Preparation of Primary Rat Hepatocyte Spheroids Utilizing the Liquid-Overlay Technique.

From PubMed via Jisc Publications RouterPublication status: ppublishFunder: BBSRC; Grant(s): BB/M503435/1Funder: MRC Skills Development Fellowship; Grant(s): MR/S019332/1Funder: University of LiverpoolFunder: MRCFunder: Liverpool Centre for Mathematics in HealthcareFunder: EPSRC; Grant(s): EP/N014499/1Herein, 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.