60. Investigation of osmotic tolerance limits for rational design of vitrification procedures

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• 作者：Allyson K. Fry ; Adam Z. Higgins ^{adam.z.higgins@oregonstate.edu}
• 刊名：Cryobiology
• 出版年：2012
• 出版时间：December, 2012
• 年：2012
• 卷：65
• 期：3
• 页码：358-359
• 全文大小：47 K

文摘

Previously, we reported a new strategy for mathematical optimization of CPA addition and removal procedures which involves minimization of a toxicity cost function while constraining cell volumes within osmotic tolerance limits. The purpose of this study was to clearly define cellular osmotic tolerance limits of bovine pulmonary artery endothelial cells for inclusion in our optimization routine. Our general experimental approach consisted of exposure to anisotonic solutions for a prescribed time period, return to isotonic solution, and measurement of cell viability. The variables investigated include the buffering system, the type of non-permeating solute, the duration of exposure to anisotonic solutions, and the temperature. Primary bovine endothelial cells cultured in 96 well plates were exposed to solutions with concentrations ranging from pure water to 4000 mOsm/kg. Viability was assessed directly before and 24 h after treating with test solutions using the resazurin-based PrestoBlue assay. The resulting hypotonic and hypertonic viability trends were fit with 3-parameter logistic models to facilitate identification of osmotic tolerance limits. The best-fit logistic models were used to determine the hypo- and hypertonic concentrations that resulted in 90 % cell yield relative to untreated cells. We first investigated the effect of the buffering system on sensitivity to osmotic damage. Cells retained greater viability after 15 min exposure to anisotonic solutions that were buffered with HEPES rather than phosphate. Thus, subsequent experiments were performed using HEPES buffered test solutions. We next investigated the effects of the type of non-permeating solute and the temperature on sensitivity to osmotic damage. Hypertonic solutions composed of NaCl were less damaging than sucrose solutions at 4 ¡ãC and 21 ¡ãC, however, sucrose solutions were less damaging than NaCl at 37 ¡ãC. Hypertonic solutions composed of either non-permeating solute resulted in significant death at 37 ¡ãC, even at relatively low concentrations. We investigated this effect further by varying exposure times from 1 to 20 min. For each solution concentration and temperature, an exponential decay model described the time-dependent loss of viability. Viability declined more rapidly with higher temperatures and greater solution concentrations. These findings indicate that it may be beneficial to use concentration- and time-dependent osmotic tolerance limits for optimization of CPA addition and removal procedures.