Several studies in the past have formed 3-dimensional (3D) spheroids of primary hepatocytes in
suspension culture. Unfortunately, primary hepatocytes in a
suspension environment tend to lose their differentiated function over time, generally due to damage from fluid shear stress and eventual spheroid settling. We have therefore created a novel
suspension culture system, by seeding H35 rat
hepatoma cells, a hepatocyte-derived cell line, in a 24-well tissue culture
polystyrene (
TCPS) plate placed atop an orbital shaker to create 3D spheroids. To provide stability to the formed spheroids, we used a long-chain
polymer,
bovine serum albumin (BSA), dissolved in the cell culture medium and/or coated on
TCPS surfaces placed in
suspension configurations. Our results demonstrate that BSA coating of culture surfaces resulted in uniform and well-defined spheroids with little spheroid settling or "flattening" of cell colonies in either static or
suspension configurations. In BSA-coated
suspension systems, spheroid size scaled with the amount of BSA dissolved in culture medium. In static uncoated cultures, the normalized rat
albumin production levels were enhanced by addition of BSA within culture medium. Thus, both addition of BSA to culture medium and application of BSA as a surface coating appear to be meaningful avenues for tailoring spheroid morphology and function. This 24-well plate
suspension culture system may be a valuable tool for high throughput investigations of liver cell behavior in a stable, uniform, 3D spheroid state.