Interstitial ice formation in cryopreserved homografts: A possible cause of tissue deterioration and calcification in vivo

Background and aim of the study: Cryopreserved valve homografts often fail in infants. Controversies are ongoing concerning the relative contributions of cryopreservation variables, immune responses, cellular viability, and d urability of the extracellular matrix to the mode of tissue failure. Methods: Tissues to be examined for patterns of ice crystal distribution we re cryopreserved. Tissue water was substituted with methanol and the tissue s cryopreserved conventionally using; dimethylsulfoxide, after which they w ere warmed and processed for light or electron microscopy. Selected specime ns were vitrified to prevent ice crystal formation, cryopreserved, and subs equently warmed and processed for light and electron microscopy. Results: Cryosubstitution of conventionally cryopreserved heart valves, whi le still frozen, demonstrated extensive extracellular ice formation, with s maller crystals in the ventricularis than in either the spongiosa or fibros a. Extracellular ice formation was prevented by vitrification, a process in which the biological system is stabilized as an amorphous solid in the abs ence of crystalline ice. Conclusion: It is proposed that the extensive ice formation observed in con ventionally cryopreserved heart valves may cause extracellular matrix damag e that predisposes the valves to calcification. Future studies will assess the above hypothesis by comparison of conventional and ice-free (vitrificat ion) cryopreservation methods in animal models of calcification.