Mbarek O, Vergé V, Hevor T
Laboratoire de Physiologie, Université d'Orléans, France.
In Vitro Cell Dev Biol Anim. 1998 May;34(5):401-11. doi: 10.1007/s11626-998-0022-0.
In primary cultures, much evidence shows the existence of different subtypes of astrocytes that are not all identified. One methodology for studying these subtypes can be their cloning. The present investigation shows a method for a direct cloning of astrocytes without previous immortalization. Astrocytes from the cerebral cortex of newborn rats were cultured, purified by shaking, and harvested by trypsinization. One single astrocyte was plated in a small volume of a homemade cloning medium. After getting a colony, successive platings were made using larger and larger vessels, up to 60-mm-diameter petri dishes. Then, subcultures were made. The yield of the cloning was similar to that of common eukaryotic cell clonings. All along the cloning procedure, the cells were positively immunostained with anti-glial fibrillary acidic protein antibodies. Cloned cells from some batches were spindle-shaped, looking like fibroblasts. Nevertheless, they were immunostained with anti-glial fibrillary acidic protein antibodies, unlike true fibroblasts. These spindle-shaped astrocytes were compared to cells from an astrocytoma cell line that had the same shape. The growth pattern of the astrocytoma cells was different from that of the astrocytes cloned from the primary cultures. All the types of studied cells contained glycogen. On the basis of the criteria of morphology, of glial fibrillary acidic protein immunolabeling, and of glycogen synthesis, the cloned cells kept the characteristics of astrocytes. This study shows that it is perfectly possible to get clones of astrocytes from one astrocyte without previous immortalization, giving thus a convenient material for the study of astrocyte biology.
在原代培养中，大量证据表明存在尚未完全鉴定的不同亚型的星形胶质细胞。研究这些亚型的一种方法是对其进行克隆。本研究展示了一种直接克隆星形胶质细胞而无需预先永生化的方法。从新生大鼠大脑皮层分离的星形胶质细胞进行培养，通过振荡纯化，并用胰蛋白酶消化收获。将单个星形胶质细胞接种在少量自制的克隆培养基中。获得一个集落之后，依次使用越来越大的培养器皿进行传代培养，直至直径为60毫米的培养皿。然后进行传代培养。克隆的产量与普通真核细胞克隆的产量相似。在整个克隆过程中，细胞用抗胶质纤维酸性蛋白抗体进行阳性免疫染色。某些批次的克隆细胞呈纺锤形，看起来像成纤维细胞。然而，与真正的成纤维细胞不同，它们用抗胶质纤维酸性蛋白抗体进行免疫染色。将这些纺锤形星形胶质细胞与来自具有相同形状的星形细胞瘤细胞系的细胞进行比较。星形细胞瘤细胞的生长模式与从原代培养中克隆的星形胶质细胞不同。所有研究的细胞类型都含有糖原。基于形态学、胶质纤维酸性蛋白免疫标记和糖原合成的标准，克隆细胞保持了星形胶质细胞的特征。这项研究表明，完全有可能从单个星形胶质细胞获得克隆，而无需预先永生化，从而为星形胶质细胞生物学研究提供了便利材料。
