title: Cryopreservation of encapsulated liver spheroids for a bioartificial liver: reducing latent cryoinjury using an ice nucleating agent.
abstract: BACKGROUND
  Acute liver failure has high mortality due to donor organ shortages. A bioartificial liver could "bridge the gap" to transplant or spontaneous recovery. Alginate encapsulation of HepG2 cells enables cell spheroid formation, thus providing sufficient functional biomass. Cryopreservation (CryoP) of these spheroids would allow an off-the-shelf capability for unpredictable emergency use. Cell death during CryoP often results from intracellular ice formation, after supercooling. An ice nucleating agent (INA), crystalline cholesterol, was trialled to reduce supercooling and subsequent cryoinjury.
  
  
  METHODS
  Spheroids were cooled in a controlled rate freezer in 12% dimethylsulfoxide/Celsior +/- INA, and sample temperatures were recorded throughout. Viability was assessed using fluorescent staining with image analysis, cell number by nuclei count, function using assays to detect liver-specific protein synthesis and secretion, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction, and broad-spectrum cytochrome P450 activity.
  
  
  RESULTS
  Spheroids cryopreserved without INA displayed latent cryoinjury in the first 6 h after thawing. INA reduced supercooling during CryoP and also latent cryoinjury. Cell numbers, viability, and function as measured over 72 h post-thaw were all improved when INA was present during CryoP.
authors.affiliation: Centre for Hepatology, University College Medical School, Hampstead, London. i.massie@medsch.ucl.ac.uk
authors.forename: Isobel
authors.initials: I
authors.lastname: Massie
authors.forename: Clare
authors.initials: C
authors.lastname: Selden
authors.forename: Humphrey
authors.initials: H
authors.lastname: Hodgson
authors.forename: Barry
authors.initials: B
authors.lastname: Fuller
mesh_terms: D002452:Cell Count; D002462:Cell Membrane / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002784:Cholesterol / Q000494:pharmacology*; D015925:Cryopreservation / Q000379:methods*; D005615:Freezing; D056945:Hep G2 Cells; D006801:Humans; D007053:Ice; D008099:Liver / Q000473:pathology*; D019164:Liver, Artificial; D010084:Oxidation-Reduction / Q000187:drug effects; D018874:Spheroids, Cellular / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*
chemical_list: D007053:Ice; D002784:Cholesterol