Possible Applications of Directional Solidification Techniques in Cyobiology

Abstract

During freezing of cell suspensions, the cooling rate achieved generally varies markedly throughout the bulk sample. As the cooling rate strongly affects cell survival, this may lead to a variation of cell survival with position in the sample. To avoid these problems, in many basic investigations microscopic sample volumes are used. But even in this case effects like the undercooling of the aqueous samples occur that prevent the study of cells under well defined thermal conditions. Thus the directional solidification of microscopic biological samples according to the Bridgman-technique was introduced to cryobiology. This technique allows the highly defined freezing of small samples and the independent control of the two fundamental solidification parameters, ice front velocity, v, and temperature gradient, G. The cooling rate B is simply the product of the two parameters, v and G, and can easily be adjusted. Applications of the Bridgman-technique are the study of the crystallization morphology (e. g. measurement of the solute pile up and the breakdown time, dendrite spacings, tip radius, electrical potentials, etc.) and the study of the interactions between growing ice crystals and biological cells (e. g. intracellular ice formation, encapsulation or rejection of cells). But although the advantages are obvious for microscopic samples, the Bridgman-technique cannot be adapted to the cryopreservation of bulk systems. In this case an alternative approach, the directional solidification according to the power-down technique can be applied. The aqueous sample is enclosed between two blocks which provide the temperature control. After a constant temperature gradient is established between the two blocks both block temperatures are lowered simultaneously with identical cooling rates. The ice front then grows with a constant velocity from the cold to the warm block. Freezing simulations and experimental results show that the power-down technique offers a great potential for future studies. Possible applications are the cryopreservation of delicate cell types which require uniform cooling conditions (e. g. heart muscle cells, oocytes), basic investigations on the mechanisms of cell and tissue damage during freezing (cryosurgery), and the processing of frozen samples with a defined and uniform microstructure (morphology studies in bulk samples, improved freeze-drying protocols, and processing of biomaterials with defined pore sizes).