Comparative Evaluation of Freezing Protocols and Cryopreservation Media for the Preservation of Alginate Encapsulated HepaRG Cells

Reliable storage and distribution are important for developing ready-to-use encapsulated cell products for transplantation and bioartificial liver applications. This study evaluated the cryopreservation of alginate-encapsulated HepaRG cells using a programmable controlled-rate freezing (CRF) protocol at −0.3 °C/min or conventional passive cooling at approximately −1 °C/min. Five media were tested: four commercial xeno-free formulations, including two DMSO-containing and two DMSO-free media, and a conventional formulation containing 90% fetal bovine serum and 10% DMSO. Following thawing and a 3-day recovery period, metabolic activity, albumin secretion, ammonia and lactate clearance, and phase I and phase II biotransformation activities were assessed. Cryopreservation reduced metabolic activity relative to non-frozen controls, but key hepatic functions remained detectable. The tested CRF protocol generally yielded equal or higher functional values than conventional freezing. Medium performance varied according to the endpoint assessed. Within CRF, CryoStor CS10 produced relatively high mean PrestoBlue and albumin secretion values, whereas Cellvation produced the highest mean CYP1A1/1A2 activity. These product-specific observations do not establish a general effect of DMSO. The findings support the short-term feasibility of the tested CRF protocol for this encapsulated HepaRG model but not broader clinical or drug-testing applications.