Background: Liver transplantation is the only curative option for end-stage liver disease, but its applicability is limited by donor shortages. Hepatocyte transplantation offers a promising alternative, particularly for paediatric acute liver failure, yet its success relies on the availability of viable cells. Cryopreservation enables off-the-shelf use, but long-term storage may impair cell quality. This study investigates how cryostorage duration, donor characteristics, and organ retrieval parameters affect the viability and functionality of cryopreserved primary human hepatocytes (PHHs).

Methodology: To investigate how the duration of cryostorage influences the post-thaw viability and functionality of PHHs, and to assess the impact of donor-related factors, including age, body mass index (BMI), warm ischemia time (WIT), and cold ischemia time (CIT), on overall cell quality. We conducted a retrospective analysis of 144 thawing events across 81 GMP-grade hepatocyte batches cryopreserved for up to 14 years. Donor age ranged from 3 days to 70 years. Viability and functionality were assessed using MTT assays, albumin and urea secretion, and CYP450 activity. Linear regression was used to assess correlations (p < 0.05 considered significant).

Results: Cryopreservation duration had no significant impact on hepatocyte viability or function, confirming that clinical-grade PHHs can maintain quality for up to 14 years. In contrast, donor age correlated negatively with post-thaw viability (R² = 0.035, p = 0.029), yield (R² = 0.039, p = 0.041), and metabolic activity (R² = 0.278, p = 0.008), while showing a positive correlation with induced EROD function (R² = 0.432, p = 0.020). Donor BMI showed significant negative associations with viability (R² = 0.075, p = 0.003), yield (R² = 0.069, p = 0.011), and metabolic activity (R² = 0.267, p = 0.019). CIT showed a weak negative correlation with viability (R² = 0.151, p < 0.0001), particularly below 60% viability when CIT exceeded ~10 hours.

Conclusions: Clinical-grade cryopreserved hepatocytes retain viability and function for over a decade. CIT, donor BMI, and age most affected post-thaw quality, while WIT and storage duration had minimal impact. These findings support refining donor selection to improve cryopreservation outcomes.