title: 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: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors: Centre for Hepatology at the Royal Free, UCL-Royal Free Hospital Campus, University College Medical School, Hampstead, London, United Kingdom.
authors: Isobel
authors: I
authors: Massie
authors: Clare
authors: C
authors: Selden
authors: Humphrey
authors: H
authors: Hodgson
authors: Barry
authors: B
authors: Fuller
mesh_terms: D002470:Cell Survival; D020099:Coated Materials, Biocompatible / Q000737:chemistry*; D003080:Cold Temperature; D015925:Cryopreservation / Q000379:methods*; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000166:cytology* / Q000502:physiology; D006801:Humans; D019164:Liver, Artificial; D009926:Organ Preservation / Q000379:methods*; D018874:Spheroids, Cellular / Q000166:cytology* / Q000502:physiology
chemical_list: D020099:Coated Materials, Biocompatible