title: Heart valve cryopreservation: protocol for addition of dimethyl sulphoxide and amelioration of putative amphotericin B toxicity.
abstract: OBJECTIVE
  To investigate the need for stepwise addition of dimethyl sulphoxide to heart valves and amelioration of putative amphotericin B toxicity.
  
  
  METHODS
  There were four groups: an untreated control (Group 1) and three experimental groups. For the latter, porcine heart valves were exposed to the antibiotic/antimycotic mixture used for disinfecting heart valves in the Bristol Heart Valve Bank, for 24 h at 22 degrees C. Dimethyl sulphoxide (Me2SO, 10% v/v) was added either in two steps (5% then 10%) (Group 2) or in a single step. For single-step addition, valves were either first placed in Hanks' balanced salt solution for 10 min before transfer to the cryoprotectant solution (Group 3) or immersed directly in the 10% cryoprotectant solution (Group 4). The valve leaflets were dissected from the valves and frozen in 10% Me2SO in multi-well tissue culture plates at 1 degrees C/min to -80 degrees C. After storage overnight, the valve leaflets were warmed at approximately 11 degrees C/min and the cryoprotectant was removed by single-step dilution in excess Hartmann's solution. Each leaflet was then divided into four pieces, which were placed in separate wells of a culture plate. Outgrowth of cells from the explants was monitored daily and graded according to the extent of cell growth.
  
  
  RESULTS
  After freezing and thawing, only 77% of the explants from valves placed directly into 10% Me2SO (Group 4) showed outgrowth of cells after freezing compared with 89% with two-step addition of Me2SO (Group 2) and 95% with one-step addition after the extra rinse in Hanks' solution (Group 3) (chi2, p=0.001). 92% of unfrozen control explants showed outgrowth of cells (Group 1). Only 37% of Group 4 explants reached confluence compared with 63 and 56%, respectively, of Groups 2 and 3 explants (chi2, p=0.007). The rates of cell growth in Group 2 (two-step addition of Me2SO) and Group 3 (one-step addition of Me2SO with additional Hanks' solution rinse) were similar and faster than the Group 4 (one-step addition of Me2SO without the additional Hanks' rinse).
  
  
  CONCLUSIONS
  Single-step addition of Me2SO before freezing gave similar results to two-step addition provided an additional rinse in Hanks' solution was introduced after exposure to the antibiotic/antimycotic mixture. This suggests that antibiotic/antimycotic carryover may have been harmful during freezing and that the additional rinse in Hanks before one-step addition of Me2SO, and the 5% Me2SO step in the two-step protocol, merely served to reduce this carryover.
authors.affiliation: Department of Clinical Sciences, Bristol Heart Valve Bank, University of Bristol, UK.
authors.forename: Vassilios
authors.initials: V
authors.lastname: Birtsas
authors.forename: W John
authors.initials: WJ
authors.lastname: Armitage
mesh_terms: D000666:Amphotericin B / Q000633:toxicity*; D000818:Animals; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents; D004121:Dimethyl Sulfoxide; D006351:Heart Valves / Q000166:cytology / Q000187:drug effects; D007552:Isotonic Solutions; D013552:Swine
chemical_list: D003451:Cryoprotective Agents; C072670:Hanks Balanced Salt Solution; D007552:Isotonic Solutions; D000666:Amphotericin B; D004121:Dimethyl Sulfoxide