from tissues (e.g., skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach, etc.) are most conveniently harvested by biopsy. An appropriate solution may be used to disperse or suspend the harvested cells. Such solutions will generally be balanced salt solutions (e.g., saline, phosphate-buffered saline (PBS), Hank's balanced salt solution, etc.) conveniently supplemented with fetal bovine serum or other naturally occurring factors at low concentrations (generally 5-25 mM) along with an acceptable buffer. Convenient buffers include HEPES, phosphate buffer, lactate buffer, etc. The cells may be used immediately or stored frozen for long periods (thawed and reused). In such cases, the cells are typically frozen in 10% DMSO, 50% serum, 40% buffered media, or some other solution commonly used in the art to store cells at such freezing temperatures, and thawed by methods commonly known in the art for thawing frozen cultured cells. For example, hepatocytes can be isolated by conventional methods (Berry et al., 1969, J. Cell Biol. 43:506-520), which can be adapted from human liver biopsy or autopsy material (e.g., to obtain primary human hepatocytes).

[0034] As used herein, a hydrogel scaffold is considered "biocompatible" if it does not exhibit toxicity when introduced into a subject (e.g., a human). In the context of the present disclosure, a biocompatible hydrogel scaffold preferably does not exhibit toxicity to cells of an engineered tissue construct or when implanted in a subject (e.g., a human) in vivo. Hepatotoxicity can be measured, for example, by quantifying hepatocyte apoptotic mortality (e.g., increased apoptosis is indicative of hepatotoxicity), transaminase levels (e.g., increased transaminase levels are indicative of hepatotoxicity), hepatocyte expansion (e.g., increased expansion is indicative of hepatotoxicity), hepatocyte droplet steatosis (e.g., increased steatosis is indicative of hepatotoxicity), bile cell mortality (e.g., increased bile cell mortality is indicative of hepatotoxicity), and gamma-glutamyl transpeptidase (GGT) levels (e.g., increased GGT levels are indicative of hepatotoxicity). Biocompatible hydrogel scaffolds can include, but are not limited to, fibrin and heparin.

[0035] As used herein, the term "hydrogel" refers to a network of polymer chains that is hydrophilic in nature such that the material absorbs large amounts of water or other aqueous solutions. Hydrogels can contain, for example, at least 70% v / v water, at least 80% v / v water, at least 90% v / v water, at least 95%, 96%, 97%, 98%, or even 99% or more v / v water (or other aqueous solutions). Hydrogels can contain natural or synthetic polymers, and the polymer network is often characterized by a high degree of cross-linking. Hydrogels also have a flexibility very similar to natural tissues due to their significant water content. Hydrogels are particularly useful in tissue engineering applications as scaffolds for culturing cells. In certain embodiments, the hydrogels are made of biocompatible polymers.

[0036] The term "adhesive material" refers to a material (e.g., a binder) that is incorporated into the cell mixture disclosed herein and for which the cells or microorganisms have some affinity. The material can be incorporated, for example, into a hydrogel prior to implantation of the engineered cell mixture. The material and the cells or microorganisms interact by any means including, for example, electrostatic or hydrophobic interactions, covalent bonds, or ionic bonds. Materials can include, but are not limited to, antibodies, proteins, peptides, nucleic acids, peptide aptamers, nucleic acid aptamers, sugars, proteoglycans, or cell receptors.

[0037] As used herein, the term "degrade" refers to the physiological process of biochemically degrading, digesting, and / or breaking down a molecule of interest (e.g., ammonia) and removing it from the body (e.g., by renal clearance).

[0038] As used herein, the term "angiogenically favorable" refers to conditions and / or environmental characteristics that are favorable for the formation of new blood vessels. In general, angiogenesis refers to the formation of new blood vessels in damaged or normally nonvascular tissue, or the formation of new blood vessels (e.g., arterioles, venules, and capillaries) at a higher density than normal in the tissue. For example, a site favorable for angiogenesis has approximately 3.6 blood vessels / mm 2 Larger (e.g., about 3.7 vessels / mm 2 , 3.8 vessels / mm 2 , 3.9 vessels / mm 2 , 4 vessels / mm 2 , 4.1 vessels / mm 2 , 4.2 vessels / mm 2 , 4.3 vessels / mm 2 , 4.4 vessels / mm 2 , 4.5 vessels / mm 2 , 5 vessels / mm 2 , 6 vessels / mm 2 , 7 vessels / mm 2 , 8 vessels / mm 2 , 9 vessels / mm 2 , 10 vessels / mm 2, 50 vessels / mm 2 , 100 vessels / mm 2 , 200 vessels / mm 2 , 300 vessels / mm 2 , 400 vessels / mm 2 , 500 vessels / mm 2 , 1000 vessels / mm 2 , 2000 vessels / mm 2 , 3000 vessels / mm 2 , 4000 vessels / mm 2 , or 4500 vessels / mm 2 The tissue may have a pre-existing microvessel density (greater than 100 μm).

[0039] As used herein, the terms "liver function tests" and "LFTs" refer to a liver panel (e.g., a group of blood tests that provide information about the status of a patient's liver). A liver panel can include measurements of gamma-glutamyltransferase levels, alkaline phosphatase levels, aspartate aminotransferase levels, alanine aminotransferase levels, albumin levels, bilirubin levels, prothrombin time, activated partial thromboplastin time, or combinations thereof.

[0040] As used herein, the term "age-adjusted normal values" refers to the process of normalizing data by age, which is a technique used to allow comparison of populations of subjects when their age profiles differ. As used herein, the term "normal values" refers to data that is not subjected to age normalization because the populations of subjects are similar across age profiles.

[0041] As used herein, the term "level" refers to the level of a protein compared to a reference. The reference can be any useful reference as defined herein. "Decreased level" and "increased level" of a protein means a decrease or increase in the protein level compared to the reference (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500% or more decrease or increase; a decrease or increase of about 10% compared to the reference). %, about 15%, about 20%, about 50%, about 75%, about 100%, or more than about 200% decrease or increase; about 0.01-fold, about 0.02-fold, about 0.1-fold, about 0.3-fold, about 0.5-fold, less than about 0.8-fold or less decrease or 15-fold increase; or about 1.2-fold, about 1.4-fold, about 1.5-fold, about 1.8-fold, about 2.0-fold, about 3.0-fold, about 3.5-fold, about 4.5-fold, about 5.0-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 100-fold, more than about 1000-fold or more increase). Protein levels can be expressed as mass / volume (e.g., g / dL, mg / mL, μg / mL, or ng / mL) or as a percentage of the total protein in the sample.

[0042] By "reference" is meant any useful standard used to compare protein levels for hyperammonemia. A reference can be any sample, standard, standard curve, or level used for comparison purposes. A reference can be a normal reference sample or a reference standard or level. A "reference sample" can be, for example, a control, e.g., a value of a predetermined negative control (e.g., a "normal control" or a backup sample taken from the same subject), a sample from a normal healthy subject (e.g., normal cells or normal tissues), a sample (e.g., cells or tissues) from a subject without hyperammonemia, a sample from a subject treated for hyperammonemia, or a sample of purified protein of known normal concentration (e.g., any of those described herein). By "reference standard or level" is meant a value or numerical value derived from a reference sample. By "normal control value" is meant a predetermined value indicative of a non-hyperammonemia condition, e.g., a value expected for a healthy control subject. Typically, a normal control value is expressed as a range ("X to Y"), a high threshold value ("below X"), or a low threshold value ("above X"). A subject having a measurement within the normal control value for a particular biomarker is typically referred to as "within the normal range" for that biomarker. A normal reference standard or level can be a value or value derived from a normal subject without hyperammonemia. In a preferred embodiment, the reference sample, standard, or level is matched to the subject sample by at least one of the following criteria: age, weight, sex, stage of disease, and general condition. A standard curve of levels of purified proteins (e.g., any of those described herein) within the normal reference range can also be used as a reference.

[0043] Detailed Description The present disclosure provides compositions and methods that can be used to treat hyperammonemia (e.g., in subjects with urea cycle disorders, organic acidemias, congenital lactic acidosis, fatty acid oxidation disorders, dibasic amino acid deficiencies, transient hyperammonemia, Reye's syndrome, severe perinatal asphyxia, viral-associated hyperammonemia, infections, drug-induced hyperammonemia, liver disease, acute liver failure, acute exacerbation of chronic liver failure, transjugular intrahepatic portosystemic shunt-induced hyperammonemia, hepatic encephalopathy, cirrhosis, end-stage liver disease, parenteral hypercaloric nutrition, thyroid disease, Hashimoto's encephalopathy, blood disorders, gastric bypass surgery, gastrointestinal bleeding, malnutrition, generalized seizures, organ transplantation, and / or renal failure) or reduce ammonia levels in a subject in need thereof. According to the compositions and methods described herein, a subject (e.g., a human) with hyperammonemia can be implanted with one or more engineered tissue constructs comprising a population of hepatic cells and, optionally, a population of stromal cells (e.g., fibroblasts).

[0044] The present disclosure is based, at least in part, on the discovery that engineered tissue constructs comprising hepatocytes and optionally stromal cells (e.g., fibroblasts) can be implanted and used in methods of treating hyperammonemia, thereby addressing significant unmet medical needs associated with hyperammonemia symptoms present in patients with urea cycle disorders, organic acidemias, congenital lactic acidosis, fatty acid oxidation disorders, dibasic amino acid deficiency, transient hyperammonemia, Reye's syndrome, severe perinatal asphyxia, viral-associated hyperammonemia, infections, drug-induced hyperammonemia, liver disease, acute liver failure, acute exacerbation of chronic liver failure, transjugular intrahepatic portosystemic shunt-induced hyperammonemia, hepatic encephalopathy, liver cirrhosis, end-stage liver disease, parenteral hypercaloric nutrition, thyroid disease, Hashimoto's encephalopathy, blood disorders, gastric bypass surgery, gastrointestinal bleeding, malnutrition, generalized seizures, organ transplant, or renal failure. The present disclosure is also based, at least in part, on the inventors' surprising discovery that implantation of engineered tissue constructs significantly reduces ammonia levels.

[0045] The following sections describe engineered tissue constructs, as well as various implantation sites that can be used with the compositions and methods of the present disclosure and parameters used for clinical monitoring following implantation of engineered tissue constructs.

[0046] Hyperammonemia The urea cycle is a cycle of biochemical reactions that produces urea from ammonia, a product of protein catabolism. The urea cycle includes five major enzymes, including carbamoyl phosphate synthase 1 (CPS1), ornithine transcarbamoylase (OTC), argininosuccinate synthase (ASS1), argininosuccinate lyase (ASL), and arginase 1 (ARG1), but other enzymes (e.g., N-acetylglutamate synthase (NAGS)) as well as mitochondrial amino acid transporters (e.g., ornithine translocase (ORNT1) and citrin) are also required. The urea cycle primarily occurs within the mitochondria of hepatocytes. Urea produced by the liver enters the bloodstream, travels to the kidneys, and is eventually excreted in the urine. Genetic defects in any of the urea cycle enzymes or transporters can cause hyperammonemia (elevated blood ammonia), or accumulation of cycle intermediates. Ammonia can then travel through the blood to the brain and cause cerebral edema, seizures, coma, long-term disability in survivors, and / or death.

[0047] For example, the enzyme OTC is a key component of the urea cycle, catalyzing the formation of citrulline from carbamoyl phosphate and ornithine, which is crucial in removing excess ammonia from the body. Deficient function of OTC can lead to hyperammonemia, which can damage the brain by a variety of mechanisms.

[0048] Hyperammonemia is an associated symptom of many diseases and disorders, including, for example, urea cycle disorders (e.g., CPS1 deficiency, OTC deficiency, ASS1 deficiency, ASL deficiency, ARG1 deficiency, NAGS deficiency, ORNT1 deficiency, and citrin deficiency), organic acidemias, congenital lactic acidosis, fatty acid oxidation disorders, dibasic amino acid deficiencies, transient hyperammonemia, Reye's syndrome, severe perinatal asphyxia, viral-associated hyperammonemia, infections (urinary tract infection or proteus deficiency), and chronic kidney disease. mirabilis, E. coli, or Klebsiella infections), drug-induced hyperammonemia (e.g., hyperammonemia caused by the drugs valproic acid, topiramate, carbamazepine, salicylates, sulfadiazine, carbonic anhydrase inhibitors, carbonic anhydrase inhibitors with valproic acid, or chemotherapy), liver disease (e.g., biliary obstruction, alpha-1 antitrypsin deficiency, Wilson's disease, cystic fibrosis, galactosemia, or tyrosinemia), acute liver failure, acute exacerbation of chronic liver failure, transjugular intrahepatic portosystemic shunt-induced hyperammonemia, hepatic encephalopathy, cirrhosis, end-stage liver disease, parenteral hypercaloric nutrition, thyroid disease, Hashimoto's encephalopathy, blood disorders (e.g., multiple myeloma or acute leukemia), gastric bypass surgery, gastrointestinal bleeding, malnutrition, generalized seizures, organ transplant, and renal failure, among others.

[0049] Engineered tissue constructs The engineered tissue constructs described herein comprise a population of hepatic cells and, optionally, a population of stromal cells (eg, fibroblasts).

[0050] In another aspect, the cell composition is provided in the form of aggregates of hepatic cell population and optional stromal cell population. In some embodiments, the hepatic cell population and optional stromal cell population are mixed under conditions that allow the two cell populations to form aggregates. In some embodiments, the hepatic cell population and optional stromal cell population are mixed using tissue fabrication techniques. In some embodiments, the hepatic cell population and optional stromal cell population are co-cultured. In some embodiments, the hepatic cell population and optional stromal cell population are mixed using tissue fabrication techniques. In some embodiments, the hepatic cell population and optional stromal cell population are co-cultured. In some embodiments, the hepatic cell population and optional stromal cell population are co-cultured by hanging drop, microwell molding, non-adherent surface, spheroid suspension culture using spinner flask, vertical wheel bioreactor, horizontal wheel bioreactor, or microfluidic spheroid system.

[0051] In other aspects, the compositions provided herein can include additional components, including, but not limited to, growth factors, ligands, cytokines, drugs, etc. In some embodiments, the cell mixture can include molecules that induce additional microenvironmental cues (e.g., small molecules or growth factors that stimulate or enhance proliferation and expansion of the cell population).

[0052] The properties of the cell aggregates of the present disclosure can be varied to suit a particular application. In certain embodiments, the density of the cell aggregates can be varied. In certain embodiments, cell aggregates of different diameters can be fabricated. In certain embodiments, the overall network organization of one or more cell aggregates can be defined, for example, by number, three-dimensional organization, alignment, diameter, density, etc.

[0053] In certain embodiments, the engineered cell composition may include one or more bioactive substances. Examples of bioactive substances(s) include, but are not limited to, hormones, neurotransmitters, growth factors, hormone, neurotransmitter or growth factor receptors, interferons, interleukins, chemokines, cytokines, colony stimulating factors, chemotactic factors, extracellular matrix components, and adhesion molecules, ligands and peptides; for example, growth hormone, parathyroid hormone, bone morphogenetic proteins, transforming growth factor alpha, TGF-beta1, TGF-beta2, fibroblast growth factors, granulocyte / macrophage colony stimulating factors, epidermal growth factors, platelet-derived growth factors, insulin-like growth factors, scatter factor / hepatocyte growth factors, fibrin, dextran, matrix metalloproteinases, collagens, fibronectin, vitronectin, hyaluronic acid, RGD-containing peptides or polypeptides, angiopoietins, and vascular endothelial cell growth factors.

[0054] In certain embodiments, the engineered cell mixtures disclosed herein include one or more adhesive materials to facilitate maintenance of the desired phenotype of the transplanted cells in vivo, including, but not limited to, antibodies, proteins, peptides, nucleic acids, peptide aptamers, nucleic acid aptamers, sugars, proteoglycans, or cell receptors.

[0055] The type of adhesive material (e.g., extracellular matrix (ECM) material, sugars, proteoglycans, etc.) is determined, in part, by the type(s) of cell being cultured (e.g., hepatocytes and stromal cells (e.g., fibroblasts)). ECM molecules found within the native microenvironment of the cells are useful for maintaining the function of either primary cells, progenitor cells, and / or cell lines.

[0056] In some embodiments, the engineered tissue construct further comprises a biocompatible hydrogel scaffold. For example, in some embodiments, the biocompatible scaffold is fibrin. In some embodiments, the biocompatible scaffold comprises a synthetic heparin mimetic. In particular, the synthetic polymers of the present disclosure may, in some embodiments, comprise an amount of negative charge similar to the amount of negative charge present in heparin. Thus, the synthetic polymers of the present disclosure may mimic the functional properties of heparin. For example, the synthetic polymers of the present disclosure have the potential to bind various bioactive agents (e.g., growth factors) that naturally bind to heparin. Thus, the synthetic polymers of the present disclosure, and hydrogels comprising the synthetic polymers described herein, may bind various bioactive agents (e.g., growth factors) to prevent the diffusion of the bioactive agents and maintain a high concentration of the bioactive agents locally, which may then act on cells and promote various cellular functions.

[0057] In some embodiments, the volume of the implant is 0.1 mL to 5 L (e.g., 0.2 mL to 5 L, 0.3 mL to 5 L, 0.4 mL to 5 L, 0.5 mL to 5 L, 1 mL to 5 L, 5 mL to 5 L, 10 mL to 5 L, 100 mL to 5 L, 1 L to 5 L, 2 L to 5 L, 3 L to 5 L, or 4 L to 5 L). For example, in some embodiments, the volume of the implant is 0.2 mL to 5 L. In some embodiments, the volume of the implant is 0.3 mL to 5 L. In some embodiments, the volume of the implant is 0.4 mL to 5 L. In some embodiments, the volume of the implant is 0.5 mL to 5 L. In some embodiments, the volume of the implant is 1 mL to 5 L. In some embodiments, the volume of the implant is 5 mL to 5 L. In some embodiments, the volume of the implant is 10 mL to 5 L. In some embodiments, the volume of the implant is 100 mL to 5 L. In some embodiments, the volume of the implant is 1 mL to 5 L. In some embodiments, the volume of the implant is between 2 mL and 5 L. In some embodiments, the volume of the implant is between 3 mL and 5 L. In some embodiments, the volume of the implant is between 4 mL and 5 L.

[0058] In some embodiments, the engineered tissue construct provides a microenvironment that promotes sustained hepatocyte survival for at least three months in a subject (e.g., at least three months, at least four months, at least five months, at least six months, at least one year, at least five years, at least ten years, or for the lifetime of the patient into whom the engineered tissue construct is implanted). For example, in some embodiments, the engineered tissue construct provides a microenvironment that promotes sustained hepatocyte survival for at least four months. In some embodiments, the engineered tissue construct provides a microenvironment that promotes sustained hepatocyte survival for at least five months. In some embodiments, the engineered tissue construct provides a microenvironment that promotes sustained hepatocyte survival for at least six months. In some embodiments, the engineered tissue construct provides a microenvironment that promotes sustained hepatocyte survival for at least one year. In some embodiments, the engineered tissue construct provides a microenvironment that promotes sustained hepatocyte survival for at least five years. In some embodiments, the engineered tissue construct provides a microenvironment that promotes sustained hepatocyte survival for at least ten years.

[0059] In some embodiments, the engineered tissue construct can be of any shape (eg, cylindrical, square, or square with rounded corners). In some embodiments, the engineered tissue construct has a tortuous topography (eg, to increase surface area).

[0060] Cell populations Cell populations are optimized to maintain appropriate morphology, phenotype, and cellular function conducive to use in the methods of the present disclosure. For example, primary human hepatocytes or neonatal foreskin fibroblasts (e.g., fibroblasts) can be isolated and / or pre-cultured under conditions optimized to ensure that each selected cell initially has the desired morphology, phenotype, and cellular function, and is therefore capable of maintaining said morphology, phenotype, and / or function in vivo after implantation of the engineered tissue constructs described herein.

[0061] liver cells The engineered tissue constructs described herein comprise hepatocytes. In some embodiments, the hepatocytes are primary human hepatocytes (PHH). In some embodiments, the hepatocytes are derived from stem cells (e.g., induced pluripotent stem cells).

