Kmicikiewicz I, Kozieł E, Pelczar U, Pierściński A, Bilińska B
Laboratory of Animal Endocrinology and Tissue Culture, Institute of Zoology, Jagiellonian University, Kraków, Poland.
Acta Histochem. 1998 Jul;100(3):297-307. doi: 10.1016/S0065-1281(98)80016-0.
Culturing cells on microcarriers maximizes the ratio of surface area to culture medium volume. The aim of this study was to show microcarrier culture as a useful tool for improvement of steroidogenic activity in mouse Leydig cells. Freshly isolated cells were grown on Sephadex microcarriers, Cytodex 3, and gelatin beads (gelaspheres) that were uncoated, coated with collagen or coated with fibronectin. The cells were cultured in control or LH-supplemented medium. The effect of LH on testosterone and estradiol secretion was studied with appropriate radioimmunoassays. The activity of hydroxysteroid dehydrogenases (3 beta-HSD, 17 beta-HSD) was evaluated histochemically. Leydig cells growing on microcarriers formed colonies. The strongest response to luteinizing hormone stimulation was observed on gelatin beads coated with fibronectin and collagen. In cells growing on a fibronectin layer, very strong activity of 3 beta-HSD and 17 beta-HSD and an increase in steroid hormone levels were observed. Basal and LH-stimulated testosterone and estradiol secretion were all much higher in the microcarrier than in the monolayer culture system. We conclude that Leydig cells maintain differentiated functions more efficiently on collagen- and fibronectin-coated gelasperes than on Sephadex microcarriers and that uncoated gelaspheres are less efficient than coated ones. Pure gelaspheres are unsuitable for improvement of the hormonal and enzymatic activity of Leydig cells.
在微载体上培养细胞可使表面积与培养基体积之比最大化。本研究的目的是表明微载体培养是提高小鼠睾丸间质细胞类固醇生成活性的一种有用工具。将新鲜分离的细胞接种在葡聚糖微载体、Cytodex 3以及未包被、胶原包被或纤连蛋白包被的明胶珠（gelaspheres）上。细胞在对照培养基或添加促黄体生成素（LH）的培养基中培养。采用适当的放射免疫分析法研究LH对睾酮和雌二醇分泌的影响。用组织化学方法评估羟类固醇脱氢酶（3β-HSD、17β-HSD）的活性。在微载体上生长的睾丸间质细胞形成集落。在纤连蛋白和胶原包被的明胶珠上观察到对促黄体生成素刺激的最强反应。在纤连蛋白层上生长的细胞中，观察到3β-HSD和17β-HSD的活性非常强，类固醇激素水平升高。在微载体培养体系中，基础状态下以及LH刺激后的睾酮和雌二醇分泌均显著高于单层培养体系。我们得出结论，睾丸间质细胞在胶原和纤连蛋白包被的gelaspheres上比在葡聚糖微载体上更有效地维持分化功能，且未包被的gelaspheres比包被的效率更低。纯gelaspheres不适合用于提高睾丸间质细胞的激素和酶活性。
