Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

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 procedure^1,2,3 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) ....

Protocol

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.

Discussion

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.......

Materials

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