Cryopreservation of Encapsulated Liver Spheroids Using a Cryogen-Free Cooler: High Functional Recovery Using a Multi-Step Cooling Profile

Acute liver failure has high mortality with unpredictable onset. A bioartificial liver, comprising alginate-encapsulated HepG2 spheroids, could temporarily replace liver function but must be cryopreservable. For clinical use, contamination risks from liquid coolants for cryopreservation and storage should be minimized. A cryogen-free cooler was compared to nitrogen vapour-controlled cryopreservation of alginate-encapsulated liver cell spheroids (AELS). AELS were cooled using a multi-step, slow-cooling profile in 12% v/v Me₂SO/Celsior and stored in liquid nitrogen; temperatures were recorded throughout, and the AELS were assayed at 24, 48 and 72 hours post-warming and results compared to unfrozen control values. Viability was assessed by fluorescent staining and quantified using image analysis; cell numbers were quantified using nuclear counts, and cell function using albumin synthesis. The cryogen-free cooler performed the cooling profile as desired, apart from one step requiring a rapid cool ramp. Viability, cell numbers and function were similarly decreased in both cryopreserved groups to ∼90%, 70% and 65% of the controls respectively. This technology offers a clinic alternative to liquid nitrogen-coolant cryopreservation.