An important advantage of employing extracellular matrix (ECM)-derived biomaterials in tissue engineering is the ability to tailor the biochemical and biophysical microenvironment of the cells. This study aims to assess whether three-dimensional (3D) liver-derived ECM hydrogel (LEMgel) promotes physiological function of liver organoids generated by self-organization of human hepatocarcinoma cells together with human mesenchymal and endothelial cells. We have optimized the decellularization method to fabricate liver ECM derived from sheep to preserve the greatest content of glycosaminoglycans, collagen, laminin, and fibronectin in produced LEMgel. During gelation, complex viscoelasticity modulus of the LEMgel (3 mg/mL) increased from 186.7 to 1570.5 Pa and Tan Delta decreased from 0.27 to 0.18. Scanning electron microscopy (SEM) determined that the LEMgel had a pore size of 382 ± 71 µm. Hepatocarcinoma cells in the self-organized liver organoids in 3D LEMgel (LEMgel organoids) showed an epithelial phenotype and expressed ALB, CYP3A4, E-cadherin, and ASGPR. The LEMgel organoid had significant upregulation of transcripts of ALB, CYP3A4, CYP3A7, and TAT as well as downregulation of AFP compared to collagen type I- and hydrogel-free-organoids or organoids in solubilized LEM and 2D culture of hepatocarcinoma cells. Generated 3D LEMgel organoids had significantly more ALB and AAT secretion, urea production, CYP3A4 enzyme activity, and inducibility. In conclusion, 3D LEMgel enhanced the functional activity of self-organized liver organoids compared to traditional 2D, 3D, and collagen gel cultures. Our novel 3D LEMgel organoid could potentially be used in liver tissue engineering, drug discovery, toxicology studies, or bio-artificial liver fabrication.

There are limited data available on the effect of a physicochemical microenvironment on mesenchymal stem cell (MSC) differentiation and repopulation of the liver. Therefore, in this study nanofibers have been used to better differentiate and maintain the function and engraftment of differentiating MSCs both in vitro and in vivo. Mouse MSCs were differentiated into early (day 18) and late (day 36) hepatocyte-like cells (HLCs) in the presence or absence of ultraweb nanofibers (nano(+) and nano(-)) and their transplantation for recovery in mice with CCl(4) induced hepatic fibrosis was investigated. In the nano(+) group, hepatocyte markers-ALB and HNF4α- were elevated in a time-dependent manner; however, those were similar levels or slightly decreased in the nano(-) group from day 18 to 36. Ultrastructural studies of the differentiated cells revealed some similarities to hepatocytes. Urea production, secretion of albumin and α-fetoprotein, and metabolic activity of the CYP450 enzymes were significantly increased within in vitro differentiated HLCs on nanofibers at day 36. MSCs, early and late HLCs in both nano(-) and nano(+) culture conditions that were transplanted by an intravenous route caused a decrease in liver fibrosis when engrafted in the recipient liver and were able to differentiate into functional hepatocytes (ALB(+)), except for late HLCs in the nano(-) group. Late HLCs transplanted in the nano(+) group were more effective in rescuing liver failure, enhancing serum ALB, homing transplanted cells and undergoing functional engraftment than the other groups. These results showed that topographic properties of nanofibers enhance differentiation of HLCs from MSCs and maintain their function in long-term culture, which has implications for cell therapies.

In order to investigate the effects of bone marrow-derived MSCs (mesenchymal stem cells) in reversing liver fibrosis and to determine their possible mechanism of action, mouse MSCs were infused into the tail vein of a CCl(4) injection mouse chronic model. MSCs caused a decrease in liver fibrosis histopathologically, 4 weeks after transplantation. The reduction in liver collagen was confirmed by quantitative analysis. Moreover, lipid peroxidation in the CCl(4)/MSC group decreased significantly. Quantitative RT (reverse transcription)-PCR analysis showed administration of MSCs has a significant antifibrotic effect as evidenced by the decrease in expression of liver collagen and increase in MMP13 (matrix metalloproteinase 13) in the CCl(4)/MSC group when compared with the CCl(4) group, 4 weeks after transplantation. The expression of alphaSMA (smooth muscle actin) and TIMP1 was also down-regulated in the CCl(4)/MSC group. Additionally, the expression of MMP9 was significantly up-regulated in the CCl(4)-treated group; however, there was no significant change after MSC injection. Few engrafted cells in the recipient liver and were able to differentiate into albumin-positive cells. In conclusion, MSCs can enhance recovery of a CCl(4)-injured mouse liver through their influence in reducing collagen deposition by possibly affecting expression of MMPs.