Improved Hepatogenic Differentiation of Human Adipose Tissue‐derived MSCs Using a Silicone‐based Cell Culture System

Abstract

Conventional two‐dimensional as well as established three‐dimensional cell culture systems exhibit a high modulus of elasticity, yet, without representing the stiffness of human organs. Previously, we established a protocol for the culture of primary rat hepatocytes on a silicone of low stiffness, which sustained their physiological functions. In terms of the development of human‐based tests to evaluate the potential risk of novel drugs, the method was adjusted for use of human adipose tissue‐derived mesenchymal stromal cells (hAT‐MSC). Aim of the study was to improve hepatogenic differentiation of hAT‐MSC on fibronectin‐coated 3D silicone scaffolds with low stiffness (E‐modulus approx. 250 kPA) mimicking the stiffness of human liver including surface modifications to increase hydrophilic properties.hAT‐MSCs were cultured on 3D silicone scaffolds. Hepatogenic differentiation was initiated by the 2 step protocol. The capability of the hepatocyte‐like cells to store glycogen, to synthesize urea and albumin as well as the enzyme activities of CYP1A1 and Cyp2B1/2 were analysed and compared to the conventional 2D cell culture and hepatogenic differentiation on polystyrene (E‐modulus 103 MPa).hAT‐MSC attached to the 3D silicone scaffolds and formed cell clusters. 10 days after starting the hepatogenic differentiation until the end of the culture on day 24, the cells on 3D silicone scaffolds displayed more intensive PAS staining for glycogen as compared to the cells on 2D cultures indicating improved glycogen storage. The urea synthesis rate [nmol/10.000 cells/24h] of these cells was significant higher as compared to cells on 2D culture throughout the culture period, though declining with ongoing culture; 10 d: 2D vs 3D 0.05±0.01 vs 1.68±0.05 and 24 d: 0.03±0.007 vs 0.4±0.04, respectively. The albumin synthesis rate [pg/10.000 cells/24h] was significant higher in cells differentiated for 24 d as compared to undifferentiated cells: 48.5±11 vs 7.4±1.9. Enzyme activity of CYP1A1 after 24 d was 3‐fold higher in cells differentiated on 3D silicone scaffolds as compared to cells cultivated on 2D and the enzyme activity of CYP2B1/2 was 12‐fold higher.With these results, medically approved silicone treated with plasma was identified to improve hepatogenic differentiation of hAT‐MSC, likely by providing the appropriate three‐dimensional growth conditions. Thus, the silicone scaffolds represent an important tool for the development of cell cultures featuring organ‐like stiffness and improving physiological functions of hepatogenic differentiated MSC.Support or Funding InformationThe study was supported by the European Regional Development Fund and the Free State of Saxony (SAB) 100282466 to J.H. and P.S.