The network formation assay: a spatially standardized neurite outgrowth analytical display for neurotoxicity screening
The network formation assay: a spatially standardized neurite outgrowth analytical display for neurotoxicity screening
复制标题
DOI：
10.1039/b922193j
复制
发表时间：
2010-01-01
期刊：
影响因子：
6.1
通讯作者：
West, Jonathan
中科院分区：
文献类型：
--
作者：
Frimat, Jean-Philippe;Sisnaiske, Julia;West, Jonathan
我们提供了一个快速、可重复性和灵敏的神经毒性测试平台，该平台结合了轴突生长分析和细胞模式的优点。这种方法包括在六角形阵列中对神经元细胞进行图案化，以标准化相邻细胞节点之间的距离，从而标准化轴突互连的长度。这一功能与定义的化验坐标相结合，为快速和灵敏的分析提供了简化的显示。我们称之为网络形成试验(NFA)。为了证明这种分析方法，我们使用了一种新的细胞图案化技术，包括薄膜聚二甲基硅氧烷(PDMS)微接触打印。分化的人SH-SY5Y神经母细胞瘤细胞在阵列中高效定植，可靠产生图案率达70%以上。神经元阵列表面支持轴突生长，形成相互连接的神经元网络。暴露于丙烯酰胺，一种神经毒性的参考化合物，抑制了网络的形成。NFA的剂量-反应曲线被用来确定20%网络抑制(NI20)值为260微米，这个浓度大约比常规细胞存活率测定产生的值低10倍，表明NFA可以区分网络形成抑制效应和总的细胞毒效应。抑制丝裂原活化蛋白激酶(MAPK)ERK1/2和磷脂酰肌醇-3-激酶(PI-3K)信号通路也会在非细胞毒性浓度下产生剂量依赖性的网络形成减少。为了进一步完善分析，开发了一个模拟来管理模式占用变化对网络形成概率的影响。总而言之，这些发展和示范突显了NFA在满足神经毒理学和神经发育生物学中高通量应用的需求方面的潜力。
We present a rapid, reproducible and sensitive neurotoxicity testing platform that combines the benefits of neurite outgrowth analysis with cell patterning. This approach involves patterning neuronal cells within a hexagonal array to standardize the distance between neighbouring cellular nodes, and thereby standardize the length of the neurite interconnections. This feature coupled with defined assay coordinates provides a streamlined display for rapid and sensitive analysis. We have termed this the network formation assay (NFA). To demonstrate the assay we have used a novel cell patterning technique involving thin film poly(dimethylsiloxane) (PDMS) microcontact printing. Differentiated human SH-SY5Y neuroblastoma cells colonized the array with high efficiency, reliably producing pattern occupancies above 70%. The neuronal array surface supported neurite outgrowth, resulting in the formation of an interconnected neuronal network. Exposure to acrylamide, a neurotoxic reference compound, inhibited network formation. A dose-response curve from the NFA was used to determine a 20% network inhibition (NI20) value of 260 mu M. This concentration was approximately 10-fold lower than the value produced by a routine cell viability assay, and demonstrates that the NFA can distinguish network formation inhibitory effects from gross cytotoxic effects. Inhibition of the mitogen-activated protein kinase (MAPK) ERK1/2 and phosphoinositide-3-kinase (PI-3K) signaling pathways also produced a dose-dependent reduction in network formation at non-cytotoxic concentrations. To further refine the assay a simulation was developed to manage the impact of pattern occupancy variations on network formation probability. Together these developments and demonstrations highlight the potential of the NFA to meet the demands of high-throughput applications in neurotoxicology and neurodevelopmental biology.