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 percent 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 about 90 percent, 70 percent and 65 percent of the controls respectively. This technology offers a clinic alternative to liquid nitrogen-coolant cryopreservation.