中国修复重建外科杂志

中国修复重建外科杂志

利用微流控技术体外构建三维海马神经元网络的初步研究

查看全文

目的初步探讨利用微流控技术体外构建三维海马神经元网络的可行性。方法设计并采用标准湿法腐蚀工艺制备网络图案化的微流控芯片。取新生 SD 大鼠丘脑组织,分离培养原代海马神经元后,接种于微流控芯片上进行培养。3、5、7 d 时行免疫荧光染色,观察海马神经元生长情况;7 d 时行海马神经元网络电生理检测。结果荧光显微镜下观察显示,随培养时间延长,海马神经元数量逐渐增加,且神经元突起亦相应增多、增长,并且均匀规则分布在微流控芯片通道,提示成功构建三维海马神经元网络。培养 7 d 时可以检测到海马神经元网络的单通道及多通道自发放电信号,提示神经元网络具有初步的生物学功能。结论图案化微流控芯片可以使海马神经元按照局限路径生长,从而形成三维神经元网络,并且具有信号传导等相应的生物学功能。

ObjectiveTo preliminary study on the feasibility of constructing three-dimensional (3D) hippocampal neural network in vitro by using microfluidic technology.MethodsA network patterned microfluidic chip was designed and fabricated by standard wet etching process. The primary hippocampal neurons of neonatal Sprague Dawley rats were isolated and cultured, and then inoculated on microfluidic chip for culture. Immunofluorescence staining was used to observe the growth of hippocampal neurons at 3, 5, and 7 days of culture and electrophysiological detection of hippocampal neuron network at 7 days of culture.ResultsThe results showed that the number of hippocampal neurons increased gradually with the prolongation of culture time, and the neurite of neurons increased accordingly, and distributed uniformly and regularly in microfluidic chip channels, suggesting that the 3D hippocampal neuron network was successfully constructed in vitro. Single and multi-channel spontaneous firing signals of hippocampal neuronal networks could be detected at 7 days of culture, suggesting that neuronal networks had preliminary biological functions.ConclusionPatterned microfluidic chips can make hippocampal neurons grow along limited paths and form 3D neuron networks with corresponding biological functions such as signal transduction, which lays a foundation for further exploring the function of neuron networks in vitro.

关键词: 微流控芯片; 神经元网络; 三维培养; 海马神经元

Key words: Microfluidic chip; neuron network; three-dimensional culture; hippocampal neuron

引用本文: 孔宪敏, 田姗姗, 陈涛, 黄映辉. 利用微流控技术体外构建三维海马神经元网络的初步研究. 中国修复重建外科杂志, 2019, 33(2): 239-242. doi: 10.7507/1002-1892.201809094 复制

登录后 ,请手动点击刷新查看全文内容。 没有账号,
登录后 ,请手动点击刷新查看图表内容。 没有账号,
1. Ng AH, Uddayasankar U, Wheeler AR. Immunoassays in microfluidic systems. Anal BioanalChem, 2010, 397(3): 991-1007.
2. Xu Y, Yang X, Wang E. Review:Aptamers in microfluidic chips. Anal ChimActa, 2010, 683(1): 12-20.
3. Varghese SS, Zhu Y, Davis TJ, et al. FRET for lab-on-a-chip devices-current trends and future prospects. Lab Chip, 2010, 10(11): 1355-1364.
4. Vincent ME, Liu W, Haney EB, et al. Microfiuidic stochastic confinement enhances analysis of rate cells by isolating cells and creating high density environments for control of diffusible signals. Chem Soc Rev, 2010, 39(3): 974-984.
5. Papp K, Végh P, Hóbor R, et al. Characterization of factors influencing on-chip complement activation to optimize parallel measurement of antibody and complement proteins on antigen microarrays. J Immunol Methods, 2011, 375(1-2): 75-83.
6. Toh A, Wang Z, Yang C, et al. Engineering microfluidic concentration gradient generators for biological applications. Microfluidics and Nanofluidics, 2014, 16(1): 1-18.
7. Plessy C, Desbois L, Fujii T, et al. Population transcriptomics with single-cell resolution: a new field made possible by microfluidics: a technology for high throughput transcript counting and data-driven definition of cell types. Bioessays, 2013, 35(2): 131-140.
8. Qin J, Ye N, Liu X, et al. Microfluidic devices for the analysis of apoptosis. Electrophoresis, 2005, 26(19): 3780-3788.
9. Sia SK, Whitesides GM. Microfluidic devices fabricated in poly(dimethylsiloxane) for biological studies. Electrophoresis, 2003, 24(21): 3563-3576.
10. Sanders G, Manz A. Chip-based microsystems for genomic and proteomic analysis. TrAC Trends in Analytical Chemistry, 2000, 19(6): 364-378.
11. ShenF, KastrupCJ, Ismagilov F. Using microfluidics to understand the effect of spatial distribution of tissue factor on blood coagulation. Thrombosis Research, 2008, 122(Suppl1): S27-S30.
12. Gomez N, Lu Y, Chen S, et al. Immobilized nerve growth factor and microtopography havedistinct effects on polarization versus axon elongation in hippocampal cells in culture. Biomaterials, 2007, 28(2): 271-284.
13. Taylor AM, Blurton-Jones M, Rhee SW, et al. A microfluidic culture platform for CNSaxonal injury, regenerationand transport. Nature Methods, 2005, 2(8): 599-605.
14. Christian KM, Song HJ, Ming GL. Functions and dysfunctions of adult hippocampalneurogenesis. Annual Review of Neuroscience, 2014, 37(1): 243.
15. Parihar VK, Limoli CL. Cranial irradiation compromises neuronal architecture in the hippocampus. Proc Natl Acad Sci USA, 2013, 110(31): 12822-12827.
16. Chakraborti A, Allen A, Allen B, et al. Cranial irradiation alters dendritic spine densityand morphology in the hippocampus. PLoS One, 2012, 7(7): e40844.