The authors acknowledge support from Xona Microfluidics, LLC, the National Institute of Mental Health (R42 MH097377), and the National Institute of Neurological Disorders and Stroke (R41 NS108895, P30 NS045892). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

NOTE: Figure 1A,B shows a schematic of the pre-assembled COC chip, including the locations of the main channels or compartments, wells, and microgrooves. The compartmentalized chip can establish distinct fluidic microenvironments within a compartment as demonstrate by the isolation of food coloring dye (Figure 1C). The protocol for preparing the pre-assembled multi-compartment chip is given in Nagendran et al., Section 112.
1. Coating of multi-compartment chips for hSC-neurons
NOTE: Figure 2A shows an overview of the coating procedures.
<ol>
	<li>Dissolve poly-L-ornithine in cell culture-grade distilled water to make 600 &#181;L of solution per chip of a 20 &#181;g/mL working solution.</li>
	<li>Aspirate the remaining PBS left after preparing the chips from the wells. When aspirating, ensure that the pipet tip is away from the channel opening. 
	NOTE: The microfluidic channels must remain filled for the entirety of this procedure. Do not aspirate liquid from the channels/compartments, only the wells.</li>
	<li>Load 150 &#181;L of poly-L-ornithine working solution in the upper right well. Wait for 90 s or until liquid begins to fill the lower right well. Add 150 &#181;L of media to the lower right well and wait for 5 min to let the liquid pass through the microgrooves.</li>
	<li>Add 150 &#181;L of media to the upper left well. Wait 90 s and then add 150 &#181;L to the lower left well. Wrap the holder of the chip(s) with parafilm and place at 4 &#176;C overnight. 
	NOTE: Alternatively, place the chip and holder at 37 &#176;C for 1 h.</li>
	<li>Aspirate the media from the wells, ensuring that the pipet tip is placed away from the channel opening. Add 150 &#181;L of sterile water to the upper right well. Wait for 90 s and then add 150 &#181;L to the lower right well. Wait for 5 min to let the liquid pass through the microgrooves.</li>
	<li>Add 150 &#181;L of sterile water to the upper left well, wait 90 s and then add another 150 &#181;L to the lower left well.</li>
	<li>Repeat steps 1.5-1.6.</li>
	<li>Thaw laminin slowly at 2 &#176;C to 8 &#176;C and prepare a 10 &#181;g/mL working solution.</li>
	<li>Load 150 &#181;L of the laminin working solution to the upper right well. Wait for 90 s or until liquid begins to fill the lower right well. Pipet 150 &#181;L to the lower right well. Wait for 5 min to let the laminin go through the microgrooves.</li>
	<li>Add 150 &#181;L of laminin to the upper left well. Wait 90 s and then add 150 &#181;L to the lower left well. Incubate the chip within its holder for 2 h at 37 &#176;C.</li>
	<li>Rinse the chip with PBS (without Ca2+ and Mg2+). Load 150 &#181;L of PBS to the upper right well. Wait for 90 s or until liquid begins to fill the lower right well. Add 150 &#181;L of PBS to the lower right well and wait for 5 min to let the PBS go through the microgrooves.</li>
	<li>Pipet 150 &#181;L of PBS to the upper left well. After 90 s pipet 150 &#181;L to the lower left well.</li>
	<li>Aspirate PBS from the chip and then rinse the chip with NSC media&#160;(see Table of Materials). Load 150 &#181;L of media to the upper right well. After 90 s or when liquid passes through the right channel into the lower right well, add 150 &#181;L to the lower right well. Wait for 5 min to let the media flow through the microgrooves.</li>
	<li>Add 150 &#181;L of media to the upper left well and another 150 &#181;L to the lower left well. Incubate the chip in a 37 &#176;C incubator with 5% CO2 to pre-condition the chip until ready to seed NSCs. 
	NOTE: Chips can be pre-conditioned overnight if needed.</li>
</ol>
2. Seeding NSCs into the multicompartment chip
NOTE: This protocol used commercially purchased NSCs, unlike our previous publication which began with human embryonic stems cells5. In this protocol neural stem cells are plated directly into the plastic chips where they differentiate into neurons. The cells can be allowed to proliferate as NSCs (without differentiation) for up to 2 passages and stored in vials in liquid N2 for further use. This protocol uses NSC vials stored in liquid N2 after passage 1 or 2. Figure 2B shows an overview of the coating procedures.
