Abstract
Multi-compartment microfluidic devices have become valuable tools for experimental neuroscientists, improving the organization of neurons and access to their distinct subcellular microenvironments for measurements and manipulations. While murine neurons are extensively used within these devices, there is a growing need to culture and maintain human neurons differentiated from stem cells within multi-compartment devices. Human neuron cultures have different metabolic demands and require longer culture times to achieve synaptic maturation. We tested different channel heights (100 μm, 400 μm, and open) to determine whether greater exposure to media for nutrient exchange might improve long-term growth of NIH-approved H9 embryonic stem cells differentiated into glutamatergic neurons. Our data showed an opposite result with both closed channel configurations having greater synaptic maturation compared to the open compartment configuration. These data suggest that restricted microenvironments surrounding neurons improve growth and maturation of neurons. We next tested whether covalently bound poly-D-lysine (PDL) might improve growth and maturation of these neurons as somata tend to cluster together on PDL adsorbed surfaces after long culture periods (>30 days). We found that covalently bound PDL greatly improved the differentiation and maturation of stem cell-derived neurons within the devices. Lastly, experimental paradigms using the multi-compartment platform show that axons of human stem cell derived neurons intrinsically regenerate in the absence of inhibitory cues and that isolated axons form presynaptic terminals when presented with synaptic targets.
Highlights
Microfluidic devices offer useful platforms for neuroscience that can mimic the neuron microenvironment on a cellular level
Interests have focused on the culture of hSCderived neurons as a more representative model for studies of human neurodegenerative diseases as these cells more closely resemble those found in the human brain tissue
Approaches to compartmentalizing neurons have included the use of Campenot Chambers (Campenot, 1977), filter-based isolations (Torre and Steward, 1992), and more recently microfluidic chambers
Summary
Microfluidic devices offer useful platforms for neuroscience that can mimic the neuron microenvironment on a cellular level. This is important for mechanistic studies, such as those pertaining to the pathology of neurodegenerative disorders (Poon et al, 2011; Wu et al, 2013; Zala et al, 2013; Van Laar et al, 2018; Zhang et al, 2018) and neurotrauma (Nagendran et al, 2017; Ohtake et al, 2018). Multi-compartment devices are configured to fluidically isolate distinct segments of neurons of cell bodies, dendrites, axons, and synapses. Studies have been achieved that would not have been feasible in vivo using these microfluidic platforms, such as studies of axonal transport, biochemical analysis of axons and axonal injury/regeneration [e.g., Taylor et al, 2005, 2013; Poon et al, 2011, 2013; Wu et al, 2013; Zala et al, 2013; Bigler et al, 2017; Nagendran et al, 2017]
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