Effects of hold time after extracellular ice formation on intracellular freezing of mouse oocytes

Mazur, Péter; Pinn, Irina L.; Seki, Shinsuke; Kleinhans, F.W.; Edashige, Keisuke

doi:10.1016/j.cryobiol.2005.07.002

Cited by 17 publications

(13 citation statements)

References 11 publications

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“…The purpose of ramp 3 was to provide time for the external liquid medium, the external ice, and the supercooled water in the cell to come to near equilibrium before recooling began. If ramp 3 was omitted, the observed temperatures of IIF were about 208C higher [13].…”

Section: The Linkam Cryostage Freezing Protocols and Rampsmentioning

confidence: 97%

The Temperature and Type of Intracellular Ice Formation in Preimplantation Mouse Embryos as a Function of the Developmental Stage1

Seki

Mazur

2010

Biology of Reproduction

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Our studies the past 5 yr have concentrated on intracellular ice formation (IIF) in mature mouse oocytes at the metaphase stage of meiosis II. Here we report an analogous investigation of the temperature of intracellular ice nucleation in preimplantation embryo stages from one-cell to early morula suspended in 1 M ethylene glycol/PBS and cooled at 20 degrees C/min to -70 degrees C. Physical modeling indicates that oocytes and preimplantation embryos undergo very little osmotic shrinkage at that cooling rate. As a consequence, their interior becomes increasingly supercooled until the supercooling is abruptly terminated by IIF. Four categories of IIF were observed. The first two were 1) those undergoing IIF at temperatures well below the temperature of external ice formation (EIF; -7.2 degrees C) vs. 2) those undergoing IIF within 1 degrees C of the EIF temperature. The other two categories were those multicellular stages in which 3) all the blastomeres underwent IIF simultaneously vs. 4) those in which blastomeres underwent IIF sequentially. Embryos in categories 1 and 3 constituted the majority (80-90%), and for them, the mean IIF temperatures of one-cell, two-cell, four- to six-cell, and early eight-cell ranged from -37 degrees C to -43 degrees C, temperatures that indicate that IIF is a consequence of homogeneous nucleation. However, the IIF nucleation temperature of early morulae in categories 1 and 3 was markedly higher; namely, -23.1 +/- 1.5 degrees C. This marked rise in nucleation temperature coincides with the appearance of aquaporin 3 and gap junctions in early morulae (compacted eight-cell), and is presumably causally related.

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Section: The Linkam Cryostage Freezing Protocols and Rampsmentioning

confidence: 97%

The Temperature and Type of Intracellular Ice Formation in Preimplantation Mouse Embryos as a Function of the Developmental Stage1

Seki

Mazur

2010

Biology of Reproduction

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“…1A by showing a 2-min hold at −10°C. This hold is introduced because we have previously shown [27] that in its absence, IIF occurs at considerably higher temperatures. Some initial cell dehydration occurs during this 2 min hold.…”

Section: Methodsmentioning

confidence: 99%

Determination of the water permeability (Lp) of mouse oocytes at −25°C and its activation energy at subzero temperatures

Kleinhans

Mazur

2009

Cryobiology

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Typically, sub zero permeability measurements are experimentally difficult and infrequently reported. Here we report an approach we have applied to mouse oocytes. Interrupted cooling involves rapidly cooling oocytes (50°C/min) to an intermediate temperature above the intracellular nucleation zone, holding them for up to 40 minutes while they dehydrate, and then rapidly cooling them to −70 °C or below. If the intermediate holding temperature and holding time are well chosen, high post thaw survival of the oocytes is possible because the freezable water is removed during the hold. The length of time required for the exit of the freezable water allows the water permeability at that temperature to be determined. These experiments used 1.5 molar ethylene glycol in PBS and included a transient hold of 2 minutes for equilibration at −10°C, just below the extracellar ice formation temperature. We obtain an Lp = 1.8 × 10−3 μm min−1 atm−1 at −25 °C based on a hold time of 30 minutes yielding 80% survival and the premise that most of the freezable water is removed during the 30 min hold. If we assume that the water permeability is a continuous function of temperature and that its Ea changes at 0°C, we obtain a subzero Ea of 21 kcal/mol; higher than the suprazero value of 14 kcal/mol. A number of assumptions are required for these water loss calculations and the resulting value of Lp can vary by up to a factor of 2, depending on the choices make.

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“…3 should be able to enhance vitrification of living cells encapsulated in the microcapsules at high cooling rates (e.g., > 10,000 o C/min). This is because it can not only depress ice formation and growth in the microcapsule but also prevent ice (if any) propagation into cells from the bulk solution where ice is usually formed first (because of its much bigger volume) (Berejnov et al 2006;Fahy et al 1987;Franks et al 1983;He et al 2008b;Karlsson et al 1994;Mazur et al 2005a;Mazur et al 2005b;Toner 1993;Toner et al 1990;Yavin and Arav 2007). This hypothesis is confirmed by a recent study where the C3H10T1/2 mouse mesenchymal stem cells encapsulated in ~100 µm alginate microcapsules were vitrified using a 400 µm, thin-walled quartz microcapillary at a low-CPA concentration (1.4 M DMSO) (Zhang et al 2010).…”

Section: Conventional Vitrificationmentioning

confidence: 99%

Preservation of Embryonic Stem Cells

He¹

2011

Methodological Advances in the Culture, Manipulation and Utilization of Embryonic Stem Cells for Basic and Practical Applicatio

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Effects of hold time after extracellular ice formation on intracellular freezing of mouse oocytes

Cited by 17 publications

References 11 publications

The Temperature and Type of Intracellular Ice Formation in Preimplantation Mouse Embryos as a Function of the Developmental Stage1

The Temperature and Type of Intracellular Ice Formation in Preimplantation Mouse Embryos as a Function of the Developmental Stage1

Determination of the water permeability (Lp) of mouse oocytes at −25°C and its activation energy at subzero temperatures

Preservation of Embryonic Stem Cells

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