Low cryoprotectant concentration rapid vitrification of mouse oocytes and embryos

Liu, Jie; Lee, Gloria Y.; Biggers, John D.; Toth, Thomas L.; Toner, Mehmet

doi:10.1016/j.cryobiol.2020.10.016

Cited by 10 publications

(5 citation statements)

References 23 publications

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“…Lower CPA concentrations should allow the times for CPA soaking before cooling and oocyte re-expansion after thawing to be reduced, while reducing osmotic stress and CPA toxicity. The cooling rates achieved here of ∼600,000 °C/min via plunging in LN 2 at its boiling temperature (77 K) are more than an order of magnitude larger than those reported for human oocytes and larger than the 23,000 °C/min reported when using Cryotops 56,57 and the ∼35,000 °C/min obtained using thin wall quartz microcapillaries 58–60 . Use of “slushed” LN 2 (cooling to its melting temperature) increased cooling rates in quartz capillaries to ∼250,000 °C/min 58 , and a similar increase (to ∼4 × 10 6 °C/min) should be achievable using crystallography tools.…”

Section: Discussioncontrasting

confidence: 56%

Ice formation and its elimination in cryopreservation of bovine oocytes

Abdelhady,

Mittan-Moreau,

Crane

et al. 2023

Preprint

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Damage from ice and potential toxicity of ice-inhibiting cryoprotective agents (CPAs) are key issues in assisted reproduction using cryopreserved oocytes and embryos. We use synchrotron-based time-resolved x-ray diffraction and tools from protein cryocrystallography to characterize ice formation within bovine oocytes after cooling at rates between ∼1000 °C/min and ∼600,000°C /min and during warming at rates between 20,000 and 150,000 °C /min. Maximum crystalline ice diffraction intensity, maximum ice volume, and maximum ice grain size are always observed during warming. All decrease with increasing CPA concentration, consistent with the decreasing free water fraction. With the cooling rates, warming rates and CPA concentrations of current practice, oocytes may show no ice after cooling but always develop substantial ice fractions on warming, and modestly reducing CPA concentrations causes substantial ice to form during cooling. With much larger cooling and warming rates achieved using cryocrystallography tools, oocytes soaked as in current practice remain essentially ice free during both cooling and warming, and when soaked in half-strength CPA solution oocytes remain ice free after cooling and develop small grain ice during warming. These results clarify the roles of cooling, warming, and CPA concentration in generating ice in oocytes, establish the character of ice formed, and suggest that substantial further improvements in warming rates are feasible. Ice formation can be eliminated as a factor affecting post-thaw oocyte viability and development, allowing other deleterious effects of the cryopreservation cycle to be studied, and osmotic stress and CPA toxicity reduced.Significance StatementCryopreservation of oocytes and embryos is critical in assisted reproduction of humans and domestic animals and in preservation of endangered species. Success rates are limited by damage from crystalline ice, toxicity of cryoprotective agents (CPAs), and damage from osmotic stress. Time-resolved x-ray diffraction of bovine oocytes shows that ice forms much more readily during warming than during cooling, that maximum ice fractions always occur during warming, and that the tools and large CPA concentrations of current protocols can at best only prevent ice formation during cooling. Using tools from cryocrystallography that give dramatically larger cooling and warming rates, ice formation can be completely eliminated and required CPA concentrations substantially reduced, expanding the scope for species-specific optimization of post-thaw reproductive outcomes.

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Section: Discussioncontrasting

confidence: 56%

Ice formation and its elimination in cryopreservation of bovine oocytes

Abdelhady,

Mittan-Moreau,

Crane

et al. 2023

Preprint

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“…Vitrification is a well-established procedure, avoiding issues of ice nucleation and crystallization by taking the sample down to the temperature of LN 2 at a rate of ~ 10,000 to 50,000 °C/min [13][14][15][16]. Prior to vitrification, the oocyte or embryo is manually transferred into increasing concentrations of cryoprotectant [17,18]. To ensure success, the transfer of cells must occur within a stringent time frame to minimize exposure of the sample to these cytotoxic solutions [19].…”

