Table 1 Different methodologies used for recellularization of acellular hepatic tissue by using different cells
From: Current progress in hepatic tissue regeneration by tissue engineering
Model | Decellularization procedure | Evaluation of ECM | Recellularization procedure | Recellularized cell | Results | Day of cells survival on ECM | Refs |
|---|---|---|---|---|---|---|---|
Human | PBS: overnight 0.01% SDS: 4Â h 0.1%, 0.2% and 0.5% SDS: 1Â h for each 1% tritonX-100: 15Â min PBS: 5Â min | 99% of DNA removed, 60% of collagen and 40% of GAGs preserved | Decellularized slices digestion by 1Â mg/ml pepsin in 10Â mM HCl: 24Â h, RT, centrifuge, supernatant coated on plates Hepatic differentiation | hiPSC | Alb production at day 4, higher expression of stage-specific markers compared to control cells cultured on Matrigel and collagen 1 | 20Â days | [121] |
Porcine | 1% tritonX-100 perfusion: 3 h, 200 ml/min 1% SDS perfusion: 6 h, 200 ml/min 1% tritonX-100 perfusion: 3 h 20 l DW 40 l PBS Liver discs lyophilization | – | Rehydration of liver disk with IMDM: overnight, 370c Cell seeding on disk using negative pressure suction and hepatic induction: 14 day | Human umbilical cord mesenchymal stem cell | ND | More than 14 days | [217] |
Porcine | Trypsin–EDTA, 1 h, 370c 1% tritonX-100, 0.1% ammonium hydroxide, 8 h DW, overnight 0.1% PAA, 4% ethanol, 2 h | 98% dsDNA (< 50 ng DNA fragment < 200 bp), all cell nuclei removed, GAGs proteins, HGF, bFGF and EGF preserved, low chemoattraction | Tissue powder solubilization by Acetic acid and gelation by pepsin, coating on plate | HepG2, liver primary cells | The high amount of urea, alb production compared to control cells cultured on Collagen 1 | More than 7 days | [120] |
Human HepaRG cell | DE induced cells were differentiated to the hepatic lineage Cell culture plate washing with DW Incubation with DW: 45Â min, 370c Wash with DW: 1 time | DAPI and filamentous actin staining confirmed cell removal | Cell lines cultured on acellular matrix in hepatic induction was done using growth factors | ESC-WA07, ESC-H9, hiPSC | Response to differentiation was varied among cells, more than 90% of WA07 and H9 demonstrated the liver function | Around 20Â days | [123] |
Rat | DW: 5Â ml/min, 40 times of liver volume 1% TritonX-100 and 0.1% ammonium hydroxide: 50 times of liver volume DW | 98.9% of DNA removed, 85% of GAGs, 52% of proteins and 71% of collagen was conserved, 60% of ECM component conserved after 28Â days treatment with PBS solution | Acellular liver powder digestion by trypsin and HCl, the coating on 24 well plates, overnight gelation Cell seeding, hepatic induction by growth factor | Human BM-MSC | After 28Â days the treated cell exhibit hepatocyte phenotype resulted cells could uptake LDL and were AFP, HNF4, CYP, and Alb positive | More than 28Â days | [218] |
Porcine | 0.01% SDS perfusion: 150 l 0.1% SDS perfusion: 150 l 1% SDS perfusion: 50 l 1% tritonX100: 50 l DW: 100 | Mesh structures were preserved, no remaining of the nucleus and intact cells, the scaffold was rich in GAGs | The ECM powder homogenized in PBS and treated with collagenase 1 to form a gel. 1 ml of the resulting gel mixed with 1 * 106 cells and hepatic induction carried out in the absence of growth factors | Bone marrow-derived-MSCs | Glycogenesis and Alb production | 14 days | [124] |
Ferret | DW: 2 l, 6 ml/min TritonX-100, 0.1% ammonium hydroxide: 4 l Dw: 8 l | – | Acellular tissue minced in small discs with 8 mm diameter and placed in 48 well plates. 3–5 * 105 cells suspended in seeding solution and then transferred on top of discs. Hepatic induction using growth factors | Human liver progenitor cells | Expression pattern similar to hepatocytes (Alb+, CK19− and EpCAM−) and duct (Alb−, CK19+, and EpCAM+). The cells were negative for Alb and positive for GNF4α indicating the maturation of hepatocyte-like cells, detoxification activity | 3 weeks | [122] |
