Mechanistic Study for Drug Induced Cholestasis Using Batch-Fabricated 3D Spheroids Developed by Agarose-Stamping Method.

Cell spheroid culture can recapitulate the tissue microstructure and cellular responses in vivo. While there is a strong need to understand the modes of toxic action using the spheroid culture method, existing preparation techniques suffer from low efficiency and high cost. Herein, we developed a metal stamp containing hundreds of protrusions for batch bulk preparation of cell spheroids in each well of the culture plates. The agarose matrix imprinted by the stamp can form an array of hemispherical pits, which facilitated the fabrication of hundreds of uniformly sized rat hepatocyte spheroids in each well. Chlorpromazine (CPZ) was used as a model drug to investigate the mechanism for drug induced cholestasis (DIC) by agarose-stamping method. Hepatocyte spheroids showed a more sensitive detection of hepatotoxicity compared to 2D and Matrigel-based culture systems. Cell spheroids were also collected for staining of cholestatic protein and showed a CPZ-concentration-dependent decrease of bile acid efflux related proteins (BSEP and MRP2) and tight junction (ZO-1). In addition, the stamping system successfully delineated the DIC mechanism by CPZ that may be associated with the phosphorylation of MYPT1 and MLC2, two central proteins in the Rho-associated protein kinase pathway (ROCK), which were significantly attenuated by ROCK inhibitors. Our results demonstrated a large-scale fabrication of cell spheroids by the agarose-stamping method, with promising benefits for exploring the mechanisms for drug hepatotoxic responses.