[0062] In some embodiments, the hepatocytes described herein are obtained by a method comprising culturing and passaging PHH to obtain an expanded PHH population, or obtaining an expanded PHH population from a single PHH cell. In some embodiments, obtaining an expanded PHH population comprises culturing and passaging PHH in an appropriate cell culture medium for human cells (e.g., for 3-120 days, e.g., 4-119 days, 5-118 days, 10-117 days, 15-116 days, 20-115 days, 30-100 days, 40-90 days, 50-80 days, 60-70 days, or 65 days). See, e.g., U.S. Provisional Patent Application No. 63 / 271,441, incorporated herein by reference.

[0063] In some embodiments, the engineered tissue construct comprises a population of hepatic cells in an amount effective for treating hyperammonemia in a subject (e.g., a human). In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount effective to reduce ammonia levels in a subject (eg, a human).

[0064] In some embodiments, the density of the hepatocytes is 0.1 M / mL to 150 M / mL (e.g., 0.2 M / mL to 149 M / mL, 0.3 M / mL to 148 M / mL, 0.4 M / mL to 147 M / mL, 0.5 M / mL to 146 M / mL, 1 M / mL to 145 M / mL, 5 M / mL to 140 M / mL, 10 M / mL to 100 M / mL, 20 M / mL to 50 M / mL, or 30 M / mL to 40 M / mL). For example, in some embodiments, the density of the hepatocytes is 0.2 M / mL to 149 M / mL. In some embodiments, the density of the hepatocytes is 0.3 M / mL to 148 M / mL. In some embodiments, the density of the hepatocytes is 0.4 M / mL to 147 M / mL. In some embodiments, the density of the hepatocytes is 0.5 M / mL to 146 M / mL. In some embodiments, the density of the hepatocytes is 1 M / mL to 145 M / mL. In some embodiments, the density of the hepatocytes is 5 M / mL to 140 M / mL. In some embodiments, the density of the hepatocytes is 10 M / mL to 100 M / mL. In some embodiments, the density of the hepatocytes is 20 M / mL to 50 M / mL. In some embodiments, the density of the hepatocytes is 30 M / mL to 40 M / mL. In some embodiments, the density of the hepatocytes is 3 M / mL to 12 M / mL (e.g., 4 M / mL to 11 M / mL, 5 M / mL to 10 M / mL, 6 M / mL to 9 M / mL, or 7 M / mL to 8 M / mL). For example, in some embodiments, the density of the hepatocytes is 4 M / mL to 11 M / mL. In some embodiments, the density of the hepatocytes is 5 M / mL to 10 M / mL. In some embodiments, the density of the hepatocytes is 6 M / mL to 9 M / mL. In some embodiments, the density of the hepatocytes is 7 M / mL to 8 M / mL.

[0065] In some embodiments, the density of the hepatocytes is 0.1 M / mL. In some embodiments, the density of the hepatocytes is 0.2 M / mL. In some embodiments, the density of the hepatocytes is 0.3 M / mL. In some embodiments, the density of the hepatocytes is 0.4 M / mL. In some embodiments, the density of the hepatocytes is 0.5 M / mL. In some embodiments, the density of the hepatocytes is 0.6 M / mL. In some embodiments, the density of the hepatocytes is 0.7 M / mL. In some embodiments, the density of the hepatocytes is 0.8 M / mL. In some embodiments, the density of the hepatocytes is 0.9 M / mL. In some embodiments, the density of the hepatocytes is 1 M / mL. In some embodiments, the density of the hepatocytes is 2 M / mL. In some embodiments, the density of the hepatocytes is 3 M / mL. In some embodiments, the density of the hepatocytes is 4 M / mL. In some embodiments, the density of the hepatocytes is 5 M / mL. In some embodiments, the density of the hepatocytes is 6 M / mL. In some embodiments, the density of the hepatocytes is 7 M / mL. In some embodiments, the density of the hepatocytes is 8 M / mL. In some embodiments, the density of the hepatocytes is 9M / mL. In some embodiments, the density of the hepatocytes is 10M / mL. In some embodiments, the density of the hepatocytes is 11M / mL. In some embodiments, the density of the hepatocytes is 12M / mL. In some embodiments, the density of the hepatocytes is 13M / mL. In some embodiments, the density of the hepatocytes is 14M / mL. In some embodiments, the density of the hepatocytes is 15M / mL. In some embodiments, the density of the hepatocytes is 16M / mL. In some embodiments, the density of the hepatocytes is 17M / mL. In some embodiments, the density of the hepatocytes is 18M / mL. In some embodiments, the density of the hepatocytes is 19M / mL. In some embodiments, the density of the hepatocytes is 20M / mL. In some embodiments, the density of the hepatocytes is 21M / mL. In some embodiments, the density of the hepatocytes is 22M / mL. In some embodiments, the density of the hepatocytes is 23M / mL.In some embodiments, the density of the hepatocytes is 24M / mL. In some embodiments, the density of the hepatocytes is 25M / mL. In some embodiments, the density of the hepatocytes is 26M / mL. In some embodiments, the density of the hepatocytes is 27M / mL. In some embodiments, the density of the hepatocytes is 28M / mL. In some embodiments, the density of the hepatocytes is 29M / mL. In some embodiments, the density of the hepatocytes is 30M / mL. In some embodiments, the density of the hepatocytes is 31M / mL. In some embodiments, the density of the hepatocytes is 32M / mL. In some embodiments, the density of the hepatocytes is 33M / mL. In some embodiments, the density of the hepatocytes is 34M / mL. In some embodiments, the density of the hepatocytes is 35M / mL. In some embodiments, the density of the hepatocytes is 36M / mL. In some embodiments, the density of the hepatocytes is 37M / mL. In some embodiments, the density of the hepatocytes is 38M / mL. In some embodiments, the density of the hepatocytes is 39M / mL. In some embodiments, the density of the hepatocytes is 40M / mL. In some embodiments, the density of the hepatocytes is 41 M / mL. In some embodiments, the density of the hepatocytes is 42 M / mL. In some embodiments, the density of the hepatocytes is 43 M / mL. In some embodiments, the density of the hepatocytes is 44 M / mL. In some embodiments, the density of the hepatocytes is 45 M / mL. In some embodiments, the density of the hepatocytes is 46 M / mL. In some embodiments, the density of the hepatocytes is 47 M / mL. In some embodiments, the density of the hepatocytes is 48 M / mL. In some embodiments, the density of the hepatocytes is 49 M / mL. In some embodiments, the density of the hepatocytes is 50 M / mL. In some embodiments, the density of the hepatocytes is 60 M / mL. In some embodiments, the density of the hepatocytes is 70 M / mL. In some embodiments, the density of the hepatocytes is 80 M / mL. In some embodiments, the density of the hepatocytes is 90 M / mL. In some embodiments, the density of the hepatocytes is 100 M / mL.In some embodiments, the density of the hepatocytes is 150 M / mL.

[0066] In some embodiments, the hepatocytes have a quantifiable efficacy (eg, the ability to decompose ammonia within a quantifiable range). I. Percentage of hepatocytes compared to total liver mass In some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount equivalent to 0.5%-30% (e.g., 0.5%-30%, 0.6%-30%, 0.7%-30%, 0.8%-30%, 0.9%-30%, 1%-30%, 2%-30%, 3%-30%, 4%-30%, 5%-30%, 10%-30%, or 20%-30%) of the subject's total liver mass. For example, in some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount equivalent to 0.6%-30% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount equivalent to 0.7%-30% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount equivalent to 0.8%-30% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount corresponding to 0.9%-30% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount corresponding to 1%-30% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount corresponding to 2%-30% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount corresponding to 3%-30% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount corresponding to 4%-30% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount corresponding to 5%-30% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount corresponding to 6%-30% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount corresponding to 7%-30% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatocyte cell population in an amount corresponding to between 8% and 30% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatocyte cell population in an amount corresponding to between 9% and 30% of the subject's total liver mass.In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount corresponding to 10%-30% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount corresponding to 11%-30% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount corresponding to 12%-30% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount corresponding to 13%-30% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount corresponding to 14%-30% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount corresponding to 15%-30% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount corresponding to 16%-30% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount corresponding to 17%-30% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount corresponding to 18%-30% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount corresponding to 19%-30% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount corresponding to 20%-30% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount corresponding to 21%-30% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount corresponding to 22%-30% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount corresponding to 23%-30% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount corresponding to 24%-30% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount corresponding to 25%-30% of the subject's total liver mass.In some embodiments, the engineered tissue construct comprises a hepatocyte cell population in an amount corresponding to 26%-30% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatocyte cell population in an amount corresponding to 27%-30% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatocyte cell population in an amount corresponding to 28%-30% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatocyte cell population in an amount corresponding to 29%-30% of the subject's total liver mass.

[0067] In some embodiments, the engineered tissue construct comprises a hepatocyte cell population in an amount equivalent to 0.5% of the subject's total liver mass. For example, in some embodiments, the engineered tissue construct comprises a hepatocyte cell population in an amount equivalent to 0.6% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatocyte cell population in an amount equivalent to 0.7% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatocyte cell population in an amount equivalent to 0.8% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatocyte cell population in an amount equivalent to 0.9% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatocyte cell population in an amount equivalent to 1% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatocyte cell population in an amount equivalent to 2% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatocyte cell population in an amount equivalent to 3% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatocyte cell population in an amount equivalent to 4% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount equivalent to 5% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount equivalent to 6% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount equivalent to 7% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount equivalent to 8% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount equivalent to 9% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount equivalent to 10% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount equivalent to 11% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount equivalent to 12% of the subject's total liver mass.In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount equivalent to 13% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount equivalent to 14% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount equivalent to 15% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount equivalent to 16% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount equivalent to 17% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount equivalent to 18% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount equivalent to 19% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount equivalent to 20% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount equivalent to 21% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount equivalent to 22% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount equivalent to 23% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount equivalent to 24% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount equivalent to 25% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount equivalent to 26% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount equivalent to 27% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount equivalent to 28% of the subject's total liver mass. In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount equivalent to 29% of the subject's total liver mass.

[0068] II. Percentage of hepatocytes relative to the mass of the remnant liver In some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount equivalent to 0.5%-20% (e.g., 0.5%-20%, 0.6%-20%, 0.7%-20%, 0.8%-20%, 0.9%-20%, 1%-20%, 2%-20%, 3%-20%, 4%-20%, 5%-20%, or 10%-20%) of the mass of the subject's remaining liver. For example, in some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount equivalent to 0.6%-20% of the mass of the subject's remaining liver. In some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount equivalent to 0.7%-20% of the mass of the subject's remaining liver. In some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount equivalent to 0.8%-20% of the mass of the subject's remaining liver. In some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount corresponding to 0.9%-20% of the mass of the subject's remaining liver. In some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount corresponding to 1%-20% of the mass of the subject's remaining liver. In some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount corresponding to 2%-20% of the mass of the subject's remaining liver. In some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount corresponding to 3%-20% of the mass of the subject's remaining liver. In some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount corresponding to 4%-20% of the mass of the subject's remaining liver. In some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount corresponding to 5%-20% of the mass of the subject's remaining liver. In some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount corresponding to 6%-20% of the mass of the subject's remaining liver. In some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount corresponding to 7%-20% of the mass of the subject's remaining liver. In some embodiments, the engineered tissue construct comprises a hepatocyte cell population in an amount corresponding to 8%-20% of the mass of the subject's remaining liver. In some embodiments, the engineered tissue construct comprises a hepatocyte cell population in an amount corresponding to 9%-20% of the mass of the subject's remaining liver. In some embodiments, the engineered tissue construct comprises a hepatocyte cell population in an amount corresponding to 10%-20% of the mass of the subject's remaining liver.In some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount corresponding to 11%-20% of the mass of the subject's remnant liver. In some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount corresponding to 12%-20% of the mass of the subject's remnant liver. In some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount corresponding to 13%-20% of the mass of the subject's remnant liver. In some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount corresponding to 14%-20% of the mass of the subject's remnant liver. In some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount corresponding to 15%-20% of the mass of the subject's remnant liver. In some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount corresponding to 16%-20% of the mass of the subject's remnant liver. In some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount corresponding to 17%-20% of the mass of the subject's remnant liver. In some embodiments, the engineered tissue construct comprises a hepatocyte population in an amount corresponding to 18%-20% of the mass of the subject's remnant liver. In some embodiments, the engineered tissue construct comprises a population of hepatic cells in an amount equivalent to 19%-20% of the mass of the subject's remnant liver.

[0069] In some embodiments, the engineered tissue construct comprises a hepatocyte cell population in an amount equivalent to 0.5% of the mass of the subject's remaining liver. For example, in some embodiments, the engineered tissue construct comprises a hepatocyte cell population in an amount equivalent to 0.6% of the mass of the subject's remaining liver. In some embodiments, the engineered tissue construct comprises a hepatocyte cell population in an amount equivalent to 0.7% of the mass of the subject's remaining liver. In some embodiments, the engineered tissue construct comprises a hepatocyte cell population in an amount equivalent to 0.8% of the mass of the subject's remaining liver. In some embodiments, the engineered tissue construct comprises a hepatocyte cell population in an amount equivalent to 0.9% of the mass of the subject's remaining liver. In some embodiments, the engineered tissue construct comprises a hepatocyte cell population in an amount equivalent to 1% of the mass of the subject's remaining liver. In some embodiments, the engineered tissue construct comprises a hepatocyte cell population in an amount equivalent to 2% of the mass of the subject's remaining liver. In some embodiments, the engineered tissue construct comprises a hepatocyte cell population in an amount equivalent to 3% of the mass of the subject's remaining liver. In some embodiments, the engineered tissue construct comprises a hepatocyte cell population in an amount equivalent to 4% of the mass of the subject's remaining liver. In some embodiments, the engineered tissue construct comprises a hepatocyte cell population in an amount equivalent to 5% of the mass of the subject's remaining liver. In some embodiments, the engineered tissue construct comprises a hepatocyte cell population in an amount equivalent to 6% of the mass of the subject's remaining liver. In some embodiments, the engineered tissue construct comprises a hepatocyte cell population in an amount equivalent to 7% of the mass of the subject's remaining liver. In some embodiments, the engineered tissue construct comprises a hepatocyte cell population in an amount equivalent to 8% of the mass of the subject's remaining liver. In some embodiments, the engineered tissue construct comprises a hepatocyte cell population in an amount equivalent to 9% of the mass of the subject's remaining liver. In some embodiments, the engineered tissue construct comprises a hepatocyte cell population in an amount equivalent to 10% of the mass of the subject's remaining liver. In some embodiments, the engineered tissue construct comprises a hepatocyte cell population in an amount equivalent to 11% of the mass of the subject's remaining liver. In some embodiments, the engineered tissue construct comprises a hepatocyte cell population in an amount equivalent to 12% of the mass of the subject's remaining liver.In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount equivalent to 13% of the mass of the subject's remaining liver. In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount equivalent to 14% of the mass of the subject's remaining liver. In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount equivalent to 15% of the mass of the subject's remaining liver. In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount equivalent to 16% of the mass of the subject's remaining liver. In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount equivalent to 17% of the mass of the subject's remaining liver. In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount equivalent to 18% of the mass of the subject's remaining liver. In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount equivalent to 19% of the mass of the subject's remaining liver. In some embodiments, the engineered tissue construct comprises a hepatic cell population in an amount equivalent to 20% of the mass of the subject's remaining liver.

[0070] III. Hepatocyte numbers in engineered tissue constructs In some embodiments, the engineered tissue construct comprises about 3×10 5 ~1.8×10 11 (For example, about 4 × 10 5 ~Approx. 1.8×10 11 , about 5×10 5 ~Approx. 1.8×10 11 , about 6×10 5 ~Approx. 1.8×10 11 , about 7×10 5 ~Approx. 1.8×10 11 , about 8×10 5 ~Approx. 1.8×10 11 , about 9×10 5 ~Approx. 1.8×10 11 , about 1×10 6 ~Approx. 1.8×10 11 , about 2×10 6 ~Approx. 1.8×10 11 , about 3×10 6 ~Approx. 1.8×10 11 , about 4×10 6 ~Approx. 1.8×10 11 , about 5×106 ~Approx. 1.8×10 11 , about 6×10 6 ~Approx. 1.8×10 11 , about 7×10 6 ~Approx. 1.8×10 11 , about 8×10 6 ~Approx. 1.8×10 11 , about 9×10 6 ~Approx. 1.8×10 11 , about 1×10 7 ~Approx. 1.8×10 11 , about 2×10 7 ~Approx. 1.8×10 11 , about 3×10 7 ~Approx. 1.8×10 11 , about 4×10 7 ~Approx. 1.8×10 11 , about 5×10 7 ~Approx. 1.8×10 11 , about 6×10 7 ~Approx. 1.8×10 11 , about 7×10 7 ~Approx. 1.8×10 11 , about 8×10 7 ~Approx. 1.8×10 11 , about 9×10 7 ~Approx. 1.8×10 11 , about 1×10 8 ~Approx. 1.8×10 11 , about 2×10 8 ~Approx. 1.8×10 11 , about 3×10 8 ~Approx. 1.8×10 11 , about 4×10 8 ~Approx. 1.8×10 11 , about 5×10 8 ~Approx. 1.8×10 11 , about 6×10 8 ~Approx. 1.8×10 11 , about 7×10 8 ~Approx. 1.8×10 11 , about 8×10 8 ~Approx. 1.8×10 11 , about 9×10 8 ~Approx. 1.8×10 11 , about 1×10 9 ~Approx. 1.8×10 11 , about 2×10 9 ~Approx. 1.8×10 11 , about 3×109 ~Approx. 1.8×10 11 , about 4×10 9 ~Approx. 1.8×10 11 , about 5×10 9 ~Approx. 1.8×10 11 , about 6×10 9 ~Approx. 1.8×10 11 , about 7×10 9 ~Approx. 1.8×10 11 , about 8×10 9 ~Approx. 1.8×10 11 , about 9×10 9 ~Approx. 1.8×10 11 , about 1×10 10 ~Approx. 1.8×10 11 , about 2×10 10 ~Approx. 1.8×10 11 , about 3×10 10 ~Approx. 1.8×10 11 , about 4×10 10 ~Approx. 1.8×10 11 , about 5×10 10 ~Approx. 1.8×10 11 , about 6×10 10 ~Approx. 1.8×10 11 , about 7×10 10 ~Approx. 1.8×10 11 , about 8×10 10 ~Approx. 1.8×10 11 , about 9×10 10 ~Approx. 1.8×10 11 , or approximately 1 × 10 11 ~Approx. 1.8×10 11 ) hepatocyte population. For example, in some embodiments, the engineered tissue construct comprises about 4×10 5 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 5 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 5 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 5 ~Approx. 1.8×10 11In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 8×10 5 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 5 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 6 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 6 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 6 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 6 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 6 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 6 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 6 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 8×10 6 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 6 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 7 ~Approx. 1.8×10 11In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 7 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 7 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 7 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 7 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 7 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 7 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 8×10 7 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 7 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 8 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 8 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 8 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 8 ~Approx. 1.8×10 11In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 8 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 8 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 8 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 8×10 8 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 8 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 9 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 9 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 9 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 9 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 9 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 9 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 9 ~Approx. 1.8×10 11In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 8×10 9 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 9 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 10 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 10 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 10 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 10 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 10 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 10 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 10 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 8×10 10 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 10 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 11 ~Approx. 1.8×10 11 The amount of hepatocytes includes the hepatocyte population.

[0071] In some embodiments, the engineered tissue construct comprises about 3×10 5 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 5 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 5 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 5 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 5 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 8×10 5 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 5 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 6 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 6 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 6 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 6 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 6 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 6 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 6 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 8×10 6 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 6 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 7In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 7 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 7 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 7 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 7 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 7 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 7 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 8×10 7 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 7 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 8×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 8In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 8×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 8×10 10In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1.8×10 11 The amount of hepatocytes includes the hepatocyte population.

