Hepatocyte Cocultures with Endothelial Cells and Fibroblasts on Micropatterned Fibrous Mats to Promote Liver-Specific Functions and Capillary Formation Capabilities

Abstract

The maintenance of hepatocyte phenotype and functions remains as a great challenge in the generation of functional liver tissue and in vitro model for drug metabolism studies. The use of hepatocyte coculture systems plays essential roles in the establishment of cell-cell and cell-extracellular matrix communications similar to native liver tissues. In the current study, micropatterned electrospun fibrous mats were created to load hepatocytes, fibroblasts, and endothelial cells (ECs), which were precisely assembled to establish their spatially controlled coculture for mimicking the in vivo structure of hepatic lobules. Hepatocytes formed compact polyhedral spheroids with an average diameter of 80-100 μm, reorganized actin filaments in the cell-cell contact regions, and well-developed bile canaliculi. Compared with hepatocytes cultured alone, the coculture of hepatocytes with either fibroblasts or ECs led to significantly higher albumin secretion, urea synthesis and cytochrome P-450 expression, which were dramatically improved by the coculture of hepatocytes with both fibroblasts and ECs. The cocultured ECs well spread on patterned regions with little organized filamentous actin, and significantly higher densities and deeper penetration into patterned scaffolds were determined for ECs after coculture with fibroblasts and hepatocytes compared with those after cultured alone or coculture with either fibroblasts or hepatocytes. A Matrigel overlay assay showed that the capabilities of ECs to form capillary tubes were significantly enhanced by micropatterned coculture with fibroblasts and hepatocytes. Thus, the coculture of hepatocytes, fibroblasts, and ECs on micropatterned fibrous mats helps both hepatocytes in the maintenance of hepatic functions and ECs in the formation of capillary-like structures. It is suggested that the micropatterned coculture model described here not only provides functional hepatic tissues for predictions of drug metabolism profiles, but also will enable investigations on more complex and physiological cell-cell communications.

Human Stem Cell-Derived Endothelial-Hepatic Platform for Efficacy Testing of Vascular-Protective Metabolites from Nutraceuticals

“…Conversely, we also investigated whether hPSC‐ECs could, in turn, impact the metabolic function of hPSC‐HEPs in a coculture setting. It has been reported that endothelial cells could improve hepatic function [] and provide some levels of hepatoprotection from acetaminophen toxicity []. In our study, cocultured hPSC‐HEPs had comparable albumin levels with monocultured hPSC‐HEPs (supplemental online Fig.…”

“…Continuous replenishment of metabolites in coculture setup recapitulated the systemic setting of liver paracrine effects on the vasculatures. Conversely, endothelial cells are known to improve hepatic function by promoting cell viability, synthesis of albumin and urea, and efficiency of the drug transporter system []. Our hPSC‐ECs could, in turn, increase the metabolizing CYP enzyme activity in hPSC‐HEPs.…”

“…Multicellular coculture models [, , ] are gaining momentum for various applications. A recent study described the use of human umbilical vein endothelial cells to stabilize hepatoblastoma C3A cells and modulate drug‐induced hepatotoxicity [].…”

Efficient and robust differentiation of endothelial cells from human induced pluripotent stem cells via lineage control with VEGF and cyclic AMP

“…The importance of endothelial cells (ECs) has already been shown in the formation of various organs such as heart[1–3], liver[4–7], kidney[8], bone[9], and skin among many others[10–13]. Thus, efficient EC preparation methods that provide scalable and stable supply are necessary for three-dimensional (3D) tissue engineering and organ regeneration.…”

Polymers in Tissue Engineering

“…Three dimensional stacked polycarbonate membranes with hepatocytes showed functional liver units in vitro [143]. Functional liver units were also observed using micropatterned Poly(ethylene glycol)-poly(dl-lactide) and lactosylated poly(dl-lactide) electrospun fibrous mats [144]. For larger structures, like the bile duct or vascular structures, polymers may be formed into sheets and then tube structures.…”