Hepatocyte Culture On3D Electrospun Fibrous Scaffolds As An In Vitro Model For Drug Testing

During drug research and development process, an in vitro liver model is usually used to predict the in vivo metabolism and hepatoxicity of new chemical entities. Good results contribute to reduced attrition and enhanced efficiency and success in the R&D process. However, existing models cannot maintain the polarity and hepatic functions of primary hepatocytes well, especially the function of phase I (C YP450) and phase II enzyme activity. Results based on these models are quite different from those of in vivo, and would lead to poor judgments. In order to get hepatocyte viability and hepatic functions better maintained in vitro, the current work aims to propose a surface modified3D electrospun scaffold for primary hepatocyte culture.Electrospun fibrous mats have been used as tissue culture scaffolds, mimicking the physical structure of extracellular matrix(ECM). But a regular electrospun scaffold has typical pore size of several micrometers, which would hinder cell infiltration, thus, a2D surface is presented and different from the3D environment in vivo. In this work, a rotating frame cylinder as the collector was used to generate high porosity and large pore-sized fibrous mats. Three scaffolds possessing pore diameters of20,60and100μm were obtained by changing the rotating speed. Scaffolds were surface modified with monoamine terminated galactose and Acp-GRGDS to enhance cell attachment, which better mimicking the biological properties of ECM. Blending electrospinning was used to get a poly (styrene-co-maleic acid)/polystyrene (PSMA/PS) scaffold. By changing the ratio of PSMA/PS for electrospining, different fibrous mats were obtained and used for preparing galactosylated scaffolds with a series of galactose densities. By controlling the amount of Acp-GRGDS added for surface modification, RGD grafted scaffolds with a series of group densities were prepared. By adding Acp-GRGDS first and then galactose, hybrid grafted scaffolds with different density ratios of galactose and RGD were obtained.Primary hepatocytes were cultured on different scaffolds. Hepatocytes infiltrated into inner space of large pore-sized scaffold and aggregated with recovered polarity, presenting extensive cell-cell contact and cell-scaffold interaction spatially. Hepatocyte polarity recovered. During the15days’culture, hepatocyte synthesis functions and enzyme activity were determined at several time points of interest. Hepatocytes on scaffolds with a medium pore diameter of60μm and galactose/RGD (group density ratio of1:1000) grafting showed superior level of functions. In vitro investigation on metabolism and hepatoxicity of model drugs using hepatocytes on different scaffolds was conducted with or without enzyme inducer/inhibitor. CYP3A11and phase II enzyme specific drugs were used, namely, testosterone and acetaminophen for metabolism study, and amiodarone and acetaminophen for hepatoxicity testing. Hepatocytes cultured on hybrid grafted scaffold showed higher enzyme activity, good response to inducer/inhibitor and sensitivity to drug-induced toxicity, exhibiting in vivo-in vitro correlation to a certain extent. The proposed culture model may have potential applications for in vitro drug testing.