Tethered primary hepatocyte spheroids on polystyrene multi-well plates for high-throughput drug safety testing

Hepatocyte spheroids are useful models for mimicking liver phenotypes in vitro because of their three-dimensionality. However, the lack of a biomaterial platform which allows the facile manipulation of spheroid cultures on a large scale severely limits their application in automated high-throughput drug safety testing. In addition, there is not yet a robust way of controlling spheroid size, homogeneity and integrity during extended culture. This work addresses these bottlenecks to the automation of hepatocyte spheroid culture by tethering 3D hepatocyte spheroids directly onto surface-modified polystyrene (PS) multi-well plates. However, polystyrene surfaces are inert toward functionalization, and this makes the uniform conjugation of bioactive ligands very challenging. Surface modification of polystyrene well plates is achieved herein using a three-step sequence, resulting in a homogeneous distribution of bioactive RGD and galactose ligands required for spheroid tethering and formation. Importantly, treatment of polystyrene tethered spheroids with vehicle and paradigm hepatotoxicant (chlorpromazine) treatment using an automated liquid handling platform shows low signal deviation, intact 3D spheroidal morphology and Z' values above 0.5, and hence confirming their amenability to high-throughput automation. Functional analyses performance (i.e. urea and albumin production, cytochrome P450 activity and induction studies) of the polystyrene tethered spheroids reveal significant improvements over hepatocytes cultured as collagen monolayers. This is the first demonstration of automated hepatotoxicant treatment on functional 3D hepatocyte spheroids tethered directly on polystyrene multi-well plates, and will serve as an important advancement in the application of 3D tethered spheroid models to high throughput drug screening.