Section: Introductionmentioning

confidence: 99%

Vitrification within a nanoliter volume: oocyte and embryo cryopreservation within a 3D photopolymerized device

Yagoub

Lim

Tan

et al. 2022

J Assist Reprod Genet

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Purpose Vitrification permits long-term banking of oocytes and embryos. It is a technically challenging procedure requiring direct handling and movement of cells between potentially cytotoxic cryoprotectant solutions. Variation in adherence to timing, and ability to trace cells during the procedure, affects survival post-warming. We hypothesized that minimizing direct handling will simplify the procedure and improve traceability. To address this, we present a novel photopolymerized device that houses the sample during vitrification. Methods The fabricated device consisted of two components: the Pod and Garage. Single mouse oocytes or embryos were housed in a Pod, with multiple Pods docked into a Garage. The suitability of the device for cryogenic application was assessed by repeated vitrification and warming cycles. Oocytes or early blastocyst-stage embryos were vitrified either using standard practice or within Pods and a Garage and compared to non-vitrified control groups. Post-warming, we assessed survival rate, oocyte developmental potential (fertilization and subsequent development) and metabolism (autofluorescence). Results Vitrification within the device occurred within ~ 3 nL of cryoprotectant: this volume being ~ 1000-fold lower than standard vitrification. Compared to standard practice, vitrification and warming within our device showed no differences in viability, developmental competency, or metabolism for oocytes and embryos. The device housed the sample during processing, which improved traceability and minimized handling. Interestingly, vitrification-warming itself, altered oocyte and embryo metabolism. Conclusion The Pod and Garage system minimized the volume of cryoprotectant at vitrification—by ~ 1000-fold—improved traceability and reduced direct handling of the sample. This is a major step in simplifying the procedure.

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“…Application of these tools to bovine oocytes allows ice-free diffraction to be obtained from cold samples even when using 50% strength vitrification solution. The cooling rates achieved of ~600,000 C/min are ~20 times larger than the 23,000 C/min reported when using Cryotops 62,63 and the ~35,000 C/min obtained using thin wall quartz microcapillaries [64][65][66] . Use of "slushed" nitrogen (cooled to its melting temperature) increased cooling rates in quartz capillaries to ~250,000 C/min 64 .…”

Section: Discussionmentioning

confidence: 66%

Ice formation and its elimination in cryopreservation of oocytes

Abdelhady,

Mittan-Moreau,

Crane

et al. 2024

Preprint

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Damage from ice and toxicity of ice-inhibiting cryoprotective agents (CPAs) are key issues in assisted reproduction of humans, domestic animals, and endangered species using cryopreserved oocytes and embryos. Ice formed in bovine oocytes (similar in size to oocytes of humans and most other mammals) after rapid cooling and during rapid warming were examined using synchrotron-based time-resolved x-ray diffraction. Using cooling rates, warming rates and CPA concentrations of current practice, oocytes show no ice after cooling but always develop large ice fractions — consistent with crystallization of most free water — during warming, so most ice-related damage must occur during warming. Increasing cooling rates allows oocytes soaked as in current practice to remain essentially ice free during both cooling and warming. Much larger convective warming rates are demonstrated and will allow routine ice-free cryopreservation with smaller CPA concentrations. These results clarify the roles of cooling, warming, and CPA concentration in generating ice in oocytes and establish the structure and grain size of ice formed. Ice formation can be eliminated as a factor affecting post-thaw oocyte viability and development in many species, improving outcomes and allowing other deleterious effects of the cryopreservation cycle to be independently studied.

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Low cryoprotectant concentration rapid vitrification of mouse oocytes and embryos

Cited by 10 publications

References 23 publications

Ice formation and its elimination in cryopreservation of bovine oocytes

Ice formation and its elimination in cryopreservation of bovine oocytes

Vitrification within a nanoliter volume: oocyte and embryo cryopreservation within a 3D photopolymerized device

Ice formation and its elimination in cryopreservation of oocytes

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