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This study presents a theoretical model to develop efficient ISF procedures, on …


Biology Articles » Cryobiology » Cryobiology of Rat Embryos II: A Theoretical Model for the Development of Interrupted Slow Freezing Procedures » Figures

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- Cryobiology of Rat Embryos II: A Theoretical Model for the Development of Interrupted Slow Freezing Procedures

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FIG. 1. A schematic diagram showing that the intracellular solute concentration reaches the critical concentration ([CPA]c) at different temperatures (A, B, and C) for different cooling conditions a, b, and c, respectively. The figure represents three situations in which the initial CPA concentrations are the same but the cooling rates vary

Figure 1

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FIG. 2. Panel A represents changes in sample temperature (top) and cooling rate (bottom) when 0.25-ml straws are plunged into LN2. Panel B represents temperature (top) and warming rate (bottom) when 0.25-ml straws are placed into a 37°C water bath

Figure 2

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FIG. 3. The solid line shows measured intracellular nucleation temperatures of rat zygotes in 1.5 M DMSO cooled at 10°C/min. The data represent the percentage of zygotes with IIF from three experimental runs (a total of 28 rat zygotes). The dotted line shows the corresponding predicted values of IIF in rat zygotes calculated using Mazur's model of IIF [11]. The dot-dash line shows the IIF predictions for mouse oocytes in the presence of glycerol for comparison (redrawn from Karlsson et al. [7])

Figure 3

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FIG. 4. A diagram showing the plane of initial CPA concentration and cooling rate divided into three regions. In region I (dotted), no combination of initial CPA concentration ([CPA]0) and cooling rate (B1) allows the intracellular solute concentration ([S]i) to reach the critical concentration ([CPA], 40%) necessary for intracellular vitrification to occur during plunging into LN2. In region II (crossed), the combinations of [CPA]0 and B1 allow the [S]i to reach 40%, but based upon Mazur's IIF model [11], IIF is predicted to occur before plunging. In region III, the combinations of [CPA]0 and B1 allow the [S]i to reach 40%, there is no IIF during slow cooling, and recrystallization is not predicted during warming

Figure 4

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FIG. 5. Plunging temperatures for each combination of initial CPA concentration ([CPA]0) and cooling rate (B1) in region III of Figure 4. A three-dimensional plot (A) shows the trend of how the optimal plunging temperature changes with different [CPA]0 and B1 values. The gray level coded plot (B) of plunging temperatures shows the exact values (indicated in °C). The points from region I and II are plotted in white, and only data in the black box in B were included in A. The areas labeled {alpha}, ß, and {gamma} provide reference points between A and B

Figure 5

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FIG. 6. A contour graph of the maximum intracellular solute concentration the cell can achieve during slow cooling from the seeding temperature to the Tend. The numbers represent the percentage of the CPA concentration

Figure 6

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FIG. 7. Comparison of the current model predictions of optimum combinations of initial CPA concentrations ([CPA]0) and cooling rates (B1) that would be predicted to avoid IIF in rat zygotes (region C) shown with similar predictions from Karlsson et al. [17] for mouse oocytes in the presence of glycerol (regions A, C, and D). Region C is consistently predicted by both models. Region D is eliminated by the current model due to low [CPA]0, which does not allow the intracellular solute concentration to reach the critical concentration (). Region B is predicted (by Karlsson et al.) as a supercritical region in which the final crystallized volume fraction would exceed 10-3, indicating IIF. M, Molar

Figure 7

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FIG. 8. Duration of the slow cooling step for different cooling rates (B1) and initial CPA concentrations ([CPA]0). Duration decreases as the cooling rate increases (A). For each B1, there is a corresponding [CPA]0, which minimizes the duration (B)

 

Figure 8

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