Multi-compartment neuron-glia co-culture platform for localized CNS axon-glia interaction study

Abstract

Formation of myelin sheaths by oligodendrocytes (OLs) in the central nervous system (CNS) is essential for rapid nerve impulse conduction. Reciprocal signaling between axons and OLs orchestrates myelinogenesis but remains largely elusive. In this study, we present a multi-compartment CNS neuron-glia microfluidic co-culture platform. The platform is capable of conducting parallel localized drug and biomolecule treatments while carrying out multiple co-culture conditions in a single device for studying axon-glia interactions at a higher throughput. The "micro-macro hybrid soft-lithography master fabrication" (MMHSM) technique enables a large number of precisely replicated PDMS devices incorporating both millimeter and micrometer scale structures to be rapidly fabricated without any manual reservoir punching processes. Axons grown from the neuronal somata were physically and fluidically isolated inside the six satellite axon/glia compartments for localized treatments. Astrocytes, when seeded and co-cultured after the establishment of the isolated axons in the satellite axon/glia compartments, were found to physically damage the established axonal layer, as they tend to grow underneath the axons. In contrast, oligodendrocyte progenitor cells (OPCs) could be co-cultured successfully with the isolated axons and differentiated into mature myelin basic protein-expressing OLs with processes aligning to neighboring axons. OPCs inside the six axon/glia compartments were treated with a high concentration of ceramide (150 μM) to confirm the fluidic isolation among the satellite compartments. In addition, isolated axons were treated with varying concentrations of chondroitin sulfate proteoglycan (CSPG, 0-25 μg ml(-1)) within a single device to demonstrate the parallel localized biomolecular treatment capability of the device. These results indicate that the proposed platform can be used as a powerful tool to study CNS axonal biology and axon-glia interactions with the capacity for localized biomolecular treatments.

Fig. 1

(A) 3D illustration of the multi-compartment neuron-glia co-culture microsystem capable of carrying out multiple localized axon treatments in parallel (Inset: Cross-sectional view of the truncated cone shaped soma compartment). (B) Illustration showing the isolation of axons from neuronal somata for localized axon-glia interaction studies. (C) Photographic image of the neuron-glia co-culture platform (20 × 20 × 4 mm³) filled with seven different color dyes for visualization.

Fig. 2

Fabrication and assembly steps for the multi-compartment neuron-glia co-culture microsystem. The PDMS device having both the macroscale reservoirs and the microscale axon-guiding channels is replicated by a single step PDMS soft-lithography process using the ‘MMHSM’ technique. The final PDMS devices are sterilized and assembled on a PDL coated 6-well culture plates for cell culture.

Fig. 3

(A) SEM images of the multi-compartment PDMS neuron-glia co-culture device fabricated by the ‘MMHSM’ technique. Scale bar = 20 μm. (B) 3D reconstructed optical profilometry image of the axon-guiding microchannels connecting the soma and the axon/glia compartment. (C) Average height and width of the axon-guiding microchannels on the PMMA master, PDMS master and the PDMS device (mean ± SD). (D) Changes in surface roughness throughout the fabrication process (mean ± SD).

Fig. 4

(A-B) Calcein-AM (green) stained images of neurons confined inside the soma compartment at DIV 1. (C) Histogram showing the distances of the closest neuron cell from the axon-guiding microchannel inlets. (D) Close-up view of axons crossing into the neighboring axon/glia compartment from the soma compartment. More than 90% of channels were filled with axons after two weeks of culture. (E) Reconstructed image of isolated axons in an axon/glia compartment immunostained for NF. White dotted box delineates the area (0.8 × 1.6 mm²) analyzed for ACR. (F) Close-up view of dense axonal layer formed inside the axon/glia compartment at DIV 17. (G) ACR analysis of the multi-compartment neuron-glia co-culture platform showing device-to-device repeatability and axon/glia compartment-to-compartment variations within a single device. (H) Isolated axons inside the six axon/glia compartments of a single device (Stained for NF = red).

Fig. 5

(A) Schematic illustration showing fluidic level difference between the soma compartment and the axon/glia compartments for fluidic isolation. Minute flow from the soma compartment toward the axon/glia compartments prevent localized treatments to isolated axons from diffusing into the soma compartment. (B) Calcein-AM stained images of OLs inside the axon/glia compartments without ceramide treatment and with ceramide treatment (150 μM) after 48 hours. Scale bars = 20 μm.

Fig. 6

Isolated axons inside the six axon/glia compartments screened with six different concentrations of CSPG. Scale bar = 100 μm.

Fig. 7

(A) OPCs uniformly distributed inside an axon/glia compartment. (B) Close-up view of differentiating OPCs inside the axon/glia compartment after two days of co-culture.

Fig. 8

Immunostained images of axons and OLs inside the axon/glia compartment at DIV 29. (A) OPCs co-cultured on top of isolated axonal layer successfully differentiated into mature MBP-expressing OLs. (B) OL processes aligning with axons inside the axon/glia compartment to form myelin-like sheaths. (axon: NF-red, mature OL: MBP-green). Scale bars = 20 μm.

Fig. 9

Images showing co-cultured axons and glial cells at DIV 27. Isolated axons co-cultured with (A-D) OPCs and (E-H) OPCs and astrocytes. Co-cultured astrocytes physically damaged pre-established axonal layer while forming a layer on the substrate. Axons were stained for NF (red) and mature OLs were stained for MBP (green). Scale bars = 100 μm.

Fig. 10

Astrocytes promoted OPC differentiation. Percentage of MBP expressing OLs from total number of OPCs, based on initial seeding density, was analyzed by immunocytochemistry and cell counting. More OPCs developed into mature-OLs when cultured in the presence of astrocytes (mean ± SD, n = 5, p < 0.05).