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Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control
Published on: April 5, 2021
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Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control
Inn Chuan Ng1, Li Zhang2, Narelle Nichola Yi Ying Shen3
1Department of Physiology & The Institute for Digital Medicine (WisDM), National University of Singapore.
Journal of Visualized Experiments : Jove
|April 19, 2021
Summary
This study presents an improved collagenase perfusion protocol for reliable isolation of primary rat hepatocytes. The enhanced method ensures high cell viability, yield, and functionality for liver disease research and toxicity testing.
Area of Science:
- Hepatology
- Cell Biology
- Toxicology
Background:
- Primary hepatocytes are crucial for liver disease research and in vitro toxicity testing.
- Traditional collagenase perfusion methods for hepatocyte isolation are technically demanding and prone to variability.
- Challenges include portal vein cannulation, contamination, and maintaining consistent perfusion conditions.
Purpose of the Study:
- To present a detailed protocol for an improved two-step collagenase perfusion procedure.
- To enhance reliability and reduce operational difficulty in primary rat hepatocyte isolation.
- To implement multiparameter perfusion control for consistent results.
Main Methods:
- Utilized a modified two-step collagenase perfusion technique.
- Incorporated a special intravenous catheter and standardized sterile disposable tubing.
- Implemented temperature control and a real-time monitoring and alarm system for perfusion parameters.
- Employed an integrated, compact perfusion system for aseptic operation within a laminar flow hood.
Main Results:
- Successfully and reliably isolated primary rat hepatocytes.
- Achieved high cell viability (85%-95%).
- Obtained consistent high cell yield (2-5 x 10^8 cells per 200-300 g rat).
- Demonstrated preserved hepatocyte functionality, including albumin and urea synthesis and cytochrome P450 (CYP) activity.
Conclusions:
- The improved protocol ensures consistent, high-quality primary rat hepatocyte isolation.
- The method mitigates variability and operational challenges associated with traditional techniques.
- This optimized procedure supports robust liver disease research and in vitro toxicity assessments.
