一种新型高通量三维脑细胞芯片的研制及农药神经毒性评价应用

Development of a novel high throughput brain-on-chip with 3D structure and its application in evaluation of pesticide-induced-neurotoxicity

摘要:

为了模拟体内脑组织细胞的三维生长环境，高效开展脑疾病和有关药物的研究工作，文中设计和制造了一种新型高通量三维脑细胞芯片。芯片由多孔滤网和三维脑细胞颗粒构成，装载于常规的96孔细胞培养板上使用。多孔滤网和细胞颗粒模具采用计算机建模、3D打印阳模、琼脂糖-PDMS两次翻模等技术制造，三维脑细胞颗粒是将小鼠胚胎脑细胞与海藻酸钠的混悬液浇灌和凝固于细胞颗粒模具，再将形成的水凝胶切割而成。用装载好的脑细胞芯片进行农药神经毒性测定，以0、10、30、50、100、200 μmol/L的毒死蜱或吡虫啉暴露胚胎脑细胞颗粒，培养完毕移去多孔滤网，方便地将细胞颗粒与培养液进行分离，然后检测细胞增殖、乙酰胆碱酯酶活力和乳酸脱氢酶释放以评估毒性。研究结果表明，在装载到96孔细胞培养板的多孔滤网中，胚胎脑细胞能够在水凝胶颗粒中正常生长增殖。在农药神经毒性测试中，毒死蜱与吡虫啉都呈现了量效相关的细胞生长增殖抑制，也表现出乙酰胆碱酯酶活力抑制、乳酸脱氢酶释放的毒性效应，但吡虫啉的作用明显弱于毒死蜱。文中构建了一种新型脑细胞芯片，可以结合酶标仪方便快速地检测药物对脑神经组织的毒性效应，也可以应用于药效和疾病机制研究。

Abstract:

We designed and fabricated a novel high throughput brain-on-chip with three dimensional structure with the aim to simulate the in vivo three-dimensional growth environment for brain tissues. The chip consists of a porous filter and 3D brain cell particles, and is loaded into a conventional 96-well plate for use. The filter and the particle molds were fabricated by using computer modeling, 3D printing of positive mold and agarose-PDMS double reversal mold. The 3D cell particles were made by pouring and solidifying a suspension of mouse embryonic brain cells with sodium alginate into a cell particle mold, and then cutting the resulting hydrogel into pieces. The loaded brain-on-chip was used to determine the neurotoxicity of pesticides. The cell particles were exposed to 0, 10, 30, 50, 100 and 200 μmol/L of chlorpyrifos or imidacloprid, separated conveniently from the medium by removing the porous filter after cultivation. Subsequently, cell proliferation, acetylcholinesterase activity and lactate dehydrogenase release were determined for toxicity evaluation. The embryonic brain cells were able to grow and proliferate normally in the hydrogel particles loaded into the filter in a 96-well plate. Pesticide neurotoxicity test showed that both chlorpyrifos and imidacloprid presented dose-dependent inhibition on cell growth and proliferation. Moreover, the pesticides showed inhibition on acetylcholinesterase activity and increase release of lactate dehydrogenase. However, the effect of imidacloprid was significantly weaker than that of chlorpyrifos. In conclusion, a novel brain-on-chip was developed in this study, which can be used to efficiently assess the drug neurotoxicity, pharmacodynamics, and disease mechanism by combining with a microtiterplate reader.