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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 b y 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 µm) 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.
Part 7: Representative Results:-When experiments are carried out properly, neuron cells loaded to the soma compartment locate close to microchannel inlets and axonal layer start to form inside the axon/glia compartments after 1 week of culture. Sign of neuron cell aggregation inside the soma compartment can be an indication that cells are not healthy. When loading oligodendrocytes after two weeks of neuron culture, axon/glia compartments should be covered with dense layer of axons and oligodendrocytes should be loaded on top of the axonal layer.
We have developed a multi-compartment co-culture platform for studying mammalian CNS axon-glia interaction. CNS axons were successfully isolated from neuronal cell bodies/dendrites, and oligodendrocytes were successfully co-cultured inside the device. Neuron density can be varied by applications, but too low or too high concentration can cause cells to die. Also, it is important to change only half of culture media so that the cells or axonal layer on the substrate are not damaged. We expect that this system will be a powerful tool for studying CNS axon-glia signaling networks in vitro and provide a path toward a high-throughput platform for screening growth factors or potential drug candidates that promote myelination and myelin repair.
This work was supported by the National Institutes of Health / National Institute of Mental Health (NIH/NIMH) grant #1R21MH085267
Disclosures: “I have nothing to disclose”
Jaewon Park, Department of Electrical and Computer Engineering, Texas A&M University, Email: jwpark/at/ece.tamu.edu.
Hisami Koito, Department of Veterinary Integrative Biosciences, Texas A&M University, Email: hkoito/at/cvm.tamu.edu.
Jianrong Li, Department of Veterinary Integrative Biosciences, Texas A&M University, Email: jrli/at/cvm.tamu.edu.
Arum Han, Department of Electrical and Computer Engineering, Texas A&M University, Email: arum.han/at/ece.tamu.edu.