Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue.
Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue.
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DOI：
10.1083/jcb.85.3.890
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发表时间：
1980-06
期刊：
影响因子：
--
通讯作者：
de Vellis J
中科院分区：
文献类型：
--
作者：
McCarthy KD;de Vellis J
发展了一种新的方法来制备几乎纯的星形胶质细胞和少突胶质细胞分离培养。该方法的基础是：(A)从出生后的大鼠大脑制备的培养物中没有存活的神经元，(B)培养物中星形胶质细胞和少突胶质细胞的分层，以及(C)当在37℃的眼眶摇床上摇动培养物15-18h时，覆盖的少突胶质细胞选择性地脱离，制备的材料看起来超过98%的纯度，并含有大约1-2x10(7)活细胞(20-40毫克细胞蛋白)。用三种方法表征了这两种培养类型。首先，用电子显微镜检查每个制剂(星形胶质细胞和少突胶质细胞的混合培养和分离培养)中的细胞，并评估每个制剂的纯度。其次，检测了2‘，3’-环核苷酸3‘-磷酸水解酶(EC 3.1.4.37)和甘油磷酸脱氢酶(EC 1.1.1.8)两种少突胶质细胞标志物。第三，考察了不同培养类型环磷酸腺苷的积累规律。除了这些研究外，我们还考察了脑提取物和二丁酰cAMP对每种制剂的大体形态和超微结构的影响。这些药物诱导星形胶质细胞突起的形成，对少突胶质细胞没有明显的形态影响。总而言之，结果表明，星形胶质细胞和少突胶质细胞的培养基本上是纯的，可以制备和维持。这些准备工作将大大有助于检查这两类主要中枢神经系统细胞的生物化学、生理学和药理学。
A novel method has been developed for the preparation of nearly pure separate cultures of astrocytes and oligodendrocytes. The method is based on (a) the absence of viable neurons in cultures prepared from postnatal rat cerebra, (b) the stratification of astrocytes and oligodendrocytes in culture, and (c) the selective detachment of the overlying oligodendrocytes when exposed to sheer forces generated by shaking the cultures on an orbital shaker for 15--18 h at 37 degrees C. Preparations appear greater than 98% pure and contain approximately 1-2 x 10(7) viable cells (20--40 mg of cell protein). Three methods were used to characterize these two culture t ypes. First, electron microscopic examination was used to identify the cells in each preparation (mixed and separated cultures of astrocytes and oligodendrocytes) and to assess the purity of each preparation. Second, two oligodendroglial cell markers, 2',3'-cyclic nucleotide 3'- phosphohydrolase (EC 3.1.4.37) and glycerol phosphate dehydrogenase (EC 1.1.1.8) were monitored. Third, the regulation of cyclic AMP accumulation in each culture type was examined. In addition to these studies, we examined the influence of brain extract and dibutyryl cAMP on the gross morphology and ultrastructure of each preparation. These agents induced astroglial process formation without any apparent morphological effect on oligodendrocytes. Collectively, the results indicate that essentially pure cultures of astrocytes and of oligodendrocytes can be prepared and maintained. These preparations should significantly aid in efforts to examine the biochemistry, physiology, and pharmacology of these two major classes of central nervous system cells.