I am working on the isolation of primary hepatocytes from mouse liver using a two-step collagenase perfusion method. I am using 0.05% Collagenase-B (Roche) in Gibco Hepatocyte Wash Medium for digestion. The procedure works well for male mice, and I obtain hepatocytes with good viability. However, when isolating hepatocytes from female mice, I consistently fail to get viable cells.

I suspect there might be sex-based physiological differences affecting digestion efficiency or hepatocyte integrity. Has anyone else observed this issue?

I would appreciate any insights, troubleshooting tips, or alternative protocols that have worked for female mouse hepatocyte isolation.

Thank you!