<ol>
	<li>Thaw NSCs according to manufacturer&#8217;s instructions. 
	NOTE: This procedure applies to H9-derived NSCs. Other cell lines will require cell density optimization.</li>
	<li>Count the cell concentration using a hemocytometer and adjust using NSC media to get a concentration of 7 x 106 cells per mL.</li>
	<li>Aspirate media from each well of the chip. Avoid aspirating media from the main channels.</li>
	<li>Pipet 5 &#181;L of the cell solution to the upper right well, followed by 5 &#181;L to the lower right well (70,000 cells total). Ensure that the cells are pipetted towards the main channel openings. Use a microscope to check that cells have entered the channel. Wait 5 min for cells to adhere to the bottom of the chip. 
	NOTE: Either or both compartments can be used to load cells.</li>
	<li>Pipet ~150 &#181;L of NSC media to the upper right well and then 150 &#181;L to the lower right well. Repeat on the left side. Incubate at 5% CO2, 37 &#176;C within a suitable humidified container.</li>
	<li>After 48 hours, aspirate NSC media from the wells and replace with neural differentiation media by adding 150 &#181;L to each top well and each bottom well.</li>
	<li>Culture neurons within a 5% CO2, 37 &#176;C incubator within a suitable humidified container. 
	NOTE: Media should be replaced every 2-3 days. Because of the small volumes used in the chip, evaporation even within a humidified incubator can substantially impact cell viability. Use a suitable secondary container to provide additional humidity for long-term culture of cells with the chip (see Table of Materials).</li>
</ol>
3. Viral fluorescent labeling of hSC-neurons within the chip
NOTE: Multiple virus can be used to label neurons within the chip. Instructions below describe the use of G-deleted Rabies-mCherry or &#8211;eGFP virus. Potentially infectious materials must be handled according to applicable rules and regulations, and may require additional training and institutional approval.
<ol>
	<li>Warm ~400 &#181;L of fresh neural differentiation media per chip to 37 &#176;C.</li>
	<li>Remove 50 &#181;L of media from one well of the axon compartment into a centrifuge tube and then add 100,000 viral units of modified rabies virus to the tube. 
	NOTE: Properly dispose spent pipet tips and centrifuge tubes containing virus.</li>
	<li>Remove the remaining neural differentiation media from the axonal compartment and place into a centrifuge tube. Store at 37 &#176;C. 
	NOTE: Make sure that the channels remain filled with liquid.</li>
	<li>Mix the 50 &#181;L of virus solution with 150 &#181;L of warmed media. Pipet 100 &#181;L to one well of the axon compartment and 100 &#181;L to the other axon compartment well. Incubate for 2 h within a 37 &#176;C incubator. 
	NOTE: The difference in volumes between the axonal and somatic compartments helps ensure the virus stays in the axonal compartment during infection/incubation times.</li>
	<li>Withdraw and dispose of virus-containing media. 
	CAUTION: Do not remove liquid within the enclosed microfluidic channels.</li>
	<li>Gently pipet 75 &#181;L of fresh media to one axon compartment well and let the media flow through the channel into other axon well.</li>
	<li>Remove media from the axon compartment and dispose properly.</li>
	<li>Repeat steps 3.6 and 3.7.</li>
	<li>Fill the axon compartment with the saved media. Pipet an additional 50 &#181;L fresh media, if needed, and then return the chip to the incubator. This additional fresh media is to count towards loss of media during liquid transfer and evaporation during the process. 
	NOTE: Visible fluorescent labeling using these modified rabies viruses occurs by 48 h and up to 8 days after which cell death of infected neurons generally occurs due to virus toxicity. Neuron imaging can be performed as with other standard culture platforms (e.g., glass bottom dishes), but special consideration is needed to maintain sufficient humidity to prevent evaporative losses.</li>
</ol>
4. Fluidic isolation, axotomy, and immunostaining within the chip
<ol>
	<li>Follow fluidic isolation, axotomy, and immunostaining with the chip as described in Nagendran et al.12.</li>
</ol>

