微流控法制备载卵母细胞水凝胶微球及其玻璃化保存  

Vitrification Preservation of Oocyte Hydrogel Microspheres Prepared by Microfluidics

作　　者：张慧 张宇琪 胡剑麟 周新丽[1] ZHANG Hui;ZHANG Yu-Qi;HU Jian-Lin;ZHOU Xin-Li(Institute of Biothermal Technology,School of Health Science and Engineering,University of Shanghai for Science and Technology,Shanghai 200093,China;Department of Reproductive Medicine,Shanghai General Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200080,China)

机构地区：[1]上海理工大学健康科学与工程学院生物系统热科学研究所,上海200093 [2]上海交通大学医学院附属第一人民医院辅助生殖医学科,上海200080

出　　处：《生物化学与生物物理进展》2024年第4期969-980,共12页Progress In Biochemistry and Biophysics

基　　金：上海市肿瘤能量治疗技术与器械协同创新中心资助项目。

摘　　要：目的通过微流控法制备载卵母细胞海藻酸钠微球,在低浓度保护剂下实现卵母细胞玻璃化保存。方法采用流动聚焦型微流控芯片,通过调整芯片结构、海藻酸钠溶液浓度和流速比,制备大小均匀、空包率低、低温耐受的载卵母细胞海藻酸钠水凝胶微球。在低浓度低温保护剂下将微球玻璃化保存,复温后检测存活率,采用细胞松弛素B和氯化锶孤雌激活卵母细胞,与Cryotop玻璃化法对比卵母细胞存活率和卵裂率、囊胚率。结果制备的海藻酸钠微球在冷冻复温前后的体积稳定且结构完整,在将卵母细胞包封在海藻酸钠水凝胶中后,空包率低,存活率、卵裂率和囊胚率与新鲜组相比无显著差异。在低浓度低温保护剂10%DMSO+10%乙二醇(EG)+0.5 mol/L海藻糖中玻璃化冻存后卵母细胞的存活率达到92.48%,卵裂率70.80%,囊胚率20.42%,与高浓度保护剂15%DMSO+15%EG+0.5 mol/L海藻糖中Cryotop玻璃化法相比无显著性差异。结论本文设计制作了三通道内部交联芯片并用于卵母细胞玻璃化保存的微流控系统,可生成大小均匀、空包率低、低温耐受的载卵母细胞海藻酸钠水凝胶微球,在低浓度保护剂下实现玻璃化保存,为卵母细胞玻璃化保存方法提供新思路。Objective This study aimed to develop a novel method for encapsulating oocytes in sodium alginate hydrogel using microfluidics,then to vitrify these encapsulated oocytes in a single-step process with low concentrations of cryoprotectants.Methods We utilized a flow-focusing microfluidic chip to generate sodium alginate hydrogel microspheres.The influence of various parameters,including throat structure,cross-linking method,sodium alginate concentrations,and flow rate ratios on the stability diameter,and coefficient of variation of microspheres were examined.To further investigate the cold-resistance of these microspheres,we used cryomicroscopy to observe changes in volume and morphology of microspheres during cooling and warming processes.We used microfluidic chip to encapsulate oocytes in sodium alginate hydrogel microspheres,the empty rate of microspheres and loss rate of oocytes were determined.After releasing from microspheres and parthenogenetic activation with cytochalasin B and strontium chloride,the survival,cleavage and blastocyst rates were evaluated during in vitro maturation.Finally,oocytes encapsulated in sodium alginate microspheres were vitrified with low concentrations of cryoprotectants.We compared the survival and development capability of the oocytes with the Cryotop method.Results When the throat of the microfluidic chip measures 300μm in length and 120μm in width,microspheres can be uniformly formed at the throat of the chip.Sodium alginate generates microspheres with a wide size distribution when cross-linking outside the chip,while internal cross-linking within the chip results in more uniform microspheres.The stability of microsphere formation is significantly improved with the use of a three-channel internal cross-linking chip.At a flow rate of 2μl/min and with 1%sodium alginate,the microfluidic chip can consistently and uniformly produce microspheres.Under flow rate ratios of 10,15,and 20,the average microsphere diameters are 262.71μm,193.63μm,and 156.63μm,respectively.The sodium al

关 键 词：微流控 卵母细胞 微囊化 玻璃化保存 低温保护剂 

分 类 号：Q2[生物学—细胞生物学] Q81