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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Published: April 5th, 2021

DOI:

10.3791/62289

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

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.

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.

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) ....

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All procedures and animal housing were carried out under protocol numbers R15-0027 and R19-0669 in accordance with the requirements of the Institutional Animal Care and Use Committee (IACUC) of the National University of Singapore.

1. Preparation of solutions and surgical instruments

  1. Prepare buffers and cell culture media in Table 1 using ultrapure water.
  2. Pre-warm the calcium-free buffer and collagenase buffer to 37 °C in a water bath before use.

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A surgeon could tell whether liver perfusion is going on smoothly by observing the outcome after certain steps. The first outcome can be observed upon cannulation, cutting of the infrahepatic IVC, and restoring the perfusion flow rate. The liver should have completely changed color from dark red to brown, while maintaining its volume. If the liver looks slightly deflated and has a reddish tint or blotches of red, it means that the perfusion flow rate was set wrongly (too low), or the portal vein was not cannulated correc.......

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There are a few points that are particularly important to observe for two-step collagenase perfusion procedure in general. Firstly, special care must be given when resecting the liver. Ensure that the gastrointestinal tract is not damaged as leakage of the contents will result in bacterial contamination. In addition, avoid damaging the Glisson's capsule, which covers the surface of the liver during the animal procedure. If the tear is large enough, it might allow premature release of disassociated hepatocytes into th.......

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This work is supported in part by MOE ARC (MOE2017-T2-1-149); NUHS Innovation Seed Grant 2017 (NUHSRO/2017/051/InnovSeed/02); Mechanobiology Institute of Singapore (R-714-106-004-135); and Institute of Bioengineering and Nanotechnology, Biomedical Research Council, Agency for Science, Technology and Research (A*STAR) (Project Numbers IAF-PP H18/01/a0/014, IAF-PP H18/01/a0/K14 and MedCaP-LOA-18-02) funding to Hanry Yu. Ng Chan Way is a research scholar of the National University of Singapore. We would like to thank Confocal Microscopy Unit & Flow Cytometry Unit of the National University of Singapore for help and advice in hepatocyte purity analysis.

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Name Company Catalog Number Comments
Material/Equipment
1 mL syringe Nipro
27G needle Nipro
Black braided silk non-absorbable, non-sterile surgical suture Look SP117
Bochem 18/10 stainless steel forceps, sharp tip contain bent round tip Bochem 10333511
Disposable Perfusion Set Vasinfuse BPF-112
Floating circular 1.5 mL microcentrifuge tube rack Sigma-Aldrich R3133
German Standard Tissue Forceps, Serrated / 1×2 teeth , 14.5cm Walentech
Greiner Cellstar aspirating pipette Merck GN710183
Haemocytometer
Integrated Perfusion System Vasinfuse IPS-001
Iris Scissors curved, stainless, 11cm Optimal Medical Products Pte Ltd CVD
Light microscope with 10X lens Olympus
Mesh Sheet 100µM Nylon Spectra-Teknic(s) Pte Ltd 06630-75
Operating Scissors, BL/BL, 13cm Optimal Medical Products Pte Ltd STR – BL/BL
Operating Scissors, SH/BL, 13cm Optimal Medical Products Pte Ltd STR – SH/BL
Reverse force hemostatic clip Shanghai Jin Zhong Pte Ltd XEC230
Water bath Grant
Reagents/Chemicals
10X Phosphate buffered saline (PBS) Sigma-Aldrich
Bovine serum albumin (BSA) Sigma-Aldrich A9056
CaCl2·2H2O Merck 137101
Collagenase Type IV Gibco 17104019
Dexamethasone TCI D1961
DMEM Gibco 31600-034
Glutamax Gibco 35050061
HEPES Invitrogen 11344-041
Insulin Sigma-Aldrich 1-9278
KCl VWR VWRC26764.298
KH2PO4 Sigma-Aldrich P5379
Linoleic acid Sigma-Aldrich L9530
NaCl Sigma-Aldrich S5886
NaHCO3 Sigma-Aldrich S8875
NaOH Merck 106462
Penicillin-Streptomycin Sigma-Aldrich P4333
Type I bovine collagen Advanced BioMatrix 5005-100ml
William’s E Media Sigma-Aldrich W1878

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