<ol>
	<li>Taylor, A. M., Wu, J., Tai, H. C., Schuman, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+translation+of+beta-catenin+regulates+synaptic+vesicle+dynamics.">Axonal translation of beta-catenin regulates synaptic vesicle dynamics.</a> The Journal of Neuroscience. 33 (13), 5584-5589 (2013).</li><li>Virlogeux, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstituting+Corticostriatal+Network+on-a-Chip+Reveals+the+Contribution+of+the+Presynaptic+Compartment+to+Huntington&#8217;s+Disease.">Reconstituting Corticostriatal Network on-a-Chip Reveals the Contribution of the Presynaptic Compartment to Huntington&#8217;s Disease.</a> Cell Reports. 22 (1), 110-122 (2018).</li><li>Neto, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmentalized+Microfluidic+Platforms:+The+Unrivaled+Breakthrough+of+&#60;em&#62;In+Vitro&#60;/em&#62;+Tools+for+Neurobiological+Research.">Compartmentalized Microfluidic Platforms: The Unrivaled Breakthrough of &#60;em&#62;In Vitro&#60;/em&#62; Tools for Neurobiological Research.</a> The Journal of Neuroscience. 36 (46), 11573-11584 (2016).</li><li>Pinto, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+proteasome+controls+presynaptic+differentiation+through+modulation+of+an+on-site+pool+of+polyubiquitinated+conjugates.">The proteasome controls presynaptic differentiation through modulation of an on-site pool of polyubiquitinated conjugates.</a> The Journal of Cell Biology. 212 (7), 789-801 (2016).</li><li>Bigler, R. L., Kamande, J. W., Dumitru, R., Niedringhaus, M., Taylor, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Messenger+RNAs+localized+to+distal+projections+of+human+stem+cell+derived+neurons.">Messenger RNAs localized to distal projections of human stem cell derived neurons.</a> Scientific Reports. 7 (1), 611 (2017).</li><li>Nagendran, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distal+axotomy+enhances+retrograde+presynaptic+excitability+onto+injured+pyramidal+neurons+via+trans-synaptic+signaling.">Distal axotomy enhances retrograde presynaptic excitability onto injured pyramidal neurons via trans-synaptic signaling.</a> Nature Communications. 8 (1), 625 (2017).</li><li>Van Laar, V. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+compartmentalized+axonal+mitochondrial+biogenesis:+Mitochondrial+DNA+replication+increases+in+distal+axons+as+an+early+response+to+Parkinson&#39;s+disease-relevant+stress.">Evidence for compartmentalized axonal mitochondrial biogenesis: Mitochondrial DNA replication increases in distal axons as an early response to Parkinson&#39;s disease-relevant stress.</a> The Journal of Neuroscience. , (2018).</li><li>del R&#237;o, J. A., Ferrer, I., Gav&#237;n, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+cellular+prion+protein+in+interneuronal+amyloid+transmission.">Role of cellular prion protein in interneuronal amyloid transmission.</a> Progress in Neurobiology. 165-167, 87-102 (2018).</li><li>Jia, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MiR-34a+Regulates+Axonal+Growth+of+Dorsal+Root+Ganglia+Neurons+by+Targeting+FOXP2+and+VAT1+in+Postnatal+and+Adult+Mouse.">MiR-34a Regulates Axonal Growth of Dorsal Root Ganglia Neurons by Targeting FOXP2 and VAT1 in Postnatal and Adult Mouse.</a> Molecular Neurobiology. , (2018).</li><li>Taylor, A. M., Dieterich, D. C., Ito, H. T., Kim, S. A., Schuman, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+local+perfusion+chambers+for+the+visualization+and+manipulation+of+synapses.">Microfluidic local perfusion chambers for the visualization and manipulation of synapses.</a> Neuron. 66 (1), 57-68 (2010).</li><li>Menon, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+E3+Ubiquitin+Ligase+TRIM9+Is+a+Filopodia+Off+Switch+Required+for+Netrin-Dependent+Axon+Guidance.">The E3 Ubiquitin Ligase TRIM9 Is a Filopodia Off Switch Required for Netrin-Dependent Axon Guidance.</a> Developmental cell. 35 (6), 698-712 (2015).</li><li>Nagendran, T., Poole, V., Harris, J., Taylor, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Pre-Assembled+Plastic+Microfluidic+Chips+for+Compartmentalizing+Primary+Murine+Neurons.">Use of Pre-Assembled Plastic Microfluidic Chips for Compartmentalizing Primary Murine Neurons.</a> Journal of visualized experiments : JoVE. (141), (2018).</li><li>Taylor, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidic+culture+platform+for+CNS+axonal+injury,+regeneration+and+transport.">A microfluidic culture platform for CNS axonal injury, regeneration and transport.</a> Nature Methods. 2 (8), 599-605 (2005).</li><li>Taylor, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+mRNA+in+uninjured+and+regenerating+cortical+mammalian+axons.">Axonal mRNA in uninjured and regenerating cortical mammalian axons.</a> The Journal of Neuroscience. 29 (15), 4697-4707 (2009).</li><li>Oh, J. H., Jung, C. R., Lee, M. O., Kim, J., Son, M. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=+Comparative+analysis+of+human+embryonic+stem+cellderived+neural+stem+cells+as+an+in+vitro+human+model."> Comparative analysis of human embryonic stem cellderived neural stem cells as an in vitro human model.</a> International Journal of Molecular Medicine. 41 (2), 783-790 (2018).</li><li>Lazar, M., Miles, L. M., Babb, J. S., Donaldson, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+deficits+in+young+adults+with+High+Functioning+Autism+and+their+impact+on+processing+speed.">Axonal deficits in young adults with High Functioning Autism and their impact on processing speed.</a> Neuroimage Clinical. 4, 417-425 (2014).</li><li>DePaul, M. A., Lin, C. Y., Silver, J., Lee, Y. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatory+repair+strategy+to+promote+axon+regeneration+and+functional+recovery+after+chronic+spinal+cord+injury.">Combinatory repair strategy to promote axon regeneration and functional recovery after chronic spinal cord injury.</a> Scientific Reports. 7 (1), 9018 (2017).</li></ol>

