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

Jonathan A Kyffin,Christopher R Cox,Steven D Webb,Pratibha Mistry,Parveen Sharma,Helen E Colley,Craig Murdoch,Joseph Leedale

doi:10.1002/cptx.87

Abstract

Herein, 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

Highlights

We describe the hepatocyte isolation procedure in detail, as well as the main immunofluorescence protocol used to characterize the properties of the resultant Primary rat hepatocytes (PRHs) spheroids
We describe the basic culture conditions required for the maintenance of the PRH spheroids for extended periods when implementing the liquid-overlay technique (LOT) (Basic Protocol 3), and the specific immunofluorescence protocol for the staining and imaging of bile cannalicular-like structures via P-glycoprotein (P-gp) immunostaining (Basic Protocol 4)
Have been established for a number of years, but to our knowledge, no definitive studies have described conditions showing the implementation of LOT and agarose-coated ultralow attachment (ULA) plates for the formation of primary rat liver spheroids

Summary

INTRODUCTION

This article describes an efficient method to generate and culture primary rat hepatocyte (PRH) spheroids using the liquid-overlay technique (LOT). This article begins with the standard protocol for the production of agarose-coated, ultralow attachment (ULA) plates (Basic Protocol 1), followed by the complete PRH isolation procedure using livers of male Wistar rats (Basic Protocol 2). PRIMARY RAT HEPATOCYTE ISOLATION PROCEDURE This procedure describes the principal methodology for the isolation of hepatocytes from the whole liver of a young adult male Wistar rat, weighing between 175 and 200 g, and the subsequent purification steps for these parenchymal cells This protocol is a modified version of the two-step collagenase perfusion technique described originally by Seglen (1976).

Weigh out into three separate 5-ml bijou bottles

36. Calculate the number of viable cells in the preparation

Background

Literature Cited