Santos Sónia Sá, Leite Sofia B, Sonnewald Ursula, Carrondo Manuel J T, Alves Paula M
Animal Cell Technology Laboratory, Instituto de Biologia Experimental e Tecnológica/Instituto de Tecnologia Química e Biológica (IBET/ITQB), Oeiras, Portugal.
J Neurosci Res. 2007 Nov 15;85(15):3386-97. doi: 10.1002/jnr.21409.
We report a study on neural metabolism of long-term three-dimensional cultures of rat embryonic brain cells in stirred vessels. Our experimental setup was optimized to keep viable aggregate cultures with neuronal maintenance for up to 44 days. Results show that aggregate size and shape could be hydrodynamically controlled depending on the impeller design, avoiding necrotic centers or significant losses in cell viability. Aggregates were composed mainly of neurons until day 16, whereas an effective growth of the glial population was observed after day 21. Cell metabolic status was evaluated by quantification of several metabolites in the culture medium; amino acid metabolism was used as a marker of metabolic interrelationships between neural cell types. Furthermore, (13)C-NMR spectroscopy was used on day 31 to explore specific metabolic pathways: incubation with [1-(13)C]glucose for 45 hr produced an increase in label incorporation in extracellular alanine, lactate, and glutamine, reflecting mainly astrocytic metabolism. The contribution of anaplerotic vs. oxidative pathways for glutamine synthesis was determined: a 92% reduction in the pyruvate carboxylase flux during the first 41 hr of incubation suggested a decrease in the need for replacing tricarboxylic acid cycle intermediates. We believe that our data corroborate the aggregating cultures as an attractive system to analyze brain cell metabolism being a valuable tool to model metabolic fluxes for in vitro brain diseases.
我们报告了一项关于搅拌容器中大鼠胚胎脑细胞长期三维培养的神经代谢研究。我们优化了实验装置，以维持具有神经元活性的聚集培养物长达44天。结果表明，根据叶轮设计，可以通过流体动力学控制聚集体的大小和形状，避免坏死中心或细胞活力的显著损失。直到第16天，聚集体主要由神经元组成，而在第21天后观察到胶质细胞群体有效生长。通过对培养基中几种代谢物进行定量来评估细胞代谢状态；氨基酸代谢被用作神经细胞类型之间代谢相互关系的标志物。此外，在第31天使用(13)C-NMR光谱来探索特定的代谢途径：用[1-(13)C]葡萄糖孵育45小时后，细胞外丙氨酸、乳酸和谷氨酰胺中的标记掺入增加，这主要反映了星形胶质细胞的代谢。确定了谷氨酰胺合成中回补途径与氧化途径的贡献：在孵育的前41小时内，丙酮酸羧化酶通量降低了92%，这表明替换三羧酸循环中间产物的需求减少。我们认为，我们的数据证实了聚集培养物是分析脑细胞代谢的一个有吸引力的系统，是模拟体外脑部疾病代谢通量的一个有价值的工具。
