Assessment of functional markers of liver in hapatocytes sheet that produced br a temperature responsive polymer compared with the sheet

Abstract

Abstract Introduction: Liver tissue engineering via cell sheet technology would open new doors for the treatment of patients with liver failure. The obtained extracellular matrix (ECM) from decellularized tissues along with temperature-responsive polymer (pNIPAAm) could provide an intelligent surface for developing hepatocyte cell sheets. The aim of this study was the investigation of structural and function cues of hepatocyte-like cell sheets on the surface of pNIPAAm and dECM. Methods and materials: The rat livers were extracted and decellularized. Its efficacy was analyzed using histological staining, scanning electron microscope (SEM), and western blotting. pN hydrogel and dECM + pN hydrogel (1:3 and 2:3 ratios) were fabricated, and scaffold architecture was characterized by FT-IR. Each well of culturing plates was coated separately with AT-MSCs. We generated three in vitro microenvironments models including I: pNIPAAm hydrogel (pN hydrogel), II: decellularized ECM incorporated into pNIPAAm hydrogel (dECM +pN hydrogel), and III: decellularized ECM scaffold (dECM scaffold). Then the AT-MSCs were differentiated into hepatocyte-like cells (HLCs). After recellularization, patterns of differentiation, and expression of hepatogenic markers were investigated via biochemical assays and qRT-PCR at different time points. Results: Production of dense and intact cell sheets was obtained in dECM + pN hydrogel, as opposed to pN hydrogel and dECM scaffold. Also, a statistically significant difference of HLCs functionality in dECM+pN hydrogel was confirmed after evaluation of the expression of hepatocyte markers including, alpha-fetoprotein, cytokeratin 18, cytochrome P450-2E1, and phosphoenolpyruvate carboxykinase. Conclusion: dECM + pN hydrogel was able to preserve hepatocyte function in cell sheets owing to the high level of albumin, urea, hepatogenic markers, and glycogenesis potential of HLCs. Accordingly, dECM incorporated in pN hydrogel could remodel microenvironments to guide the AT-MSCs into conducive differentiation and proliferation to give rise to multilayer sheets of cells in their own ECM.