Introduction: Cell transplantation therapy has garnered considerable attention as a promising alternative to organ transplantation. Our research has focused on hepatocytes derived from non–heart-beating donors (NHBDs), with prior reports on the efficacy of two-layer preservation methods for cardiac death donor livers and the utility of microencapsulation for cryopreserving hepatocytes. In this study, we conducted a fundamental investigation into a simple and broadly applicable approach for hepatocyte isolation using an oxygenated perfusion solution (pO2 approximately 500 mmHg) and evaluated its effects on cell viability and function.

Materials and Methods: Male SD rats were euthanized to induce cardiac arrest, followed by 30 minutes of warm ischemia. Livers were then processed for hepatocyte isolation and culture using either:

Group 1: Hanks’/collagenase solution, or

Group 2: Oxygenated Hanks’/collagenase solution.

A control group consisted of hepatocytes isolated from living rat donors. We assessed hepatocyte viability immediately after isolation and evaluated functional capacity by measuring albumin production over time in culture.

Results: Post-isolation cell viability was 85.2 ± 3.8% in the control group, 56.1 ± 3.2% in Group 1, and 65.7 ± 4.3% in Group 2. Oxygenated perfusion yielded significantly higher viability compared with the non-oxygenated solution. Regarding albumin production, Group 2 showed lower levels than the control group on days 3, 5, and 7; however, values were significantly higher than those in Group 1 at these time points. Notably, on days 1, 10, and 14, albumin production in Group 2 exceeded both Group 1 and the control group, showing statistical significance. Morphological assessments revealed that after day 7 of culture, Group 2 maintained a greater number of viable hepatocytes compared with Group 1.

Conclusion: Oxygenated perfusion solutions significantly enhance post-isolation hepatocyte viability and prolong functional maintenance compared with conventional non-oxygenated preparations in hepatocytes from NHBDs subjected to warm ischemia. This simple yet effective modification to the isolation protocol may broaden the applicability of marginal donor livers for cell transplantation therapy, contributing to an expanded donor pool and improved clinical feasibility.