Bertoncello I, Bradley T R, Chamley W A, Hodgson G S
J Cell Physiol. 1982 Nov;113(2):224-30. doi: 10.1002/jcp.1041130207.
Normal, primary explanted, bovine granulosa cells grow reproducibly in agar culture as anchorage-independent clones. Epidermal growth factor (EGF) and rat erythrocytes are effective stimulators of colony formation, and when both are added to the culture medium at optimal concentrations, there is an enhancement of colony numbers and colony size, indicative of an independent, and operationally additive, mode of action for the two factors. The ability of cells propagated from agar clones to secrete progesterone, and to augment progesterone secretion 4-fold in the presence of 1 mM dbcAMP is proof that colonies originate from and are composed of functional granulosa cells. Maximal colony numbers are present at day 10 of incubation, and colony forming cells undergo self-renewal as assessed by the ability of cells from primary colonies to reclone in agar. Absolute cloning efficiency, however, is dependent on a number of factors. Inherent variability exists in cloning efficiency of granulosa cells from individual follicles. Quantitative and qualitative clonal growth was improved at an osmolality of less than 300 mOsm when compared with higher osmolalities. Cl-1 medium and the alpha modification of Eagle's medium were equally effective in supporting agar clonogenic growth, whereas both Ham's F12 and NCTC 135 media exhibited poor clonogenic growth supporting properties. The substitution of agarose for agar did not affect colony numbers but colonies grown in the presence of agarose tended to be smaller and more uniform in size.
正常的、原代移植的牛颗粒细胞在琼脂培养中可重复生长，形成不依赖贴壁的克隆。表皮生长因子（EGF）和大鼠红细胞是集落形成的有效刺激物，当二者以最佳浓度添加到培养基中时，集落数量和集落大小均会增加，这表明这两种因子具有独立且在作用上可叠加的作用方式。从琼脂克隆中传代培养的细胞分泌孕酮的能力，以及在1 mM二丁酰环磷腺苷（dbcAMP）存在下孕酮分泌增加4倍，证明集落源自功能性颗粒细胞且由其组成。培养第10天时集落数量达到最大值，通过原代集落细胞在琼脂中再次克隆的能力评估，集落形成细胞可自我更新。然而，绝对克隆效率取决于多种因素。来自单个卵泡的颗粒细胞克隆效率存在固有变异性。与较高渗透压相比，渗透压低于300 mOsm时，定量和定性克隆生长均得到改善。Cl-1培养基和伊格尔培养基的α改良型在支持琼脂克隆生长方面同样有效，而哈姆氏F12培养基和NCTC 135培养基的克隆生长支持特性较差。用琼脂糖替代琼脂不影响集落数量，但在琼脂糖存在下生长的集落往往较小且大小更均匀。
