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

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 alginate hydrogel microspheres maintained their volume and structural integrity during the cooling and warming processes.Using a three-channel internal cross-linking microfluidic chip to encapsulate oocytes,at a flow rate ratio of 10,the empty rate is 32.28%,and the cell loss rate is 11.09%.After encapsulation and subsequent release,the oocyte survival rate(96.99%),cleavage rate(88.71%),and blastocyst formation rate(26.29%)showed no significant differences compared to the fresh group.After the microspheres were vitrified using a low concentration of cryoprotectant(10%DMSO+10%ehylene glycol(EG)+0.5 mol/L trehalose),the survival rate,cleavage rate,and blastocyst rate were 92.48%,70.80%,and 20.42%,respectively.No significant difference was observed when compared to the Cryotop method using a higher concentration of cryoprotectant solution(15%DMSO+15%EG+0.5 mol/L trehalose).Conclusion We designed and fabricated a microfluidic system with three-channel internal cross-linking chips used for oocyte vitrification preservation.The microfluidic system can generate oocytes-loaded sodium alginate hydrogel microspheres with uniform size,low empty rate,and good cold-resistance.The method successfully reduced the concentration of cryoprotectants in a single-step vitrification process,the developmental capability of oocytes during in vitro maturation were comparable with Cryotop method.Unlike the Cryotop method,the oocytes encapsulated in hydrogel does not come into contact with liquid nitrogen,eliminating the risk of cross-contamination.This study provides a novel approach to oocyte vitrification.