S.P. and T.N. declare no competing financial interests. V.P. is an employee of Xona Microfluidics, LLC. J.H. is a member of Xona Microfluidics, LLC. A.M.T. is an inventor of the microfluidic chamber/chip (US 7419822 B2, EPO 2719756 B1) and is a member of Xona Microfluidics, LLC.

The preassembled multi-compartment COC chip is an easy-to-use compartmentalized platform to differentiate and maintain human NSCs into neurons for long-term (&#62;4 weeks). In this protocol, we demonstrate the differentiation of human NSCs into glutamatergic neurons, retrograde label neurons, perform immunocytochemistry, visualize dendritic spine morphology and perform axotomy. These chips are compatible with high resolution imaging and there is no autofluorescence with the COC12.
COC multi-compartment chips are functionally equivalent to silicone-based compartmentalized devices, and have benefits and drawbacks as described previously12. Table 1 compares multi-compartment COC chips and silicone devices for culturing hSC-neurons. The COC compartmentalized chips provide a better hydrophilic surface for attachment and maintenance of stem cells over a long culture period. PDMS-based devices need to be assembled and attached to glass coverslips. The hydrophobic nature of the PDMS devices causes aggregation of stem cells5; this leads to both challenges in imaging at the cellular level and greater susceptibility to physical damage due the movement of cell aggregates during media changes. The plastic chip overcomes these challenges. COC is gas impermeable, unlike PDMS, so air pockets trapped or formed within the channels must be removed by the user. The pre-coating solution reduces the possibility for air to become trapped in the channels. This solution consists of ethanol and other agents. A previously published protocol for culturing murine neurons within these plastic chips provides additional details about pipetting cells and media within the chips12. NSCs are more fragile that murine neurons, so must be handled more gently. It is also critical to mix the stem cells thoroughly before plating by gently pipetting them up and down.
Use of neurons derived from in vitro differentiated human stem cells is becoming increasingly popular in medicine and research. These neurons are important for research and clinical applications for many CNS disorders, including neurodegenerative diseases and traumatic brain injuries. These neurons closely resemble human fetal neurons15. In the future, appropriately aged neurons could be generated from stem cells to mimic age-related neuronal function and used in conjunction with these compartmentalized devices. These devices will facilitate research in diseases affecting axon health and function such as axon deficits in neurons of patients diagnosed with autism spectrum disorders and axonal regeneration after injury16,17.

