[Obtaining a primary mouse hepatocyte culture]

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.