Improved culture of human stem cell neurons in XonaChips™

The XonaChip™ offers advantages for culturing neurons differentiated from human stem cells. Differentiated neurons attach and distribute more evenly in the XonaChip™ than in silicone-based devices, resulting in healthy cultures that can be maintained for 4-5 weeks or more.
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Introduction

Neurons differentiated from human stem cells are increasingly used in neuroscience. The unique extreme polarization of these and other post-mitotic neurons demands an approach to measure and manipulate distinct neuronal compartments. Microfabricated multicompartment devices, pioneered by Xona scientists, have become well-established and well-used research tools for neuroscientists in the last 10-15 years 1-5. These devices compartmentalize neurons and provide a method to physically and chemically manipulate subcellular regions of neurons, including somata, dendrites, axons, and synapses 6-8.

To provide an easy-to-use and fully assembled device, Xona has developed plastic XonaChipsTM 8 (see Introducing XonaChips™ for more details). In this current TechNote, researchers at UNC-Chapel Hill differentiated human neural stem cells into glutamatergic neurons and found that XonaChips improve the long-term culture of these neurons over previous silicone-based compartmentalized devices.

Results & Discussion

Human neural stem cells (NSCs) were differentiated into neurons and neuronal projections entered the axonal compartment after one week (7-10 days) in the chip with differentiation media (Figure 1). The resulting neurons attached and distributed more evenly within the somatic compartment of the chip than in PDMS devices. Neurons in the XonaChips™ had healthy bundled axons and neurons were maintained within the chips for 4-5 weeks.

Figure 1. Human stem cell differentiated neurons grown in XonaChips™ and side-by-side comparison with PDMS compartmentalized devices.

Methods

XonaChipsTM were prepared according to the protocol. XC Pre-CoatTM was first used to ensure even wetting of the chip. XC Pre-CoatTM is included with each XonaChipTM order. The chip was then coated with Poly-L-Ornithine (20 µg/ml) and laminin (10 µg/ml) before pre-conditioning with NSC media. Poly(dimethylsiloxane) (PDMS) or silicone-based compartmentalized devices were prepared according to Xona’s silicone-based devices protocol. These devices were also coated with Poly-L-Ornithine and laminin before pre-conditioning with NSC media.

H9-derived human neural stem cells (ThermoFisher Scientific, 510088) were thawed according to the manufacturer’s instructions. Approximately 70,000 NSCs were seeded into the right compartment of the XonaChipTM. The same number were seeded into the right compartment of the PDMS compartmentalized device. Images were acquired with an inverted phase contrast microscope.

Conclusion

In summary, the XonaChip™ is a fully assembled multicompartment device that is easy to use and results in healthy, long-lasting cultures. Importantly, as shown previously, these chips allow microenvironments to be established as with our SND series devices and are equally compatible with high-resolution fluorescence microscopy.

About Xona Microfluidics, LLC

Xona Microfluidics, LLC is a California LLC based in Temecula, California with R&D facilities in Research Triangle Park, North Carolina. More information can be found at xonamicrofluidics.com.

If you are interested in testing a XonaChipTM contact us at info@xona.us

 

 

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2          Bigler, R. L., Kamande, J. W., Dumitru, R., Niedringhaus, M. & Taylor, A. M. Messenger RNAs localized to distal projections of human stem cell derived neurons. Sci Rep. 7 (1), 611, doi:10.1038/s41598-017-00676-w, (2017).

3          Nagendran, T. et al. Distal axotomy enhances retrograde presynaptic excitability onto injured pyramidal neurons via trans-synaptic signaling. Nat Commun. 8 (1), 625, doi:10.1038/s41467-017-00652-y, (2017).

4          Jia, L. et al. MiR-34a Regulates Axonal Growth of Dorsal Root Ganglia Neurons by Targeting FOXP2 and VAT1 in Postnatal and Adult Mouse. Molecular Neurobiology. doi:10.1007/s12035-018-1047-3, (2018).

5          Van Laar, V. S. et al. Evidence for compartmentalized axonal mitochondrial biogenesis: Mitochondrial DNA replication increases in distal axons as an early response to Parkinson’s disease-relevant stress. The Journal of Neuroscience. doi:10.1523/jneurosci.0541-18.2018, (2018).

6          Taylor, A. M. et al. A microfluidic culture platform for CNS axonal injury, regeneration and transport. Nat Methods. 2 (8), 599-605, doi:10.1038/nmeth777, (2005).

7          Taylor, A. M., Dieterich, D. C., Ito, H. T., Kim, S. A. & Schuman, E. M. Microfluidic local perfusion chambers for the visualization and manipulation of synapses. Neuron. 66 (1), 57-68, doi:10.1016/j.neuron.2010.03.022, (2010).

8          Nagendran, T., Poole, V., Harris, J. & Taylor, A. M. Use of Pre-Assembled Plastic Microfluidic Chips for Compartmentalizing Primary Murine Neurons Journal of visualized experiments : JoVE.  (in press).