Taylor Anne M, Rhee Seog Woo, Tu Christina H, Cribbs David H, Cotman Carl W, Jeon Noo Li
Department of Biomedical Engineering, University of California at Irvine, Irvine, California 92697.
Langmuir. 2003 Mar 4;19(5):1551-1556. doi: 10.1021/la026417v.
This paper describes and characterizes a novel microfabricated neuronal culture device. This device combines microfabrication, microfluidic, and surface micropatterning techniques to create a multicompartment neuronal culturing device that can be used in a number of neuroscience research applications. The device is fabricated in poly(dimethylsiloxane), PDMS, using soft lithography techniques. The PDMS device is placed on a tissue culture dish (polystyrene) or glass substrate, forming two compartments with volumes of less than 2 μL each. These two compartments are separated by a physical barrier in which a number of micron-size grooves are embedded to allow growth of neurites across the compartments while maintaining fluidic isolation. Cells are plated into the somal (cell body) compartment, and after 3-4 days, neurites extend into the neuritic compartment via the grooves. Viability of the neurons in the devices is between 50 and 70% after 7 days in culture; this is slightly lower than but comparable to values for a control grown on tissue culture dishes. Healthy neuron morphology is evident in both the devices and controls. We demonstrate the ability to use hydrostatic pressure to isolate insults to one compartment and, thus, expose localized areas of neurons to insults applied in soluble form. Due to the high resistance of the microgrooves for fluid transport, insults are contained in the neuritic compartment without appreciable leakage into the somal compartment for over 15 h. Finally, we demonstrate the use of polylysine patterning in combination with the microfabricated device to facilitate identification and visualization of neurons. The ability to direct sites of neuronal attachment and orientation of neurite outgrowth by micropatterning techniques, combined with fluidically isolated compartments within the culture area, offers significant advantages over standard open culture methods and other conventional methods for manipulating distinct neuronal microenvironments.
本文描述并表征了一种新型的微加工神经元培养装置。该装置结合了微加工、微流体和表面微图案化技术，以创建一种多隔室神经元培养装置，可用于多种神经科学研究应用。该装置采用软光刻技术在聚二甲基硅氧烷（PDMS）中制造。将PDMS装置放置在组织培养皿（聚苯乙烯）或玻璃基板上，形成两个隔室，每个隔室的体积小于2μL。这两个隔室由一个物理屏障隔开，屏障中嵌入了许多微米尺寸的凹槽，以允许神经突穿过隔室生长，同时保持流体隔离。将细胞接种到胞体（细胞体）隔室中，3 - 4天后，神经突通过凹槽延伸到神经突隔室。培养7天后，装置中神经元的存活率在50%至70%之间；这略低于在组织培养皿上生长的对照值，但与之相当。在装置和对照中都能明显看到健康的神经元形态。我们展示了利用静水压力将损伤隔离到一个隔室的能力，从而使神经元的局部区域暴露于以可溶形式施加的损伤。由于微凹槽对流体传输的高阻力，损伤在神经突隔室中被限制，在超过15小时内不会明显泄漏到胞体隔室中。最后，我们展示了将聚赖氨酸图案化与微加工装置结合使用，以促进神经元的识别和可视化。通过微图案化技术引导神经元附着位点和神经突生长方向的能力，结合培养区域内流体隔离的隔室，相对于标准的开放培养方法和其他用于操纵不同神经元微环境的传统方法具有显著优势。
