This protocol describes a procedure for 3D culturing of neurons using Matrigel® within XonaChips®. Results using this method are illustrated using E18 rat neurons.
Download Post as PDF

Introduction

3D neuronal cultures have been an immense interest for researchers as they recapitulate the 3D complex microenvironment as seen in vivo. Among different approaches for culturing neurons in 3D, the Matrigel® matrix provides a promising environment for growing neurons2-5, because it is rich in structural proteins such as laminin, collagen, etc.—proteins that are found in the brain. This TechNote provides a protocol for 3D culturing of neurons within XonaChips® using Corning® Matrigel® matrix. For more information on cyclic olefin copolymer XonaChips®1,2, see the Introducing XonaChips® TechNote.

Protocol

  1. Place XonaChip® in a humidified secondary container such as a ChipTray™.
  2. Perform the coating procedures using XC Pre-Coat and XC PDL as described in the XonaChip® protocol for culturing murine derived primary neurons1.
  3. Dissociate rat neurons in Neurobasal media (NBM) supplemented with 1x Glutamax, 1x B27, and 1x Antibiotic- antimycotic. Use 90,000 cells per device.
  4. Prepare 10 µL of Matrigel®-cell mixture per device by adding 2 µL of Matrigel® to 90,000 cells and NBM resulting in a 20% Matrigel®-cell solution.
    NOTE: Slowly thaw Matrigel® on ice in a 4 °C refrigerator overnight or follow manufacturer’s guidelines. All steps should be completed on ice with consumables and reagents pre-chilled.
  5. Aspirate media from each well of the XonaChip®. Avoid aspirating from the main channels.
  6. Prepare 20% Matrigel® solution without cells in NBM and add 5 µL to the upper left well and 5 µL to the lower left well (axonal compartment).
  7. Pipette 5 µL of the Matrigel®-cell solution to the upper right well, followed by 5 µL to the lower right well of the XonaChip® using pre-chilled pipettes. Place the ChipTray™ containing the XonaChip® in the incubator at 5% CO2, 37 °C for 30 minutes.
    NOTE: Ensure that the Matrigel-cell solution is pipetted towards the main channel openings. Use a microscope to check the attachment off cells that entered the channel.
  8. Add ~150 µL of NBM media to the upper right well and then 150 µL to the lower right well. Add media on the left side as well. Incubate the XonaChip® at 5% CO2, 37 °C within ChipTrays™
  9. RReplace media with fresh pre-warmed NBM media. Incubate at 5% CO2, 37 °C.

Results

Neurons were dissociated from E18 rat combined cortex-hippocampus tissue and grown using the above protocol within two compartment XonaChips® (XC450) for 12 days (Fig. 1). Healthy neuron growth was evident including growth and isolations of axons. At 12 days in-vitro (DIV), neurons were fixed and immunostained with the dendritic marker, Map2, and synaptic marker, synapsin1. Z-stack images of fluorescently labeled neurons within the chamber were acquired using a Zeiss LSM 780 (40X 1.4 NA oil immersion objective) microscope. The 3D image was processed from the z-stack series using Imaris software. Both XonaChips® and Xona’s silicone devices are equally compatible for growing 3D neuron cultures using Corning® Matrigel® matrix.

Conclusion

In summary, culturing neurons in 3D serves to bridge the gap between traditional 2D cell culture approaches and in vivo animal models such as transgenic mice. 3D neuron cultures in these microfluidic devices provide high reproducibility and reliability for drug screening studies and may help to develop highly organized in vitro assays to mimic and treat brain disorders. 3D Matrigel® cultures in XonaChips® are compatible for high-resolution fluorescence microscopy imaging.

About Xona Microfluidics Inc

Xona Microfluidics, Inc is a life sciences company based in Research Triangle Park, North Carolina. More information can be found at xonamicrofluidics.com.

If you are interested in testing XonaChips® contact us at info@xona.us

References

  1. Nagendran, T., Poole, V., Harris, J. & Taylor, A. M. Use of Pre-Assembled Plastic Microfluidic Chips for Compartmentalizing Primary Murine Neurons. Vis. Exp. 2018; (141), e58421, doi:10.3791/58421.
  2. Paranjape, S.R., Nagendran, T., Poole, V., Harris, J., and Taylor, A.M. Compartmentalization of Human Stem Cell-Derived Neurons within Pre-Assembled Plastic Microfluidic Chips. J. Vis. Exp. 2019; (147), e59250, doi:10.3791/59250.
  3. Carla D’ Avanzo et al., Alzheimer’s in 3D culture: Challenges and perspectives. Bioessays 2015; 37: 1139–1148.
  4. Joseph park et al., A 3D human triculture system modeling neurodegeneration and neuroinflammation in Alzheimer’s disease. Nature Neuroscience 2018; 941–951.
  5. Leonardo D’Aiuto et al., Generation of three-dimensional human neuronal cultures: application to modeling CNS viral infections. Stem Cell Research & Therapy 2018; 9:134.
  6. Young Hye Kim et al., A 3D human neural cell culture system for modeling Alzheimer’s disease. Nat Protoc. 2015; 10(7): 985–1006
WordPress Image Lightbox Plugin