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[Obtaining a primary mouse hepatocyte culture]
Abstract:
A method of isolating and culturing mouse hepatocytes is described. The mouse liver was subsequently perfused in situ with Ca-free and collagenase containing solutions, mechanically dispersed and purified by centrifugation. The suspension obtained contained up to 10(7) parenchymal cells, whose viability being not less than 80-90%. In the suspension, the mean diameter of hepatocytes was 25 micron. On being plated on plastic Petry dishes in concentrations of 1-5.10(5)/ml and in the presence of serum, hepatocytes were attached to the substrate in 2-3 hours to form cell monolayers. The cultured hepatocytes maintained their viability for 5-7 days.
Insights
This study details a new method for isolating and culturing mouse hepatocytes, achieving high viability and cell monolayer formation. The technique ensures reliable primary hepatocyte cultures for research applications.
Area of Science:
- Hepatocyte isolation and cell culture techniques.
- Mouse liver perfusion and cell dispersion.
- Primary cell culture and monolayer formation.
Context:
- Hepatocytes are crucial for drug metabolism and toxicology studies.
- Efficient isolation and culture of primary hepatocytes are essential for in vitro research.
- Existing methods may have limitations in yield or viability.
Purpose:
- To describe a robust method for isolating and culturing mouse hepatocytes.
- To optimize cell dispersion and purification for high-purity hepatocyte suspension.
- To establish conditions for stable hepatocyte attachment and monolayer formation in culture.
Summary:
- A novel in situ liver perfusion method using Ca-free and collagenase solutions isolates mouse hepatocytes.
- Mechanical dispersion and centrifugation yield a high-purity parenchymal cell suspension (up to 10^7 cells) with 80-90% viability.
- Plating at 1-5x10^5 cells/ml with serum allows rapid attachment and monolayer formation, maintaining viability for 5-7 days.
Impact:
- Provides a reliable protocol for obtaining viable primary mouse hepatocytes.
- Facilitates advanced research in liver physiology, toxicology, and drug development.
- Enables consistent in vitro studies using primary hepatocyte models.