Three dimensional neuronal cell cultures more accurately model voltage gated calcium channel functionality in freshly dissected nerve tissue.
Three dimensional neuronal cell cultures more accurately model voltage gated calcium channel functionality in freshly dissected nerve tissue.
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DOI：
10.1371/journal.pone.0045074
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Kisaalita W
中科院分区：
文献类型：
--
作者：
Lai Y;Cheng K;Kisaalita W
研究表明，在传统平面上培养的神经元细胞可能表现出夸大的电压门控钙通道（VGCC）功能。为了更好地理解这一现象，我们在二维（2D）平面和三维（3D）合成聚l -乳酸（PLLA）和聚苯乙烯（PS）聚合物支架上培养取自小鼠颈上神经节（SCG）的原代神经元细胞。将这些2D和3d培养的细胞与新鲜解剖的SCG组织中的细胞进行比较，在高K+去极化的情况下，细胞内钙的增加。在3d培养和新鲜解剖的细胞中，钙的增加是相同的，但在2d培养的细胞中，钙的增加明显更高。这一发现确立了3d培养细胞的生理学相关性。为了阐明夸张的2d培养细胞功能背后的机制，我们获得了转录酶表达和相关膜蛋白分布（caveolin-1）。我们的研究结果支持这样一种观点，即2D培养的SCG细胞的VGCC功能被夸大可能是由于膜结构的差异，其特征是独特组织的腔泡脂筏。在临床前药物发现研究中使用3d培养细胞的实际意义是，这种平台将更有效地消除假阳性命中，从而提高筛选活动的总体产量。
It has been demonstrated that neuronal cells cultured on traditional flat surfaces may exhibit exaggerated voltage gated calcium channel (VGCC) functionality. To gain a better understanding of this phenomenon, primary neuronal cells harvested from mice superior cervical ganglion (SCG) were cultured on two dimensional (2D) flat surfaces and in three dimensional (3D) synthetic poly-L-lactic acid (PLLA) and polystyrene (PS) polymer scaffolds. These 2D- and 3D-cultured cells were compared to cells in freshly dissected SCG tissues, with respect to intracellular calcium increase in response to high K+ depolarization. The calcium increases were identical for 3D-cultured and freshly dissected, but significantly higher for 2D-cultured cells. This finding established the physiological relevance of 3D-cultured cells. To shed light on the mechanism behind the exaggerated 2D-cultured cells’ functionality, transcriptase expression and related membrane protein distributions (caveolin-1) were obtained. Our results support the view that exaggerated VGCC functionality from 2D cultured SCG cells is possibly due to differences in membrane architecture, characterized by uniquely organized caveolar lipid rafts. The practical implication of use of 3D-cultured cells in preclinical drug discovery studies is that such platforms would be more effective in eliminating false positive hits and as such improve the overall yield from screening campaigns.
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