Human stem cell differentiated neurons (hSC-neurons) are increasingly used for biological research. These neurons, which can be derived from human source material, are of great interest for translational research, including the study of traumatic brain injuries and neurodegenerative disorders such as Alzheimer&#8217;s disease. Thus, tools to improve and facilitate the study of hSC-neurons are in demand.
To study the unique polarized morphology of neurons, many researchers use multi-compartmentalized microfluidic devices1,2,3,4,5,6,7,8,9,10,11. These devices enable measurements and manipulations of long projection neurons with unique subcellular access. Multi-compartmentalized microfluidic devices consist of two parallel microfluidic compartments separated by microgrooves, which guide axonal growth. Neurons or neural stem cells (NSCs) are plated in the somatodendritic compartment, and then adhere to the bottom of the compartment surface after minutes. Differentiated neurons grow and extend their axons/projections through the microgroove region into an adjacent and isolated axonal compartment. In the past, these devices were exclusively made using poly (dimethylsiloxane) (PDMS) replica molding. PDMS devices have many drawbacks previously described12, including persistent hydrophobicity and the need to assemble to a glass coverslip immediately prior to use. Pre-assembled injection molded chips overcome many of these drawbacks and are sold commercially (see Table of Materials)12. The compartments of these chips are permanently made hydrophilic and the entire chip is injection molded in optically transparent cyclic olefin copolymer (COC).
This protocol demonstrates how to use this COC chip to differentiate human NSCs into excitatory neurons, and to separate and fluidically isolate their long neuronal projections. For this demonstration, neurons were differentiated from NIH-approved H9 stem cells. Similar procedures can be used to differentiate human induced pluripotent stem cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor hydrazide 488</td>
			<td>ThermoFisher Scientific</td>
			<td>A10436</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor secondary antibodies</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>anti_beta-tubulin III</td>
			<td>Aves</td>
			<td>TUJ</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>anti-vGAT antibody</td>
			<td>Synaptic Systems</td>
			<td>131 003</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>anti-vGlut1 antibody</td>
			<td>NeuroMab</td>
			<td>75-066</td>
			<td>clone N28/9, 1:100</td>
		</tr>
		<tr>
			<td>complete neural stem cell media:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;REC HU EGF 
			10 UG BIOSOURCE (TM)&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>PHG0314</td>
			<td>20ng/mL&#160;</td>
		</tr>
		<tr>
			<td>REC HU FGF BASIC 
			10 UG BIOSOURCE (TM)&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>PHG0024</td>
			<td>20ng/mL&#160;</td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement (100X)</td>
			<td>ThermoFisher Scientific</td>
			<td>35050061</td>
			<td>2mM&#160;</td>
		</tr>
		<tr>
			<td>KnockOut DMEM/F-12</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>12660012</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro Neural Supplement</td>
			<td>ThermoFisher Scientific</td>
			<td>A1050801</td>
			<td>2%</td>
		</tr>
		<tr>
			<td>Epifluorescence imaging system</td>
			<td>EVOS Fluorescence imaging system</td>
			<td>AMF4300</td>
			<td>10x objective</td>
		</tr>
		<tr>
			<td>fluorinated ethylene propylene film</td>
			<td>American Durafilm</td>
			<td>50A</td>
			<td>0.5 mil thickness</td>
		</tr>
		<tr>
			<td>Fluoromount G</td>
			<td>ThermoFisher Scientific</td>
			<td>00-4958-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco DPBS without Calcium and Magnesium</td>
			<td>ThermoFisher Scientific</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>GIBCO HUMAN NSC (H9) KIT 
			COMBO KIT 
			&#160; 
			&#160;</td>
			<td>Gibco</td>
			<td>N7800200</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Laminin</td>
			<td>ThermoFisher Scientific</td>
			<td>23017015</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Pasteur pipettes</td>
			<td>Sigma-Aldrich</td>
			<td>CLS7095D5X SIGMA</td>
			<td>5.75 in length</td>
		</tr>
		<tr>
			<td>H9-DERIVED HU NEURAL STEM CELL 
			1E6 CELLS/VIAL; 1 ML&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>510088</td>
			<td></td>
		</tr>
		<tr>
			<td>hibernate-E Medium</td>
			<td>ThermoFisher Scientific</td>
			<td>A1247601</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator, 5% CO2 37 &#176;C</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laser scanning confocal imaging system</td>
			<td>Olympus</td>
			<td>FV3000RS</td>
			<td>30x silicone oil objective</td>
		</tr>
		<tr>
			<td>modified rabies virus</td>
			<td>Salk Institute for Biological Studies</td>
			<td>G-deleted Rabies-eGFP</td>
			<td>Material Transfer Agreement required</td>
		</tr>
		<tr>
			<td>Mr. Frosty&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>5100-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Neural differentiation media</td>
			<td></td>
			<td></td>
			<td>Per 100 mL.&#160;</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (100x)</td>
			<td>ThermoFisher Scientific</td>
			<td>15240112</td>
			<td>1mL (100X)</td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma Aldrich</td>
			<td>A8960</td>
			<td>200mM&#160;</td>
		</tr>
		<tr>
			<td>BDNF</td>
			<td>ThermoFisher Scientific</td>
			<td>PHC7074</td>
			<td>40 ng/mL&#160;</td>
		</tr>
		<tr>
			<td>Gibco B27 Plus Supplement (50X)</td>
			<td>FisherScientific</td>
			<td>A3582801</td>
			<td>2mL (50X)</td>
		</tr>
		<tr>
			<td>Gibco CultureOne Supplement (100X)&#160;</td>
			<td>FisherScientific</td>
			<td>A3320201</td>
			<td>1mL (100X)</td>
		</tr>
		<tr>
			<td>Gibco Neurobasal Plus Medium</td>
			<td>FisherScientific</td>
			<td>A3582901</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro Accutase Cell Dissociation Reagent</td>
			<td>ThermoFisher Scientific</td>
			<td>A1110501</td>
			<td></td>
		</tr>
		<tr>
			<td>Taylor Wharton Liquid N2 dewar</td>
			<td>FisherScientific</td>
			<td>20HCB11M</td>
			<td></td>
		</tr>
		<tr>
			<td>triton X-100</td>
			<td>ThermoFisher Scientific</td>
			<td>28314</td>
			<td></td>
		</tr>
		<tr>
			<td>XC pre-coat&#160;</td>
			<td>Xona Microfluidics, LLC</td>
			<td>XC Pre-Coat</td>
			<td>included with XonaChips</td>
		</tr>
		<tr>
			<td>XonaChip</td>
			<td>Xona Microfluidics, LLC</td>
			<td>XC450</td>
			<td>450 &#181;m length microgroove barrier</td>
		</tr>
		<tr>
			<td>Humidifier Tray</td>
			<td>Xona Microfluidics, LLC</td>
			<td>humidifier tray</td>
			<td></td>
		</tr>
	</tbody>
</table>

