Three‐Dimensional Cell Culture of Rat Hepatocytes in Sub‐microliter Hydrogel Wells

Abstract

Sub-microliter hydrogel wells were used to culture 3D (three-dimension) micro-tissues using H35rat hepatoma cells. These micro-tissues behave more like in-vivo tissues than traditional monolayer cell cultures. Duraform Polyamide resin molds were created using the Computer-Aided Design (CAD) software SolidWorksTM and a 3D printing process Selective Laser Sintering (SLS). The molds are autoclavable and reusable multiple times to create the miniature hydrogel wells using 2% agarose (well size:1.1 mm deep and 0.6 mm wide with a rounded bottom). In these hydrogels wells, hepatocytes self-assemble into three dimensional micro-tissues, where cells attach to each other and proliferate as clusters, also called spheroids. Different cell seeding concentrations were investigated and it was found that an initial seeding concentration of 50,000 cells/mL in a volume of 70 microliters was most optimum for cell proliferation to produce the 3D spheroids. Further, it was observed that 3D cell growth was promoted when the wells were pre-coated overnight with 2% bovine serum albumin (BSA). Micro-tissue culture of hepatocytes could find applications in drug screening, disease testing and toxicology studies with the 3D tissues. This work was supported by a grant from National Science Foundation.