The temperature of intracellular ice formation in mouse oocytes vs. the unfrozen fraction at that temperature

Mazur, Péter; Pinn, Irina L.; Kleinhans, F.W.

doi:10.1016/j.cryobiol.2007.02.001

Cited by 18 publications

(14 citation statements)

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“…Some of the 1X data in the Mouse Oocytes-Variable R section also appear in the Constant R section. All the data in the Constant R section come from Table 4 of [4]; however, some of the values for U, m s , and m CPA , here differ slightly from those in [4]. This is because, here, the means were calculated by Eq 1 in the footnote of Table 1, whereas in [4], they were calculated by Eq 2.…”

Section: Tablementioning

confidence: 77%

“…As a consequence, the weight ratios of CPA to salt (R values) varied in each solution, and different isopleths of the ternary phase diagram were applicable. The middle third of Table 4 summarizes the data in [4]. In that case, the external solutions were prepared to hold the R value constant in each set.…”

Section: Nih-pa Author Manuscriptmentioning

confidence: 99%

“…There again, if the highest value of U (0.105) is omitted, the ratio drops from 1.9 to 1.6, equal to or lower than the ratios for the three concentration properties. As we have pointed out [1,4,5], there is reasoning to believe that IIF at temperatures below −40°C is due to homogeneous nucleation of the intracellular supercooled water rather than due to the attainment of a critically low U value. …”

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confidence: 99%

“…Consequently, it is not clear whether IIF was correlated with unfrozen fraction or with the NaCl concentration. That confounding was partly uncoupled in a subsequent paper [4] which determined the flash temperatures in EG/PBS or glycerol/PBS solutions in which the CPA and salt concentrations were both varied so as to hold the R value constant. The results with this approach suggest that the unfrozen fraction is the critical factor for IIF.…”

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confidence: 99%

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Relationship between intracellular ice formation in oocytes of the mouse and Xenopus and the physical state of the external medium—A revisit

Mazur

Kleinhans

2008

Cryobiology

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We have previously reported that intracellular ice formation (IIF) in mouse oocytes suspended in glycerol/PBS solutions or ethylene glycol (EG)/PBS solutions and rapidly cooled to −50°C or below occurs at temperatures where a critical fraction of the external water remains unfrozen (Cryobiology 51, 2005, 29-53; 54, 2007, 223-233). For mouse oocytes in PBS or glycerol/PBS that fraction is 0.06; for oocytes in EG that fraction was calculated to be 0.13, more than double. The fractions unfrozen are computed from ternary phase diagrams. In the previous publication, we used the EG data of Woods et al. (Cryobiology 38,1999, 403-407). Since then, we have determined that ternary phase diagrams for EG/NaCl/water synthesized by summing binary phase data for EG/water NaCl/water gives substantially different curves, which seem more realistic (Cryobiology 54, 2007, 212-222). Unfrozen fractions at the temperatures of IIF computed from these synthesized phase diagrams are about half of those calculated from the Woods et al. data, and are in close agreement with the computations for glycerol; i.e., IIF occurs when about 92-94% of the external water is frozen. A parallel paper was published by Guenther et al. (Cryobiology 52, 2006, 401-416) on IIF in oocytes of the frog Xenopus. It too examined whether the temperatures of IIF were related to the unfrozen fractions at those temperatures. It also used the Woods et al. ternary phase data to calculate the unfrozen fractions for EG solutions. As reported here, once again the values of these unfrozen fractions are substantially different from those calculated using synthesized phase diagrams. With the latter, the unfrozen fractions at IIF become very similar for EG and glycerol. KeywordsIntracellular ice formation; oocytes; mouse; Xenopus; unfrozen fraction; phase diagrams Our laboratory has recently reported on several variables that influence the temperature at which intracellular ice forms in mouse oocytes and in oocytes of the frog Xenopus. [5,1] that are cooled rapidly enough to ensure that they undergo intracellular ice formation (IIF); namely 20°C/min and 10°C/min, respectively. One major variable affecting the temperature of IIFin mouse oocytes was the concentration of ethylene glycol (EG) or glycerol present in the *Corresponding author. Fax: +1 865 974 8027 e-mail address: pmazur@utk.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. The bottom three lines of Table 1 and the bottom half of Table 2 show the computed values for the right-hand five columns based on the synthesized ternary phase diagrams [3]. The chief conclus...

