Isolation and culture of ADSCs
All cell culture experiments involving human ADSCs were approved by the Human Research Ethics committees of the E-Da Hospital (Kaohsiung, Taiwan) (Ethic No. EMRP51104N) and carried out in accordance with the approved guidelines. Informed consent was obtained from all subjects. Human adipose tissue was harvested from subcutaneous fat obtained during abdominal liposuction surgery. The fat tissues were washed with PBS twice and cut into small pieces (1-2 mm3). Subsequently, the tissues were digested with collagenase type I in Hank’s balanced salt solution at 37°C. Fetal bovine serum was then added, and resultant cells were centrifuged at 800 g for 10 min and collected. The cell pellet was washed with PBS and treated with ammonium-chloride-potassium lysing buffer solution to lyse red blood cells. These cells were centrifuged at 400 g for 10 minutes and resuspended in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS and 1% penicillin-streptomycin (Thermo Fisher Scientific). The supernatant and debris were removed by changing the fresh medium after 48 hours incubation. The purity of ADSCs was characterized by determining surface markers CD29(+), CD105(+), CD90(+), CD31 (-), CD34 (-) and CD14 (-) by using a flow cytometer (Accuri C6; BD, USA).
Fabrication of collagen fibers-based 3D spheroids
A stirring bioreactor was used to produce the collagen fiber-based 3D cell spheroids (Figure 1B). Briefly, collagen monomers (1 mL, 0.01 mg/mL) were reconstituted at 37°C for 3 hours to produce collagen fibers (intended to act both as the adherent and ECM component). A total of 1.5 ´ 105 cells/mL of ADSCs (passage 2) or mouse hepatocytes (AML12; ATCC, VA, USA) and reconstituted collagen fibers were added into the stirring bioreactor at 40 rpm. The stirring bioreactor was then placed into an incubator at 37°C to enable stable cell spheroid formation. 3D spheroids (ADSC/hepatocyte spheroids, ADSC spheroids, and hepatocyte spheroids) were harvested after cultivation for a predetermined period. The sphericity and size of spheroids were examined. The 3D spheroids generated by using a commercially available EZSHERE dish were used as control.
Cell viability assay
The viability of cells in collagen fibers-based 3D spheroids was examined using a live/dead cell assay (Thermo Fisher Scientific). Briefly, an assay solution was prepared by mixing 1 mL of PBS containing 2.5 mL/mL of 4 mM ethidium homodimer-1 (EthD-1) and 1 mL/mL of 2 mM calcein-AM solution. This assay solution (100 mL) was added to the culture and the mixture was placed at 37°C in 5% CO2 for 15 minutes. The culture medium was removed, and the sample was observed using a fluorescence microscope using excitation filters of 494 nm (calcein-AM) and 528 nm (EthD-1) (Olympus IX71, Japan).
Albumin and cytokine enzyme-linked immunosorbent assay (ELISA)
To examine the level of albumin and cytokines secreted by 3D spheroids, the medium from cultured samples was collected. The secreted protein level of albumin, vascular endothelial growth factor (VEGF), and hepatic growth factor (HGF) in the collected culture medium were quantified using ELISA kits (BioVision, Milpitas, CA, USA) following manufacturers’ instructions.
Animal studies were approved by the Institutional Animal Care and Use Committee of I-Shou University, Taiwan (AUP-ISU-106-50-05) and carried out in accordance with the approved guidelines. The animal studies are reported in accordance and comply with the ARRIVE guidelines. Sprague-Dawley rats (male, 6 weeks old) were supplied by National Laboratory Animal Center (NLAC) Taiwan and housed in standard cages, with free access to food and water in a temperature-controlled environment under a 12-h light (50 lux illumination) and 12-h dark (< 10 lux illumination) cycle.
Experimental design and cell implantation
Animal experiments were performed over 14 weeks (Figure 3A). Liver cirrhosis was induced by TAA. TAA working solution (80 mg/mL; Sigma-Aldrich, St. Louis, MO, USA) was prepared with PBS. A total of 19 rats were used to establish the liver cirrhosis rat model and 3 rats served as the normal control. Rats were intraperitoneally injected three doses of 100 μL of TAA solution (80 mg/mL in PBS) per week for a total of 8 weeks. Rats were anesthetized using ZoletilÒ (intraperitoneal administration of 40 mg/kg of tiletamine with 50 mg/kg of zolazepam,) and xylazine (10 mg/kg), and randomly allocated into the following groups (n=3/group): Group A- implanted with 3D ADSC/hepatocyte spheroids, Group B- implanted with ADSC spheroids, Group C- implanted with hepatocyte spheroids, and Group D- no treatment, served as a negative control group. Surgery in rats was performed by a surgeon and monitored by experienced veterinarians and 3D cell spheroids were implanted into one lesion site (three spheroids/rat). During operation, clinical signs of pain, salivation, or abnormal behavior were carefully monitored . Any issues arising from the injection, resulted in exclusion from the study. Subsequently, randomly selected rats from each group were sacrificed at weeks 4 and 6 after 3D cell spheroids implantation. The liver and serum were harvested for histopathological and biochemical analysis.
At weeks 4 and 6 post cell spheroids implantation, the rat livers tissues from all groups were harvested and fixed in 10% neutral-buffered formalin. The liver tissues were then dehydrated in graded ethanol solutions, cleared in xylene, embedded in paraffin blocks, and cut into 3 μm-thick sections. H&E staining was performed for the general histopathological examination of the livers. Masson’s trichrome staining was conducted to assess changes in the collagen content of liver tissue. ImageJ software (Version 1.50; National Institutes of Health, USA) was used to measure the collagen content in each group . Three microscopic fields were taken under 100× magnification from each liver tissue for ImageJ analysis.
Blood biochemical parameters, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin, and g- glutamyltransferase (g-GT) were assayed to evaluate rat liver function. Rat serums were isolated from whole blood sample and subjected to ALT, AST, g-GT and total bilirubin biochemical analysis according to the manufacturer’s instructions.
All values are expressed as the mean ± standard error of the mean (SEM). Comparisons among multiple groups were analyzed by one-way ANOVA followed by Tukey’s multiple comparison. A p value of <0.05 was deemed statistically significant. All statistical analyses were performed using SPSS, version 17.0.