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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

ModelDecellularization procedureEvaluation of ECMRecellularization procedureRecellularized cellResultsDay of cells survival on ECMRefs
HumanPBS: overnight
0.01% SDS: 4 h
0.1%, 0.2% and 0.5% SDS: 1 h for each
1% tritonX-100: 15 min
PBS: 5 min
99% of DNA removed, 60% of collagen and 40% of GAGs preservedDecellularized slices digestion by 1 mg/ml pepsin in 10 mM HCl: 24 h, RT, centrifuge, supernatant coated on plates
Hepatic differentiation
hiPSCAlb production at day 4, higher expression of stage-specific markers compared to control cells cultured on Matrigel and collagen 120 days[121]
Porcine1% 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 cellNDMore than 14 days[217]
PorcineTrypsin–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 chemoattractionTissue powder solubilization by Acetic acid and gelation by pepsin, coating on plateHepG2, liver primary cellsThe high amount of urea, alb production compared to control cells cultured on Collagen 1More 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: 45 min, 370c
Wash with DW: 1 time
DAPI and filamentous actin staining confirmed cell removalCell lines cultured on acellular matrix in hepatic induction was done using growth factorsESC-WA07, ESC-H9, hiPSCResponse to differentiation was varied among cells, more than 90% of WA07 and H9 demonstrated the liver functionAround 20 days[123]
RatDW: 5 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 28 days treatment with PBS solutionAcellular liver powder digestion by trypsin and HCl, the coating on 24 well plates, overnight gelation
Cell seeding, hepatic induction by growth factor
Human BM-MSCAfter 28 days the treated cell exhibit hepatocyte phenotype resulted cells could uptake LDL and were
AFP, HNF4, CYP, and Alb positive
More than 28 days[218]
Porcine0.01% 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 GAGsThe 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 factorsBone marrow-derived-MSCsGlycogenesis and Alb production14 days[124]
FerretDW: 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 factorsHuman liver progenitor cellsExpression 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 activity3 weeks[122]
PorcineHeparinized PBS: 6 h
SDS 0.1%: 72 h
PBS: 12 h
0.1% PAA: 0.5 h
99.4% of DNA removed, 65% of collagen and GAGs, as well as more than 40% of growth factors, was preservedDecellularized cubic liver powder solubilization in 3 mg pepsin/0.1 M HCl: 72 h, RT, 120 rpm, the resulting solution was concentrated tenfolds by addition of 1/10 of the total volume of 1 M NaCl and PBS10X.
Preparation of 0%, 2%, 5%, 10% and 20% of ECM in hepatic differentiation media
Porcine IPSCsAlb protein expression and secretion into the media increased fourfold and twofold, respectively, in comparison to control groups20 days[14]
RatHeparinized 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 destructionThe recellularization carried out 4 times through injection of 5 * 106 cell/0.15 ml/min with 30 min intervals. Hepatic induction without using growth factorsHepatocyte induced porcine IPSCsAlb and urea secretion5 days[14]
RatSaline: 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 preservedThe 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 shakerHuman iPSC-derived hepatocyteThe 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 period14 days[118]
Porcine1% 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 observedThe 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 factorsBone-marrow-derived MSCsEfficient 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 cultures23 days[219]
Rat2% sodium deoxycholate: 2 ml/min, 4 h
DW: detergent washing
3% triton-100: 4 h
DW containing 0.02% sodium azide and 5 mM EDTA: 72 h
1% PAA: 1 h
PBS: 500 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 removedPretreatment of whole liver scaffolds with collagen and GAGs for 60 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 7 days and stopped at day 11Human fetal liver progenitor cells: hFL4TERT and SV40The 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 markers11 days[131]