Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Inn Chuan Ng; Li Zhang; Narelle Nichola Yi Ying Shen; Yun Ting Soong; Chan Way Ng; Phoebe Kang Sheing Koh; Yan Zhou; Hanry Yu

doi:10.3791/62289

Method Article

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

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DOI:

10.3791/62289

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April 5th, 2021

Inn Chuan Ng^*¹ , Li Zhang^*² , Narelle Nichola Yi Ying Shen³ , Yun Ting Soong⁴ , Chan Way Ng⁵ , Phoebe Kang Sheing Koh¹ , Yan Zhou³ , Hanry Yu¹^,³^,⁴^,⁵^,⁶

¹Department of Physiology & The Institute for Digital Medicine (WisDM), National University of Singapore, ²College of Agriculture and Biology, Zhongkai University of Agriculture and Engineering, ³Mechanobiology Institute, National University of Singapore, ⁴Institute of Bioengineering and Nanotechnology, A*STAR, ⁵NUS Graduate School for Integrative Sciences and Engineering, ⁶CAMP, Singapore-MIT Alliance for Research and Technology

^* These authors contributed equally

Summary

This protocol details the use of a special intravenous catheter, standardized sterile disposable tubing, temperature control complemented by real-time monitoring, and an alarm system for two-step collagenase perfusion procedure to improve the consistency in the viability, yield, and functionality of isolated primary rat hepatocytes.

Abstract

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure for primary hepatocyte isolation is technically challenging, especially in portal vein cannulation. The procedure is also prone to occasional contamination and variations in perfusion conditions due to difficulties in the assembly, optimization, or maintenance of the perfusion setup. Here, a detailed protocol for an improved two-step collagenase perfusion procedure with multiparameter perfusion control is presented. Primary rat hepatocytes were successfully and reliably isolated by taking the necessary technical precautions at critical steps of the procedure, and by reducing the operational difficulty and mitigating the variability of perfusion parameters through the adoption of a special intravenous catheter, standardized sterile disposable tubing, temperature control, and real-time monitoring and alarm system. The isolated primary rat hepatocytes consistently exhibit high cell viability (85%-95%), yield (2-5 x 10⁸ cells per 200-300 g rat) and functionality (albumin, urea and CYP activity). The procedure was complemented by an integrated perfusion system, which is compact enough to be set up in the laminar flow hood to ensure aseptic operation.

Introduction

Primary hepatocytes are important tools for liver-related basic research, disease treatment, and application such as drug testing. The current gold standard for primary hepatocyte isolation is the two-step collagenase perfusion procedure¹^,²^,³ introduced by Seglen in the 1970s⁴. However, this procedure is technically challenging and has a high failure rate when performed by novice surgeons. Even when a perfusion is considered successful, drastic differences in hepatocyte viability (typically 60%-95%) and yield (0.5-5 x 10⁸ per 200-300 g rat) ....