Healy Mae W, Dolitsky Shelley N, Villancio-Wolter Maria, Raghavan Meera, Tillman Alexandra R, Morgan Nicole Y, DeCherney Alan H, Park Solji, Wolff Erin F
Program in Reproductive and Adult Endocrinology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Department of Obstetrics and Gynecology, Walter Reed National Military Medical Center, Bethesda, MD 20889, USA.
Micromachines (Basel). 2021 Mar 4;12(3):261. doi: 10.3390/mi12030261.
We hypothesized that the creation of a 3-dimensional ovarian follicle, with embedded granulosa and theca cells, would better mimic the environment necessary to support early oocytes, both structurally and hormonally. Using a microfluidic system with controlled flow rates, 3-dimensional two-layer (core and shell) capsules were created. The core consists of murine granulosa cells in 0.8 mg/mL collagen + 0.05% alginate, while the shell is composed of murine theca cells suspended in 2% alginate. Somatic cell viability tests and hormonal assessments (estradiol, progesterone, and androstenedione) were performed on days 1, 6, 13, 20, and 27. Confocal microscopy confirmed appropriate compartmentalization of fluorescently-labeled murine granulosa cells to the inner capsule and theca cells to the outer shell. Greater than 78% of cells present in capsules were alive up to 27 days after collection. Artificially constructed ovarian follicles exhibited intact endocrine function as evidenced by the production of estradiol, progesterone, and androstenedione. Oocytes from primary and early secondary follicles were successfully encapsulated, which maintained size and cellular compartmentalization. This novel microfluidic system successfully encapsulated oocytes from primary and secondary follicles, recapitulating the two-compartment system necessary for the development of the mammalian oocyte. Importantly, this microfluidic system can be easily adapted for sterile, high throughput applications.
我们推测,构建一种包含颗粒细胞和卵泡膜细胞的三维卵巢卵泡,在结构和激素方面能更好地模拟支持早期卵母细胞所需的环境。利用具有可控流速的微流体系统,创建了三维双层(核心和外壳)胶囊。核心由处于0.8 mg/mL胶原蛋白+0.05%藻酸盐中的小鼠颗粒细胞组成,而外壳由悬浮在2%藻酸盐中的小鼠卵泡膜细胞组成。在第1、6、13、20和27天进行了体细胞活力测试和激素评估(雌二醇、孕酮和雄烯二酮)。共聚焦显微镜证实了荧光标记的小鼠颗粒细胞在内层胶囊、卵泡膜细胞在外层外壳中的适当分隔。收集后长达27天,胶囊中超过78%的细胞存活。人工构建的卵巢卵泡表现出完整的内分泌功能,雌二醇、孕酮和雄烯二酮的产生证明了这一点。来自初级和早期次级卵泡的卵母细胞被成功封装,其大小和细胞分隔得以维持。这种新型微流体系统成功封装了来自初级和次级卵泡的卵母细胞,重现了哺乳动物卵母细胞发育所需的双室系统。重要的是,这种微流体系统可以很容易地适用于无菌、高通量应用。