High-yield isolation of primary human hepatocytes from small liver samples

Area of Science:

• Hepatology
• Cell Biology
• Drug Development

Background:

• Primary human hepatocytes (PHH) isolation is crucial for research but traditional perfusion methods are resource-intensive.
• Existing non-perfusion methods often yield insufficient numbers of viable hepatocytes and lack purity assessment.

Purpose of the Study:

• To develop and optimize a non-perfusion protocol for isolating high yields of viable PHH from non-perfusable liver specimens.
• To provide a practical alternative to resource-intensive perfusion techniques.

Main Methods:

• Optimized protocol involves slicing liver tissue into 350 μm sections using a vibratome.
• A two-step digestion using ethylenediaminetetraacetic acid (EDTA) and collagenase was employed.
• Cell yield and viability were assessed via propidium iodide staining; cell populations were characterized by immunofluorescent imaging.

Main Results:

• The optimized protocol yielded approximately 1.17 × 10⁶ viable PHH per gram of tissue, a 2-fold increase over other non-perfusion methods.
• Achieved an average hepatocyte viability of 80%, surpassing published non-perfusion methods.
• Demonstrated functional integrity through glycogen staining and albumin secretion; effective with steatotic liver tissue.

Conclusions:

• The developed non-perfusion protocol successfully isolates viable and functional PHH from various liver samples.
• This method serves as a practical alternative to perfusion, expanding the utility of PHH in research and drug development.