title: Cryoprotective agent toxicity interactions in human articular chondrocytes.
abstract: BACKGROUND
  Vitrification is a method of cryopreservation by which cells and tissues can be preserved at low temperatures using cryoprotective agents (CPAs) at high concentrations (typically ≥6.0 M) to limit the harmful effects of ice crystals that can form during cooling processes. However, at these concentrations CPAs are significantly cytotoxic and an understanding of their toxicity characteristics and interactions is important. Therefore, single-CPA and multiple-CPA solutions were evaluated for their direct and indirect toxicities on chondrocytes.
  
  
  METHODS
  Chondrocytes were isolated from human articular cartilage samples and exposed to various single-CPA and multiple-CPA solutions of five common CPAs (dimethyl sulfoxide (DMSO), ethylene glycol (EG), propylene glycol (PG), glycerol (Gy) and formamide (Fm)) at both 6.0 and 8.1 M concentrations at 0 °C for 30 min. Chondrocyte survival was determined using a fluorescent cell membrane integrity assay. The data obtained was statistically analyzed and regression coefficients were used to represent the indirect toxicity effect which a specific combination of CPAs exerted on the final solution's toxicity.
  
  
  RESULTS
  Multiple-CPA solutions were significantly less toxic than single-CPA solutions (P<0.01). The indirect toxicity effects between CPAs were quantifiable using regression analysis. Cell survival rates of approximately 40% were obtained with the four-CPA combination solution DMSO-EG-Gy-Fm. In the multiple-CPA combinations, PG demonstrated the greatest degree of toxicity and its presence within a combination solution negated any benefits of using multiple lower concentration CPAs.
  
  
  CONCLUSIONS
  Multiple-CPA solutions are less cytotoxic than single-CPA solutions of the same total concentration. PG was the most toxic CPA when used in combinations. The highest chondrocyte survival rates were obtained with the 6.0 M DMSO-EG-Gy-Fm combination solution.
authors.affiliation: Division of Orthopaedic Surgery, Department of Surgery, University of Alberta, Edmonton, AB, Canada T6G 2B7. almansoori@ualberta.ca
authors.forename: K A
authors.initials: KA
authors.lastname: Almansoori
authors.forename: V
authors.initials: V
authors.lastname: Prasad
authors.forename: J F
authors.initials: JF
authors.lastname: Forbes
authors.forename: G K
authors.initials: GK
authors.lastname: Law
authors.forename: L E
authors.initials: LE
authors.lastname: McGann
authors.forename: J A W
authors.initials: JA
authors.lastname: Elliott
authors.forename: N M
authors.initials: NM
authors.lastname: Jomha
mesh_terms: D002358:Cartilage, Articular / Q000166:cytology / Q000187:drug effects*; D002462:Cell Membrane / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects*; D003080:Cold Temperature; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000633:toxicity*; D004121:Dimethyl Sulfoxide / Q000633:toxicity; D004347:Drug Interactions; D019855:Ethylene Glycol / Q000633:toxicity; D005456:Fluorescent Dyes; D005559:Formamides / Q000633:toxicity; D005990:Glycerol / Q000633:toxicity; D006801:Humans; D019946:Propylene Glycol / Q000633:toxicity; D012044:Regression Analysis; D058989:Vitrification
chemical_list: D003451:Cryoprotective Agents; D005456:Fluorescent Dyes; D005559:Formamides; C031066:formamide; D019946:Propylene Glycol; D019855:Ethylene Glycol; D005990:Glycerol; D004121:Dimethyl Sulfoxide