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

Purpose: Isolation of primary human hepatocytes (PHH) from liver specimens typically relies on the two-step perfusion method, which requires large samples, substantial resources, specialised expertise and suitable vessels for cannulation. Although non-perfusion methods exist, they yield low numbers of hepatocytes and inadequately assess hepatocyte purity. We compared and optimised these methods to develop an improved technique that isolates high yields of viable PHH from non-perfusable liver specimens. Method: In the optimised protocol, non-cancerous resected liver tissue (mean weight: 8.5 ± 2.0 g, SEM) was sliced into 350 μm sections using a vibratome and subjected to a two-step isolation digestion with ethylenediaminetetraacetic acid (EDTA) and collagenase. Cell yield and viability were assessed using propidium iodide staining. Cell populations were characterised by immunofluorescent imaging. Results: The optimised protocol yielded 1.17 ± 0.2 × 10 Conclusions: Our non-perfusion protocol permits the isolation of viable and functional PHH from a diverse range of liver samples. This advancement provides a practical alternative to perfusion methods and will extend the use of PHH in research and drug development. Supplementary Information: The online version contains supplementary material available at 10.1007/s44164-025-00097-4.