Isolation and Primary Culture of Rat Hepatic Cells

Summary

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 × 10⁸ cells per preparation with cell viability between 88 ~ 96%.

Abstract

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 ¹ and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 ². Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 μm pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Protocol

1. Preparation

All buffers are freshly prepared using sterile technique and filter sterilized using a Corning 0.22 μm filter.

1. Prepare Perfusion buffer I by adding the following to Hank's Balanced Salt Solution (HBSS, without Ca²⁺ and Mg²⁺, see Table 1): Mg²⁺ (MgCl₂) to 0.9 mM, EDTA to 0.5 mM, and HEPES to 25 mM.
2. Prepare Perfusion buffer II by adding the following to HBSS (with Ca²⁺ and Mg²⁺, see Table 1): HEPES to 25 mM.
3. Prepare Perfusion buffer II plus collagenaseII: Disso....

Discussion

Primary culture of hepatocytes is an in vitro model widely used to study various aspects of liver physiology and pathology. For example, primary culture is used to assess the expression and function of drug-metabolizing enzymes including cytochromes P450, drug metabolism, drug-drug interactions, and the mechanisms of cytotoxicity and genotoxicity ^3-7. The protocol describing isolation and culture of rat hepatic cells is adapted from the previous reports of Aiken et al. ⁸, a.......