compartmentalization of human stem cell-derived neurons within pre-assembled plastic microfluidic chips

Use of microfluidic devices to compartmentalize cultured neurons has become a standard method in neuroscience. This protocol shows how to use a pre-assembled multi-compartment chip made in a cyclic olefin copolymer (COC) to compartmentalize neurons differentiated from human stem cells. The footprint of these COC chips are the same as a standard microscope slide and are equally compatible with high resolution microscopy. Neurons are differentiated from human neural stem cells (NSCs) into glutamatergic neurons within the chip and maintained for 5 weeks, allowing sufficient time for these neurons to develop synapses and dendritic spines. Further, we demonstrate multiple common experimental procedures using these multi-compartment chips, including viral labeling, establishing microenvironments, axotomy, and immunocytochemistry.

After one week (7-10 days) in the chip with differentiation media, NSCs differentiate into neurons and neuronal projections enter the axonal compartment (Figure 3). Within the chip, neurons attach and distribute evenly within the somatic compartment. In comparison, neurons in PDMS devices clump/aggregate as early as 5 days post addition of differentiation media, leading to compromised cell health as can be seen in the Figure 3B (day 13 magnified image). Neurons in the chips look healthy with bundled axons. Healthy neurons can be maintained within the chips for 4-5 weeks.
To visualize neuron maturation and dendritic spine development, modified rabies virus was delivered to the axonal compartment at Day 29 to retrograde label neurons, including dendritic spines, with mCherry fluorescent protein. Four days after rabies virus infection, neurons extending axons into the axonal compartment expressed mCherry. The neurons at differentiation day 33 showed formation of dendritic spines (Figure 4). Visualization of dendritic spines demonstrates that NSC derived neurons differentiated within the chips form mature synapses.
The multi-compartment chip is also compatible with immunocytochemistry to visualize the cellular localization of protein. After 26 days of maintaining neurons in differentiation media, neurons were labeled for the excitatory synaptic marker, vGlut1 (Figure 5). These results show that virally labeled neurons co-localize with vGlut1 (Figure 5E) and neuron specific marker, &#946;-tubulin III (Figure 5F-H).
The ability to create distinct microenvironments isolated to axons was also demonstrated using Alexa Fluor 488 hydrazide, a low molecular weight fluorescent dye (Figure 6).
Axon injury studies are commonly performed within compartmentalized devices. A proof of principle experiment was performed to selectively injure axons of differentiated neurons with the pre-assembled COC chip (Figure 7). Results were equivalent to silicone compartmentalized devices6,13,14.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59250/59250fig1.jpg" /> 
Figure 1: The pre-assembled COC, multicompartment microfluidic chip. (A) A drawing of the chip identifying the upper and lower wells. The size of the chip is 75 mm x 25 mm, the size of standard microscope slides. (B) A zoomed in region showing the channels and microgrooves separating the channels. Additional details are provided in Nagendran et al.12. (C) This photograph illustrates the creation of isolated microenvironments within each compartment using food coloring dye. This entire figure has been reproduced from 12. <a href="https://www.jove.com/files/ftp_upload/59250/59250fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59250/59250fig2.jpg" /> 
Figure 2: Plastic compartmentalized chip coating and NSC cell plating timeline. (A) Plastic multicompartment chips were coated with a pre-coating solution, and then poly-L-ornithine and laminin before pre-conditioning with NSC media. (B) NSCs were plated with 7 x 104 cells in the somatic compartment of the chip. The cells were grown in NSC media for 24 h and then the media was replaced with neural differentiation media. Differentiated neurons were observed by 7-10 days post differentiation. <a href="https://www.jove.com/files/ftp_upload/59250/59250fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59250/59250fig3.jpg" /> 
Figure 3: hSC-neuron growth comparison in chips and PDMS devices. (A) A phase contrast image of hSC-neurons grown at 13 days after differentiation in the plastic multicompartment chip. (B) A zoomed in region of hSC-neurons cultured within the white box in (A) and an equivalent region within a PDMS device (right). hSC-neurons within the chips attach well. Aggregated neuron clusters form in PDMS-based devices. Representative of 2 independent experiments. <a href="https://www.jove.com/files/ftp_upload/59250/59250fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59250/59250fig4.jpg" /> 
