Isolation and Purification of Primary Human Liver Cells – Impact of Liver Pathology on the Outcome

Introduction: Large quantities of primary human hepatocytes are required for in vitro models enabling the investigation of metabolism and cytotoxicity of xenobiotics as well as studies in the field of regenerative medicine. Since availability is still scarce, liver samples from resected liver specimen are used as one possible cell source although isolation outcome is often unsatisfying.

Aim: We analyzed the impact of liver pathology on the outcome of the isolation and purification of liver cells obtained from resected liver specimen.

Method: Samples were taken from patients undergoing partial hepatectomy for various primary (Group 1, n=12) or secondary tumors (Group 2, n=11). Hepatocyte isolation was performed by a modified two-step enzymatic perfusion technique using collagenase P. After isolation, the resulting hepatocyte suspension was immediately purified by density gradient centrifugation using different concentrations of Percoll solution (density 1.124) ranging from 10–50%. Cell number and viability were determined by Trypan blue exclusion test.

Results: When the original cell suspension is subjected to a 25% Percoll gradient, we found the most satisfying degree of separation of dead and viable cells. Using this approach we increased the viability from 53.4±4.2% (mean±SE) to 81.9±2.5% (p<0.01). Regarding the liver pathology, there were no significant differences in the degree of purification. Hepatocyte purity in group 1 improved from 47.8±5.1% to 82.0±4.2% compared to a raise from 59.6±6.7% to 81.8±2.8% in group 2. Significant differences between group 1 and 2 were observed concerning the number of viable cells surviving Percoll purification. For group 1 only 48.6±7.4% of the cells survived purification procedure whereas survival in group 2 was 74.8±7.3%. When subdividing group 1 into HCC (n=5) and Klatskin-tumors (n=7), in the HCC group 67.9±11.7%, whereas in the group of Klatskin-tumors 34.7±5.7% of the viable cells survived.

Conclusion: Liver cells isolated from specimens resected for secondary tumors showed the best results regarding isolation and purification outcome and suggests that this tissue represents the most valuable cell source. Samples from specimen with primary tumors, especially Klatskin-tumors, are less yielding. However, using purification procedures, even those samples can be seen as useful cell sources.

Key words

liver cell isolation and purification - liver pathology - primary human liver cells