Structural, ultrastructural and functional studies of human cardiac valve allografts that suffered an increment of the cryostorage temperature.

Human cardiac valve allografts (HVAs) suffer injuries during the cryopreservation period. Here, we described structural, ultrastructural and functional damages suffered by HVAs after an increment of their cryostorage temperature (100 degree C). Two experimental groups of pulmonary and aortic HVAs were compared: cryopreserved (HVAcryo) and cryopreserved with temperature changes (HVAΔT). Transmission electron microscopy (TEM) was used to analyze valve fibroblasts and extracellular matrix morphology. Total collagen amount was estimated using two different methods and fibroblast viability was assessed measuring their oxygen consumption rate. Porcine heart grafts valves were used to set the techniques. Disorganized collagen network was seen in HVAΔT by TEM. Fibroblasts showed damages in the cellular membrane and many secretor vesicles. Mitochondria and chromatin were also altered. HVAΔT had less amount of collagen and fibroblasts showed an oxygen consumption rate markedly diminished compared to HVAcryo. The increment of 100 degree C suffered by HVAs caused damages that made them unsuitable for clinical purposes.

Morphological and biochemical analysis of human cardiac valve allografts after an increment of the cryostorage temperature.

Cryopreserved human cardiac valve allografts could suffer lethal damages if the temperature is elevated during cryostorage. This work describes the functional and morphological alterations suffered by human cardiac valve allografts after a gradual increment of the cryostorage temperature from -147 degrees C to -47 degrees C due to a technical failure. Three experimental groups of human pulmonary and aortic allografts were compared: fresh, cryopreserved (-147 degrees C) and cryopreserved with temperature changes from -147 degrees C up to -47 degrees C and back to -147 degrees C. Fibroblast functionality was studied to asses the degree of valvular damages. Collagen network was also analyzed with bright light field and polarized microscopy; an immunohistochemistry for procollagen I was performed and the MTT colorimetric assay was used to evaluate fibroblast mitochondrial enzymatic activity. Porcine heart grafts valves were used to set the MTT colorimetric assay. With bright light field microscopy, disorganized collagen network was seen together with interstitial edema in cryopreserved groups. Polarized microscopy showed that fresh allografts had abundant collagen type I and III, cryopreserved group had less amount of collagen type I and in allografts that suffered cryopreservation temperature elevation collagen type I synthesis could not been demonstrated. Procollagen I was present in fibroblast cytoplasm of fresh group, but it was diminished in cryopreserved group and was absent in the group that suffered temperature elevation. Temperature changes during the cryopreservation period of human cardiac valve allografts induced fibroblast activity reduction. When the cryopreservation temperature is elevated during cryostorage, fibroblasts lost their functionality and the allografts may be not suitable for transplant.