Quantification of residual cryoprotectants and cytotoxicity in thawed bovine ovarian tissues after slow freezing or vitrification
Quantification of residual cryoprotectants and cytotoxicity in thawed bovine ovarian tissues after slow freezing or vitrification
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DOI：
10.1093/humrep/deab274
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发表时间：
2021-12-20
影响因子：
6.1
通讯作者：
Suzuki, Nao
中科院分区：
文献类型：
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作者：
Sugishita, Yodo;Meng, Lingbo;Suzuki, Nao
研究问题：在缓慢冷冻或玻璃化冷冻后，解冻/加温的卵巢组织中残留了多少冷冻保护剂？在解冻/升温后，需要在介质中至少扩散清洗60分钟，以显著减少缓慢冷冻或玻璃化冷冻的卵巢组织中残留的冷冻保护剂。目前已知的卵巢组织冷冻保存(OTC)方法多为常规方法，而玻璃化冷冻法因其实用性而广受欢迎。玻璃化冷冻的主要问题是，在移植时，加热的组织中残留了多少具有潜在毒性的冷冻保护剂。研究设计、大小、持续时间这是一项动物研究，使用的是20个牛卵巢的卵巢组织。本研究的持续时间为2018年至2020年。对象/材料、地点、方法取20只牛卵巢组织，随机分为新鲜(未冷冻)对照组、1.5M二甲基亚砜(DMSO)缓冻组、1.5M 1，2-丙二醇组(PROH)和35%乙二醇组(EG)。分别于冷冻(解冻前)、解冻后0min(解冻后即刻)、介质扩散洗涤后30、60和120min，用气相色谱法测定解冻/加温后组织中残留的冷冻保护剂浓度。然后，观察新鲜(未冷冻)对照组、DMSO或PROH缓冻组和玻璃化冷冻组卵巢组织(1 mm×1 mm×1 mm)原始卵泡、颗粒细胞、细胞器和基质细胞的超微结构变化。用实时定量聚合酶链式反应检测DNA损伤感受器聚腺苷二磷酸核糖聚合酶-1(PARP1)和凋亡前体caspase-3(CASP3)在体外培养24或48h的解冻/温热卵巢组织中的表达，并用免疫组织化学方法检测caspase-3(AC3)活化的caspase-3(AC3)在DNA双链断裂中的蛋白表达。主要结果和Chance的作用随着扩散洗涤时间的延长，冰冻保护剂的残留浓度降低。洗涤60min后，DMSO、PROH和EG的冷冻保护剂残留量差异不显著(P>；0.05)。洗涤对组织完整性无影响，AC3、γ-H_2AX阳性细胞百分率未见明显升高，表明组织安全、质量好，可用于移植。大规模数据N/A限制，谨慎的理由由于这项研究是以牛的卵巢组织进行的，对人类的概括性可能是有限的。超过120分钟的卵巢组织的潜在变化没有被调查。这项研究结果的更广泛的意义这项研究解决了人们对EG在加热的卵巢组织中的细胞毒性的担忧，并可能为设计标准的OTC玻璃化冷冻方案提供见解。研究资金/竞争利益(S)这项研究由日本科学促进会资助的科学研究补助金(B)资助。
STUDY QUESTION How much residual cryoprotectant remains in thawed/warmed ovarian tissues after slow freezing or vitrification? SUMMARY ANSWER After thawing/warming, at least 60 min of diffusion washing in media was necessary to significantly reduce the residual cryoprotectants in ovarian tissues frozen by slow freezing or vitrification. WHAT IS KNOWN ALREADY Ovarian tissue cryopreservation (OTC) by slow freezing has been the conventional method; while the vitrification method has gained popularity for its practicality. The main concern about vitrification is how much potentially toxic residual cryoprotectant remains in the warmed tissues at the time of transplantation. STUDY DESIGN, SIZE, DURATION This was an animal study using the ovarian tissues from 20 bovine ovaries. The duration of this study was from 2018 to 2020. PARTICIPANTS/MATERIALS, SETTING, METHODS Ovarian cortex tissues were prepared from 20 bovine ovaries and assigned randomly to groups of fresh (non-frozen) control, slow freezing with 1.5 M dimethyl sulfoxide (DMSO), 1.5 M 1,2-propanediol (PROH) and vitrification with 35% ethylene glycol (EG). The residual cryoprotectant concentrations in thawed/warmed tissues were measured by gas chromatography at the following time points: frozen (before thawing/warming), 0 min (immediately after thawing/warming), 30, 60 and 120 min after diffusion washing in media. Next, the ultrastructural changes of primordial follicles, granulosa cells, organelles and stromal cells in the ovarian tissues (1 mm x 1 mm x 1 mm) were examined in fresh (non-frozen) control, slow freezing with DMSO or PROH and vitrification with EG groups. Real-time quantitative PCR was carried out to examine the expressions of poly (ADP-ribose) polymerase-1 (PARP1), a DNA damage sensor and caspase-3 (CASP3), an apoptosis precursor, in thawed/warmed ovarian tissues that were washed for either 0 or 120 min and subsequently in tissues that were ex vivo cultured for 24 or 48 h. The same set of tissues were also used to analyze the protein expressions of gamma H2A histone family member X (gamma H2AX) for DNA double-strand breaks and activated caspase-3 (AC3) for apoptosis by immunohistochemistry. MAIN RESULTS AND THE ROLE OF CHANCE The residual cryoprotectant concentrations decreased with the extension of diffusion washing time. After 60 min washing, the differences of residual cryoprotectant between DMSO, PROH and EG were negligible (P > 0.05). This washing did not affect the tissue integrity or significantly elevate the percentage of AC3 and gamma H2AX positive cells, indicating that tissues are safe and of good quality for transplantation. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION Since the study was performed with ovarian tissues from bovines, generalizability to humans may be limited. Potential changes in ovarian tissue beyond 120 min were not investigated. WIDER IMPLICATIONS OF THE FINDINGS This study addresses concerns about the cytotoxicity of EG in warmed ovarian tissues and could provide insights when devising a standard vitrification protocol for OTC. STUDY FUNDING/COMPETING INTEREST(S) The study was funded by a Grant-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science to N.S.