Cryopreservation demands that lethal intracellular freezing (IIF) not occur. There are two routes to its prevention. One is referred to as equilibrium slow cooling. In this procedure, cells are cooled slowly enough so that they lose nearly all their water osmotically before reaching the nucleation temperature at which IIF becomes possible. Many cell types can be easily and successfully cryopreserved by this method, but many others can not, one example being mouse and human oocytes. The second route to avoiding IIF is to subject cells to vitrification procedures, which convert cell water into a glass. To achieve vitrification, the belief is that cells have to be suspended in high concentrations of a permeating CPA and cooled and warmed at high rates, with a reciprocal relation between CPA concentration and rate. This has led us into an investigation in mouse oocytes of the effects of cooling and warming rates and holding temperatures on the growth of intracellular ice by recrystallization during warming. We have found that this lethal process occurs over a range of at least -80?C to -50?C with a large increase in rapidity with increasing temperature. Companion studies with """"""""vitrified"""""""" oocytes indicate that their survival depends almost entirely on the warming rate with cooling rate having relatively little effect. A new specific aim will pursue further studies on this matter. An important question is how does external ice cross the cell membrane to initiate ice nucleation within the cell. A corollary in multi-cellular systems is how does internal ice in one cell propagate to its neighbors? To investigate these questions, we are proposing in this supplemental application to intensify our study of whether two types of pores in two types of cell systems serve as routes for ice transmission. The two types of pores are those in aquaporins and those in gap junctions. The first cell type is the mouse 8-cell/morula embryo. Early 8-cell embryos possess neither aquaporins or gap junctions;late 8-cell (= early morula) embryos possess both. The second cell type are tissue-culture cells;namely, hamster V79 cells and human neuroblastoma cells. By appropriate transfection, both can be obtained with or without functional aquaporins or gap junctions. An important element of the latter work will be the involvement of Dr. David Spray (Einstein College of Medicine) as a collaborator. Dr. Spray is a world authority on gap junctions. In April, 2007, NCRR held a workshop to review the status of the cryopreservation of sperm, oocytes, and embryos of important laboratory research animals, including the zebrafish;however, to date, all attempts to cryopreserve their embryos have failed. For several reasons, immature oocytes may be more amenable. One reason is that their permeability to water and CPA is considerably higher than that of mature oocytes or embryos. Consequently, we propose in a new specific aim to investigate IIF in the immature oocytes. This study will be strongly enhanced by the addition of Dr. Mary Hagedorn (Smithsonian Institution) as a co-investigator. Her expertise is zebrafish cryobiology, an area in which she has made major contributions.
(provided by applicant): The ability to preserve cells by freezing to low temperatures has important implications and applications to assisted reproduction and tissue transplantation in medicine, to improving agricultural productivity, to the maintenance of the gemplasm of genetically important laboratory animals far more cost effectively that its maintenance by living colonies, and to the preservation of the germplasm of endangered species.
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