Notter M F, Hansen J T, Okawara S, Gash D M
Department of Neurobiology and Anatomy, University of Rochester Medical School, NY 14642.
Exp Brain Res. 1989;76(1):38-46. doi: 10.1007/BF00253621.
In order to maintain a chronic supply of growth factor for medulla cells in vitro, chromaffin cells from rat, African green monkeys and man were co-cultured with C6 glioma cells, which secrete growth factors that sustain sympathetic neurons in vitro. The response of chromaffin cells to coculture was compared to treatment of medullary cells with nerve growth factor (NGF) alone. Dispersed chromaffin cell preparations were obtained by a trypsin-collagenase procedure, and subjected to differential plating on collagen-coated surfaces. With both human and monkey tissue, non-chromaffin cells did attach to the culture plates and an enriched chromaffin cell population could be replated. Rat adrenal medulla cells survived very poorly in vitro and were not enriched in this procedure. Cultured human and monkey chromaffin cells survived as epithelial cells (50%) and showed neuritic outgrowth on 55 to 66% of the cells after eight days when treated with nerve growth factor (NGF). These cells showed strong catecholamine histofluorescence, tyrosine hydroxylase (TH) and dopamine beta hydroxylase (DBH) immunoreactivity. In contrast, only ten percent of adult rat chromaffin cells survived in culture, although NGF treatment rescued an additional 20% of the cells and induced neuritic outgrowth after one week in vitro. C6 glioma cells were treated with mitomycin C bromodeoxyuridine to inhibit mitosis and were plated with the various medulla cells in a one to one ratio. Both human and monkey chromaffin cells expressed extensive and enhanced neuritic arborization within eight days of co-culture, (64-82% respectively) and exhibited intimate contact with the glioma cells as seen at the ultrastructural level. Importantly, survival of adult rat adrenal medulla cells was enhanced to 50% or more with 40% of the cells extending neurites when co-cultured with glioma cells for seven days. Chromaffin cells from all three species reacted for TH, DBH and PNMT in co-culture and were histo-fluorescent. The majority of these cells were also immunoreactive for serotonin and enkephalin, while only 37% of chromaffin cells indicated the presence of NPY. These data indicate that adrenal medulla can be maintained in vitro as the neuronal phenotype when co-cultured with growth factor producing cells and that this strategy may be useful for in vivo transplantation studies.
为了在体外维持髓质细胞生长因子的长期供应，将大鼠、非洲绿猴和人的嗜铬细胞与C6胶质瘤细胞共培养，C6胶质瘤细胞可分泌在体外维持交感神经元的生长因子。将嗜铬细胞对共培养的反应与单独用神经生长因子（NGF）处理髓质细胞的反应进行比较。通过胰蛋白酶 - 胶原酶法获得分散的嗜铬细胞制剂，并在胶原包被的表面进行差异铺板。对于人和猴的组织，非嗜铬细胞确实附着在培养板上，富集的嗜铬细胞群体可以重新铺板。大鼠肾上腺髓质细胞在体外存活很差，在此过程中未得到富集。培养的人和猴嗜铬细胞作为上皮细胞存活（50%），在用神经生长因子（NGF）处理八天后，55%至66%的细胞出现神经突生长。这些细胞显示出强烈的儿茶酚胺组织荧光、酪氨酸羟化酶（TH）和多巴胺β羟化酶（DBH）免疫反应性。相比之下，成年大鼠嗜铬细胞在培养中只有10%存活，尽管NGF处理在体外一周后挽救了另外20%的细胞并诱导了神经突生长。用丝裂霉素C溴脱氧尿苷处理C6胶质瘤细胞以抑制有丝分裂，并以1:1的比例与各种髓质细胞一起铺板。在共培养八天内，人和猴嗜铬细胞均表现出广泛且增强的神经分支（分别为64% - 82%），并且在超微结构水平上显示出与胶质瘤细胞的紧密接触。重要的是，成年大鼠肾上腺髓质细胞与胶质瘤细胞共培养七天后，存活率提高到50%或更高，40%的细胞长出神经突。来自所有三个物种的嗜铬细胞在共培养中对TH、DBH和PNMT有反应，并且具有组织荧光。这些细胞中的大多数对5 - 羟色胺和脑啡肽也有免疫反应性，而只有37%的嗜铬细胞显示有神经肽Y。这些数据表明，肾上腺髓质与产生生长因子的细胞共培养时可在体外维持为神经元表型，并且这种策略可能对体内移植研究有用。
