基于微流控芯片模拟体内受精及早期胚胎发育环境的输卵管模型构建

Construction of microfluidics-based fallopian tube model for mimicking fertilization and early embryo culture in vivo

背景:胚胎受精和早期胚胎培养是辅助生殖技术中重要的一部分,然而近几十年来胚胎培养技术却基本没有更新,因此胚胎受精和早期胚胎培养的条件成为了限制辅助生殖技术发展的一个瓶颈。目的:构建基于微流控芯片来模拟体内受精及早期胚胎发育环境的仿真输卵管模型。方法:采用软光刻法制作微流控芯片,使芯片微通道在形状上符合输卵管的解剖结构;组织消化贴壁法进行小鼠输卵管原代上皮细胞的培养和提纯;用角蛋白免疫荧光法对提纯后的小鼠输卵管原代上皮细胞进行鉴定,并将鉴定后的上皮细胞种植在微流控芯片通道内壁上以模拟输卵管生化环境;将芯片接入自动换液装置以模拟输卵管液流环境。结果与结论:①这款输卵管模型呈圆柱状,长度为2 cm,直径为1 cm,在形状上与体内输卵管峡部的解剖学特征比较相符合;②角蛋白免疫荧光结果为阳性,提示组织消化贴壁法可分离培养出小鼠输卵管原代上皮细胞;③提纯后的小鼠输卵管原代上皮细胞种植在模型内壁,为胚胎的受精和早期培养提供了与体内微环境类似的生化环境。微流控芯片接入自动换液装置后,通道内的代谢废物能被及时带走,新的营养物质得以补充,实现了对输卵管真实流体环境的模拟;④研究将微流控芯片应用于辅助生殖技术,通过模拟体内受精及早期胚胎发育环境,实现了输卵管解剖学和生化环境的的重建,构建了以输卵管为模型的器官芯片,为进一步改善辅助生殖技术和提高受精率和优胚率奠定基础。

BACKGROUND:With the increasing proportion of infertility in the population,more and more attentions have been paid on assisted reproductive techniques.Fertilization and early embryo culture are the significant parts of assisted reproductive techniques;however,they remain unchanged in the last few decades.OBJECTIVE:To design a novel microfluidics-based fallopian tube model that can mimic the microenvironment of fertilization and early embryo culture in vivo.METHODS:Microfluidic device was manufactured by soft lithography method to mimic the anatomical characteristic of fallopian tube in vivo.Mouse oviduct primary epithelial cells were cultured and purified by explants culture method,and then the purified cells were identified by keratin immunofluorescence method.Epithelial cells were then loaded into the channel to mimic the biochemical environment of fallopian tube in vivo.The chip was connected to the automatic liquid changing device to mimic the liquid environment of fallopian tube in vivo.RESLUTS AND CONCLUSION:(1)The channel of this model is cylindrical with 2 cm of height and 1 cm of diameter,which were in accordance with the anatomical characteristic of the isthmus of fallopian tube in shape.(2)The keratin immunofluorescence was positive,which indicated that mouse oviduct primary epithelial cells can be obtained by explants culture method.(3)The cells were loaded into the channel to cover the wall of channel,which provided a biochemical microenvironment similar to that in vivo for fertilization and early embryo culture.After the chip was connected to the automatic liquid changing device,metabolic waste could be taken away and nutrient substance can be replenished in time,which mimics the real fluid environment in vivo.(4)This study combined microfluidics technology and assisted reproductive techniques to design a novel fallopian tube model,which mimics the micro-environment of fertilization and early embryo culture in vivo.This study has laid a foundation for further improvement of assisted reproductive techniques and the rate of fertilization and embryo optimization.