[0072] IIIa. Age-dependent hepatocyte numbers in engineered tissue constructs IIIai. Neonatal (e.g., 0-30 days old) In some embodiments, the engineered tissue constructs are implanted into neonates and approximately 3×10 5 ~Approx. 3×10 10 (For example, about 4 × 10 5 ~Approx. 3×10 10 , about 5×10 5 ~Approx. 3×10 10 , about 6×10 5 ~Approx. 3×10 10 , about 7×10 5 ~Approx. 3×10 10 , about 8×10 5 ~Approx. 3×10 10 , about 9×10 5 ~Approx. 3×10 10 , about 1×10 6 ~Approx. 3×10 10 , about 2×10 6 ~Approx. 3×10 10 , about 3×10 6 ~Approx. 3×10 10 , about 4×10 6 ~Approx. 3×10 10 , about 5×10 6 ~Approx. 3×10 10 , about 6×10 6 ~Approx. 3×10 10 , about 7×10 6 ~Approx. 3×10 10 , about 8×10 6 ~Approx. 3×10 10 , about 9×10 6 ~Approx. 3×10 10 , about 1×10 7 ~Approx. 3×10 10 , about 2×107 ~Approx. 3×10 10 , about 3×10 7 ~Approx. 3×10 10 , about 4×10 7 ~Approx. 3×10 10 , about 5×10 7 ~Approx. 3×10 10 , about 6×10 7 ~Approx. 3×10 10 , about 7×10 7 ~Approx. 3×10 10 , about 8×10 7 ~Approx. 3×10 10 , about 9×10 7 ~Approx. 3×10 10 , about 1×10 8 ~Approx. 3×10 10 , about 2×10 8 ~Approx. 3×10 10 , about 3×10 8 ~Approx. 3×10 10 , about 4×10 8 ~Approx. 3×10 10 , about 5×10 8 ~Approx. 3×10 10 , about 6×10 8 ~Approx. 3×10 10 , about 7×10 8 ~Approx. 3×10 10 , about 8×10 8 ~Approx. 3×10 10 , about 9×10 8 ~Approx. 3×10 10 , about 1×10 9 ~Approx. 3×10 10 , about 2×10 9 ~Approx. 3×10 10 , about 3×10 9 ~Approx. 3×10 10 , about 4×10 9 ~Approx. 3×10 10 , about 5×10 9 ~Approx. 3×10 10 , about 6×10 9 ~Approx. 3×10 10 , about 7×10 9 ~Approx. 3×10 10 , about 8×10 9 ~Approx. 3×10 10 , about 9×10 9 ~Approx. 3×10 10 , about 1×10 10~Approx. 3×10 10 , or about 2 × 10 10 ~Approx. 3×10 10 ) hepatocyte population. For example, in some embodiments, the engineered tissue construct comprises about 4×10 5 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 5 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 5 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 5 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 8×10 5 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 5 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 6 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 6 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 6 ~Approx. 3×10 10 , about 4×10 6 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 6 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 6 ~Approx. 3×10 10In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 6 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 8×10 6 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 6 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of 1×10 7 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 7 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 7 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 7 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 7 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 7 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 7 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 8×10 7 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 7 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 8 ~Approx. 3×1010 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 8 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 8 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 8 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 8 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 8 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 8 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 8×10 8 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 8 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 9 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 9 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 9 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 9 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×109 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 9 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 9 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 8×10 9 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 9 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 10 ~Approx. 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 10 ~Approx. 3×10 10 The amount of hepatocytes includes the hepatocyte population.

[0073] In some embodiments, the engineered tissue construct comprises about 3×10 5 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 5 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 5 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 5 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 5 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 8×10 5 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 5 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×106 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 6 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 6 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 6 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 6 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 6 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 6 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 8×10 6 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 6 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 7 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 7 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 7 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 7 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 7 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 7 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 7 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 8×10 7 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×107 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 8×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 8×109 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 10 The amount of hepatocytes includes the hepatocyte population.

[0074] IIIaii. Infants (e.g., 1 year old) In some embodiments, the engineered tissue construct is implanted into an infant, and approximately 2×10 7 ~about 6×10 10 (For example, about 3 × 10 7 ~about 6×10 10 , about 4×10 7 ~about 6×10 10 , about 5×10 7 ~about 6×10 10 , about 6×10 7 ~about 6×10 10 , about 7×10 7 ~about 6×10 10 , about 8×10 7 ~about 6×10 10 , about 9×10 7 ~about 6×10 10 , about 1×10 8 ~about 6×10 10 , about 2×10 8 ~about 6×10 10 , about 3×10 8 ~about 6×10 10 , about 4×10 8 ~about 6×10 10 , about 5×10 8 ~about 6×10 10 , about 6×10 8 ~about 6×10 10 , about 7×10 8 ~about 6×10 10 , about 8×10 8 ~about 6×10 10 , about 9×10 8~about 6×10 10 , about 1×10 9 ~about 6×10 10 , about 2×10 9 ~about 6×10 10 , about 3×10 9 ~about 6×10 10 , about 4×10 9 ~about 6×10 10 , about 5×10 9 ~about 6×10 10 , about 6×10 9 ~about 6×10 10 , about 7×10 9 ~about 6×10 10 , about 8×10 9 ~about 6×10 10 , about 9×10 9 ~about 6×10 10 , about 1×10 10 ~about 6×10 10 , about 2×10 10 ~about 6×10 10 , about 3×10 10 ~about 6×10 10 , about 4×10 10 ~about 6×10 10 , or about 5 × 10 10 ~about 6×10 10 ) hepatocyte population. For example, in some embodiments, the engineered tissue construct comprises about 3×10 7 ~about 6×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 7 ~about 6×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 7 ~about 6×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 7 ~about 6×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 7 ~about 6×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 8×107 ~about 6×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 7 ~about 6×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 8 ~about 6×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 8 ~about 6×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 8 ~about 6×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 8 ~about 6×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 8 ~about 6×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 8 ~about 6×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 8 ~about 6×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 8×10 8 ~about 6×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 8 ~about 6×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 9 ~about 6×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 9 ~about 6×10 10In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 9 ~about 6×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 9 ~about 6×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 9 ~about 6×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 9 ~about 6×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 9 ~about 6×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 8×10 9 ~about 6×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 9 ~about 6×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 10 ~about 6×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 10 ~about 6×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 10 ~about 6×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 10 ~about 6×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 10 ~about 6×10 10 The amount of hepatocytes includes the hepatocyte population.

[0075] In some embodiments, the engineered tissue construct comprises about 2×10 7 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 7 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 7 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 7 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 7 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 7 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 8×10 7 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 7 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 8×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 8In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 8×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 10 The amount of hepatocytes includes the hepatocyte population.

[0076] IIIaiii. Children (e.g., 5 years old) In some embodiments, the engineered tissue construct is implanted into a child and is approximately 3.5×10 7 ~Approx. 1.05×10 11 (For example, about 4 × 10 7 ~Approx. 1.05×10 11 , about 5×10 7 ~Approx. 1.05×10 11 , about 6×10 7 ~Approx. 1.05×10 11 , about 7×10 7 ~Approx. 1.05×10 11 , about 8×10 7 ~Approx. 1.05×10 11 , about 9×10 7 ~Approx. 1.05×10 11 , about 1×10 8 ~Approx. 1.05×10 11 , about 2×10 8 ~Approx. 1.05×10 11 , about 3×10 8 ~Approx. 1.05×10 11 , about 4×10 8 ~Approx. 1.05×10 11 , about 5×10 8 ~Approx. 1.05×10 11 , about 6×10 8 ~Approx. 1.05×10 11 , about 7×10 8 ~Approx. 1.05×10 11 , about 8×10 8 ~Approx. 1.05×10 11 , about 9×10 8 ~Approx. 1.05×10 11 , about 1×10 9 ~Approx. 1.05×10 11 , about 2×10 9 ~Approx. 1.05×10 11 , about 3×10 9 ~Approx. 1.05×10 11 , about 4×10 9 ~Approx. 1.05×10 11 , about 5×10 9 ~Approx. 1.05×10 11 , about 6×10 9 ~Approx. 1.05×10 11 , about 7×10 9 ~Approx. 1.05×10 11 , about 8×10 9~Approx. 1.05×10 11 , about 9×10 9 ~Approx. 1.05×10 11 , about 1×10 10 ~Approx. 1.05×10 11 , about 2×10 10 ~Approx. 1.05×10 11 , about 3×10 10 ~Approx. 1.05×10 11 , about 4×10 10 ~Approx. 1.05×10 11 , about 5×10 10 ~Approx. 1.05×10 11 , about 6×10 10 ~Approx. 1.05×10 11 , about 7×10 10 ~Approx. 1.05×10 11 , about 8×10 10 ~Approx. 1.05×10 11 , about 9×10 10 ~Approx. 1.05×10 11 , about 1×10 11 ~Approx. 1.05×10 11 ) hepatocyte population. For example, in some embodiments, the engineered tissue construct comprises about 4×10 7 ~Approx. 1.05×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 7 ~Approx. 1.05×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 7 ~Approx. 1.05×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 7 ~Approx. 1.05×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatic cells in an amount of about 8×10 7 ~Approx. 1.05×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 7 ~Approx. 1.05×10 11In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 8 ~Approx. 1.05×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 8 ~Approx. 1.05×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 8 ~Approx. 1.05×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 8 ~Approx. 1.05×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 8 ~Approx. 1.05×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 8 ~Approx. 1.05×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 8 ~Approx. 1.05×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatic cells in an amount of about 8×10 8 ~Approx. 1.05×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 8 ~Approx. 1.05×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 9 ~Approx. 1.05×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 9 ~Approx. 1.05×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 9 ~Approx. 1.05×10 11In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 9 ~Approx. 1.05×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 9 ~Approx. 1.05×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 9 ~Approx. 1.05×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 9 ~Approx. 1.05×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatic cells in an amount of about 8×10 9 ~Approx. 1.05×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 9 ~Approx. 1.05×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 10 ~Approx. 1.05×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 10 ~Approx. 1.05×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 10 ~Approx. 1.05×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 10 ~Approx. 1.05×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 10 ~Approx. 1.05×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 10 ~Approx. 1.05×10 11In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 10 ~Approx. 1.05×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatic cells in an amount of about 8×10 10 ~Approx. 1.05×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 10 ~Approx. 1.05×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 11 ~Approx. 1.05×10 11 The amount of hepatocytes includes the hepatocyte population.

[0077] In some embodiments, the engineered tissue construct comprises about 3.5×10 7 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 7 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 7 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 7 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 7 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 8×10 7 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 7 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×108 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 8×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 8×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×1010 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 8×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1.05×10 11 The amount of hepatocytes includes the hepatocyte population.

[0078] IIIaiv. Children (e.g., 9 years old) In some embodiments, the engineered tissue construct is implanted into a child and is approximately 4.5×10 7 ~Approx. 1.35×10 11 (For example, about 5 × 10 7 ~Approx. 1.35×10 11 , about 6×10 7 ~Approx. 1.35×10 11 , about 7×10 7 ~Approx. 1.35×10 11 , about 8×10 7 ~Approx. 1.35×10 11 , about 9×10 7 ~Approx. 1.35×10 11 , about 1×10 8 ~Approx. 1.35×10 11 , about 2×10 8 ~Approx. 1.35×10 11 , about 3×10 8 ~Approx. 1.35×10 11 , about 4×10 8~Approx. 1.35×10 11 , about 5×10 8 ~Approx. 1.35×10 11 , about 6×10 8 ~Approx. 1.35×10 11 , about 7×10 8 ~Approx. 1.35×10 11 , about 8×10 8 ~Approx. 1.35×10 11 , about 9×10 8 ~Approx. 1.35×10 11 , about 1×10 9 ~Approx. 1.35×10 11 , about 2×10 9 ~Approx. 1.35×10 11 , about 3×10 9 ~Approx. 1.35×10 11 , about 4×10 9 ~Approx. 1.35×10 11 , about 5×10 9 ~Approx. 1.35×10 11 , about 6×10 9 ~Approx. 1.35×10 11 , about 7×10 9 ~Approx. 1.35×10 11 , about 8×10 9 ~Approx. 1.35×10 11 , about 9×10 9 ~Approx. 1.35×10 11 , about 1×10 10 ~Approx. 1.35×10 11 , about 2×10 10 ~Approx. 1.35×10 11 , about 3×10 10 ~Approx. 1.35×10 11 , about 4×10 10 ~Approx. 1.35×10 11 , about 5×10 10 ~Approx. 1.35×10 11 , about 6×10 10 ~Approx. 1.35×10 11 , about 7×10 10 ~Approx. 1.35×10 11 , about 8×10 10 ~Approx. 1.35×10 11 , about 9×10 10 ~Approx. 1.35×10 11 , about 1×10 11 ~Approx. 1.35×1011 ) hepatocyte population. For example, in some embodiments, the engineered tissue construct comprises about 4×10 7 ~Approx. 1.35×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 7 ~Approx. 1.35×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 7 ~Approx. 1.35×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 7 ~Approx. 1.35×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatic cells in an amount of about 8×10 7 ~Approx. 1.35×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 7 ~Approx. 1.35×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 8 ~Approx. 1.35×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 8 ~Approx. 1.35×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 8 ~Approx. 1.35×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 8 ~Approx. 1.35×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 8 ~Approx. 1.35×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 8 ~Approx. 1.35×10 11In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 8 ~Approx. 1.35×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatic cells in an amount of about 8×10 8 ~Approx. 1.35×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 8 ~Approx. 1.35×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 9 ~Approx. 1.35×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 9 ~Approx. 1.35×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 9 ~Approx. 1.35×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 9 ~Approx. 1.35×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 9 ~Approx. 1.35×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 9 ~Approx. 1.35×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 9 ~Approx. 1.35×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatic cells in an amount of about 8×10 9 ~Approx. 1.35×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 9 ~Approx. 1.35×10 11In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 10 ~Approx. 1.35×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 10 ~Approx. 1.35×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 10 ~Approx. 1.35×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 10 ~Approx. 1.35×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 10 ~Approx. 1.35×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 10 ~Approx. 1.35×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 10 ~Approx. 1.35×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatic cells in an amount of about 8×10 10 ~Approx. 1.35×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 10 ~Approx. 1.35×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 11 ~Approx. 1.35×10 11 The amount of hepatocytes includes the hepatocyte population.

[0079] In some embodiments, the engineered tissue construct comprises about 3.5×10 7 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 7In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 7 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 7 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 7 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 8×10 7 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 7 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 8×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 9In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 8×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 8×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1.35×1011 The amount of hepatocytes includes the hepatocyte population.

[0080] IIIav. Adults (e.g., 18 years or older) In some embodiments, the engineered tissue constructs are implanted into adults and have a cell count of approximately 9×10 7 ~Approx. 1.8×10 11 (For example, about 1×10 8 ~Approx. 1.8×10 11 , about 2×10 8 ~Approx. 1.8×10 11 , about 3×10 8 ~Approx. 1.8×10 11 , about 4×10 8 ~Approx. 1.8×10 11 , about 5×10 8 ~Approx. 1.8×10 11 , about 6×10 8 ~Approx. 1.8×10 11 , about 7×10 8 ~Approx. 1.8×10 11 , about 8×10 8 ~Approx. 1.8×10 11 , about 9×10 8 ~Approx. 1.8×10 11 , about 1×10 9 ~Approx. 1.8×10 11 , about 2×10 9 ~Approx. 1.8×10 11 , about 3×10 9 ~Approx. 1.8×10 11 , about 4×10 9 ~Approx. 1.8×10 11 , about 5×10 9 ~Approx. 1.8×10 11 , about 6×10 9 ~Approx. 1.8×10 11 , about 7×10 9 ~Approx. 1.8×10 11 , about 8×10 9 ~Approx. 1.8×10 11 , about 9×10 9 ~Approx. 1.8×10 11 , about 1×10 10 ~Approx. 1.8×10 11 , about 2×10 10 ~Approx. 1.8×10 11 , about 3×10 10 ~Approx. 1.8×1011 , about 4×10 10 ~Approx. 1.8×10 11 , about 5×10 10 ~Approx. 1.8×10 11 , about 6×10 10 ~Approx. 1.8×10 11 , about 7×10 10 ~Approx. 1.8×10 11 , about 8×10 10 ~Approx. 1.8×10 11 , about 9×10 10 ~Approx. 1.8×10 11 , or approximately 1 × 10 11 ~Approx. 1.8×10 11 ) hepatocyte population. For example, in some embodiments, the engineered tissue construct comprises about 4×10 7 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 7 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 7 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 7 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatic cells in an amount of about 8×10 7 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 7 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 8 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 8 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 8~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 8 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 8 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 8 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 8 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatic cells in an amount of about 8×10 8 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 8 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 9 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 9 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 9 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 9 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 9 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 9 ~Approx. 1.8×10 11In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 9 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatic cells in an amount of about 8×10 9 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 9 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 10 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 10 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 10 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 10 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 10 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 10 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 10 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatic cells in an amount of about 8×10 10 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 10 ~Approx. 1.8×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×1011 ~Approx. 1.8×10 11 The amount of hepatocytes includes the hepatocyte population.

[0081] In some embodiments, the engineered tissue construct comprises about 9×10 7 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 8×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 8 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 9In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 6×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 8×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 9 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 2×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 3×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 4×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 5×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 6×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 7×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in the amount of about 8×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 9×10 10 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1×10 11 In some embodiments, the engineered tissue construct comprises a population of hepatocytes in an amount of about 1.8×10 11 The amount of hepatocytes includes the hepatocyte population.

[0082] stromal cells The engineered tissue constructs described herein optionally include stromal cells (e.g., fibroblasts). In some embodiments, the stromal cells are fibroblasts. In some embodiments, the fibroblasts are human dermal fibroblasts (e.g., normal human dermal fibroblasts, neonatal foreskin fibroblasts, human lung fibroblasts, human ventricular cardiac fibroblasts, human atrial cardiac fibroblasts, human uterine fibroblasts, human bladder fibroblasts, human gingival fibroblasts, human pericardial fibroblasts, human gallbladder fibroblasts, human portal vein fibroblasts, human vas deferens fibroblasts). In some embodiments, the fibroblasts are human dermal fibroblasts. In some embodiments, the fibroblasts are normal human dermal fibroblasts. In some embodiments, the fibroblasts are neonatal foreskin fibroblasts. In some embodiments, the fibroblasts are human lung fibroblasts. In some embodiments, the fibroblasts are human ventricular cardiac fibroblasts. In some embodiments, the fibroblasts are human atrial cardiac fibroblasts. In some embodiments, the fibroblasts are human uterine fibroblasts. In some embodiments, the fibroblasts are human bladder fibroblasts. In some embodiments, the fibroblasts are human gingival fibroblasts. In some embodiments, the fibroblasts are human pericardial fibroblasts. In some embodiments, the fibroblasts are human gallbladder fibroblasts. In some embodiments, the fibroblasts are human portal vein fibroblasts. In some embodiments, the fibroblasts are vas deferens fibroblasts.

[0083] In some embodiments, the engineered tissue construct comprises an optional stromal cell (eg, fibroblast) population in an amount effective for treating hyperammonemia in a subject. In some embodiments, the engineered tissue construct comprises an optional stromal cell (eg, fibroblast) population in an amount effective to reduce ammonia levels in a subject.

