Ice-free cryopreservation, known as vitrification, is an appealing approach for banking of adherent cells and tissues because it prevents dissociation and morphological damage that may result from ice crystal formation. However, current vitrification methods are often limited by the cytotoxicity of the concentrated cryoprotective agent (CPA) solutions that are required to suppress ice formation. Recently, we described a mathematical strategy for identifying minimally toxic CPA equilibration procedures based on the minimization of a toxicity cost function. Here we provide direct experimental support for the feasibility of these methods when applied to adherent endothelial cells. We first developed a concentration- and temperature-dependent toxicity cost function by exposing the cells to a range of glycerol concentrations at 21°C and 37°C, and fitting the resulting viability data to a first order cell death model. This cost function was then numerically minimized in our state constrained optimization routine to determine addition and removal procedures for 17 molal (mol/kg water) glycerol solutions. Using these predicted optimal procedures, we obtained 81% recovery after exposure to vitrification solutions, as well as successful vitrification with the relatively slow cooling and warming rates of 50°C/min and 130°C/min. In comparison, conventional multistep CPA equilibration procedures resulted in much lower cell yields of about 10%. Our results demonstrate the potential for rational design of minimally toxic vitrification procedures and pave the way for extension of our optimization approach to other adherent cell types as well as more complex systems such as tissues and organs.
BackgroundSimple and effective cryopreservation of human oocytes would have an enormous impact on the financial and ethical constraints of human assisted reproduction. Recently, studies have demonstrated the potential for cryopreservation in an ice-free glassy state by equilibrating oocytes with high concentrations of cryoprotectants (CPAs) and rapidly cooling to liquid nitrogen temperatures. A major difficulty with this approach is that the high concentrations required for the avoidance of crystal formation (vitrification) also increase the risk of osmotic and toxic damage. We recently described a mathematical optimization approach for designing CPA equilibration procedures that avoid osmotic damage and minimize toxicity, and we presented optimized procedures for human oocytes involving continuous changes in solution composition.MethodsHere we adapt and refine our previous algorithm to predict piecewise-constant changes in extracellular solution concentrations in order to make the predicted procedures easier to implement. Importantly, we investigate the effects of using alternate equilibration endpoints on predicted protocol toxicity. Finally, we compare the resulting procedures to previously described experimental methods, as well as mathematically optimized procedures involving continuous changes in solution composition.ResultsFor equilibration with CPA, our algorithm predicts an optimal first step consisting of exposure to a solution containing only water and CPA. This is predicted to cause the cells to initially shrink and then swell to the maximum cell volume limit. To reach the target intracellular CPA concentration, the cells are then induced to shrink to the minimum cell volume limit by exposure to a high CPA concentration. For post-thaw equilibration to remove CPA, the optimal procedures involve exposure to CPA-free solutions that are predicted to cause swelling to the maximum volume limit. The toxicity associated with these procedures is predicted to be much less than that of conventional procedures and comparable to that of the corresponding procedures with continuous changes in solution composition.ConclusionsThe piecewise-constant procedures described in this study are experimentally facile and are predicted to be less toxic than conventional procedures for human oocyte cryopreservation. Moreover, the mathematical optimization approach described here will facilitate the design of cryopreservation procedures for other cell types.
Calcein is an intracellular fluorescent probe that has been used as an indicator of cell volume in several previous studies. These studies have reported two different fluorescence responses depending on the optical setup used to collect the data: wide-field microscopy has resulted in a decrease in fluorescence upon cell shrinkage, whereas confocal microscopy has been shown to yield the opposite result. In this short communication, we have investigated the effect of optical setup on detection of cell volume changes in calcein-stained endothelial cells. A confocal microscope was used to collect the fluorescence data, and the pinhole diameter was varied in order to examine the effects of optical section thickness on fluorescence response. For large pinhole diameters - which correspond to relatively thick optical sections - fluorescence intensity decreased when cells were induced to shrink. In contrast, for small pinhole diameters the fluorescence intensity increased with cell shrinkage. The transition between these two types of fluorescence responses occurred when using a pinhole diameter of 285 μm, which corresponds with an optical section thickness slightly less than the height of the cells. Our results have implications for the design and interpretation of experiments involving the use of calcein as a cell volume indicator.
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