oa Mechanism of Cardiovascular Tissue Immunogenicity Reduction by Ice-free Cryopreservation

A variety of reasons for allograft heart valve failure have been discussed in the past and most investigators have emphasized immunological issues. Standard quantitative and qualitative cellular and matrix evaluations have not helped to solve the discussion of whether remaining allogeneic cells or potentially altered extracellular matrix contributed to the observed degeneration. Preliminary data on patients treated with decellularized allografts has recently demonstrated that decellularization did not significantly improve outcome in terms of pressure gradients and structural deterioration compared to non-decellularized allografts. These early clinical results question the validity of theories suggesting that an immune reaction to the remaining donor cells in allogeneic heart valves is the sole cause of structural deterioration.Porcine and ovine pulmonary and aortic heart valves were cryopreserved using traditional cryopreservation by freezing with 10% dimethylsulfoxide or ice-free cryopreservation in an 83% cryoprotectant formulation consisting of 4.65 mol/L dimethylsulfoxide, 4.65 mol/L formamide and 3.31 mol/L 1,2-propanediol. Cell viability was assessed using a water soluble fluorometric viability oxidation-reduction (REDOX) indicator which detects metabolic activity by both fluorescing and changing color in response to chemical reduction of the growth medium. Statistical analyses were performed using a t-test or one-way analysis of variance, p values<0.05 were considered statistically significant. Viability assessment revealed that heart valve tissues were significantly less viable in ice-free cryopreserved valves compared with frozen valves, p<0.05, due to cryoprotectant cytotoxicity. Juvenile sheep studies demonstrated that ice-free cryopreserved heart valves had minimal T-cell mediated inflammation in the valve leaflet stroma compared with frozen controls. Severe valvular stenosis with right heart failure was observed in recipients of frozen valves, the echo data revealed increased velocity and pressure gradients compared to ice-free valve recipients (p=0.0403, p=0.0591). In vitro studies have demonstrated retention of hemocompatibility, biocompatibility and reduction of ice-free cryopreserved heart valve tissue immunogenicity. Based upon these observations, it is hypothesized that preservation of extracellular matrix structure due to the absence of ice and minimal cell viability due to cryoprotectant cytotoxicity combine to decrease tissue repair activity and reduced immunogenicity. Work in progress is extending ice-free cryopreservation to other cardiovascular and orthopedic tissue engineering applications including in vitro and in vivo cell repopulation.