Porcine | Heparinized PBS: 6Â h SDS 0.1%: 72Â h PBS: 12Â h 0.1% PAA: 0.5Â h | 99.4% of DNA removed, 65% of collagen and GAGs, as well as more than 40% of growth factors, was preserved | Decellularized cubic liver powder solubilization in 3Â mg pepsin/0.1Â M HCl: 72Â h, RT, 120Â rpm, the resulting solution was concentrated tenfolds by addition of 1/10 of the total volume of 1Â M NaCl and PBS10X. Preparation of 0%, 2%, 5%, 10% and 20% of ECM in hepatic differentiation media | Porcine IPSCs | Alb protein expression and secretion into the media increased fourfold and twofold, respectively, in comparison to control groups | 20Â days | [14] |
Rat | Heparinized saline: 20 ml 0.1% SDS: 3 ml/min, 15 h PBS: 12 h 0.1% PAA: 15 min | The amount of DNA was below a detectable level, more than 60% of collagen and all of the GAGs maintained, methylene blue staining revealed no vascular tree destruction | The recellularization carried out 4 times through injection of 5 * 106 cell/0.15 ml/min with 30 min intervals. Hepatic induction without using growth factors | Hepatocyte induced porcine IPSCs | Alb and urea secretion | 5 days | [14] |
Rat | Saline: 20 ml DW: 5 ml/min, 40 times of liver volume 1% tritonX-100 and 0.1% NaOH: 50 times of liver volume DW: wash out the detergent | 98.9% of DNA was declined, 47% of HGF, 48% of bFGF as well as fibronectin and laminin preserved | The tissue minced to 8 mm disks and placed on 24 well plates. Overnight incubation by Hepatocyte media, 10 * 6 cells in 20 µl media pipetted to scaffold and after 20 min 10 of scaffolds transferred to t25 flask placed on a shaker | Human iPSC-derived hepatocyte | The cell proliferation was increased, average gene expression of CYP2C9, CYP3A4 and HMGCR 5 times increased, fetal liver markers AFP and CYP3A7 decreased during the culture period | 14 days | [118] |
Porcine | 1% SDS: 36 l, 200 ml/min, 3 h DW: 200 ml/min, 3 h 1% SDS: 3 l, 200 ml/min, 3 h DW: 200 ml/min, 3 h Repetition of previous steps 1% Triton-100: 36 l DW: 36 l, 200 ml/min PBS: 36 l, 200 ml/min | Cellular components and nuclei were removed, reticular collagen fibers were observed | The 100 µl of MSCs spheroids suspension pipetted on top of DLSs under negative pressure suction following overnight incubation in culture medium at 370c. hepatic induction was done by growth factors | Bone-marrow-derived MSCs | Efficient expression of Albp-ZsGreen, Alb, drug-metabolizing enzymes, and enzymes related to fat and amino acid metabolism as well as higher secretion of urea than 2D cultures | 23 days | [219] |
Rat | 2% sodium deoxycholate: 2Â ml/min, 4Â h DW: detergent washing 3% triton-100: 4Â h DW containing 0.02% sodium azide and 5Â mM EDTA: 72Â h 1% PAA: 1Â h PBS: 500Â ml | 99.3% of DNA is removed, the amount of collagen and elastin were more than that assessed for wet tissue, 60 and 15% respectively. 17% of GAGs, 0.1% of cytokines were preserved, the intact cells and nuclei were removed | Pretreatment of whole liver scaffolds with collagen and GAGs for 60Â min followed by overnight treatment by DMEM. DMEM was exchanged with liver fetal cell medium and the recellularization of livers with 44 and 73*106 cells was completed for 7Â days and stopped at day 11 | Human fetal liver progenitor cells: hFL4TERT and SV40 | The recellularized cells were viable in four of six livers until the end of experiments. A low expression or lack of liver function markers such as Alb, CYP450, and CKs. The cells were positive for human mitochondria and endothelial markers | 11Â days | [131] |