[0084] In some embodiments, the stromal cell (e.g., fibroblast) population comprises up to 1.8×10 12 (For example, about 1 to about 1.8 × 10 12, about 10 to about 1.8 x 10 12 , about 100 to about 1.8 x 10 12 , about 1×10 3 ~Approx. 1.8×10 12 , about 2×10 3 ~Approx. 1.8×10 12 , about 3×10 3 ~Approx. 1.8×10 12 , about 4×10 3 ~Approx. 1.8×10 12 , about 5×10 3 ~Approx. 1.8×10 12 , about 6×10 3 ~Approx. 1.8×10 12 , about 7×10 3 ~Approx. 1.8×10 12 , about 8×10 3 ~Approx. 1.8×10 12 , about 9×10 3 ~Approx. 1.8×10 12 , about 1×10 4 ~Approx. 1.8×10 12 , about 2×10 4 ~Approx. 1.8×10 12 , about 3×10 4 ~Approx. 1.8×10 12 , about 4×10 4 ~Approx. 1.8×10 12 , about 5×10 4 ~Approx. 1.8×10 12 , about 6×10 4 ~Approx. 1.8×10 12 , about 7×10 4 ~Approx. 1.8×10 12 , about 8×10 4 ~Approx. 1.8×10 12 , about 9×10 4 ~Approx. 1.8×10 12 , about 1×10 5 ~Approx. 1.8×10 12 , about 2×10 5 ~Approx. 1.8×10 12 , about 3×10 5 ~Approx. 1.8×10 12 , about 4×10 5 ~Approx. 1.8×10 12 , about 5×10 5 ~Approx. 1.8×10 12 , about 6×10 5 ~Approx. 1.8×1012 , about 7×10 5 ~Approx. 1.8×10 12 , about 8×10 5 ~Approx. 1.8×10 12 , about 9×10 5 ~Approx. 1.8×10 12 , about 1×10 6 ~Approx. 1.8×10 12 , about 2×10 6 ~Approx. 1.8×10 12 , 3×10 6 ~Approx. 1.8×10 12 , 4×10 6 ~Approx. 1.8×10 12 , 5×10 6 ~Approx. 1.8×10 12 , 6×10 6 ~Approx. 1.8×10 12 , 7×10 6 ~Approx. 1.8×10 12 , 8×10 6 ~Approx. 1.8×10 12 , 9×10 6 ~Approx. 1.8×10 12 , about 1×10 7 ~Approx. 1.8×10 12 , about 2×10 7 ~Approx. 1.8×10 12 , about 3×10 7 ~Approx. 1.8×10 12 , about 4×10 7 ~Approx. 1.8×10 12 , about 5×10 7 ~Approx. 1.8×10 12 , about 6×10 7 ~Approx. 1.8×10 12 , about 7×10 7 ~Approx. 1.8×10 12 , about 8×10 7 ~Approx. 1.8×10 12 , about 9×10 7 ~Approx. 1.8×10 12 , about 1×10 8 ~Approx. 1.8×10 12 , about 2×10 8 ~Approx. 1.8×10 12 , about 3×10 8 ~Approx. 1.8×10 12 , about 4×10 8 ~Approx. 1.8×1012 , about 5×10 8 ~Approx. 1.8×10 12 , about 6×10 8 ~Approx. 1.8×10 12 , about 7×10 8 ~Approx. 1.8×10 12 , about 8×10 8 ~Approx. 1.8×10 12 , about 9×10 8 ~Approx. 1.8×10 12 , about 1×10 9 ~Approx. 1.8×10 12 , about 2×10 9 ~Approx. 1.8×10 12 , about 3×10 9 ~Approx. 1.8×10 12 , about 4×10 9 ~Approx. 1.8×10 12 , about 5×10 9 ~Approx. 1.8×10 12 , about 6×10 9 ~Approx. 1.8×10 12 , about 7×10 9 ~Approx. 1.8×10 12 , about 8×10 9 ~Approx. 1.8×10 12 , about 9×10 9 ~Approx. 1.8×10 12 , about 1×10 10 ~Approx. 1.8×10 12 , about 2×10 10 ~Approx. 1.8×10 12 , about 3×10 10 ~Approx. 1.8×10 12 , about 4×10 10 ~Approx. 1.8×10 12 , about 5×10 10 ~Approx. 1.8×10 12 , about 6×10 10 ~Approx. 1.8×10 12 , about 7×10 10 ~Approx. 1.8×10 12 , about 8×10 10 ~Approx. 1.8×10 12 , about 9×10 10 ~Approx. 1.8×10 12 , about 1×10 11 ~Approx. 1.8×10 12 , about 2×10 11 ~Approx. 1.8×1012 , about 3×10 11 ~Approx. 1.8×10 12 , about 4×10 11 ~Approx. 1.8×10 12 , about 5×10 11 ~Approx. 1.8×10 12 , about 6×10 11 ~Approx. 1.8×10 12 , about 7×10 11 ~Approx. 1.8×10 12 , about 8×10 11 ~Approx. 1.8×10 12 , about 9×10 11 ~Approx. 1.8×10 12 , or approximately 1 × 10 12 ~Approx. 1.8×10 12 ) stromal cells (e.g., fibroblasts). For example, in some embodiments, the engineered tissue construct comprises about 1 to about 1.8 x 10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 10 to about 1.8 x 10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 100 to about 1.8 x 10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 1×10 3 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 2×10 3 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a population of stromal cells (e.g., fibroblasts) in an amount of about 3×10 3 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 4×10 3 ~Approx. 1.8×10 12In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 5×10 3 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 6×10 3 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 7×10 3 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 8×10 3 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 9×10 3 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 1×10 4 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 2×10 4 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 3×10 4 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 4×10 4 ~Approx. 1.8×10 12In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 5×10 4 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 6×10 4 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 7×10 4 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 8×10 4 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 9×10 4 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 1×10 5 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 2×10 5 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 3×10 5 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 4×10 5 ~Approx. 1.8×10 12In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 5×10 5 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 6×10 5 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 7×10 5 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 8×10 5 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 9×1 0 5 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 1×10 6 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 2×10 6 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 3×10 6 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 4×10 6 ~Approx. 1.8×10 12In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 5×10 6 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 6×10 6 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 7×10 6 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 8×10 6 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 9×10 6 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 1×10 7 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 2×10 7 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 3×10 7 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 4×10 7 ~Approx. 1.8×10 12In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 5×10 7 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 6×10 7 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 7×10 7 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 8×10 7 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 9×10 7 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 1×10 8 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 2×10 8 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 3×10 8 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 4×10 8 ~Approx. 1.8×10 12In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 5×10 8 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 6×10 8 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 7×10 8 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 8×10 8 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 9×10 8 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 1×10 9 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 2×10 9 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 3×10 9 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 4×10 9 ~Approx. 1.8×10 12In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 5×10 9 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 6×10 9 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 7×10 9 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 8×10 9 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 9×10 9 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 1×10 10 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 2×10 10 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 3×10 10 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 4×10 10 ~Approx. 1.8×10 12In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 5×10 10 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 6×10 10 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 7×10 10 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 8×10 10 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 9×10 10 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 1×10 11 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 2×10 11 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 3×10 11 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 4×10 11 ~Approx. 1.8×10 12In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 5×10 11 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 6×10 11 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 7×10 11 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 8×10 11 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 9×10 11 ~Approx. 1.8×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 1×10 12 ~Approx. 1.8×10 12 The amount of interstitial cells (e.g., fibroblasts) Contains a stromal cell (e.g., fibroblast) population.

[0085] In some embodiments, the engineered tissue construct comprises 0 stromal cells (e.g., fibroblasts). In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 1 stromal cell (e.g., fibroblast). In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 10 stromal cells (e.g., fibroblasts). In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 100 stromal cells (e.g., fibroblasts). In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 1 x 103 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 2×10 3 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 3×10 3 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 4×10 3 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 5×10 3 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 6×10 3 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 7×10 3 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 8×10 3 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 9×10 3 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 1×10 4 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 2×10 4 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 3×10 4 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 4×104 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 5×10 4 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 6×10 4 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 7×10 4 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 8×10 4 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 9×10 4 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 1×10 5 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 2×10 5 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 3×10 5 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 4×10 5 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 5×10 5 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 6×10 5 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 7×105 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 8×10 5 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 9×10 5 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 1×10 6 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 2×10 6 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 3×10 6 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 4×10 6 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 5×10 6 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 6×10 6 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 7×10 6 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 8×10 6 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 9×10 6 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 1×107 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 2×10 7 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 3×10 7 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 4×10 7 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 5×10 7 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 6×10 7 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 7×10 7 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 8×10 7 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 9×10 7 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 1×10 8 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 2×10 8 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 3×10 8 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 4×108 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 5×10 8 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 6×10 8 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 7×10 8 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 8×10 8 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 9×10 8 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 1×10 9 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 2×10 9 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 3×10 9 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 4×10 9 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 5×10 9 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 6×10 9 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 7×109 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 8×10 9 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 9×10 9 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 1×10 10 Stromal cells (e.g., fibroblasts In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 2×10 10 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 3×10 10 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 4×10 10 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 5×10 10 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 6×10 10 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 7×10 10 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 8×10 10 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 9×10 10In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 1×10 11 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 2×10 11 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 3×10 11 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 4×10 11 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 5×10 11 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 6×10 11 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 7×10 11 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 8×10 11 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 9×10 11 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 1×10 12 In some embodiments, the engineered tissue construct comprises a stromal cell (e.g., fibroblast) population in an amount of about 1.8×10 12 The amount of stromal cells (e.g., fibroblasts) comprises a stromal cell (e.g., fibroblast) population.

[0086] Combination of hepatocytes and stromal cells The cell compositions disclosed herein can be provided as a suspension comprising hepatocytes and optionally stromal cells (eg, fibroblasts).

[0087] In some embodiments, the ratio of hepatocytes:stromal cells (e.g., fibroblasts) is 1:10 to 4:1 (e.g., 1:10 to 4:1, 1:10 to 3:1, 1:10 to 2:1, 1:10 to 1:1, 1:9 to 4:1, 1:9 to 3:1, 1:9 to 2:1, 1:9 to 1:1, 1:8 to 4:1, 1:8 to 3:1, 1:8 to 2:1, 1:8 to 1:1, 1:7 to 4:1, 1:7 to 3:1, 1:7 to 2:1, 1:7 to 1:1, 1:6 to 4:1, The ratios are 1:6-3:1, 1:6-2:1, 1:6-1:1, 1:5-4:1, 1:5-3:1, 1:5-2:1, 1:5-1:1, 1:4-4:1, 1:4-3:1, 1:4-2:1, 1:4-1:1, 1:3-4:1, 1:3-3:1, 1:3-2:1, 1:3-1:1, 1:2-4:1, 1:2-3:1, 1:2-2:1, 1:2-1:1, 1:1-4:1, 1:1-3:1, 1:1-2:1, and 1:0-1:1).

[0088] For example, in some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:9 to 4:1. In some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:8 to 4:1. In some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:7 to 4:1. In some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:6 to 4:1. In some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:5 to 4:1. In some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:4 to 4:1. In some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:3 to 4:1. In some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:2 to 4:1. In some embodiments, the ratio of hepatocytes:stromal cells (e.g., fibroblasts) is 1:1 to 4:1. In some embodiments, the ratio of hepatocytes:stromal cells (e.g., fibroblasts) is 1:0 to 4:1.

[0089] In some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:10 to 3:1. In some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:9 to 3:1. In some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:8 to 3:1. In some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:7 to 3:1. In some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:6 to 3:1. In some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:5 to 3:1. In some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:4 to 3:1. In some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:3 to 3:1. In some embodiments, the ratio of hepatocytes:stromal cells (e.g., fibroblasts) is 1:2 to 3:1. In some embodiments, the ratio of hepatocytes:stromal cells (e.g., fibroblasts) is 1:1 to 3:1. In some embodiments, the ratio of hepatocytes:stromal cells (e.g., fibroblasts) is 1:0 to 3:1.

[0090] In some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:10 to 2:1. In some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:9 to 2:1. In some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:8 to 2:1. In some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:7 to 2:1. In some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:6 to 2:1. In some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:5 to 2:1. In some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:4 to 2:1. In some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:3 to 2:1. In some embodiments, the ratio of hepatocytes:stromal cells (e.g., fibroblasts) is 1:2 to 2:1. In some embodiments, the ratio of hepatocytes:stromal cells (e.g., fibroblasts) is 1:1 to 2:1. In some embodiments, the ratio of hepatocytes:stromal cells (e.g., fibroblasts) is 1:0 to 2:1.

[0091] In some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:10 to 1:1. In some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:9 to 1:1. In some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:8 to 1:1. In some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:7 to 1:1. In some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:6 to 1:1. In some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:5 to 1:1. In some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:4 to 1:1. In some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:3 to 1:1. In some embodiments, the ratio of hepatocytes:stromal cells (e.g., fibroblasts) is 1:2 to 1:1. In some embodiments, the ratio of hepatocytes:stromal cells (e.g., fibroblasts) is 1:1 to 1:1. In some embodiments, the ratio of hepatocytes:stromal cells (e.g., fibroblasts) is 1:0 to 1:1.

[0092] In some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:10 to 1:0. In some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:9 to 1:0. In some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:8 to 1:0. In some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:7 to 1:0. In some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:6 to 1:0. In some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:5 to 1:0. In some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:4 to 1:0. In some embodiments, the ratio of hepatocytes:interstitial cells (e.g., fibroblasts) is 1:3 to 1:0. In some embodiments, the ratio of hepatocytes:stromal cells (eg, fibroblasts) is 1:2 to 1:0. In some embodiments, the ratio of hepatocytes:stromal cells (eg, fibroblasts) is 1:1 to 1:0.

[0093] Biocompatible hydrogel scaffolds The engineered tissue construct may include a biocompatible scaffold or matrix. The biocompatible scaffold may be liquid, gel, semi-solid, or solid at room temperature (e.g., 25°C). The biocompatible scaffold may be biodegradable or non-biodegradable. In some embodiments, the scaffold is bioabsorbable or bioreplaceable. Exemplary biocompatible scaffolds include polymers and hydrogels, such as collagen, fibrin, chitosan, MATRIGEL™, dextran (including chemically or photocrosslinkable dextran), engineered tissue matrices (e.g., submucosa), PEG hydrogels (e.g., heparin-conjugated PEG hydrogels), poly(lactic-co-glycolic acid) (PLGA), hydroxyethyl methacrylate (HEMA), gelatin, alginate, agarose, polysaccharides, hyaluronic acid (HA), peptide-based self-assembling gels, and thermoresponsive poly(NIPAAm). Many biopolymers are known to those of skill in the art (Bryant and Anseth, J. Biomed. Mater. Res. (2002) 59(1):63-72; Mann et al., Biomaterials (2001) 22(22):3045-3051; Mann et al., Biomaterials (2001) 22(5):439-444; and Peppas et al., Eur. J. Pharm. Biopharm. (2000) 50(1), 27-46; all of which are incorporated by reference). In other embodiments, the biocompatible scaffold can include a biopolymer having any of a number of growth factors, adhesion molecules, degradation sites, or bioactive agents to enhance cell viability or for any of a number of other reasons. Such molecules are well known to those of skill in the art.

[0094] In some embodiments, the PEG hydrogels can be chemically crosslinkable and / or modified with bifunctional groups. In certain embodiments, the biocompatible scaffold comprises an allogeneic component, an autologous component, or both an allogeneic and an autologous component. In certain embodiments, the biocompatible scaffold comprises a synthetic or semi-synthetic material. In certain embodiments, the biocompatible scaffold comprises a framework or support (e.g., a fibrin-derived scaffold).

[0095] Biocompatible hydrogel scaffolds suitable for use include any polymer capable of gelling in situ, e.g., those that do not require chemicals or conditions (e.g., temperature or pH) that are not cytocompatible. This includes both stable and biodegradable biopolymers.

[0096] The polymers used herein are preferably crosslinked (e.g., ionically crosslinked). In certain embodiments, the methods and constructs described herein use polymers that can be photochemically accelerated (i.e., photocrosslinked) by exposure to light of an appropriate wavelength (i.e., photopolymerizable), or that can be weakened or soluble upon exposure to light or other stimuli. Some of the polymers listed above (e.g., collagen, HA) are not inherently photosensitive, but can be made photosensitive by adding acrylate or other photosensitive groups.

[0097] In certain embodiments, the method utilizes a photoinitiator. A photoinitiator is a molecule capable of promoting the polymerization of a hydrogel when irradiated with light of an appropriate wavelength defined by the reactive groups on the molecule. In the context of the present disclosure, the photoinitiator is cytocompatible. There are several photoinitiators known that can be used with different wavelengths of light. For example, 2,2-dimethoxy-2-phenyl-acetophenone, HPK 1-hydroxycyclohexyl-phenyl ketone, and Irgacure 2959 (hydroxyl-1-[4-(hydroxyethoxy)phenyl]-2-methyl-1-propanone) are all activated by UV light (365 nm). Other crosslinkers that are activated by wavelengths of light that are cytocompatible (e.g., blue light) can also be used in the methods described herein.

[0098] In other embodiments, the method includes the use of a polymer having a non-photochemically polymerizable moiety. In certain embodiments, the non-photochemically polymerizable moiety is a Michael acceptor. Non-limiting examples of such Michael acceptor moieties include α,β-unsaturated ketones, esters, amides, sulfones, sulfoxides, and phosphonates. Further non-limiting examples of Michael acceptors include quinine and vinylpyridine. In some embodiments, the polymerization of the Michael acceptor is promoted by a nucleophile. Suitable nucleophiles include, but are not limited to, thiols, amines, alcohols, and molecules having thiol, amine, and alcohol moieties. In certain embodiments, the present disclosure features the use of thermally crosslinked polymers.

[0099] In some embodiments, the biocompatible scaffold comprises a synthetic heparin mimetic. In particular, the synthetic polymers of the present disclosure may, in some embodiments, comprise an amount of negative charge similar to the amount of negative charge present in heparin. Thus, the synthetic polymers of the present disclosure may mimic the functional properties of heparin. For example, the synthetic polymers of the present disclosure may have the potential to bind various bioactive agents (e.g., growth factors) that naturally bind to heparin. Thus, the synthetic polymers of the present disclosure, and hydrogels comprising the synthetic polymers described herein, may bind various bioactive agents (e.g., growth factors) to prevent the diffusion of the bioactive agents and maintain a high concentration of the bioactive agents locally, which may then act on cells and promote various cellular functions.

[0100] Methods for generating engineered tissue constructs The manufacturing involves two cell types: hepatocytes (e.g., primary human hepatocytes (PHH)) and optionally stromal cells (e.g., fibroblasts, e.g., human skin fibroblasts (e.g., normal human skin fibroblasts, neonatal foreskin fibroblasts, human lung fibroblasts, human ventricular cardiac fibroblasts, human atrial cardiac fibroblasts, human uterine fibroblasts, human urinary bladder fibroblasts, human gingival fibroblasts, human pericardial fibroblasts, human gallbladder fibroblasts, human portal vein fibroblasts), human vas deferens fibroblasts). A frozen master cell bank (MCB) was procured from an external source and received as cryopreserved cells. All cell types are terminally differentiated cells isolated from primary donors obtained with appropriate donor consent for therapeutic use. For example, hepatocytes (e.g., PHH) are obtained from cadaveric donors and the MCB is created via collagenase perfusion, Percoll density gradient purification, and subsequent cryopreservation. The hepatocytes are cryopreserved until the start of the manufacturing build. Prior to accepting a lot as a released MCB, release testing is performed on hepatocyte (e.g., PHH) candidate MCBs to ensure that their performance characteristics meet the characterization, release, and stability acceptance criteria. Stromal cells (e.g., fibroblasts, e.g., human skin fibroblasts (e.g., normal human skin fibroblasts, neonatal foreskin fibroblasts, human lung fibroblasts, human ventricular heart fibroblasts, human atrial heart fibroblasts, human uterine fibroblasts, human urinary bladder fibroblasts, human gingival fibroblasts, human pericardial fibroblasts, human gallbladder fibroblasts, human portal vein fibroblasts), human vas deferens fibroblasts) are isolated from a single donor, e.g., neonatal foreskin, by physically separating the dermal and epidermal layers and sequentially digesting with dispase and collagenase. After isolation, stromal cells (e.g., fibroblasts, e.g., human skin fibroblasts (e.g., normal human skin fibroblasts, neonatal foreskin fibroblasts, human lung fibroblasts, human ventricular cardiac fibroblasts, human atrial cardiac fibroblasts, human uterine fibroblasts, human urinary bladder fibroblasts, human gingival fibroblasts, human pericardial fibroblasts, human gallbladder fibroblasts, human portal vein fibroblasts, human vas deferens fibroblasts)) are minimally expanded and cryopreserved to create frozen MCBs.The frozen MCB is shipped to the manufacturing site and stromal cells (e.g., fibroblasts, e.g., human skin fibroblasts (e.g., normal human skin fibroblasts, neonatal foreskin fibroblasts, human lung fibroblasts, human ventricular cardiac fibroblasts, human atrial cardiac fibroblasts, human uterine fibroblasts, human urinary bladder fibroblasts, human gingival fibroblasts, human pericardial fibroblasts, human gallbladder fibroblasts, human portal vein fibroblasts, human vas deferens fibroblasts)) are expanded to create working cell banks (WCBs), which are then cryopreserved until the start of the manufacturing build. These WCBs are released based on specific acceptance criteria before being used in the manufacturing process.

