Extracellular Matrix Effects on Hepatocyte Functioning: A Multivariate Regression Approach
thesisposted on 27.07.2018, 00:00 by Lioudmila V Sorokina
The use of in vitro liver models represents an important step in drug discovery and disease modeling. In the United States, 11% of acute liver cases are due to idiosyncratic drug-induced liver (DILI) injury, yet presently used animal models are a poor predictor of idiosyncratic DILI. Micropatterned co-cultures (MPCCs), composed of primary human hepatocytes (PHHs) and 3T3-J2 murine embryonic fibroblasts, demonstrate stable, long-term hepatic functions by controlling substrate geometry and cell-cell interactions in vitro. Yet present MPCC model is not fully human-relevant due to the use of both animal-derived cells and substrate. Furthermore, PHHs used in MPCCs are source-limited cells. To address these challenges, MPCCs using induced pluripotent stem cells-derived human hepatocyte-like cells (iHeps) in place of PHHs were designed and tested on various extracellular matrix (ECM) proteins. In a separate study, an ECM screen was performed in a traditional MPCC format to elucidate the effects of human ECM components on cell functioning and to compare them to mixed-ECM substrates such as decellularized human and porcine liver biomatrices. The presence of collagen I at 50% concentration showed upregulation of most tested hepatocyte functions. Finally, a platform was designed for in vitro delivery of transforming growth factor beta (TGF-β), a cytokine that plays a significant role in regulation of many chronic liver diseases. Present in vitro models of TGF-β employ soluble methods of delivery, which are not physiologically relevant and cost prohibitive. Fibronectin – coated polyelectrolyte multilayers (PEMs) of chitosan and heparin were used for adsorption and delivery of TGF-β to PHHs to discern the effect of microenvironmental cues on cell functioning. Multivariate regression analysis was employed to identify the contributing variables that can be used in the design of next generation in vitro liver models. In particular, adsorbed TGF-β and the presence of co-culture upregulated PHH functions in PEMs.