Efficient Production of Live Offspring from Mouse Oocytes Vitrified with a Novel Cryoprotective Agent, Carboxylated ε-poly-L-lysine

Watanabe, Hidenobu; Kohaya, Natsuki; Kamoshita, Maki; Fujiwara, Katsuyoshi; Matsumura, Kazuaki; Hyon, Suong‐Hyu; Ito, Junya; Kashiwazaki, Naomi

doi:10.1371/journal.pone.0083613

Cited by 30 publications

(32 citation statements)

References 20 publications

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“…Our previous study showed that the vitrification of mouse oocytes with EG and COOH-PLL was successful in mouse oocytes [20]. It has been reported that higher concentration of CPA such as EG increases toxicity of cells after cryopreservation but up to 20% (w/v) of COOH-PLL did not decease the survivability of frozen-thawed cells [19].…”

Section: Discussionmentioning

confidence: 99%

Successful vitrification of pronuclear-stage pig embryos with a novel cryoprotective agent, carboxylated ε-poly-L-lysine

et al. 2017

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Vitrification is a powerful tool for the efficient production of offspring derived from cryopreserved oocytes or embryos in mammalian species including domestic animals. Genome editing technologies such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated (Cas)9 are now available even for domestic species, suggesting that the vitrification of embryos at the pronuclear stage (PN) will be more important because they could provide genomic host cells to be targeted by TALENs or CRISPR/Cas9. Although we reported the successful production of piglets derived from vitrified PN embryos by a solid-surface vitrification method with glutathione supplementation, further improvements are required. The cryoprotective agent (CPA) carboxylated ε-poly-L-lysine (COOH-PLL) was introduced in 2009. COOH-PLL reduces the physical and physiological damage caused by cryopreservation in mammalian stem cells and the vitrification of mouse oocytes and embryos. Those results suggested that vitrification of COOH-PLL may help improve the developmental ability of pig embryos vitrified at the PN stage. However, it remains unclear whether COOH-PLL is available as a CPA for the vitrification of embryos in domestic species. In this study, we evaluated COOH-PLL as a CPA with ethylene glycol (EG) and Cryotop as a device for the vitrification of PN pig embryos. Exposure to vitrification solution supplemented with COOH-PLL up to 30% did not decrease developmental ability to the 2-cell stage and the blastocyst stage. After warming, most of the vitrified embryos survived regardless of the concentration of COOH-PLL (76.0 ± 11.8% to 91.8 ± 4.6%). However, the vitrified embryos without COOH-PLL showed a lower development rate up to the blastocyst stage (1.3 ± 1.0%) compared to the fresh embryos (28.4 ± 5.0%) (p<0.05). In contrast, supplementation of 20% (w/v) COOH-PLL in the vitrification solution dramatically improved the developmental ability to blastocysts of the vitrified embryos (19.4 ± 4.6%) compared to those without COOH-PLL (p<0.05). After the transfer of embryos vitrified with 30% (v/v) EG and 20% (w/v) COOH-PLL, we successfully obtained 15 piglets from 8 recipients. Taken together, our present findings demonstrate for the first time that COOH-PLL is an effective CPA for embryo vitrification in the pig. COOH-PLL is a promising CPA for further improvements in the vitrification of oocytes and embryos in mammalian species.

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Section: Discussionmentioning

confidence: 99%

Successful vitrification of pronuclear-stage pig embryos with a novel cryoprotective agent, carboxylated ε-poly-L-lysine

et al. 2017

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“… 11 Polyampholytes have been used to successfully cryopreserve stem cells, 31 cell monolayers, 32 and mouse oocytes. 33 Structure–property relationships for these materials are still missing however. 34 …”

Section: Introductionmentioning

confidence: 99%

Post-Thaw Culture and Measurement of Total Cell Recovery Is Crucial in the Evaluation of New Macromolecular Cryoprotectants

Murray

Gibson

2020

Biomacromolecules

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The storage and transport of cells is a fundamental technology which underpins cell biology, biomaterials research, and emerging cell-based therapies. Inspired by antifreeze and ice-binding proteins in extremophiles, macromolecular (polymer) cryoprotectants are emerging as exciting biomaterials to enable the reduction and/or replacement of conventional cryoprotective agents such as DMSO. Here, we critically study post-thaw cellular outcomes upon addition of macromolecular cryoprotectants to provide unambiguous evidence that post-thaw culturing time and a mixture of assays are essential to claim a positive outcome. In particular, we observe that only measuring the viability of recovered cells gives false positives, even with non-cryoprotective polymers. Several systems gave apparently high viability but very low total cell recovery, which could be reported as a success but in practical applications would not be useful. Post-thaw culture time is also shown to be crucial to enable apoptosis to set in. Using this approach we demonstrate that polyampholytes (a rapidly emerging class of cryoprotectants) improve post-thaw outcomes across both measures, compared to poly(ethylene glycol), which can give false positives when only viability and short post-thaw time scales are considered. This work will help guide the discovery of new macromolecular cryoprotectants and ensure materials which only give positive results under limited outcomes can be quickly identified and removed.

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“…For example, superovulation of dromedary camels with two injections of FSH dissolved in hyaluronan solution produces more similar sized embryos compared to other protocols [42]. V. A novel nonpenetrating CPA, carboxylated ε-poly-L-Lysin (PLL): A Japanese group has recently reported that the addition of PLL in vitrification solutions significantly improved the survival rate of mouse oocytes and embryos [43]. Unfortunately, this product is not commercially available.…”

Section: Cytoskeleton Stabilization: Cytoskeleton-stabilizingmentioning

confidence: 99%

Current status and future direction of cryopreservation of camelid embryos

2017

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Over the past 3 decades, and similar to the horse industry, fresh embryo transfer has been widely practiced on large commercial scales in different camelid species, especially the dromedary camel and alpaca. However, the inability to cryopreserve embryos significantly reduces its broader application, and as such limits the capacity to utilize elite genetic resources internationally. In addition, cryopreservation of the semen of camelids is also difficult, suggesting an extreme sensitivity of the germplasm to cooling and freezing. As a result, genetic resources of camelids must continue to be maintained as living collections of animals. Due to concerns over disease outbreaks such as that of the highly pathogenic Middle East Respiratory Syndrome in the Middle East and Asia, there is an urgent need to establish an effective gene banking system for camelid species, especially the camel. The current review compares and summarizes recent progress in the field of camelid embryo cryopreservation, identifying four possible reasons for the slow development of an effective protocol and describing eight future directions to improve the current protocols. At the same time, the results of a recent dromedary camel embryo transfer study which produced a high morphologic integrity and survival rate of Open Pulled Straw-vitrified embryos are also discussed.

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Efficient Production of Live Offspring from Mouse Oocytes Vitrified with a Novel Cryoprotective Agent, Carboxylated ε-poly-L-lysine

Cited by 30 publications

References 20 publications

Successful vitrification of pronuclear-stage pig embryos with a novel cryoprotective agent, carboxylated ε-poly-L-lysine

Successful vitrification of pronuclear-stage pig embryos with a novel cryoprotective agent, carboxylated ε-poly-L-lysine

Post-Thaw Culture and Measurement of Total Cell Recovery Is Crucial in the Evaluation of New Macromolecular Cryoprotectants

Current status and future direction of cryopreservation of camelid embryos

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