[0101] An overview of the continuous manufacturing process of the build is shown in Figure 6. Once the manufacturing build is initiated, stromal cells (e.g., fibroblasts, e.g., human skin fibroblasts (e.g., normal human skin fibroblasts, neonatal foreskin fibroblasts, human lung fibroblasts, human ventricular cardiac fibroblasts, human atrial cardiac fibroblasts, human uterine fibroblasts, human urinary bladder fibroblasts, human gingival fibroblasts, human pericardial fibroblasts, human gallbladder fibroblasts, human portal vein fibroblasts, human vas deferens fibroblasts)) are thawed from their respective WCBs (Figure 6; step 1), expanded, and tested to measure viability and cell number. Hepatocytes (e.g., PHH) are thawed from the hepatocyte MCB and tested to measure viability and cell count (Figure 6; step 2), then optionally combined with stromal cells (e.g., fibroblasts, e.g., human skin fibroblasts (e.g., normal human skin fibroblasts, neonatal foreskin fibroblasts, human lung fibroblasts, human ventricular cardiac fibroblasts, human atrial cardiac fibroblasts, human uterine fibroblasts, human urinary bladder fibroblasts, human gingival fibroblasts, human pericardial fibroblasts, human gallbladder fibroblasts, human portal vein fibroblasts, human vas deferens fibroblasts)) at a ratio (e.g., 1:2), centrifuged, placed into an array of microwells (e.g., pyramidal microwells), and incubated for 2-3 days to promote the cells to self-assemble into multicellular liver aggregates. Liver aggregates are deemed amenable to encapsulation after microscopic confirmation of compaction.

[0102] The hepatocytes and optional interstitial aggregates are then encapsulated in a solution (e.g., fibrinogen solution) that is polymerized (e.g., with thrombin; FIG. 6; step 6). These encapsulation steps are performed within a mold (e.g., a cylindrical mold) that controls the overall dimensions of the engineered tissue construct to be approximately 2 mm in thickness and 6 mm to 100 cm in outer diameter (e.g., 7 mm to 999 mm, 8 mm to 998 mm, 9 mm to 997 mm, 10 mm to 996 mm, 20 mm to 995 mm, 30 mm to 990 mm, 40 mm to 980 mm, 60 mm to 960 mm, 90 mm to 930 mm, 100 mm to 900 mm, 200 mm to 800 mm, 300 mm to 700 mm, 400 mm to 600 mm, or 500 mm). The thickness is controlled by the volume of the cell-hydrogel suspension, targeting a thickness of approximately 2 mm.

[0103] The engineered tissue constructs of the present disclosure can be formed by the processes described herein. In some embodiments, engineered tissue constructs with defined cellular configurations in a biocompatible hydrogel scaffold can be prepared by photopatterning a PEG hydrogel containing a population of hepatic cells and fibroblast cells, resulting in a hydrogel network of 3D cellular hepatic cells and stromal cells (e.g., fibroblasts). Further control of cell orientation within these patterned domains can be achieved by utilizing dielectrophoretic patterning techniques. Dielectrophoresis (DEP) can be used alone or in conjunction with photopolymerization methods to pattern cells in relatively homogenous slabs of hydrogel.

[0104] In some embodiments, organization of cells and materials into spatial arrangements (e.g., engineered tissue constructs) can be achieved by physically constraining the cell / material arrangement by using wells or grooves, or by injecting cells into microfluidic channels or oriented cavities / pores. In certain embodiments, organization of cells can be achieved by physically positioning the cells with electric fields, magnetic tweezers, optical tweezers, ultrasound, pressure waves, or micromanipulators.

[0105] In certain embodiments, a method for fabricating engineered tissue constructs and embedding the constructs in an extracellular matrix includes (1) creating a 3D mold defined by channels or grooves; (2) suspending a cell population in liquid collagen and centrifuging the cells into the channels of the mold; (3) removing excess cell / collagen suspension to form aggregates comprising hepatocytes and stromal cells (e.g., fibroblasts); and (4) removing the aggregates from the mold by encapsulation in an extracellular matrix scaffold.

[0106] In some embodiments, a method for fabricating an engineered tissue construct comprises (1) suspending a population of cells in a naturally derived and / or synthetic scaffold, (2) disposing the suspended cells into channels of a 3D mold, and (3) allowing the cells to form one or more aggregates at least partially embedded in the naturally derived and / or synthetic scaffold. In some embodiments, the 3D mold can be created by molding, templating, photolithography, printing, deposition, sacrificial molding, stereolithography, or a combination thereof.

[0107] In some embodiments, engineered tissue constructs can be fabricated using custom 3D printer technology to extrude a lattice of carbohydrate glass filaments with predefined diameters, spacing, and orientation. For example, in some embodiments, soluble (clinical grade, sterile) fibrinogen and thrombin are combined and poured into the lattice. After the solution polymerizes into insoluble fibrin, the carbohydrate filaments dissolve, leaving channels within the fibrin. The channels can then be filled with a suspension of cells in a naturally derived or synthetic scaffold (e.g., soluble type I collagen), which subsequently polymerizes to entrap the cells within the channels.

[0108] This method allows the formation of three-dimensional scaffolds with dimensions ranging from hundreds of micrometers to tens of centimeters in length and width, and tens of micrometers to hundreds of micrometers in height. Resolutions of up to 100 micrometers are achievable with photopolymerization, and single-cell resolution (10 μm) possible with DEP. Photopolymerization devices, DEP devices, and other methods for generating three-dimensional co-cultures are described in U.S. Patent No. 8,906,684, which is incorporated herein by reference.

[0109] The cells can be cultured in vitro under various culture conditions. The cells can be expanded in culture, e.g., grown under conditions that promote their proliferation. The culture medium can be liquid or semi-solid, e.g., including agar, methylcellulose, etc. The cell population can be suspended in a suitable nutrient medium, such as Iscove's modified DMEM or RPMI 1640, which is typically supplemented with fetal bovine serum (about 5-10%), L-glutamine, thiols, particularly 2-mercaptoethanol, and antibiotics (e.g., penicillin and streptomycin). The culture can include growth factors to which the T regulatory cells respond. A growth factor, as defined herein, can be a molecule capable of promoting the survival, growth, and / or differentiation of cells in either culture or intact tissue through specific action on a transmembrane receptor. Growth factors include polypeptide and non-polypeptide factors.

[0110] The cells produced by the method described herein can be used immediately. Alternatively, cells can be frozen at liquid nitrogen temperature and stored for long periods, and can be thawed and reused. For example, cells can be frozen in 10% dimethylsulfoxide (DMSO), 50% serum, 40% buffered medium, or any other solution commonly used in the art to store cells at such freezing temperatures, and thawed by the method commonly known in the art for thawing frozen cultured cells.

[0111] Implantation of engineered tissue constructs The engineered cell composition described herein can be transplanted into a subject.Non-limiting examples of non-human subjects include non-human primates, dogs, cats, mice, rats, guinea pigs, rabbits, poultry, pigs, horses, cows, goats, or sheep.In certain embodiments, the subject can be any animal.In certain embodiments, the subject can be any mammal.In certain embodiments, the subject can be a human.

[0112] In some embodiments, the engineered tissue construct is implanted into a subject at an implantation site selected from the group consisting of the peritoneum (e.g., retroperitoneum), the peritoneal cavity (e.g., omentum or mesentery), the rectus abdominis muscle, the oblique abdominal muscle, the quadriceps muscle, extraperitoneal fat, and the renal capsule, an extraperitoneal site, a site on the surface of the liver, or an extrapleural site, or a site suitable for vascularization. For example, in some embodiments, the peritoneum is the retroperitoneum. In some embodiments, the peritoneal cavity is the omentum. In some embodiments, the peritoneal cavity is the mesentery. In some embodiments, the omentum is the greater omentum or omental pouch. In some embodiments, the mesentery is the small intestine. In some embodiments, the engineered tissue construct is implanted into a subject as a pedicled omental wrap or an omental wrap.

[0113] The engineered tissue constructs can be implanted in any suitable manner, often using a pharma- ceutically acceptable carrier. In some embodiments, the engineered tissue constructs are implanted at a tissue or organ site. In some embodiments, the engineered tissue constructs are implanted at an orthotopic site. In other embodiments, the engineered tissue constructs are implanted at an ectopic site.

[0114] In some embodiments, the engineered tissue construct is implanted into any site suitable for vascularization. In some embodiments, a site suitable for vascularization has a minimum of about 3.6 vessels / mm 2 ~about 4500 blood vessels / mm 2 (e.g., 3.7 vessels / mm 2 ~about 4000 vessels / mm 2 , 3.8 vessels / mm2~about 3500 vessels / mm 2, 3.9 vessels / mm 2 ~about 3000 blood vessels / mm 2 , 4 vessels / mm 2 ~about 2500 blood vessels / mm 2 , 5 vessels / mm 2 ~about 2000 blood vessels / mm 2 , 10 vessels / mm 2 ~about 1000 blood vessels / mm 2 , or approximately 100 vessels / mm 2 ) microvessel density.

[0115] In some embodiments, a site suitable for vascularization has approximately 3.7 vessels / mm 2 In some embodiments, a site suitable for angiogenesis may have a pre-existing microvessel density of approximately 3.8 vessels / mm 2 In some embodiments, a site suitable for angiogenesis may have a pre-existing microvessel density of approximately 3.9 vessels / mm 2 In some embodiments, a site suitable for angiogenesis may have a pre-existing microvessel density of about 4 vessels / mm 2 In some embodiments, a site suitable for angiogenesis may have a pre-existing microvessel density of about 4.1 vessels / mm 2 In some embodiments, a site suitable for angiogenesis may have a pre-existing microvessel density of approximately 4.2 vessels / mm 2 In some embodiments, a site suitable for angiogenesis may have a pre-existing microvessel density of approximately 4.3 vessels / mm 2 In some embodiments, a site suitable for angiogenesis may have a pre-existing microvessel density of approximately 4.4 vessels / mm 2 In some embodiments, a site suitable for angiogenesis may have a pre-existing microvessel density of about 4.5 vessels / mm 2 In some embodiments, a site suitable for angiogenesis may have a pre-existing microvessel density of about 5 vessels / mm 2 In some embodiments, a site suitable for angiogenesis may have a pre-existing microvessel density of about 6 vessels / mm 2In some embodiments, a site suitable for angiogenesis may have a pre-existing microvessel density of about 7 vessels / mm 2 In some embodiments, a site suitable for angiogenesis may have a pre-existing microvessel density of about 8 vessels / mm 2 In some embodiments, a site suitable for angiogenesis may have a pre-existing microvessel density of about 9 vessels / mm 2 In some embodiments, a site suitable for angiogenesis may have a pre-existing microvessel density of about 10 vessels / mm 2 In some embodiments, a site suitable for angiogenesis may have a pre-existing microvessel density of about 50 vessels / mm 2 In some embodiments, a site suitable for angiogenesis may have a pre-existing microvessel density of about 100 vessels / mm 2 In some embodiments, a site suitable for angiogenesis may have a pre-existing microvessel density of about 200 vessels / mm 2 In some embodiments, a site suitable for angiogenesis may have a pre-existing microvessel density of about 300 vessels / mm 2 In some embodiments, a site suitable for angiogenesis may have a pre-existing microvessel density of about 400 vessels / mm 2 In some embodiments, a site suitable for angiogenesis may have a pre-existing microvessel density of about 500 vessels / mm 2 In some embodiments, a site suitable for angiogenesis may have a pre-existing microvessel density of about 600 vessels / mm 2 In some embodiments, a site suitable for angiogenesis may have a pre-existing microvessel density of about 700 vessels / mm 2 In some embodiments, a site suitable for angiogenesis may have a pre-existing microvessel density of about 800 vessels / mm 2 In some embodiments, a site suitable for angiogenesis may have a pre-existing microvessel density of about 900 vessels / mm 2 In some embodiments, a site suitable for angiogenesis may have a pre-existing microvessel density of about 1000 vessels / mm2 In some embodiments, a site suitable for vascularization may have a pre-existing microvessel density of about 2000 vessels / mm 2 In some embodiments, a site suitable for vascularization may have a pre-existing microvessel density of about 3000 vessels / mm 2 In some embodiments, a site suitable for vascularization may have a pre-existing microvessel density of about 4000 vessels / mm 2 In some embodiments, a site suitable for vascularization may have a pre-existing microvessel density of approximately 4100 vessels / mm 2 In some embodiments, a site suitable for vascularization may have a pre-existing microvessel density of approximately 4200 vessels / mm 2 In some embodiments, a site suitable for angiogenesis may have a pre-existing microvessel density of approximately 4300 vessels / mm 2 In some embodiments, a site suitable for angiogenesis may have a pre-existing microvessel density of approximately 4400 vessels / mm 2 In some embodiments, a site suitable for vascularization may have a pre-existing microvessel density of about 4500 vessels / mm 2 The cells may have a greater pre-existing microvessel density. Autologous, allogeneic, or xenogeneic cells may be used. The cells may be transplanted in any physiologically acceptable medium. In one embodiment, the cells are cryopreserved in 5-20% DMSO, 5% dextrose, and autologous serum. As is well known to those skilled in the art, the dosage of the cells of the invention to be transplanted in vivo is determined with reference to various parameters including the species, age, weight, and disease state of the host. The dosage also depends on the site targeted within the subject. For example, transplantation of an engineered tissue construct into the omentum may require a different dosage than transplantation into the mesentery. The dosage is preferably selected such that the transplantation produces an effective outcome, which can be measured by molecular assays (e.g., liver function tests) or by monitoring suitable symptoms in the subject (e.g., symptoms of hyperammonemia).

[0116] In some embodiments, the method further comprises administering an immunosuppressant or immunomodulatory agent to modulate the immune response. In some embodiments, the immune response is a humoral response or an antibody-mediated response.

[0117] In some embodiments, the transplantation method prevents graft rejection or promotes graft survival. The engineered tissue constructs disclosed herein can be administered in combination with one or more additional immunosuppressive therapies, including, but not limited to, drugs that inhibit T cell activation (e.g., calcineurin inhibitors (CNIs)); systemic immunosuppressants for universal transplantation tolerance (corticosteroids, such as methylprednisolone (MEDROL® or SOLU-MEDROL®), prednisone, or prednisolone); CNIs, such as tacrolimus (PROGRAF® or Avastin®), or other anti-inflammatory drugs (e.g., cyclosporine ... STAFRAF®; cyclosporine (NEORAL®, SANDIMMUNE®, or GENGRAF®); costimulatory blockade, such as abatacept (ORENCIA®) ​​and belatacept (NULOJIX®); antimetabolites, such as mycophenolate mofetil (CELLCEPT® or MYFORTIC®); azathioprine (IMURAN®); mTORI, such as sirolimus (RAPAMUNE®); everolimus (AF INITOR®; T cell depleting monoclonal antibodies, such as muromonab-CD3 (OKT3); alemtuzumab (Campath® or LEMTRADA®); ATG (THYMOBLOBULIN® or ATGAM®); B cell depleting monoclonal antibodies, such as rituximab (RITUXAN®); proteasome inhibitors, such as bortezomib (VELCADE®); IL-2-Ra monoclonal antibodies, such as daclizumab (ZENAPAX® )); basiliximab (SIMULECT®); lymphocyte integrin blocking monoclonal antibodies, such as natalizumab (TYSABRI®); N-acetylcysteine ​​(NAC); Hepatitis B vaccine (HEPLISAV-B®); glecaprevir and pibrentasvir (MAVYRET®); sovosbuvir (VOSEVI®); obeticholic acid (OCALIVA®); elbasvir and grazoprevir (ZEPATIER®); cholic acid (CHOLBAM®);Daclatasvir (DAKLINZA®); ombitasvir, paritaprevir, and ritonavir (TECHNIVIE™); simeprevir (OLYSIO™); sovosbuvir (SOVALDI®); telaprevir (INCIVEK™); boceprevir (VICTRELIS™); tenofovir disoproxil fumarate (VIREAD®); telbivudine (TYZEKA™); entecavir (BARACLUDE™); adefovir (HEPSERA®); peginterferon alfa-2a (PEGASYS®), peginterferon alfa-2b (PEGINTRON®), or ribavirin and twinrix. Additional agents include griltazone and vitamin E.;

[0118] In some embodiments, the engineered tissue constructs disclosed herein can be administered in combination with one or more additional immunosuppressive therapies, including, but not limited to, PEGylated anti-CD28 monovalent monoclonal antibody fragments (e.g., anti-human CD28 FR104) or domain antibodies (e.g., lurizumab (BMS-931699)), T reg Examples of antibodies for expansion include IL-2Ra specific antibodies (e.g., Fc IL-2 muteins (e.g., AMG-592)), PEGylated IL-2 antibodies, humanized IgG1 anti-CD40L antagonists (e.g., AT-1501), bivalent anti-CD40L domain antibodies (e.g., letolizumab (BMS-986004)), Fc silent human IgG1 anti-CD40 antibodies (e.g., VIB4920 or iscalimab (CFZ533)), immuRifidase, or human anti-IL6 monoclonal antibodies (e.g., clazakizumab (CSL300)).

[0119] Recommended clinical parameters for monitoring after implantation of engineered tissue constructs After implantation of the engineered tissue construct, the subject exhibits a change in one or more clinical parameters. For example, in some embodiments, after implantation of the engineered tissue construct, the subject exhibits an age-adjusted normal serum ammonia level of about 80 μmol / L or less (e.g., less than about 79 μmol / L, 78 μmol / L, 77 μmol / L, 76 μmol / L, 75 μmol / L, 74 μmol / L, 73 μmol / L, 72 μmol / L, 71 μmol / L, 70 μmol / L, 69 μmol / L, 68 μmol / L, 67 μmol / L, 66 μmol / L, 65 μmol / L, 64 μmol / L, 63 μmol / L, 62 μmol / L, 61 μmol / L, 60 μmol / L, 50 μmol / L, 40 μmol / L, 30 μmol / L, 20 μmol / L, 25 μmol / L, or 10 μmol / L). Alternatively, for example, in some embodiments, after implantation of the engineered tissue construct, the subject exhibits a serum ammonia level of about 500 μmol / L or less (e.g., less than about 499 μmol / L, 488 μmol / L, 487 μmol / L, 486 μmol / L, 485 μmol / L, 480 μmol / L, 470 μmol / L, 460 μmol / L, 450 μmol / L, 400 μmol / L, 300 μmol / L, 200 μmol / L, 100 μmol / L, 50 μmol / L, 40 μmol / L, 30 μmol / L, 20 μmol / L, or 10 μmol / L).

[0120] In some embodiments, after transplantation of an engineered tissue construct, the subject exhibits an improvement in a test of gallbladder ejection fraction (e.g., a hepatobiliary iminodiacetic acid scan).As yet another example, in some embodiments, after transplantation of an engineered tissue construct, the subject exhibits a change in one or more parameters relative to baseline levels in a blood test.

[0121] Blood tests In some embodiments, after transplantation of an engineered tissue construct, the subject exhibits a change in one or more parameters in a blood test (e.g., liver function tests (LFTs), ammonia tests, or bilirubin tests). In some embodiments, after transplantation of an engineered tissue construct, the subject exhibits a change relative to baseline levels in one or more parameters in a blood test (e.g., albumin, gamma-glutamyltransferase (GGT) levels, alkaline phosphatase (ASP) levels, aspartate aminotransferase (AST) levels, alanine aminotransferase (ALT) levels, or bilirubin levels).

[0122] albumin In some embodiments, after implantation of an engineered tissue construct, the subject exhibits a change in albumin levels that can be measured by LFT, e.g., in some embodiments, after implantation of an engineered tissue construct, the subject exhibits a change in albumin levels that return to age-adjusted normal values.

