Protocols for thawing and cryoprotectant dilution of heart valves.
OBJECTIVE
To reduce the time taken for thawing and removal of cryoprotectant from heart valves.


METHODS
Three sets of experiments were carried out using porcine heart valves. The valves in all three experiments were first exposed to 10% (v/v) dimethyl sulphoxide (DMSO) by a 2-step protocol. Outcome was determined after the various experimental treatments by monitoring the outgrowth of cells from valve leaflet explants. Experiment 1-Dilution protocol. Valves exposed to 10% DMSO were subjected to 4-, 2- or 1-step dilution to remove the DMSO. Experiment 2-Warming rate. The rate of warming was increased by reducing the volume of cryoprotectant medium in which the valves were frozen. Valves were exposed to 10% DMSO, frozen in different volumes (100, 50, 25 or 0 ml) of cryoprotectant medium, and warmed in a 37 degrees C water bath. The DMSO was removed by 4-step dilution. Experiment 3-Standard vs. Modified protocol. Valves were either frozen in 100 ml 10% DMSO, thawed, and subjected to 4-step dilution (Standard) or frozen in 50 ml 10% DMSO, thawed, and the DMSO removed by single-step dilution (Modified).


RESULTS
Neither the rate of warming nor the rate of dilution of DMSO had any influence on the subsequent outgrowth of valve leaflet fibroblasts. There were no differences in the outgrowth of cells from valve leaflets cryopreserved by the Standard or Modified protocols.


CONCLUSIONS
The time taken for thawing and dilution of heart valves could be reduced from >20 min to <10 min without detriment to the viability of the leaflet fibroblasts. This should have a positive impact on valve replacement surgery as the thawing and dilution of valves are typically carried out while the patients are on cardiopulmonary bypass.
Armitage
W John
WJ
Bristol Heart Valve Bank, Department of Clinical Science, University of Bristol, Bristol, BS1 2LX, United Kingdom. w.j.armitage@bristol.ac.uk
Dale
Wayne
W
Alexander
Emily A
EA
D000818:Animals; D049109:Cell Proliferation; D015925:Cryopreservation / Q000295:instrumentation / Q000379:methods*; D003451:Cryoprotective Agents / Q000008:administration & dosage / Q000494:pharmacology*; D046508:Culture Techniques; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005615:Freezing; D006350:Heart Valve Prosthesis; D006351:Heart Valves / Q000187:drug effects* / Q000473:pathology*; D009924:Organ Culture Techniques / Q000379:methods; D013552:Swine; D013696:Temperature; D013997:Time Factors; D014021:Tissue Preservation / Q000379:methods
D003451:Cryoprotective Agents; D004121:Dimethyl Sulfoxide