Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue.

Abstract

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°C. Preparations appear >98% pure and contain -1-2 x 10' viable cells (20-40 mg of cell protein). Three methods were used to characterize these two culture types. 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.The cellular heterogeneity of the central nervous system has largely prevented investigators from describing the biochemical characteristics of welldefined cell populations within this system . To circumvent this problem, we and others have directed our efforts toward developing new tech-890 niques that could be used to prepare homogeneous populations of neurons, oligodendrocytes, or astrocytes (1,4,7,13,15,20,25). The separation of cells in primary culture has a number of advantages over other methods often used to purify brain cells (20) . As a result, we have concentrated J . CELL BIOLOGY

Axonal abnormalities in vanishing white matter

“…Astrocytes or forebrain mixed cells were isolated from cerebral hemispheres of E18 mice and cultured following previously described methods with minor modifications 27, 28, 29, 30, 31. Cultures were kept in a humidified incubator at 37°C and 7.5% CO 2 at all times.…”

TGF‐β signaling directly regulates transcription and functional expression of the electrogenic sodium bicarbonate cotransporter 1, NBCe1 (SLC4A4), via Smad4 in mouse astrocytes

“…For primary culture of mixed glia, mouse pups aged P2/P3 were used, according to McCarthy & de Vellis, 1980, as previously described (Schrödl‐Häußel et al, 2015). For obtaining cultures from Tgf‐β2 deficient mice, embryos at embryonic day 18 (E18) were used.…”

Differential expression of NDF/neuregulin receptors ErbB-3 and ErbB-4 and involvement in inhibition of neuronal differentiation

“…To correlate this with receptor expression, we generated primary cell cultures from E17 and newborn rat brains and enriched them for either neurons, astroglia, or oligodendrocytes, by using established protocols (McCarthy and de Vellis, 1980). Reverse transcriptase PCR analysis of total RNA extracted from the type-selected cultures, by using speci®c oligonucleotide primers and Southern blot analysis, identi®ed erbB-3 only in oligodendrocytic cultures, but erbB-4 was expressed, at this developmental stage, in all three types of brain cells ( Figure 5a).…”

“…Primary cell cultures of embryonic rat brains (E17) were prepared by trypsinization as previously described (McCarthy and de Vellis, 1980). The cultures were grown in Dulbecco's modi®ed Eagle's (DME) medium containing 10% fetal bovine serum (FBS), 2 mM glutamine, 50 u/ml penicillin and 50 mg/ml streptomycin.…”

“…The cultures were grown in Dulbecco's modi®ed Eagle's (DME) medium containing 10% fetal bovine serum (FBS), 2 mM glutamine, 50 u/ml penicillin and 50 mg/ml streptomycin. Astrocyte-and oligodendrocyte-enriched cultures were obtained from newborn rats using a previously described method (McCarthy and de Vellis, 1980). Neuron-enriched cultures were prepared from E17 rat brains by using a modi®cation of the previously described procedure (Barde et al, 1980).…”