Tong Zhi-Bin, Huang Ruili, Braisted John, Chu Pei-Hsuan, Simeonov Anton, Gerhold David L
Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20850, United States.
Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20850, United States.
SLAS Discov. 2024 Apr;29(3):100143. doi: 10.1016/j.slasd.2024.01.004. Epub 2024 Jan 26.
Three-dimensional (3D) cell culture in vitro promises to improve representation of neuron physiology in vivo. This inspired development of a 3D culture platform for LUHMES (Lund Human Mesencephalic) dopaminergic neurons for high-throughput screening (HTS) of chemicals for neurotoxicity. Three culture platforms, adhesion (2D-monolayer), 3D-suspension, and 3D-shaken, were compared to monitor mRNA expression of seven neuronal marker genes, DCX, DRD2, ENO2, NEUROD4, SYN1, TH, and TUBB3. These seven marker genes reached similar maxima in all three formats, with the two 3D platforms showing similar kinetics, whereas several markers peaked earlier in 2D adhesion compared to both 3D culture platforms. The differentiated LUHMES (dLUHMES) neurons treated with ziram, methylmercury or thiram dynamically increased expression of metallothionein biomarker genes MT1G, MT1E and MT2A at 6 h. These gene expression increases were generally more dynamic in 2D adhesion cultures than in 3D cultures, but were generally comparable between 3D-suspension and 3D-u plate (low binding) platforms. Finally, we adapted 3D-suspension culture of dLUHMES and neural stem cells to 1536 well plates with a HTS cytotoxicity assay. This HTS assay revealed that cytotoxicity IC values were not significantly different between adhesion and 3D-suspension platforms for 31 of 34 (91%) neurotoxicants tested, whereas IC values were significantly different for at least two toxicants. In summary, the 3D-suspension culture platform for LUHMES dopaminergic neurons supported full differentiation and reproducible assay results, enabling quantitative HTS (qHTS) for cytotoxicity in 1536 well format with a Robust Z' score of 0.68.
体外三维（3D）细胞培养有望改善体内神经元生理学的表现。这激发了用于LUHMES（隆德人类中脑）多巴胺能神经元的3D培养平台的开发，用于对化学物质进行神经毒性的高通量筛选（HTS）。比较了三种培养平台，即贴壁（2D单层）、3D悬浮和3D振荡培养，以监测七个神经元标记基因DCX、DRD2、ENO2、NEUROD4、SYN1、TH和TUBB3的mRNA表达。这七个标记基因在所有三种培养形式中都达到了相似的最大值，两个3D平台显示出相似的动力学，而与两个3D培养平台相比，有几个标记在2D贴壁培养中达到峰值的时间更早。用福美锌、甲基汞或福美双处理的分化型LUHMES（dLUHMES）神经元在6小时时动态增加了金属硫蛋白生物标志物基因MT1G、MT1E和MT2A的表达。这些基因表达的增加在2D贴壁培养中通常比在3D培养中更具动态性，但在3D悬浮和3D - u板（低结合）平台之间通常具有可比性。最后，我们将dLUHMES和神经干细胞的3D悬浮培养应用于1536孔板，并进行了HTS细胞毒性测定。该HTS测定表明，在测试的34种神经毒性剂中的31种（91%）中，贴壁和3D悬浮平台之间的细胞毒性IC值没有显著差异，而至少有两种毒性剂的IC值存在显著差异。总之，用于LUHMES多巴胺能神经元的3D悬浮培养平台支持完全分化和可重复的测定结果，能够在1536孔板中进行细胞毒性的定量HTS（qHTS），稳健Z'评分为0.68。
