A new approach to neural cell culture for long-term studies
A new approach to neural cell culture for long-term studies
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DOI：
10.1016/s0165-0270(01)00412-5
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发表时间：
2001-09-30
影响因子：
3
通讯作者：
DeMarse, TB
中科院分区：
文献类型：
--
作者：
Potter, SM;DeMarse, TB
我们开发了一种新的细胞培养方法，可以在数月内保持细胞的健康和无菌。使用传统技术，原代神经元培养物很少能存活超过 2 个月。由于蒸发而导致的培养基渗透强度的增加是导致这些培养物健康状况逐渐下降的一个重要且未被充分认识的因素。由于这一点以及空气传播病原体污染的可能性始终存在，迄今为止，对任何特定培养物进行重复或扩展实验即使不是不可能，也是很困难的。我们通过使用形成气密密封的培养皿盖克服了生存问题，并结合了透明疏水膜（氟化乙烯-丙烯），该膜选择性地渗透氧气（O-2）和二氧化碳（CO2），并且相对不渗透水蒸气。这可以防止污染并大大减少蒸发，从而允许使用非加湿培养箱。我们采用这种技术在多电极阵列上培养大鼠胚胎的分离皮质培养物。经过一年多的培养，神经元仍然表现出强大的自发电活动。密封培养皿与细胞外多电极记录和刺激相结合，可以研究培养的神经元网络的发育、适应和跨月的长期可塑性。膜密封培养皿也可用于培养许多其他易蒸发和污染的细胞类型。 (C) 2001 Elsevier Science B.V. 保留所有权利。
We have developed a new method for culturing cells that maintains their health and sterility for many months. Using conventional techniques, primary neuron cultures seldom survive more than 2 months. Increases in the osmotic strength of media due to evaporation are a large and underappreciated contributor to the gradual decline in the health of these cultures. Because of this and the ever-present likelihood of contamination by airborne pathogens, repeated or extended experiments on any given culture have until now been difficult, if not impossible. We surmounted survival problems by using culture dish lids that form a gas-tight seal, and incorporate a transparent hydrophobic membrane (fluorinated ethylene-propylene) that is selectively permeable to oxygen (O-2) and carbon dioxide (CO2), and relatively impermeable to water vapor. This prevents contamination and greatly reduces evaporation, allowing the use of a non-humidified incubator. We have employed this technique to grow dissociated cortical cultures from rat embryos on multi-electrode arrays. After more than a year in culture, the neurons still exhibit robust spontaneous electrical activity. The combination of sealed culture dishes with extracellular multi-electrode recording and stimulation enables study of development, adaptation, and very long-term plasticity, across months, in cultured neuronal networks. Membrane-sealed dishes will also be useful for the culture of many other cell types susceptible to evaporation and contamination. (C) 2001 Elsevier Science B.V. All rights reserved.