ABSTRACT
BACKGROUND@#Currently, there is an urgent need for scalable and reliable in vitro models to assess the effects oftherapeutic entities on the human liver. Hepatoma cell lines, including Huh-7, show weakly resemblance to humanhepatocytes, limiting their significance in toxicity studies. Co-culture of hepatic cells with non-parenchymal cells, and thepresence of extracellular matrix have been shown to influence the biological behavior of hepatocytes. The aim of this studywas to generate the scalable and functional hepatic micro-tissues (HMTs). @*METHODS@#The size-controllable HMTs were generated through co-culturing of Huh-7 cells by mesenchymal stem cellsand human umbilical vein endothelial cells in a composite hydrogel of liver-derived extracellular matrix and alginate, usingan air-driven droplet generator. @*RESULTS@#The generated HMTs were functional throughout a culture period of 28 days, as assessed by monitoringglycogen storage, uptake of low-density lipoprotein and indocyanine green. The HMTs also showed increased secretionlevels of albumin, alpha-1-antitrypsin, and fibrinogen, and production of urea. Evaluating the expression of genes involvedin hepatic-specific and drug metabolism functions indicated a significant improvement in HMTs compared to two-dimensional(2D) culture of Huh-7 cells. Moreover, in drug testing assessments, HMTs showed higher sensitivity tohepatotoxins compared to 2D cultured Huh-7 cells. Furthermore, induction and inhibition potency of cytochrome P450enzymes confirmed that the HMTs can be used for in vitro drug screening. @*CONCLUSION@#Overall, we developed a simple and scalable method for generation of liver micro-tissues, using Huh-7,with improved hepatic-specific functionality, which may represent a biologically relevant platform for drug studies.
ABSTRACT
BACKGROUND@#Currently, there is an urgent need for scalable and reliable in vitro models to assess the effects oftherapeutic entities on the human liver. Hepatoma cell lines, including Huh-7, show weakly resemblance to humanhepatocytes, limiting their significance in toxicity studies. Co-culture of hepatic cells with non-parenchymal cells, and thepresence of extracellular matrix have been shown to influence the biological behavior of hepatocytes. The aim of this studywas to generate the scalable and functional hepatic micro-tissues (HMTs). @*METHODS@#The size-controllable HMTs were generated through co-culturing of Huh-7 cells by mesenchymal stem cellsand human umbilical vein endothelial cells in a composite hydrogel of liver-derived extracellular matrix and alginate, usingan air-driven droplet generator. @*RESULTS@#The generated HMTs were functional throughout a culture period of 28 days, as assessed by monitoringglycogen storage, uptake of low-density lipoprotein and indocyanine green. The HMTs also showed increased secretionlevels of albumin, alpha-1-antitrypsin, and fibrinogen, and production of urea. Evaluating the expression of genes involvedin hepatic-specific and drug metabolism functions indicated a significant improvement in HMTs compared to two-dimensional(2D) culture of Huh-7 cells. Moreover, in drug testing assessments, HMTs showed higher sensitivity tohepatotoxins compared to 2D cultured Huh-7 cells. Furthermore, induction and inhibition potency of cytochrome P450enzymes confirmed that the HMTs can be used for in vitro drug screening. @*CONCLUSION@#Overall, we developed a simple and scalable method for generation of liver micro-tissues, using Huh-7,with improved hepatic-specific functionality, which may represent a biologically relevant platform for drug studies.
ABSTRACT
Objective: Recent advances in cell therapy have encouraged researchers to provide an alternative for treatment and restoration of damaged liver through using hepatocytes. However, these cells quickly lose their functional capabilities in vitro. Here, we aim to use the secretome of mesenchymal stromal cells [MSCs] to improve in vitro maintenance conditions for hepatocytes
Materials and Methods: In this experimental study, following serum deprivation, human adipose tissue-derived MSCs [hAT-MSCs] were cultured for 24 hours under normoxic [N] and hypoxic [H] conditions. Their conditioned media [CM] were subsequently collected and labeled as N-CM [normoxia] and H-CM [hypoxia]. Murine hepatocytes were isolated by perfusion of mouse liver with collagenase, and were cultured in hepatocyte basal [William's] medium supplemented with 4% N-CM or H-CM. Untreated William's and hepatocyte-specific media [HepZYM] were used as controls. Finally, we evaluated the survival and proliferation rates, as well as functionality and hepatocyte-specific gene expressions of the cells
Results: We observed a significant increase in viability of hepatocytes in the presence of N-CM and H-CM compared to HepZYM on day 5, as indicated by MTS [3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]- 2H-tetrazolium] assay. Indocyanine green [ICG] uptake of hepatocytes in the H-CM and HepZYM groups on days 3 and 5 also suggested that H-CM maintained the hepatocytes at about the same level as the hepatocyte-specific medium. The HepZYM group had significantly higher levels of albumin [Alb] and urea secretion compared to the other groups [P<0.0001]. However, there were no significant differences in cytochrome activity and cytochrome gene expression profiles among these groups. Finally, we found a slightly, but not significantly higher concentration of vascular endothelial growth factor [VEGF] in the H-CM group compared to the N-CM group [P=0.063]
Conclusion: The enrichment of William's basal medium with 4% hAT-MSC-H-CM improved some physiologic parameters in a primary hepatocyte culture
ABSTRACT
Hepatocyte-like cells [HLCs] are generated from either various human pluripotent stem cells [hPSCs] including induced pluripotent stem cells [iPSCs] and embryonic stem cells [ESCs], or direct cell conversion, mesenchymal stem cells as well as other stem cells like gestational tissues. They provide potential cell sources for biomedical applications. Liver transplantation is the gold standard treatment for the patients with end stage liver disease, but there are many obstacles limiting this process, like insufficient number of donated healthy livers. Meanwhile, the number of patients receiving a liver organ transplant for a better life is increasing. In this regard, HLCs may provide an adequate cell source to overcome these shortages. New molecular engineering approaches such as CRISPR/Cas system applying in iPSCs technology provide the basic principles of gene correction for monogenic inherited metabolic liver diseases, as another application of HLCs. It has been shown that HLCs could replace primary human hepatocytes in drug discovery and hepatotoxicity tests. However, generation of fully functional HLCs is still a big challenge; several research groups have been trying to improve current differentiation protocols to achieve better HLCs according to morphology and function of cells. Large-scale generation of functional HLCs in bioreactors could make a new opportunity in producing enough hepatocytes for treating end-stage liver patients as well as other biomedical applications such as drug studies. In this review, regarding the biomedical value of HLCs, we focus on the current and efficient approaches for generating hepatocyte-like cells in vitro and discuss about their applications in regenerative medicine and drug discovery