[0123] For example, if the subject is a human infant (e.g., 6-12 months of age), the subject's albumin level may be about 30-55 U / L (e.g., 31-55 U / L, 32-55 U / L, 33-55 U / L, 34-55 U / L, 35-55 U / L, 36-55 U / L, 37-55 U / L, 38-55 U / L, 39-55 U / L, 40-55 U / L, 41-55 U / L, 42-55 U / L, 43-55 U / L, 44-55 U / L, 45-55 U / L, 46-55 U / L, 47-55 U / L, 48-55 U / L, 49-55 U / L, 50-55 U / L, 51-55 U / L, 52-55 U / L, 53-55 U / L, 54-55 U / L, 55-55 U / L, 56-55 U / L, 57-55 U / L, 58-55 U / L, 59-55 U / L, 60-60 U / L, 61-60 U / L, 62-60 U / L, 63-60 U / L, 64-60 U / L, 65-60 U / L, 66-60 U / L, 67-60 U / L, 68-60 U / L, 69-70 U / L, 70-70 U / L, 71-70 U / L, 72-70 U / L, 73-70 U / L, 74-70 U / L, 75-70 U / L, 76-70 U / L, A subject is determined to have an albumin level that has returned to age-adjusted normal when it is within the normal range of 43-55 U / L, 44-55 U / L, 45-55 U / L, 46-55 U / L, 47-55 U / L, 48-55 U / L, 49-55 U / L, 50-55 U / L, 51-55 U / L, 52-55 U / L, 53-55 U / L, or 54-55 U / L).

[0124] Alternatively, for example, if the subject is a human between 1-45 years of age, the subject is determined to exhibit an albumin level that has returned to age-adjusted normal when the subject's albumin level is within the normal range of about 40-50 U / L (e.g., about 41-50 U / L, 42-50 U / L, 43-50 U / L, 44-50 U / L, 45-50 U / L, 46-50 U / L, 47-50 U / L, 48-50 U / L, or 49-50 U / L).

[0125] If the subject is a human between 46 and 90 years of age, the subject is determined to exhibit an albumin level that has returned to age-adjusted normal when the subject's albumin level is within the normal range of about 35-50 U / L (e.g., about 36-50 U / L, 37-50 U / L, 38-50 U / L, 39-50 U / L, 40-50 U / L, 41-50 U / L, 42-50 U / L, 43-50 U / L, 44-50 U / L, 45-50 U / L, 46-50 U / L, 47-50 U / L, 48-50 U / L, or 49-50 U / L).

[0126] Gamma-glutamyltransferase In some embodiments, after implantation of an engineered tissue construct, the subject exhibits a change in GGT levels that can be measured by LFT, e.g., in some embodiments, after implantation of an engineered tissue construct, the subject exhibits a change in GGT levels that return to age-adjusted normal values.

[0127] For example, if the subject is a human infant (e.g., 6-12 months of age), the subject's GGT level may be between about 1-39 U / L (e.g., 2-39 U / L, 3-39 U / L, 4-39 U / L, 5-39 U / L, 6-39 U / L, 7-39 U / L, 8-39 U / L, 9-39 U / L, 10-39 U / L, 11-39 U / L, 12-39 U / L, 13-39 U / L, 14-39 U / L, 15-39 U / L, 16-39 U / L, 17-39 U / L, 18-39 U / L, 19-39 U / L, 20-39 U / L, 21-39 U / L, 22-39 U / L, 23-39 U / L, 24-39 U / L, 25-39 U / L, 26-39 U / L, 27-39 U / L, 28-39 U / L, 29-39 U / L, 30-39 U / L, 31-39 U / L, 32-39 U / L, 33-39 U / L, 34-39 U / L, 35-39 U / L, 36-39 U / L, 37-39 U / L, 38-39 U / L, 39-39 U / L, 39-39 U / L, 39-39 U / L, 40-40 U / L, 41-40 U / L, 42-40 U / L, 43-40 U / L, 44-40 U / L, 45-40 U / L, 46-40 U / L A subject is determined to exhibit a GGT level that has returned to age-adjusted normal when the GGT level is within the normal range of 39U / L, 21-39U / L, 22-39U / L, 23-39U / L, 24-39U / L, 25-39U / L, 26-39U / L, 27-39U / L, 28-39U / L, 29-39U / L, 30-39U / L, 31-39U / L, 32-39U / L, 33-39U / L, 34-39U / L, 35-39U / L, 36-39U / L, 37-39U / L, or 38-39U / L).

[0128] Alternatively, for example, if the subject is a human child between 1 and 5 years of age, the subject is determined to exhibit a GGT level that has returned to age-adjusted normal when the subject's GGT level is within the normal range of about 3-22 U / L (e.g., about 3-22 U / L, 4-22 U / L, 5-22 U / L, 6-22 U / L, 7-22 U / L, 8-22 U / L, 9-22 U / L, 10-22 U / L, 11-22 U / L, 12-22 U / L, 13-22 U / L, 14-22 U / L, 15-22 U / L, 16-22 U / L, 17-22 U / L, 18-22 U / L, 19-22 U / L, 20-22 U / L, and 21-22 U / L).

[0129] Alkaline phosphatase In some embodiments, after transplantation of an engineered tissue construct, the subject exhibits a change in ASP levels that can be measured by LFTs, e.g., in some embodiments, after transplantation of an engineered tissue construct, the subject exhibits a change in ASP levels, where the ASP levels return to age-adjusted normal values.

[0130] For example, if the subject is a human, the subject's ASP level may be about 50-300 U / L (e.g., about 51-300 U / L, about 52-300 U / L, about 53-300 U / L, about 54-300 U / L, about 55-300 U / L, about 56-300 U / L, about 57-300 U / L, about 58-300 U / L, about 59-300 U / L, about 60-300 U / L, about 65-300 U / L, about 70-300 U / L, about 75-70 U / L, about 76-70 U / L, about 77-70 U / L, about 78-70 U / L, about 79-80 U / L, about 81-80 U / L, about 82-80 U / L, about 83-80 U / L, about 84-80 U / L, about 85-80 U / L, about 86-80 U / L, about 87-80 U / L, about 88-80 U / L, about 89-90 U / L, about 90-90 U / L, about 91-90 U / L, about 92-90 U / L, about 93-90 U / L, about 94-90 U / L, about 95-90 U / L, about 96-90 U / L, about 97-90 U / L, about 98-90 U / L, about 99-90 U / L, about 100-100 U / L, about 101-102 U / L, about 103-104 U / L, about 104-105 U / L, about 105-106 U / L, about A subject is determined to exhibit an ASP level that has returned to age-adjusted normal when the ASP level is within the normal range of about 80-300 U / L, about 90-300 U / L, about 100-300 U / L, about 125-300 U / L, about 150-300 U / L, about 175-300 U / L, about 200-300 U / L, about 225-300 U / L, about 250-300 U / L, or about 275-300 U / L).

[0131] Aspartate aminotransferase In some embodiments, after implantation of an engineered tissue construct, the subject exhibits a change in AST levels, which can be measured by LFTs, e.g., in some embodiments, after implantation of an engineered tissue construct, the subject exhibits a change in AST levels, which return to age-adjusted normal values.

[0132] For example, if the subject is a human, the subject's AST level is less than 50 U / L (e.g., 51 U / L, 52 U / L, 53 U / L, 54 U / L, 55 U / L, 56 U / L, 57 U / L, 58 U / L, 59 U / L, 60 U / L, 61 U / L, 62 U / L, 63 U / L, 64 U / L, 65 U / L, 66 U / L, 67 U / L, 68 U / L, 69 U / L A subject is determined to exhibit an AST level that has returned to age-adjusted normal when the AST level is within the normal range of less than 70 U / L, 75 U / L, 80 U / L, 85 U / L, 90 U / L, 100 U / L, 110 U / L, 120 U / L, 130 U / L, 140 U / L, 150 U / L, 200 U / L, 300 U / L, 400 U / L, and 500 U / L.

[0133] Alanine aminotransferase In some embodiments, after transplantation of an engineered tissue construct, the subject exhibits a change in ALT levels that can be measured by LFT, for example, in some embodiments, after transplantation of an engineered tissue construct, the subject exhibits a change in ALT levels, where the ALT levels return to age-adjusted normal values.

[0134] For example, if the subject is a human, the subject's ALT level is less than 50 U / L (e.g., 51 U / L, 52 U / L, 53 U / L, 54 U / L, 55 U / L, 56 U / L, 57 U / L, 58 U / L, 59 U / L, 60 U / L, 61 U / L, 62 U / L, 63 U / L, 64 U / L, 65 U / L, 66 U / L, 67 U / L, 68 U / L, 69 U / L A subject is determined to exhibit an ALT level that has returned to age-adjusted normal when the ALT level is within the normal range of <0.05, 70 U / L, 75 U / L, 80 U / L, 85 U / L, 90 U / L, 100 U / L, 110 U / L, 120 U / L, 130 U / L, 140 U / L, 150 U / L, 200 U / L, 300 U / L, 400 U / L, and 500 U / L).

[0135] Bilirubin In some embodiments, after implantation of an engineered tissue construct, the subject exhibits a change in total bilirubin levels, which can be measured by bilirubin testing. For example, in some embodiments, after implantation of an engineered tissue construct, the subject exhibits a change in total bilirubin levels, with the total bilirubin levels returning to an age-adjusted normal value of less than about 1.2 mg / dL (e.g., less than about 1.2 mg / dL, 1.1 mg / dL, 1 mg / dL, 0.9 mg / dL, 0.8 mg / dL, 0.7 mg / dL, 0.6 mg / dL, 0.5 mg / dL, 0.4 mg / dL, 0.3 mg / dL, 0.2 mg / dL, or 0.1 mg / dL).

[0136] In some embodiments, after implantation of an engineered tissue construct, the subject exhibits a change in direct bilirubin levels, which can be measured by bilirubin testing. For example, in some embodiments, after implantation of an engineered tissue construct, the subject exhibits a change in direct bilirubin levels, with the direct bilirubin levels returning to an age-adjusted normal value of less than about 1.7 mg / dL (e.g., less than about 1.6 mg / dL, 1.5 mg / dL, 1.4 mg / dL, 1.3 mg / dL, 1.2 mg / dL, 1.1 mg / dL, 1 mg / dL, 0.9 mg / dL, 0.8 mg / dL, 0.7 mg / dL, 0.6 mg / dL, 0.5 mg / dL, 0.4 mg / dL, 0.3 mg / dL, 0.2 mg / dL, or 0.1 mg / dL).

[0137] In some embodiments, after implantation of an engineered tissue construct, the subject exhibits a change in bilirubin levels that can be measured by bilirubin testing. For example, in some embodiments, after implantation of an engineered tissue construct, the subject exhibits a change in bilirubin levels that return to an age-adjusted normal value of less than about 1 mg / dL (less than about 0.9 mg / dL, 0.8 mg / dL, 0.7 mg / dL, 0.6 mg / dL, 0.5 mg / dL, 0.4 mg / dL, 0.3 mg / dL, 0.2 mg / dL, or 0.1 mg / dL).

[0138] kit The compositions described herein can be provided in a kit for use in treating hyperammonemia (e.g., in subjects with urea cycle disorders, organic acidemias, congenital lactic acidosis, fatty acid oxidation disorders, dibasic amino acid deficiencies, transient hyperammonemia, Reye's syndrome, severe perinatal asphyxia, viral-associated hyperammonemia, infections, drug-induced hyperammonemia, liver disease, acute liver failure, acute exacerbation of chronic liver failure, transjugular intrahepatic portosystemic shunt-induced hyperammonemia, hepatic encephalopathy, liver cirrhosis, end-stage liver disease, parenteral hypercaloric nutrition, thyroid disease, Hashimoto's encephalopathy, blood disorders, gastric bypass surgery, gastrointestinal bleeding, malnutrition, generalized seizures, organ transplant, and / or renal failure). The kit can include one or more engineered tissue constructs described herein. The kit can include a package insert instructing a user of the kit (e.g., a physician) to perform any one of the methods described herein. The kit can optionally include a surgical or another device for administering the composition. In some embodiments, the kit can include one or more additional therapeutic agents. EXAMPLES

[0139] The following examples are presented to provide one of ordinary skill in the art with an illustration of how the compositions and methods described herein can be used and evaluated, and are intended to be purely illustrative of the invention and are not intended to limit the scope of what the inventors regard as their invention.

[0140] Example 1. In vivo evaluation of implantation of engineered tissue constructs To quantify the effectiveness of the engineered tissue constructs in reducing ammonia levels, studies were performed with three groups of mice. All groups contained eight female mice (N=8) aged 4-8 weeks. Group 1 consisted of control (B6EiC3SnF1 / J) mice. Group 2 consisted of mice carrying the X-chromosome sparse fur-abnormal skin and hair mutation (Otc spf-ash ) was used to generate hypomorphic transgenic mice carrying the Otc spf-ashThe ) mutation reduces the activity of ornithine transcarbamylase (OTC) in the liver, a key enzyme in the urea cycle. The reduction in hepatic OTC activity in this chronic hyperammonemia model mouse is usually 5-10% compared to wild-type mice. This reduction in OTC activity results in increased plasma ammonia levels. Mice in groups 1 (healthy control) and 2 (unhealthy control) did not undergo surgery. Group 3 was treated with the same (OTC) mutation as group 2. spf-ash ) transgenic mice, while Group 3 was administered the immunosuppressant composition every 2 days (Figure 1). The immunosuppressant composition was administered every 2 days for 6 days prior to Day 1 of the study. One day prior to the start of the study, blood was drawn from all three groups to obtain baseline plasma ammonia concentration measurements. On Day 0, Group 3 received 1.41 x 10 6 primary human hepatocytes and 2.82 x 10 6 Two engineered tissue constructs consisting of normal human dermal fibroblasts were implanted. The first of five NH4Cl exposures was performed on day 4. 7.5 mmol / kg NH4Cl was administered intraperitoneally to all three groups. On the day before each NH4Cl exposure, all animals had their bladders emptied, urine discarded, and an overnight fast was initiated. Blood samples (50 μl) were collected before each NH4Cl exposure, 20 min, and 40 min after each NH4Cl exposure. The other four NH4Cl exposures were performed on days 11, 18, 25, and 32. Baseline urine collections were performed on days 7, 14, 21, and 28. Mice were euthanized on day 32 (day 1). After euthanasia, the whole liver of each animal was harvested, half of the liver was fixed for histological analysis, and the other half was snap frozen in liquid nitrogen for evaluation of OTC activity.

[0141] result Mice implanted with engineered tissue constructs showed levels of human albumin (ng / mL) over time (Figure 2). Clinical observations of all animals were performed starting on day 1 prior to ammonia challenge administration, once a week thereafter, and prior to anesthesia (Figure 3).

[0142] Group 3 mice implanted with engineered tissue constructs (B6EiC3Sn a / A-Otcspf-ash / J(spf ash Group 1: healthy unoperated wild-type control (B6EiC3SnF1 / J) mice; Group 2: unhealthy unoperated transgenic B6EiC3Sna / A-Otc mice spf-ash / J(spf ash ) mice showed remarkable resilience to ammonia exposure compared to non-human mice (Figure 3). Quantitative evaluation of behavior was performed for all animals according to the behavioral scoring system shown in Table 1. Behavioral scores were assessed for 5 min starting 15 min after intraperitoneal injection of 7.5 mmol / kg NH4Cl. The scoring system was based on the appearance of ataxia (A), seizures (S), and abnormal responses to sound (R). Ataxia was quantified by gently pulling the mouse's tail and observing its gait. A score of 2 was assigned if the mouse was able to walk normally, a score of 1 was assigned if it stumbled, and a score of 0 indicated that the mouse was unable to walk. Seizures were classified as spontaneous myoclonus or tonic-clonic movements. Finally, hyperresponsiveness to sound was quantified by ringing a 100-db bell 5-6 times in succession and observing the mouse's behavior.

[0143] Based on this scoring system, normal mice would be expected to receive a score of 7 (A2S2R3). Severely affected mice would receive a score of 1 (A0S1R0). Any mice that died during exposure automatically received a score of zero. Mice that exhibited tonic-clonic seizures always died after the seizure. Scoring was performed by two different observers blinded to treatment. Interrater reliability of this scoring system was verified in preliminary tests by Spearman correlation, correlation = 0.95, p < 0.0001, n = 17 observations.

[0144] [Table 1]

[0145] Assessment of serum ammonia concentrations revealed that mice in group 3 implanted with engineered tissue constructs had significantly lower ammonia levels 20 minutes after each exposure compared to wild-type, group 1, unoperated healthy control mice and group 2, transgenic, unoperated unhealthy control mice (Figure 4), with ammonia reduction sustained up to day 35 (Figure 5).

[0146] Example 2. Treatment of hyperammonemia in human patients by implanting engineered tissue constructs containing hepatocytes and stromal cells The engineered tissue constructs are used to treat a pediatric patient (1 year old) with hyperammonemia. After administering a regimen of immunosuppressants, the patient is administered approximately 2 × 10 7 ~Approx. 6×10 10 Administer engineered tissue constructs containing hepatocytes (e.g., primary human hepatocytes) and stromal cells (e.g., fibroblasts) (e.g., human dermal fibroblasts or neonatal foreskin fibroblasts) (hepatocyte:stromal cells (e.g., fibroblasts) ratio of 1:10 to 4:1). Implant the engineered tissue constructs into the small intestinal mesentery. Evaluate the patients one month after introducing the implants. A blood sample is taken and the serum ammonia level is measured to determine a level below about 50 μmol / L (e.g., about 49 μmol / L, 48 μmol / L, 47 μmol / L, 46 μmol / L, 45 μmol / L, 44 μmol / L, 43 μmol / L, 42 μmol / L, 41 μmol / L, 40 μmol / L, 39 μmol / L, 38 μmol / L, 37 μmol / L, 36 μmol / L, 35 μmol / L, 34 μmol / L, 33 μmol / L, 32 μmol / L, 36 μmol / L, 37 μmol / L, 38 μmol / L, 37 μmol / L, 36 μmol / L, 35 μmol / L, 36 μmol / L, 37 μmol / L, 37 μmol / L, 37 μmol / L, 37 μmol / L, 38 ... 1 μmol / L, 30 μmol / L, 29 μmol / L, 28 μmol / L, 27 μmol / L, 26 μmol / L, 25 μmol / L, 24 μmol / L, 23 μmol / L, 22 μmol / L, 21 μmol / L, 20 μmol / L, 19 μmol / L, 18 μmol / L, 17 μmol / L, 16 μmol / L, 15 μmol / L, 14 μmol / L, 13 μmol / L, 12 μmol / L, 11 μmol / L, or less than 10 μmol / L). Additionally, patients show a significant improvement in gallbladder ejection fraction.

[0147] Example 3. Treatment of urea cycle disorders in human patients by implanting engineered tissue constructs containing hepatocytes and stromal cells The engineered tissue construct is used to treat pediatric patients (1 year old) with urea cycle disorders. After administering a regimen of immunosuppressants, the patient is administered an engineered tissue construct containing hepatocytes (e.g., primary human hepatocytes) and stromal cells (e.g., fibroblasts) (e.g., human dermal fibroblasts or neonatal foreskin fibroblasts) in an amount equivalent to 0.5%-30% of the mass of the subject's remnant liver (hepatocyte:stromal cell (e.g., fibroblast) ratio of 1:10-4:1). The engineered tissue construct is implanted into the omentum. The patient is evaluated one month after introduction of the implant. The patient shows a notable improvement in liver function based on improvement in blood levels of one or more of the following: gamma-glutamyltransferase, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, or albumin.

