Woman's Hospital, University Hospital of Basel, Basel, Switzerland.
Tissue Eng Part A. 2010 Jun;16(6):2063-73. doi: 10.1089/ten.TEA.2009.0684.
The development of techniques allowing the growth of primordial follicles to mature follicles in vitro has much potential for both reproductive medicine and developmental research. However, human primordial and preantral follicles fail to grow after isolation from the surrounding ovarian stroma. Granulosa cells, which normally undergo apoptosis after ovulation, contain a subpopulation of ovarian follicular cells remaining viable in vitro over prolonged periods when cultured in the presence of leukemia-inhibiting factor. However, when cultured as monolayers, they progressively lose all their characteristics, such as follicle-stimulating hormone receptor and cytochrome P450-aromatase. Here, we describe a three-dimensional culture system containing type I collagen, which, together with leukemia-inhibiting factor, allowed the survival and growth of a subpopulation of granulosa cells isolated from mature ovarian follicles and supported them to proliferate into spherical structures exhibiting steroidogenic capacity, as demonstrated by P450-aromatase and 3beta-hydroxysteroid dehydrogenase. After transplantation into the ovaries of immunodeficient mice, these cells became localized preferentially within antral follicles and the prolonged expression of follicle-stimulating hormone receptor was confirmed as well. With this optimization of the culture conditions, an environment was created, which acts as a niche closely mimicking the development of early ovarian follicles in vitro.
技术的发展使得原始卵泡在体外成熟为成熟卵泡成为可能，这对生殖医学和发育研究都有很大的潜力。然而，人类原始卵泡和窦前卵泡从周围卵巢基质中分离出来后无法生长。颗粒细胞在排卵后通常会凋亡，但在白血病抑制因子存在的情况下培养时，会有一小部分卵巢滤泡细胞在体外长时间保持存活。然而，当它们被培养成单层时，会逐渐失去所有的特征，如卵泡刺激素受体和细胞色素 P450 芳香化酶。在这里，我们描述了一种包含 I 型胶原的三维培养系统，该系统与白血病抑制因子一起，允许从成熟卵巢卵泡中分离的颗粒细胞亚群存活和生长，并支持它们增殖成具有甾体生成能力的球形结构，这可以通过 P450 芳香化酶和 3β-羟甾脱氢酶来证明。将这些细胞移植到免疫缺陷小鼠的卵巢中后，它们优先定位于窦卵泡内，并证实了卵泡刺激素受体的长期表达。通过对培养条件的优化，创造了一种环境，该环境作为一个小生境，在体外非常接近地模拟了早期卵巢卵泡的发育。
