Kyffin, Jonathan A.Cox, Christopher R.Leedale, JosephColley, Helen E.Murdoch, CraigMistry, PratibhaWebb, Steven D.Sharma, Parveen2019-10-022019-10-022019-09-13Kyffin, J. A., Cox, C. R., Leedale, J., Colley, H. E., Murdoch, C., Mistry, P., Webb, S. D., & Sharma, P. (2019). Preparation of primary rat hepatocyte spheroids utilizing the liquid-overlay technique. Current Protocols in Toxicology, 81(1), e87. https://doi.org/10.1002/cptx.871934-925410.1002/cptx.87http://hdl.handle.net/10034/622663Herein, we describe a protocol for the preparation and analysis of primary isolated rat hepatocytes in a 3D cell culture format described as spheroids. The hepatocyte cells spontaneously self-aggregate into spheroids without the need for synthetic extracellular matrices or hydrogels. Primary rat hepatocytes (PRHs) are a readily available source of primary differentiated liver cells and therefore conserve many of the required liver-specific functional markers, and elicit the natural in vivo phenotype when compared with common hepatic cells lines. We describe the liquid-overlay technique which provides an ultra-low attachment surface on which PRHs can be cultured as spheroids. © 2019 The Authors. Basic Protocol 1: Preparation of agarose-coated plates Basic Protocol 2: Primary rat hepatocyte isolation procedure Basic Protocol 3: Primary rat hepatocyte spheroid culture Basic Protocol 4: Immunofluorescent analysis of PRH spheroids. [Abstract copyright: © 2019 The Authors.]https://creativecommons.org/licenses/by/4.0/bile canaliculiimmunofluorescenceliquid-overlay techniqueliver spheroidsprimary rat hepatocytesArticle1934-9262Current Protocols in Toxicology2019-10-02