Figure 4: Human NSC derived neurons show dendritic spine morphology. Retrograde labeled mCherry neuron at differentiation day 33 grown within the chip. The magnified region outlined in red, highlights the presence of dendritic spines, which provide evidence supporting the development of mature glutamatergic synapses. Red arrows point towards dendritic spines. <a href="https://www.jove.com/files/ftp_upload/59250/59250fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59250/59250fig5.jpg" /> 
Figure 5: Human NSC derived neurons cultured within chips exhibit excitatory synapses. Immunostaining was performed at differentiation day 26. Neuron imaging was performed in the somatic compartment. A) vGlut1 (green) and (B) DAPI (blue) immunolabeling. (C) mCherry-labeled neurons (red) retrograde labeled using a modified rabies virus. (D) A merged fluorescent micrograph of (A-C). (E) Dendritic spines and vGlut1 positive puncta colocalized with mCherry-positive dendrites, shown in a zoomed in region from (D). Immunofluorescence micrographs of (F) neuron specific marker, &#946;-tubulin III (magenta), and (G) vGlut1 (green). (H) Overlay of &#946;-tubulin III, and vGlut1. <a href="https://www.jove.com/files/ftp_upload/59250/59250fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59250/59250fig6.jpg" /> 
Figure 6: Virally transduced mCherry neurons extend projections into an axon localized microenvironment established within the preassembled COC chip. (A) mCherry labeled neuron extend axons through the microgrooves of the chip and into an isolated axon compartment. Isolation of the axon compartment is visualized using Alexa Fluor 488 hydrazide. Imaging of neurons occurred at differentiation day 26 and 3 days after infection with the modified rabies virus. (B) The fluorescence image in (A) is merged with a DIC image. Note the location of the microgrooves. <a href="https://www.jove.com/files/ftp_upload/59250/59250fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/59250/59250fig7.jpg" /> 
Figure 7: Axotomy performed within the COC multicompartment chip. (A) mCherry labeled neurons at differentiation day 33 were imaged before axotomy. &#8216;Fire&#8217; color look-up table. (B) Immediately after axotomy, showing that axons are completely severed. <a href="https://www.jove.com/files/ftp_upload/59250/59250fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Plastic multi-compartment chips</td>
			<td>PDMS multi-compartment devices</td>
		</tr>
		<tr>
			<td>isolate axons</td>
			<td>isolate axons</td>
		</tr>
		<tr>
			<td>establish microenvironments</td>
			<td>establish microenvironments</td>
		</tr>
		<tr>
			<td>axotomize neurons</td>
			<td>axotomize neurons</td>
		</tr>
		<tr>
			<td>optically transparent</td>
			<td>optically transparent</td>
		</tr>
		<tr>
			<td>compatible with high resolution imaging</td>
			<td>compatible with high resolution imaging</td>
		</tr>
		<tr>
			<td>compatible with fluorescence microscopy</td>
			<td>compatible with fluorescence microscopy</td>
		</tr>
		<tr>
			<td>fully assembled</td>
			<td>assembly to substrate required</td>
		</tr>
		<tr>
			<td>healthy axons &#62;21 days</td>
			<td>healthy axons &#62;14 days</td>
		</tr>
		<tr>
			<td>hydrophilic culturing surface</td>
			<td>hydrophobic</td>
		</tr>
		<tr>
			<td>gas impermeable</td>
			<td>gas permeable</td>
		</tr>
		<tr>
			<td>rounded microgrooves and channels</td>
			<td>straight microgrooves</td>
		</tr>
		<tr>
			<td>not compatible with laser ablation</td>
			<td>Can be used for laser ablation when PDMS chambers are assembled on special laser ablation compatible slides.&#160;</td>
		</tr>
		<tr>
			<td>device cannot be altered to remove top&#160;</td>
			<td>top is removable for staining within microgrooves</td>
		</tr>
		<tr>
			<td>not compatible with mineral oil-based immersion oils (silicone-based oils are fine)</td>
			<td>compatible with mineral oil-based immersion oils</td>
		</tr>
		<tr>
			<td>&#160;impermeable to small molecules and organic solvents&#160;</td>
			<td>absorption of small molecules &#38; organic solvents</td>
		</tr>
		<tr>
			<td>No leakage issues with the chip&#160;</td>
			<td>coating with poly-L-ornithine and laminin make the devices leaky</td>
		</tr>
		<tr>
			<td>differentiated neurons remain evenly distributed (&#62; 4 weeks) at the tested cell densities</td>
			<td>differentiated neurons begin to aggregate after 3-4 days in culture at the tested cell densities&#160;</td>
		</tr>
	</tbody>
</table>
Table 1: Comparision of multi-compartment COC chips and silicone devices for culturing neurons