[0148] Example 4. Treatment of organic acidemias in human patients by implanting engineered tissue constructs containing hepatocytes and stromal cells The engineered tissue constructs are used to treat neonatal patients (1 day old) with organic acidemia. After administering a regimen of immunosuppressants, the patients are administered approximately 3 × 10 5 ~Approx. 3×10 10 (For example, 1×10 6 ~Approx. 1×10 10 , or 1 × 10 7 ~Approx. 1×10 9 , or approximately 1 × 10 8) Administer engineered tissue constructs containing hepatocytes (e.g., primary human hepatocytes) and stromal cells (e.g., fibroblasts) (e.g., human dermal fibroblasts or neonatal foreskin fibroblasts) (hepatocyte:stromal cells (e.g., fibroblasts) ratio of 1:10 to 4:1). Implant the engineered tissue construct into the omental pouch. Evaluate the patient one month after introducing the implant. A blood sample is taken and the serum ammonia level is measured to determine a level below about 50 μmol / L (e.g., about 49 μmol / L, 48 μmol / L, 47 μmol / L, 46 μmol / L, 45 μmol / L, 44 μmol / L, 43 μmol / L, 42 μmol / L, 41 μmol / L, 40 μmol / L, 39 μmol / L, 38 μmol / L, 37 μmol / L, 36 μmol / L, 35 μmol / L, 34 μmol / L, 33 μmol / L, 32 μmol / L, 36 μmol / L, 37 μmol / L, 38 μmol / L, 37 μmol / L, 36 μmol / L, 35 μmol / L, 36 μmol / L, 37 μmol / L, 37 μmol / L, 37 μmol / L, 37 μmol / L, 38 ... 1 μmol / L, 30 μmol / L, 29 μmol / L, 28 μmol / L, 27 μmol / L, 26 μmol / L, 25 μmol / L, 24 μmol / L, 23 μmol / L, 22 μmol / L, 21 μmol / L, 20 μmol / L, 19 μmol / L, 18 μmol / L, 17 μmol / L, 16 μmol / L, 15 μmol / L, 14 μmol / L, 13 μmol / L, 12 μmol / L, 11 μmol / L, or less than 10 μmol / L). Additionally, the patient shows a significant improvement in liver function based on improvement in blood levels of one or more of gamma-glutamyltransferase, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, or albumin.

[0149] Example 5. Treatment of congenital lactic acidosis in human patients by implanting engineered tissue constructs containing hepatocytes and stromal cells The engineered tissue constructs are used to treat a patient (age 18) with congenital lactic acidosis. After administering a regimen of immunosuppressants, the patient received approximately 9 × 10 7 ~Approx. 1.8×10 11 (For example, 1×10 8 ~Approx. 1×10 11 or 1×10 9 ~Approx. 1×10 10) Administer engineered tissue constructs containing hepatocytes (e.g., primary human hepatocytes) and stromal cells (e.g., fibroblasts) (e.g., human dermal fibroblasts or neonatal foreskin fibroblasts) (hepatocyte:stromal cells (e.g., fibroblasts) ratio of 1:10 to 4:1). Implant the engineered tissue construct into the small intestinal mesentery. Evaluate the patient one month after introducing the implant. A blood sample is taken and the serum ammonia level is measured to determine a level below about 50 μmol / L (e.g., about 49 μmol / L, 48 μmol / L, 47 μmol / L, 46 μmol / L, 45 μmol / L, 44 μmol / L, 43 μmol / L, 42 μmol / L, 41 μmol / L, 40 μmol / L, 39 μmol / L, 38 μmol / L, 37 μmol / L, 36 μmol / L, 35 μmol / L, 34 μmol / L, 33 μmol / L, 32 μmol / L, 36 μmol / L, 37 μmol / L, 38 μmol / L, 37 μmol / L, 36 μmol / L, 35 μmol / L, 36 μmol / L, 37 μmol / L, 37 μmol / L, 37 μmol / L, 37 μmol / L, 38 ... 1 μmol / L, 30 μmol / L, 29 μmol / L, 28 μmol / L, 27 μmol / L, 26 μmol / L, 25 μmol / L, 24 μmol / L, 23 μmol / L, 22 μmol / L, 21 μmol / L, 20 μmol / L, 19 μmol / L, 18 μmol / L, 17 μmol / L, 16 μmol / L, 15 μmol / L, 14 μmol / L, 13 μmol / L, 12 μmol / L, 11 μmol / L, or less than 10 μmol / L). Additionally, the patient shows a significant improvement in liver function based on improvement in blood levels of one or more of gamma-glutamyltransferase, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, or albumin.

[0150] Example 6. Treatment of fatty acid oxidation disorders in human patients by implanting engineered tissue constructs containing hepatocytes and stromal cells The engineered tissue constructs are used to treat a pediatric patient (age 9) with a fatty acid oxidation disorder. After administering a regimen of immunosuppressants, the patient is administered approximately 4.5 x 10 7 ~Approx. 1.35×10 11 (For example, 5×10 7 ~Approx. 1×10 11 , 1×10 8 ~Approx. 1×10 10 , or approximately 1 × 10 9) Administer engineered tissue constructs containing hepatocytes (e.g., primary human hepatocytes) and stromal cells (e.g., fibroblasts) (e.g., human dermal fibroblasts or neonatal foreskin fibroblasts) (hepatocyte:stromal cells (e.g., fibroblasts) ratio of 1:10 to 4:1). Implant the engineered tissue construct into the small intestinal mesentery. Evaluate the patient one month after introducing the implant. A blood sample is taken and the serum ammonia level is measured to determine a level below about 50 μmol / L (e.g., about 49 μmol / L, 48 μmol / L, 47 μmol / L, 46 μmol / L, 45 μmol / L, 44 μmol / L, 43 μmol / L, 42 μmol / L, 41 μmol / L, 40 μmol / L, 39 μmol / L, 38 μmol / L, 37 μmol / L, 36 μmol / L, 35 μmol / L, 34 μmol / L, 33 μmol / L, 32 μmol / L, 36 μmol / L, 37 μmol / L, 38 μmol / L, 37 μmol / L, 36 μmol / L, 35 μmol / L, 36 μmol / L, 37 μmol / L, 37 μmol / L, 37 μmol / L, 37 μmol / L, 38 ... 1 μmol / L, 30 μmol / L, 29 μmol / L, 28 μmol / L, 27 μmol / L, 26 μmol / L, 25 μmol / L, 24 μmol / L, 23 μmol / L, 22 μmol / L, 21 μmol / L, 20 μmol / L, 19 μmol / L, 18 μmol / L, 17 μmol / L, 16 μmol / L, 15 μmol / L, 14 μmol / L, 13 μmol / L, 12 μmol / L, 11 μmol / L, or less than 10 μmol / L). Additionally, the patient shows a significant improvement in liver function based on improvement in blood levels of one or more of gamma-glutamyltransferase, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, or albumin.

[0151] Example 7. Treatment of dibasic amino acid deficiency in human patients by transplanting engineered tissue constructs containing hepatocytes and stromal cells The engineered tissue construct is used to treat a patient (age 60) with dibasic amino acid deficiency. After administering a regimen of immunosuppressants, the patient is administered an engineered tissue construct containing hepatocytes (e.g., primary human hepatocytes) and stromal cells (e.g., fibroblasts) (e.g., human dermal fibroblasts or neonatal foreskin fibroblasts) in an amount equivalent to 0.5%-30% of the subject's total liver mass (hepatocyte:stromal cell (e.g., fibroblast) ratio of 1:10-4:1). The engineered tissue construct is implanted into the small intestinal mesentery. The patient is evaluated one month after introduction of the implant. A blood sample is taken and the serum ammonia level is measured to determine a level below about 50 μmol / L (e.g., about 49 μmol / L, 48 μmol / L, 47 μmol / L, 46 μmol / L, 45 μmol / L, 44 μmol / L, 43 μmol / L, 42 μmol / L, 41 μmol / L, 40 μmol / L, 39 μmol / L, 38 μmol / L, 37 μmol / L, 36 μmol / L, 35 μmol / L, 34 μmol / L, 33 μmol / L, 32 μmol / L, 36 μmol / L, 37 μmol / L, 38 μmol / L, 37 μmol / L, 36 μmol / L, 35 μmol / L, 36 μmol / L, 37 μmol / L, 37 μmol / L, 37 μmol / L, 37 μmol / L, 38 ... 1 μmol / L, 30 μmol / L, 29 μmol / L, 28 μmol / L, 27 μmol / L, 26 μmol / L, 25 μmol / L, 24 μmol / L, 23 μmol / L, 22 μmol / L, 21 μmol / L, 20 μmol / L, 19 μmol / L, 18 μmol / L, 17 μmol / L, 16 μmol / L, 15 μmol / L, 14 μmol / L, 13 μmol / L, 12 μmol / L, 11 μmol / L, or less than 10 μmol / L). Additionally, the patient shows a significant improvement in liver function based on improvement in blood levels of one or more of gamma-glutamyltransferase, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, or albumin.

[0152] Example 8. Treatment of transient hyperammonemia in human patients by implanting engineered tissue constructs containing hepatocytes and stromal cells The engineered tissue constructs are used to treat a patient (age 30) with transient hyperammonemia. After administering a regimen of immunosuppressants, the patient received approximately 9×10 7 ~Approx. 1.8×10 11 (For example, 1×108 ~Approx. 1×10 11 or 1×10 9 ~Approx. 1×10 10 ) Administer engineered tissue constructs containing hepatocytes (e.g., primary human hepatocytes) and stromal cells (e.g., fibroblasts) (e.g., human dermal fibroblasts or neonatal foreskin fibroblasts) (hepatocyte:stromal cells (e.g., fibroblasts) ratio of 1:10 to 4:1). Implant the engineered tissue construct into the omentum. Evaluate the patient one month after introducing the implant. A blood sample is taken and the serum ammonia level is measured to determine a level below about 50 μmol / L (e.g., about 49 μmol / L, 48 μmol / L, 47 μmol / L, 46 μmol / L, 45 μmol / L, 44 μmol / L, 43 μmol / L, 42 μmol / L, 41 μmol / L, 40 μmol / L, 39 μmol / L, 38 μmol / L, 37 μmol / L, 36 μmol / L, 35 μmol / L, 34 μmol / L, 33 μmol / L, 32 μmol / L, 36 μmol / L, 37 μmol / L, 38 μmol / L, 37 μmol / L, 36 μmol / L, 35 μmol / L, 36 μmol / L, 37 μmol / L, 37 μmol / L, 37 μmol / L, 37 μmol / L, 38 ... 1 μmol / L, 30 μmol / L, 29 μmol / L, 28 μmol / L, 27 μmol / L, 26 μmol / L, 25 μmol / L, 24 μmol / L, 23 μmol / L, 22 μmol / L, 21 μmol / L, 20 μmol / L, 19 μmol / L, 18 μmol / L, 17 μmol / L, 16 μmol / L, 15 μmol / L, 14 μmol / L, 13 μmol / L, 12 μmol / L, 11 μmol / L, or less than 10 μmol / L). Additionally, the patient shows a significant improvement in liver function based on improvement in blood levels of one or more of gamma-glutamyltransferase, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, or albumin.

[0153] Example 9. Treatment of Reye's syndrome in human patients by implanting engineered tissue constructs containing hepatocytes and stromal cells The engineered tissue construct is used to treat a patient (age 40) with Reye's syndrome. After administering a regimen of immunosuppressants, the patient is administered an engineered tissue construct containing hepatocytes (e.g., primary human hepatocytes) and stromal cells (e.g., fibroblasts) (e.g., human dermal fibroblasts or neonatal foreskin fibroblasts) in an amount equivalent to 0.5%-20% of the mass of the subject's remaining liver (hepatocyte:stromal cell (e.g., fibroblast) ratio of 1:10-4:1). The engineered tissue construct is implanted into the omentum. The patient is evaluated one month after introduction of the implant. A blood sample is taken and the serum ammonia level is measured to determine a level below about 50 μmol / L (e.g., about 49 μmol / L, 48 μmol / L, 47 μmol / L, 46 μmol / L, 45 μmol / L, 44 μmol / L, 43 μmol / L, 42 μmol / L, 41 μmol / L, 40 μmol / L, 39 μmol / L, 38 μmol / L, 37 μmol / L, 36 μmol / L, 35 μmol / L, 34 μmol / L, 33 μmol / L, 32 μmol / L, 36 μmol / L, 37 μmol / L, 38 μmol / L, 37 μmol / L, 36 μmol / L, 35 μmol / L, 36 μmol / L, 37 μmol / L, 37 μmol / L, 37 μmol / L, 37 μmol / L, 38 ... 1 μmol / L, 30 μmol / L, 29 μmol / L, 28 μmol / L, 27 μmol / L, 26 μmol / L, 25 μmol / L, 24 μmol / L, 23 μmol / L, 22 μmol / L, 21 μmol / L, 20 μmol / L, 19 μmol / L, 18 μmol / L, 17 μmol / L, 16 μmol / L, 15 μmol / L, 14 μmol / L, 13 μmol / L, 12 μmol / L, 11 μmol / L, or less than 10 μmol / L). Additionally, the patient shows a significant improvement in liver function based on improvement in blood levels of one or more of gamma-glutamyltransferase, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, or albumin.

[0154] Example 10. Treatment of severe perinatal asphyxia in human patients by transplanting engineered tissue constructs containing hepatocytes and stromal cells The engineered tissue constructs are used to treat a pediatric patient (1 year old) with perinatal asphyxia. After administering a regimen of immunosuppressants, the patient is administered approximately 2 × 10 7 ~Approx. 6×10 10 (For example, 1×10 8 ~Approx. 1×1010 , or approximately 1 × 10 9 ) Administer engineered tissue constructs containing hepatocytes (e.g., primary human hepatocytes) and stromal cells (e.g., fibroblasts) (e.g., human dermal fibroblasts or neonatal foreskin fibroblasts) (hepatocyte:stromal cells (e.g., fibroblasts) ratio of 1:10 to 4:1). Implant the engineered tissue construct into the omentum. Evaluate the patient one month after introducing the implant. A blood sample is taken and the serum ammonia level is measured to determine a level below about 50 μmol / L (e.g., about 49 μmol / L, 48 μmol / L, 47 μmol / L, 46 μmol / L, 45 μmol / L, 44 μmol / L, 43 μmol / L, 42 μmol / L, 41 μmol / L, 40 μmol / L, 39 μmol / L, 38 μmol / L, 37 μmol / L, 36 μmol / L, 35 μmol / L, 34 μmol / L, 33 μmol / L, 32 μmol / L, 36 μmol / L, 37 μmol / L, 38 μmol / L, 37 μmol / L, 36 μmol / L, 35 μmol / L, 36 μmol / L, 37 μmol / L, 37 μmol / L, 37 μmol / L, 37 μmol / L, 38 ... 1 μmol / L, 30 μmol / L, 29 μmol / L, 28 μmol / L, 27 μmol / L, 26 μmol / L, 25 μmol / L, 24 μmol / L, 23 μmol / L, 22 μmol / L, 21 μmol / L, 20 μmol / L, 19 μmol / L, 18 μmol / L, 17 μmol / L, 16 μmol / L, 15 μmol / L, 14 μmol / L, 13 μmol / L, 12 μmol / L, 11 μmol / L, or less than 10 μmol / L). Additionally, the patient shows a significant improvement in liver function based on improvement in blood levels of one or more of gamma-glutamyltransferase, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, or albumin.

[0155] Example 11. Treatment of viral-associated hyperammonemia in human patients by implanting engineered tissue constructs containing hepatocytes and stromal cells The engineered tissue constructs are used to treat a patient (age 55) with viral-associated hyperammonemia. After administering a regimen of immunosuppressants, the patient received approximately 9×10 7 ~Approx. 1.8×10 11 (For example, 1×10 8 ~Approx. 1×10 11 or 1×10 9~Approx. 1×10 10 ) Administer engineered tissue constructs containing hepatocytes (e.g., primary human hepatocytes) and stromal cells (e.g., fibroblasts) (e.g., human dermal fibroblasts or neonatal foreskin fibroblasts) (hepatocyte:stromal cells (e.g., fibroblasts) ratio of 1:10 to 4:1). Implant the engineered tissue construct into the omentum. Evaluate the patient one month after introducing the implant. A blood sample is taken and the serum ammonia level is measured to determine a level below about 50 μmol / L (e.g., about 49 μmol / L, 48 μmol / L, 47 μmol / L, 46 μmol / L, 45 μmol / L, 44 μmol / L, 43 μmol / L, 42 μmol / L, 41 μmol / L, 40 μmol / L, 39 μmol / L, 38 μmol / L, 37 μmol / L, 36 μmol / L, 35 μmol / L, 34 μmol / L, 33 μmol / L, 32 μmol / L, 36 μmol / L, 37 μmol / L, 38 μmol / L, 37 μmol / L, 36 μmol / L, 35 μmol / L, 36 μmol / L, 37 μmol / L, 37 μmol / L, 37 μmol / L, 37 μmol / L, 38 ... 1 μmol / L, 30 μmol / L, 29 μmol / L, 28 μmol / L, 27 μmol / L, 26 μmol / L, 25 μmol / L, 24 μmol / L, 23 μmol / L, 22 μmol / L, 21 μmol / L, 20 μmol / L, 19 μmol / L, 18 μmol / L, 17 μmol / L, 16 μmol / L, 15 μmol / L, 14 μmol / L, 13 μmol / L, 12 μmol / L, 11 μmol / L, or less than 10 μmol / L). Additionally, the patient shows a significant improvement in liver function based on improvement in blood levels of one or more of gamma-glutamyltransferase, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, or albumin.

[0156] Example 12. Treatment of drug-induced hyperammonemia in human patients by implanting engineered tissue constructs containing hepatocytes and stromal cells The engineered tissue constructs are used to treat a patient (age 25) with drug-induced hyperammonemia. After administering a regimen of immunosuppressants, the patient received approximately 9 × 10 7 ~Approx. 1.8×10 11 (For example, 1×10 8 ~Approx. 1×10 11 or 1×10 9 ~Approx. 1×10 10) Administer engineered tissue constructs containing hepatocytes (e.g., primary human hepatocytes) and stromal cells (e.g., fibroblasts) (e.g., human dermal fibroblasts or neonatal foreskin fibroblasts) (hepatocyte:stromal cells (e.g., fibroblasts) ratio of 1:10 to 4:1). Implant the engineered tissue construct into the small intestinal mesentery. Evaluate the patient one month after introducing the implant. A blood sample is taken and the serum ammonia level is measured to determine a level below about 50 μmol / L (e.g., about 49 μmol / L, 48 μmol / L, 47 μmol / L, 46 μmol / L, 45 μmol / L, 44 μmol / L, 43 μmol / L, 42 μmol / L, 41 μmol / L, 40 μmol / L, 39 μmol / L, 38 μmol / L, 37 μmol / L, 36 μmol / L, 35 μmol / L, 34 μmol / L, 33 μmol / L, 32 μmol / L, 36 μmol / L, 37 μmol / L, 38 μmol / L, 37 μmol / L, 36 μmol / L, 35 μmol / L, 36 μmol / L, 37 μmol / L, 37 μmol / L, 37 μmol / L, 37 μmol / L, 38 ... 1 μmol / L, 30 μmol / L, 29 μmol / L, 28 μmol / L, 27 μmol / L, 26 μmol / L, 25 μmol / L, 24 μmol / L, 23 μmol / L, 22 μmol / L, 21 μmol / L, 20 μmol / L, 19 μmol / L, 18 μmol / L, 17 μmol / L, 16 μmol / L, 15 μmol / L, 14 μmol / L, 13 μmol / L, 12 μmol / L, 11 μmol / L, or less than 10 μmol / L). Additionally, the patient shows a significant improvement in liver function based on improvement in blood levels of one or more of gamma-glutamyltransferase, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, or albumin.

[0157] Example 13. Treatment of liver disease in human patients by transplanting engineered tissue constructs containing hepatocytes and stromal cells The engineered tissue constructs are used to treat a patient (age 70) with liver disease. After administering a regimen of immunosuppressants, the patient receives approximately 9×10 7 ~Approx. 1.8×10 11 (For example, 1×10 8 ~Approx. 1×10 11 or 1×10 9 ~Approx. 1×10 10) Administer engineered tissue constructs containing hepatocytes (e.g., primary human hepatocytes) and stromal cells (e.g., fibroblasts) (e.g., human dermal fibroblasts or neonatal foreskin fibroblasts) (hepatocyte:stromal cells (e.g., fibroblasts) ratio of 1:10 to 4:1). Implant the engineered tissue construct into the omental pouch. Evaluate the patient one month after introducing the implant. A blood sample is taken and the serum ammonia level is measured to determine a level below about 50 μmol / L (e.g., about 49 μmol / L, 48 μmol / L, 47 μmol / L, 46 μmol / L, 45 μmol / L, 44 μmol / L, 43 μmol / L, 42 μmol / L, 41 μmol / L, 40 μmol / L, 39 μmol / L, 38 μmol / L, 37 μmol / L, 36 μmol / L, 35 μmol / L, 34 μmol / L, 33 μmol / L, 32 μmol / L, 36 μmol / L, 37 μmol / L, 38 μmol / L, 37 μmol / L, 36 μmol / L, 35 μmol / L, 36 μmol / L, 37 μmol / L, 37 μmol / L, 37 μmol / L, 37 μmol / L, 38 ... 1 μmol / L, 30 μmol / L, 29 μmol / L, 28 μmol / L, 27 μmol / L, 26 μmol / L, 25 μmol / L, 24 μmol / L, 23 μmol / L, 22 μmol / L, 21 μmol / L, 20 μmol / L, 19 μmol / L, 18 μmol / L, 17 μmol / L, 16 μmol / L, 15 μmol / L, 14 μmol / L, 13 μmol / L, 12 μmol / L, 11 μmol / L, or less than 10 μmol / L). Additionally, the patient shows a significant improvement in liver function based on improvement in blood levels of one or more of gamma-glutamyltransferase, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, or albumin.

