Enhancing the Maturity and Diclofenac Metabolism Ability of Mesenchymal Stem Cell-Derived Human Hepatocytes In Vitro Using Microfluidics Technology.

Human stem cell-derived hepatocyte-like cells (HLCs) represent a powerful tool for testing the efficacy and safety of novel therapies. However, most traditional 2D in vitro models yield HLCs with unpaired hepatic functions, hampering HLCs' adoption in the non-clinical drug development process. Here, we design a novel hepatic perfused microphysiological system (HLC-chip) that upon the optimization of the cell chamber architecture, cell inoculation strategy, and surface coating shows to improve the maturity of human HLCs derived from mesenchymal stem cell (MSC). The HLC-chip is microfabricated by photolithography and soft lithography techniques, based on polydimethylsiloxane (PDMS) molding. In particular, the optimized square-shaped HLC-chip design with seven inlets sealed against a collagen-coated polystyrene surface enables the homogeneous distribution of HLCs displaying the typical hepatic morphology. Additionally, HLCs can be maintained in the HLC-chip up to 10 days under perfusion, being positive for the hepatic markers HNF-4a, CK-18, OATP-C, and MRP2, while presenting increased ammonia detoxification ability. Likewise, upon GC-MS analysis, the 11.96- and 6.85-fold augment of diclofenac glucuronidation products in the HLC-chip and 2D cultures, respectively, demonstrate the enhanced biotransformation competence of cells. This study supports the generation of high-quality data from complex in vitro HLC systems and its usefulness for drug metabolism and toxicology studies.