The regenerative capacity of hepatocyte organoids following long-term cryopreservation in Republic of Korea

Background: Hepatocyte organoids (HOs) are more functionally versatile than bile duct epithelial cell organoids, but have a shorter lifespan. This limitation has been improved by optimizing culture methods, but could remain a barrier to their wider application in research and therapy, especially for use at the appropriate time.Methods: This study aimed to prolong the lifespan of pig HOs and assess their viability and functionality after a year of cryopreservation. Genes involved in apoptosis (TP53, P21, CASP8) and liver function (ALB, CYP3A29, EPCAM) were analyzed to evaluate the impact of adipose-derived mesenchymal stem cell (A-MSC) co-culture before and after freezing on cryoresistance. HOs were cut into fragments, cryopreserved for a year, and cultured alone or co-cultured with A-MSCs in Matrigel post-thawing.Results: After thawing, co-cultured HOs exhibited a higher development rate than those cultured alone. P21 expression increased irrespective of pre-freezing culture conditions. The ALB and CYP3A29 expression patterns resembled those of non-frozen HOs, with similar effects from co-culture. EPCAM expression surged post-freezing.Conclusion: This study demonstrates that HOs maintain liver function post-preservation, showing increased EPCAM expression and enhanced regenerative capacity against cryoinjury. These findings suggest that HOs may be a valuable cell source for drug development in animals and research on medications for human diseases.