Konishi Yoshihiro, Lindholm Kristina, Yang Li-Bang, Li Rena, Shen Yong
Haldeman Laboratory of Molecular and Cellular Neurobiology, Sun Health Research Institute, Sun City, AZ 85351, USA.
Am J Pathol. 2002 Nov;161(5):1567-76. doi: 10.1016/S0002-9440(10)64435-5.
Isolation and culture of mature neurons from affected brain regions during diseased states provide a well-suited in vitro model system to study age-related neurodegeneration under dynamic conditions at cellular levels. We have developed a novel technique to isolate living neurons from rapidly autopsied human elderly brains, and have succeeded in keeping them alive in vitro. Specifically, the parietal cortex blocks were fractionated by density gradients and further enriched for neurons by an immunomagnetic sorting DNA-linker technique. The postmortem interval averaged 2.6 hours. After isolation and purification of neurons using this technology, the cells were maintained in vitro for 2 weeks. Our evaluation revealed that 80% of the isolated cells were neurons and they exhibited neurotransmitter phenotypes (glutamate and gamma-aminobutyric acid) as well as glutamate receptors. Studies on cell viability and calcium influx suggest that these isolated living cortical neurons still retain their typical neuronal functions. Our present study demonstrates that neurons isolated from human elderly brain autopsies can survive in vitro and maintain their functional properties. Our study has opened an opportunity to apply such neurons to dynamic pharmacological studies of neurological disorders at the single-cell level.
在疾病状态下从受影响的脑区分离和培养成熟神经元，为在细胞水平的动态条件下研究与年龄相关的神经退行性变提供了一个非常合适的体外模型系统。我们开发了一种新技术，可从快速尸检的人类老年大脑中分离活神经元，并成功使其在体外存活。具体而言，通过密度梯度对顶叶皮质块进行分级分离，并通过免疫磁珠分选DNA连接技术进一步富集神经元。平均尸检间隔时间为2.6小时。使用该技术分离和纯化神经元后，将细胞在体外培养2周。我们的评估显示，80%的分离细胞为神经元，它们表现出神经递质表型（谷氨酸和γ-氨基丁酸）以及谷氨酸受体。对细胞活力和钙内流的研究表明，这些分离的活皮质神经元仍保留其典型的神经元功能。我们目前的研究表明，从人类老年大脑尸检中分离的神经元能够在体外存活并维持其功能特性。我们的研究为在单细胞水平将此类神经元应用于神经疾病的动态药理学研究提供了机会。