[0158] Example 14. Treatment of acute liver failure in human patients by transplanting engineered tissue constructs containing hepatocytes and stromal cells The engineered tissue constructs are used to treat a patient (age 45) with acute liver failure. After administering a regimen of immunosuppressants, the patient received approximately 9×10 7 ~Approx. 1.8×10 11 (For example, 1×10 8 ~Approx. 1×10 11 or 1×10 9 ~Approx. 1×10 10) Administer engineered tissue constructs containing hepatocytes (e.g., primary human hepatocytes) and stromal cells (e.g., fibroblasts) (e.g., human dermal fibroblasts or neonatal foreskin fibroblasts) (hepatocyte:stromal cells (e.g., fibroblasts) ratio of 1:10 to 4:1). Implant the engineered tissue construct into the omental pouch. Evaluate the patient one month after introducing the implant. A blood sample is taken and the serum ammonia level is measured to determine a level below about 50 μmol / L (e.g., about 49 μmol / L, 48 μmol / L, 47 μmol / L, 46 μmol / L, 45 μmol / L, 44 μmol / L, 43 μmol / L, 42 μmol / L, 41 μmol / L, 40 μmol / L, 39 μmol / L, 38 μmol / L, 37 μmol / L, 36 μmol / L, 35 μmol / L, 34 μmol / L, 33 μmol / L, 32 μmol / L, 36 μmol / L, 37 μmol / L, 38 μmol / L, 37 μmol / L, 36 μmol / L, 35 μmol / L, 36 μmol / L, 37 μmol / L, 37 μmol / L, 37 μmol / L, 37 μmol / L, 38 ... 1 μmol / L, 30 μmol / L, 29 μmol / L, 28 μmol / L, 27 μmol / L, 26 μmol / L, 25 μmol / L, 24 μmol / L, 23 μmol / L, 22 μmol / L, 21 μmol / L, 20 μmol / L, 19 μmol / L, 18 μmol / L, 17 μmol / L, 16 μmol / L, 15 μmol / L, 14 μmol / L, 13 μmol / L, 12 μmol / L, 11 μmol / L, or less than 10 μmol / L). Additionally, the patient shows a significant improvement in liver function based on improvement in blood levels of one or more of gamma-glutamyltransferase, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, or albumin.

[0159] Example 15. Treatment of acute exacerbations of chronic liver failure in human patients by transplanting engineered tissue constructs containing hepatocytes and stromal cells The engineered tissue construct is used to treat a patient (75 years old) with acute exacerbation of chronic liver failure. After administering a regimen of immunosuppressants, the patient is administered an engineered tissue construct containing hepatocytes (e.g., primary human hepatocytes) and stromal cells (e.g., fibroblasts) (e.g., human dermal fibroblasts or neonatal foreskin fibroblasts) in an amount equivalent to 0.5%-30% of the subject's total liver mass (hepatocyte:stromal cell (e.g., fibroblast) ratio of 1:10-4:1). The engineered tissue construct is implanted into the omental pouch. The patient is evaluated one month after introduction of the implant. A blood sample is taken and the serum ammonia level is measured to determine a level below about 50 μmol / L (e.g., about 49 μmol / L, 48 μmol / L, 47 μmol / L, 46 μmol / L, 45 μmol / L, 44 μmol / L, 43 μmol / L, 42 μmol / L, 41 μmol / L, 40 μmol / L, 39 μmol / L, 38 μmol / L, 37 μmol / L, 36 μmol / L, 35 μmol / L, 34 μmol / L, 33 μmol / L, 32 μmol / L, 36 μmol / L, 37 μmol / L, 38 μmol / L, 37 μmol / L, 36 μmol / L, 35 μmol / L, 36 μmol / L, 37 μmol / L, 37 μmol / L, 37 μmol / L, 37 μmol / L, 38 ... 1 μmol / L, 30 μmol / L, 29 μmol / L, 28 μmol / L, 27 μmol / L, 26 μmol / L, 25 μmol / L, 24 μmol / L, 23 μmol / L, 22 μmol / L, 21 μmol / L, 20 μmol / L, 19 μmol / L, 18 μmol / L, 17 μmol / L, 16 μmol / L, 15 μmol / L, 14 μmol / L, 13 μmol / L, 12 μmol / L, 11 μmol / L, or less than 10 μmol / L). Additionally, the patient shows a significant improvement in liver function based on improvement in blood levels of one or more of gamma-glutamyltransferase, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, or albumin.

[0160] Example 16. Treatment of transjugular intrahepatic portosystemic shunt-induced hyperammonemia in human patients by implanting engineered tissue constructs containing hepatocytes and stromal cells The engineered tissue constructs are used to treat a patient (age 80) with transjugular intrahepatic portosystemic shunt-induced hyperammonemia. After administering a regimen of immunosuppressants, the patient received approximately 9 × 10 7~Approx. 1.8×10 11 (For example, 1×10 8 ~Approx. 1×10 11 or 1×10 9 ~Approx. 1×10 10 ) Administer engineered tissue constructs containing hepatocytes (e.g., primary human hepatocytes) and stromal cells (e.g., fibroblasts) (e.g., human dermal fibroblasts or neonatal foreskin fibroblasts) (hepatocyte:stromal cells (e.g., fibroblasts) ratio of 1:10 to 4:1). Implant the engineered tissue construct into the omentum. Evaluate the patient one month after introducing the implant. A blood sample is taken and the serum ammonia level is measured to determine a level below about 50 μmol / L (e.g., about 49 μmol / L, 48 μmol / L, 47 μmol / L, 46 μmol / L, 45 μmol / L, 44 μmol / L, 43 μmol / L, 42 μmol / L, 41 μmol / L, 40 μmol / L, 39 μmol / L, 38 μmol / L, 37 μmol / L, 36 μmol / L, 35 μmol / L, 34 μmol / L, 33 μmol / L, 32 μmol / L, 36 μmol / L, 37 μmol / L, 38 μmol / L, 37 μmol / L, 36 μmol / L, 35 μmol / L, 36 μmol / L, 37 μmol / L, 37 μmol / L, 37 μmol / L, 37 μmol / L, 38 ... 1 μmol / L, 30 μmol / L, 29 μmol / L, 28 μmol / L, 27 μmol / L, 26 μmol / L, 25 μmol / L, 24 μmol / L, 23 μmol / L, 22 μmol / L, 21 μmol / L, 20 μmol / L, 19 μmol / L, 18 μmol / L, 17 μmol / L, 16 μmol / L, 15 μmol / L, 14 μmol / L, 13 μmol / L, 12 μmol / L, 11 μmol / L, or less than 10 μmol / L). Additionally, the patient shows a significant improvement in liver function based on improvement in blood levels of one or more of gamma-glutamyltransferase, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, or albumin.

[0161] Example 17. Treatment of hepatic encephalopathy in human patients by transplanting engineered tissue constructs containing hepatocytes and stromal cells The engineered tissue constructs are used to treat a pediatric patient (age 9) with hepatic encephalopathy. After administering a regimen of immunosuppressants, the patient received approximately 4.5 x 10 7 ~Approx. 1.35×10 11 (For example, 5×107 ~Approx. 1×10 11 , 1×10 8 ~Approx. 1×10 10 , or approximately 1 × 10 9 ) Administer engineered tissue constructs containing hepatocytes (e.g., primary human hepatocytes) and stromal cells (e.g., fibroblasts) (e.g., human dermal fibroblasts or neonatal foreskin fibroblasts) (hepatocyte:stromal cells (e.g., fibroblasts) ratio of 1:10 to 4:1). Implant the engineered tissue construct into the omentum. Evaluate the patient one month after introducing the implant. A blood sample is taken and the serum ammonia level is measured to determine a level below about 50 μmol / L (e.g., about 49 μmol / L, 48 μmol / L, 47 μmol / L, 46 μmol / L, 45 μmol / L, 44 μmol / L, 43 μmol / L, 42 μmol / L, 41 μmol / L, 40 μmol / L, 39 μmol / L, 38 μmol / L, 37 μmol / L, 36 μmol / L, 35 μmol / L, 34 μmol / L, 33 μmol / L, 32 μmol / L, 36 μmol / L, 37 μmol / L, 38 μmol / L, 37 μmol / L, 36 μmol / L, 35 μmol / L, 36 μmol / L, 37 μmol / L, 37 μmol / L, 37 μmol / L, 37 μmol / L, 38 ... 1 μmol / L, 30 μmol / L, 29 μmol / L, 28 μmol / L, 27 μmol / L, 26 μmol / L, 25 μmol / L, 24 μmol / L, 23 μmol / L, 22 μmol / L, 21 μmol / L, 20 μmol / L, 19 μmol / L, 18 μmol / L, 17 μmol / L, 16 μmol / L, 15 μmol / L, 14 μmol / L, 13 μmol / L, 12 μmol / L, 11 μmol / L, or less than 10 μmol / L). Additionally, the patient shows a significant improvement in liver function based on improvement in blood levels of one or more of gamma-glutamyltransferase, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, or albumin.

[0162] Example 18. Treatment of liver cirrhosis in human patients by implanting engineered tissue constructs containing hepatocytes and stromal cells The engineered tissue constructs are used to treat a patient (70 years old) with cirrhosis of the liver. After administering a regimen of immunosuppressants, the patient receives approximately 9×10 7 ~Approx. 1.8×10 11 (For example, 1×10 8 ~Approx. 1×1011 or 1×10 9 ~Approx. 1×10 10 ) Administer engineered tissue constructs containing hepatocytes (e.g., primary human hepatocytes) and stromal cells (e.g., fibroblasts) (e.g., human dermal fibroblasts or neonatal foreskin fibroblasts) (hepatocyte:stromal cells (e.g., fibroblasts) ratio of 1:10 to 4:1). Implant the engineered tissue construct into the omentum. Evaluate the patient one month after introducing the implant. A blood sample is taken and the serum ammonia level is measured to determine a level below about 50 μmol / L (e.g., about 49 μmol / L, 48 μmol / L, 47 μmol / L, 46 μmol / L, 45 μmol / L, 44 μmol / L, 43 μmol / L, 42 μmol / L, 41 μmol / L, 40 μmol / L, 39 μmol / L, 38 μmol / L, 37 μmol / L, 36 μmol / L, 35 μmol / L, 34 μmol / L, 33 μmol / L, 32 μmol / L, 36 μmol / L, 37 μmol / L, 38 μmol / L, 37 μmol / L, 36 μmol / L, 35 μmol / L, 36 μmol / L, 37 μmol / L, 37 μmol / L, 37 μmol / L, 37 μmol / L, 38 ... 1 μmol / L, 30 μmol / L, 29 μmol / L, 28 μmol / L, 27 μmol / L, 26 μmol / L, 25 μmol / L, 24 μmol / L, 23 μmol / L, 22 μmol / L, 21 μmol / L, 20 μmol / L, 19 μmol / L, 18 μmol / L, 17 μmol / L, 16 μmol / L, 15 μmol / L, 14 μmol / L, 13 μmol / L, 12 μmol / L, 11 μmol / L, or less than 10 μmol / L). Additionally, the patient shows a significant improvement in liver function based on improvement in blood levels of one or more of gamma-glutamyltransferase, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, or albumin.

[0163] Example 19. Treatment of end-stage liver disease in human patients by transplanting engineered tissue constructs containing hepatocytes and stromal cells The engineered tissue constructs are used to treat a patient (age 40) with end-stage liver disease. After administering a regimen of immunosuppressants, the patient receives approximately 9×10 7 ~Approx. 1.8×10 11 (For example, 1×10 8 ~Approx. 1×10 11 or 1×10 9~Approx. 1×10 10 ) hepatocytes (e.g., 1 × 10 8 ~Approx. 1×10 11 or 1×10 9 ~Approx. 1×10 10 ) Administer engineered tissue constructs containing hepatocytes (e.g., primary human hepatocytes) and stromal cells (e.g., fibroblasts) (e.g., human dermal fibroblasts or neonatal foreskin fibroblasts) (hepatocyte:stromal cells (e.g., fibroblasts) ratio of 1:10 to 4:1). Implant the engineered tissue construct into the omentum. Evaluate the patient one month after introducing the implant. A blood sample is taken and the serum ammonia level is measured to determine a level below about 50 μmol / L (e.g., about 49 μmol / L, 48 μmol / L, 47 μmol / L, 46 μmol / L, 45 μmol / L, 44 μmol / L, 43 μmol / L, 42 μmol / L, 41 μmol / L, 40 μmol / L, 39 μmol / L, 38 μmol / L, 37 μmol / L, 36 μmol / L, 35 μmol / L, 34 μmol / L, 33 μmol / L, 32 μmol / L, 36 μmol / L, 37 μmol / L, 38 μmol / L, 37 μmol / L, 36 μmol / L, 35 μmol / L, 36 μmol / L, 37 μmol / L, 37 μmol / L, 37 μmol / L, 37 μmol / L, 38 ... 1 μmol / L, 30 μmol / L, 29 μmol / L, 28 μmol / L, 27 μmol / L, 26 μmol / L, 25 μmol / L, 24 μmol / L, 23 μmol / L, 22 μmol / L, 21 μmol / L, 20 μmol / L, 19 μmol / L, 18 μmol / L, 17 μmol / L, 16 μmol / L, 15 μmol / L, 14 μmol / L, 13 μmol / L, 12 μmol / L, 11 μmol / L, or less than 10 μmol / L). Additionally, the patient shows a significant improvement in liver function based on improvement in blood levels of one or more of gamma-glutamyltransferase, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, or albumin.

[0164] Other embodiments All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0165] While the invention has been described with reference to specific embodiments, it will be understood that the invention is capable of further modifications, and this application is generally intended to cover any variations, uses, or adaptations of the invention in accordance with the principles of the invention, including departures from the invention which come within known or customary practice in the art to which this invention pertains and which may be applicable to the essential characteristics as hereinbefore described, and which comply with the scope of the appended claims.

[0166] Other embodiments are within the scope of the claims.

Claims

1. 1. One or more engineered tissue constructs comprising a hepatic cell population and a stromal cell population for use in treating hyperammonemia in a subject having a urea cycle disorder, organic acidemia, congenital lactic acidosis, fatty acid oxidation disorder, dibasic amino acid deficiency, transient hyperammonemia, Reye's syndrome, severe perinatal asphyxia, viral-associated hyperammonemia, infection, drug-induced hyperammonemia, liver disease, acute liver failure, acute exacerbation of chronic liver failure, transjugular intrahepatic portosystemic shunt-induced hyperammonemia, hepatic encephalopathy, cirrhosis, end-stage liver disease, parenteral hypercaloric nutrition, thyroid disease, Hashimoto's encephalopathy, blood disorder, gastric bypass surgery, gastrointestinal bleeding, malnutrition, generalized seizures, organ transplant, or renal failure, wherein said use comprises transplanting said one or more engineered tissue constructs into said subject.

2. One or more engineered tissue constructs comprising a population of hepatic cells and a population of stromal cells for use in reducing ammonia levels in a subject in need thereof, said use comprising transplanting said one or more engineered tissue constructs into said subject.

3. 3. The one or more engineered tissue constructs of Claim 1 or 2, wherein the hepatocyte cell population comprises hepatocytes in an amount corresponding to 0.5% to 30% or 0.5% to 20% of the subject's total liver mass.

4. The hepatocyte population is 3×10 ５ ~1.8 x 10 １１ 3. The one or more engineered tissue constructs of claim 1 or 2, wherein the one or more engineered tissue constructs comprise up to 1.8 x 10<12> hepatocytes, or the stromal cell population comprises up to 1.8 x 10<12> stromal cells, or both.

5. 3. The one or more engineered tissue constructs of claim 1 or 2, wherein the hepatocytes are primary human hepatocytes, the stromal cells are fibroblasts, or both.

6. 6. The one or more engineered tissue constructs of claim 5, wherein said fibroblasts are selected from the group consisting of normal human skin fibroblasts and neonatal foreskin fibroblasts. (a) a ratio of hepatocytes to stromal cells of 1:10 to 4:1; (b) the volume of the engineered tissue construct is between 0.1 mL and 5 L; and (c) the density of hepatocytes is 0.1 M / mL to 150 M / mL; 3. The one or more engineered tissue constructs of claim 1 or 2, wherein the one or more engineered tissue constructs are at least one of:

8. 3. The one or more engineered tissue constructs of claim 1 or 2, wherein the engineered tissue construct further comprises a biocompatible hydrogel scaffold.

9. 10. The one or more engineered tissue constructs of claim 8, wherein the biocompatible scaffold comprises fibrin.

10. 3. The one or more engineered tissue constructs of Claim 1 or 2, wherein the engineered tissue construct is for implantation into the subject at an implantation site selected from the group consisting of the peritoneum, peritoneal cavity, rectus abdominis muscle, oblique abdominal muscle, quadriceps muscle, extraperitoneal fat, and kidney capsule, an extraperitoneal site, a site on the surface of the liver, or an extrapleural site, or a site suitable for vascularization. (a) the peritoneum is retroperitoneal, or (b) the peritoneal cavity is the omentum or mesentery; 11. One or more engineered tissue constructs according to claim 10.

12. 12. The one or more engineered tissue constructs of claim 11, wherein the omentum is the greater omentum or omental pouch.

13. 12. The one or more engineered tissue constructs of claim 11, wherein the mesentery is a small intestinal mesentery.

14. The engineered tissue construct has a blood vessel density of approximately 3.6 vessels / mm ２ 3. The one or more engineered tissue constructs of claim 1 or 2, for implantation into the subject at an implantation site having greater microvascular density.

15. 3. The one or more engineered tissue constructs of claim 1 or 2, wherein the subject is a human.

16. 3. The one or more engineered tissue constructs of claim 1 or 2, wherein after implantation of the engineered tissue construct, the subject exhibits a serum ammonia level of about 50 μmol / L or less.

17. 3. The one or more engineered tissue constructs of claim 1 or 2, wherein after implantation of the engineered tissue construct, the subject exhibits a change in one or more parameters relative to baseline levels in a blood test.

18. (a) the blood test is a liver function test, and / or 18. The one or more engineered tissue constructs of claim 17, wherein (b) the one or more parameters comprise levels of gamma-glutamyltransferase, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, or albumin.

19. 3. The one or more engineered tissue constructs of claim 1 or 2, wherein after implantation of the engineered tissue construct, the subject shows improvement in a test of gallbladder ejection fraction.

20. 20. The one or more engineered tissue constructs of claim 19, wherein the test is a hepatobiliary iminodiacetic acid scan.

21. (a) The infection is a urinary tract infection or an infection caused by Proteus mirabilis, E. coli, or Klebsiella; (b) the drug-induced hyperammonemia is caused by the drugs valproic acid, topiramate, carbamazepine, salicylate, sulfadiazine, carbonic anhydrase inhibitors, carbonic anhydrase inhibitors with valproic acid, or chemotherapy; (c) the liver disease is biliary atresia, alpha-1 antitrypsin deficiency, Wilson's disease, cystic fibrosis, galactosemia, or tyrosinemia, or (d) the one or more engineered tissue constructs of claim 1, wherein the hematological disorder is multiple myeloma or acute leukemia.

22. 10. A kit comprising an engineered tissue construct, the kit further comprising a package insert instructing a user of the kit to transplant the engineered tissue construct into the subject according to the use of claim 1 or 2.

Images