Freeze Substitution has historically been used for processing small pieces of tissue for microscopic examination. These small pieces of tissue have usually been ultra-rapidly frozen by either metal mirror, high pressure or immersion fixation. In today's expanding field of cryobiology, freeze substitution is being employed to better understand the effects of ice formation and possible deformation of cell structure in tissues that have been processed by controlled-rate freezing. This technique is of extreme importance in laboratories focused on developing methods of preservation by freezing tissues, such as heart valves or veins, which are intended, for clinical applications.

Overview: Many laboratories use methods of substitution, which incorporate acetone as the ice dissolution solvent. We have done comparative studies using both acetone and methanol as a substitution media at -80° C and -90° C. Theoretically, either of the two methods should give satisfactory results provided one uses extremely small tissue samples and follows the accepted guidelines for substitution. Since our protocol requires the use of relatively large samples, which are encased in a voluminous amount of media it is obligatory to adhere strictly to a methanol (-90° C) substitution regimen to be assured complete substitution has taken place (Table 1). Porcine heart valves were dissected into segments consisting of the aortic wall and leaflet. Segments were exposed to a variety of new cryoprotective agents (CPAs) and placed in the center of a parafilm sleeve with 1.5ml of CPA and cooled at 1° C/min to −80°and stored in the vapor phase of liquid nitrogen. Samples were processed for either routine histological studies or freeze-substituted to obtain profiles of ice formation.