Broussard J R, Thibodeaux J K, Myers M W, Roussel J D, Hansel W, Godke R A
Department of Dairy, Louisiana State University, Baton Rouge, USA.
J Anim Sci. 1995 Nov;73(11):3287-93. doi: 10.2527/1995.73113287x.
The development of a serum-free culture system for bovine granulosa cells that would allow for cellular proliferation without induction of steroidogenesis would provide researchers with an important in vitro tool for determining differentiation mechanisms during folliculogenesis. The objective of the present study was to determine the effect of a commercially prepared serum substitute and a medium supplement on proliferation and progesterone production by bovine granulosa cells. Granulosa cells were obtained by aspirating the follicular fluid of follicles 2 to 8 mm in diameter. For each experiment, growth curves to determine the proliferative and steroidogenic response of granulosa cells to several different medium additions were constructed. Cells were counted on d 1, 2, 4, 6, and 8 of culture to determine cell concentration and the media harvested to determine progesterone content. In Exp. 1, granulosa cells were seeded at an initial rate of 5.0 x 10(5) for 48 h in serum-supplemented medium then allotted to one of five treatments including medium alone or medium containing fetal bovine serum (FBS; 1%), Gibco BRL media supplement-x (GMS-X; 1%), fatty acid-free bovine serum albumin (FAF-BSA; 4 mg/mL), or SerXtend (5%). For Exp. 2 and 3, granulosa cells were plated in serum-supplemented medium for either 48 or 24 h and seeded at either 5.0 x 10(5) or 2.5 x 10(5) cells/flask, respectively, and cultured in different concentrations of SerXtend. All treatment media supported granulosa cell proliferation to some extent; the SerXtend treatment provided the highest proliferation rate at all concentrations above .3125%. Also, during the proliferative stage of the growth curve, cells in the SerXtend treatment produced lower amounts of progesterone compared with cells in the other treatments. In summary, granulosa cells may be propagated in vitro in a serum-free environment without inducing progesterone production.
开发一种用于牛颗粒细胞的无血清培养系统，该系统能使细胞增殖而不诱导类固醇生成，这将为研究人员提供一个重要的体外工具，用于确定卵泡发生过程中的分化机制。本研究的目的是确定一种商业制备的血清替代品和一种培养基补充剂对牛颗粒细胞增殖和孕酮产生的影响。通过抽吸直径为2至8毫米卵泡的卵泡液获得颗粒细胞。对于每个实验，构建生长曲线以确定颗粒细胞对几种不同培养基添加物的增殖和类固醇生成反应。在培养的第1、2、4、6和8天对细胞进行计数以确定细胞浓度，并收集培养基以确定孕酮含量。在实验1中，颗粒细胞以5.0×10⁵的初始接种率在补充血清的培养基中培养48小时，然后分配到五种处理之一，包括单独的培养基或含有胎牛血清（FBS；1%）、Gibco BRL培养基补充剂-x（GMS-X；1%）、无脂肪酸牛血清白蛋白（FAF-BSA；4毫克/毫升）或SerXtend（5%）的培养基。对于实验2和3，颗粒细胞分别在补充血清的培养基中培养48或24小时，并分别以5.0×10⁵或2.5×10⁵个细胞/瓶的接种率接种，然后在不同浓度的SerXtend中培养。所有处理培养基在一定程度上都支持颗粒细胞增殖；在所有高于0.3125%的浓度下，SerXtend处理提供了最高的增殖率。此外，在生长曲线的增殖阶段，与其他处理的细胞相比，SerXtend处理的细胞产生的孕酮量较低。总之，颗粒细胞可以在无血清环境中体外增殖而不诱导孕酮产生。