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Section: Tablementioning

confidence: 77%

Section: Nih-pa Author Manuscriptmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Relationship between intracellular ice formation in oocytes of the mouse and Xenopus and the physical state of the external medium—A revisit

Mazur

Kleinhans

2008

Cryobiology

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“…One indication of normality is their morphological appearance. A second is that they shrink or swell in anisosmotic solutions of glycerol/PBS or EG/PBS in quantitative accord with that calculated from fundamental osmotic equations (For example, see Table 7 of Mazur et al [14]. Thirdly, M. Kasai (personal communication) has found that 84% of ICR oocytes vitrified in the same medium and by the same procedure developed to the 2-cell stage after dezonation and in vitro fertilization, a percentage nearly equal to the 89% for untreated controls.…”

Section: Source Of Oocytesmentioning

confidence: 86%

Kinetics and activation energy of recrystallization of intracellular ice in mouse oocytes subjected to interrupted rapid cooling

Seki

Mazur

2008

Cryobiology

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Intracellular ice formation (IIF) is almost invariably lethal. In most cases, it results from the too rapid cooling of cells to below −40°C, but in some cases it is manifested, not during cooling, but during warming when cell water that vitrified during cooling first devitrifies and then recrystallizes during warming. Recently, Mazur et al. [Cryobiol. 55 (2007) 158] dealt with one such case in mouse oocytes. It involved rapidly cooling the oocytes to −25°C, holding them 10 min, rapidly cooling them to −70°C, and warming them slowly until thawed. No IIF occurred during cooling but intracellular freezing, as evidenced by blackening of the cells, became detectable at −56°C during warming and was complete by −46°C. The present study differs in that the oocytes were warmed rapidly from −70°C to temperatures between −65°C and −50°C and held for 3 to 60 min. This permitted us to determine the rate of blackening as function of temperature. That in turn allowed us to calculate the activation energy (Ea) for the blackening process; namely, 27.5 kcal/mole. This translates to about a quadrupling of the blackening rate for every 5° rise in temperature. These data then allowed us to compute the degree of blackening as a function of temperature for oocytes warmed at rates ranging from 10 to 10,000°C/min. A 10-fold increase in warming rate increased the temperature at which a given degree of blackening occurred by 8°C. These findings have significant implications both for cryobiology and cryo-electron microscopy. KeywordsIce; intracellular; recrystallization; activation energy; oocytes; mouse The major cause of death in cells subjected to freezing is the formation of intracellular ice (IIF). Or put differently, to survive freezing, a cell must be cryopreserved in ways that avoid or minimize IIF. In a recent study, Mazur et al. [13] investigated intracellular freezing in mouse oocytes subjected to an unorthodox cooling regime referred to as interrupted rapid cooling. In this procedure, cells are cooled rapidly to a temperature above that at which intracellular ice can form, held various times at that temperature, and then cooled rapidly to −70°C or below. The rationale is the following: Since after the initial rapid cool, the cells are unfrozen, they are by definition supercooled, and because their water is supercooled, it will leave the cells osmotically in response to the lower chemical potential outside. If they are held at this intermediate temperature for sufficient time, they will lose most of their freezable water and *Corresponding author. Fax +1 865 974-8027, E-mail address: E-mail: pmazur@utk.edu (P. Mazur). ✧ Research supported by NIH Grant R01-RR18470Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the prod...

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Measurement of cooling and warming rates in vitrification‐based plant cryopreservation protocols

Teixeira

González‐Benito

García

2014

Biotechnology Progress

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Cryopreservation protocols include the use of additives and pretreatments aimed to reduce the probability of ice nucleation at all temperatures, mainly through micro-viscosity increase. Still, there is a risk of ice formation in the temperature region comprised between the equilibrium freezing (Tf ) and the glass transition (TG ) temperatures. Consequently, fast cooling and warming, especially in this region, is a must to avoid ice-derived damage. Vitrification and droplet-vitrification techniques, frequently used cryopreservation protocols based in fast cooling, were studied, alongside with the corresponding warming procedures. A very fast data acquisition system, able to read very low temperatures, down to that of liquid nitrogen, was employed. Cooling rates, measured between -20°C and -120°C, ranged from ca. 5°C s(-1) to 400°C s(-1) , while warming rates spanned from ca. 2°C s(-1) to 280°C s(-1) , for the different protocols and conditions studied. A wider measuring window (0°C to -150°C) produced lower rates for all cases. The cooling and warming rates were also related to the survival observed after the different procedures. Those protocols with the faster rates yielded the highest survival percentages.

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The temperature of intracellular ice formation in mouse oocytes vs. the unfrozen fraction at that temperature

Cited by 18 publications

References 18 publications

Relationship between intracellular ice formation in oocytes of the mouse and Xenopus and the physical state of the external medium—A revisit

Relationship between intracellular ice formation in oocytes of the mouse and Xenopus and the physical state of the external medium—A revisit

Kinetics and activation energy of recrystallization of intracellular ice in mouse oocytes subjected to interrupted rapid cooling

Measurement of cooling and warming rates in vitrification‐based plant cryopreservation protocols

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