Vision Research Center and Departments of Ophthalmology and Basic Medical Science, University of Missouri - Kansas City, School of Medicine, 2411 Holmes St., Kansas City, MO 64108, United States.
J Neurosci Methods. 2012 Jan 15;203(1):141-5. doi: 10.1016/j.jneumeth.2011.09.007. Epub 2011 Sep 24.
Drug discovery and development efforts critically rely on cell-based assays for high-throughput screening. These assay systems mostly utilize immortalized cell lines, such as human embryonic kidney cells, and can provide information on cytotoxicity and cell viability, permeability and uptake of compounds as well as receptor pharmacology. While this approach has proven extremely useful for single-target pharmacology, there is an urgent need for neuropharmacological studies to screen novel drug candidates in a cellular environment resembles neurons in vivo more closely, in order to gain insight into the involvement of multiple signaling pathways. Primary cultured neuronal cells, such as cortical neurons, have long been used for basic research and low-throughput screening and assay development, and may thus be suitable candidates for the development of neuropharmacological high-throughput screening approaches. We here developed and optimized protocols for the use of primary cortical neuronal cells in high-throughput assays for neuropharmacology and neuroprotection, including calcium mobilization, cytotoxicity and viability as well as ion channel pharmacology. Our data show low inter-experimental variability and similar reproducibility as conventional cell line assays. We conclude that primary neuronal cultures provide a viable alternative to cell lines in high-throughput assay systems by providing a cellular environment more closely resembling physiological conditions in the central nervous system.
药物发现和开发工作严重依赖基于细胞的高通量筛选方法。这些检测系统大多使用永生化细胞系，如人胚肾细胞，可以提供有关细胞毒性和细胞活力、化合物的通透性和摄取以及受体药理学的信息。虽然这种方法在单靶标药理学方面已被证明非常有用，但迫切需要神经药理学研究在更接近体内神经元的细胞环境中筛选新型药物候选物，以深入了解多种信号通路的参与。原代培养神经元细胞，如皮质神经元，长期以来一直用于基础研究和低通量筛选以及检测方法的开发，因此可能是开发神经药理学高通量筛选方法的合适候选物。我们在这里开发并优化了用于高通量神经药理学和神经保护检测的原代皮质神经元细胞的方案，包括钙动员、细胞毒性和活力以及离子通道药理学。我们的数据显示，实验间的变异性低，与传统细胞系检测方法的重现性相似。我们得出结论，原代神经元培养物通过提供更接近中枢神经系统生理条件的细胞环境，为高通量检测系统中的细胞系提供了可行的替代方法。
