A multi-compartment CNS neuron-glia Co-culture microfluidic platform.
A multi-compartment CNS neuron-glia Co-culture microfluidic platform.
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DOI：
10.3791/1399
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发表时间：
2009-09
期刊：
影响因子：
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通讯作者：
Jaewon Park;H. Koito;Jianrong Li;A. Han
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作者：
Jaewon Park;H. Koito;Jianrong Li;A. Han
我们提出了一种用于体外中枢神经系统轴突-胶质细胞相互作用研究的新型多室神经元共培养微系统平台，能够并行进行多达六个独立实验以实现更高的通量。我们开发了一种新的制造方法，通过单一的软光刻工艺来创建在同一器件内具有微观和宏观结构的微流体器件，从而实现具有良好可重复性的大规模制造。多隔室微流体共培养平台由一个用于神经元的体细胞隔室和六个用于少突胶质细胞（OL）的轴突/神经胶质隔室组成。体细胞室和轴突/神经胶质细胞室通过轴突引导微通道阵列连接，这些微通道充当物理屏障，将神经元体细胞限制在体细胞室中，同时允许轴突生长到轴突/神经胶质细胞室中。装载到轴突/神经胶质区室中的 OL 只能与轴突相互作用，而不能与神经元胞体或树突相互作用，从而实现局部轴突-神经胶质相互作用研究。微通道还实现了隔室之间的流体隔离，允许在单个设备上进行六个独立的实验，以获得更高的通量。使用聚二甲基硅氧烷 (PDMS) 的软光刻是生物医学微型设备中常用的技术。这些设备上的储液器通常通过手动冲压来限定。虽然简单，但该过程的对准不良和耗时使得该过程不适合必须重复创建大量储层的情况。新开发的方法不需要手动打孔储层，克服了这些限制。首先，使用微铣床在聚甲基丙烯酸甲酯（PMMA）块上制作七个储液器（深度：3.5 mm）。然后，在玻璃基板上制造的脊微结构阵列在 PMMA 块上热压印，以限定连接体细胞和轴突/神经胶质细胞室的微通道。该过程导致宏观尺度储层（3.5 毫米）和微观尺度通道（2.5 微米）在单个 PMMA 母模内重合。使用软光刻获得用作模具母版的 PDMS 复制品，并从此母版复制最终的 PDMS 器件。将来自 E16-18 大鼠的原代神经元装入体细胞室并培养两周。细胞培养一周后，轴突穿过微通道并在轴突/神经胶质室内形成仅轴突的网络层。轴突在六个轴突/神经胶质区室中均匀生长，并且在体外共培养 14 天时，将来自 P1-2 大鼠的 OL 添加到轴突/神经胶质区室中。
We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability. The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput. Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 microm) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master. Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.