Storage Temperatures for Cold-Chain Delivery in Cell Therapy: A Study of Alginate-Encapsulated Liver Cell Spheroids Stored at −80°C or −170°C for Up to 1 Year

Abstract

A bioartificial liver comprising alginate-encapsulated liver cell spheroids (ELS) could bridge the gap to transplant or spontaneous recovery in acute liver failure, but will be required for emergency use, necessitating cryopreservation. A cryopreservation protocol has been developed, but beyond this, the feasibility of cold-chain storage is considered here. Cryopreservation will be increasingly required for timely delivery of tissue and bioengineered products, and significant, but often, over-looked factors that impact on cost and ease of clinical application are the storage temperature and useful preservation time. Storage in the vapor phase of liquid nitrogen (∼-170°C) is the gold standard, but for safety and economic purposes, storing ELS in electric freezers at -80°C may be preferable. ELS were cryopreserved using an optimized protocol and stored at either -80°C or at -170°C for up to 1 year. ELS were removed from storage after 1, 2, 3, 6, 9, or 12 months, and recovery was assessed 24 h postwarming. Cell recovery was assessed using viability (fluorescent staining with image analysis), cell number (nuclei count), and functional (hepatospecific protein enzyme-linked immunosorbent assay) assays. Viability, the viable cell number, and function of ELS stored at -170°C were maintained at similar values throughout the year. In contrast, ELS stored at -80°C exhibited decreased viability, viable cell numbers, and function by as early as 1 month. Progressive deterioration was subsequently observed. After 12 months of storage at -80°C, viable cell recovery of ELS was ∼15% that of ELS stored at -170°C. While convenience and cost might support the use of -80°C for storage of multicellular bioengineered products such as ELS, results indicate rapid deterioration in functional recoveries after only a few weeks. This study demonstrates that storage temperature is an important consideration in regenerative medicine and caution should be applied by limiting storage at -80°C to only a few weeks.