Watch this Scientific Journal Video about Compartmentalization of Human Stem Cell-Derived Neurons within Pre-Assembled Plastic Microfluidic Chips at JoVE.com

Compartmentalization of Human Stem Cell-Derived Neurons within Pre-Assembled Plastic Microfluidic Chips

Unterteilung menschlicher Stammzellen-abgeleiteter Neuronen in vormontierte Mikrofluidchips aus Kunststoff

This protocol demonstrates the use of compartmentalized microfluidic chips, injection molded in a cyclic olefin copolymer to cultured neurons differentiated from human stem cells. These chips are preassembled and easier-to-use than traditional compartmentalized poly(dimethylsiloxane) devices. Multiple common experimental paradigms are described here, including viral labeling, fluidic isolation, axotomy, and immunostaining.

Use of microfluidic devices to compartmentalize cultured neurons has become a standard method in neuroscience. This protocol shows how to use a ...

compartmentalization-human-stem-cell-derived-neurons-within-pre

Research

JoVE Journal

Neuroscience

65.8K Aufrufe.  UNC Neuroscience Center. Durch die abteilungalisierten mikrofluidischen Geräte können Neuronen in ihrer stark polarisierten Form untersucht werden. Diese Geräte sind sowohl für die grundlagende als auch für die translationale neurowissenschaftliche Forschung von entscheidender Bedeutung. Dieses Protokoll beschreibt, wie zyklische Olefin-Copolymerchips verwendet werden, um Neuronen zu unterteilen, die sich von menschlichen Stammzellen unterscheiden.Diese COC-Chips produzieren gesündere Kulturen differenzi...

Serie: Compartmentalization of Human Stem Cell-Derived Neurons within Pre-Assembled Plastic Microfluidic Chips

Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, cell-based model that produces functioning synapses and undergoes physiological responses to intoxication. Here we describe a simple and robust method to efficiently differentiate murine embryonic stem cells (ESCs) into defined lineages of synaptically active, networked neurons. Following an 8 day differentiation protocol, mouse embryonic stem cell-derived neurons (ESNs) rapidly express and compartmentalize neurotypic proteins, form neuronal morphologies and develop intrinsic electrical responses. By 18 days after differentiation (DIV 18), ESNs exhibit active glutamatergic and &#947;-aminobutyric acid (GABA)ergic synapses and emergent network behaviors characterized by an excitatory:inhibitory balance. To determine whether intoxication with CNTs functionally antagonizes synaptic neurotransmission, thereby replicating the in vivo pathophysiology that is responsible for clinical manifestations of botulism or tetanus, whole-cell patch clamp electrophysiology was used to quantify spontaneous miniature excitatory post-synaptic currents (mEPSCs) in ESNs exposed to tetanus neurotoxin (TeNT) or botulinum neurotoxin (BoNT) serotypes /A-/G. In all cases, ESNs exhibited near-complete loss of synaptic activity within 20 hr. Intoxicated neurons remained viable, as demonstrated by unchanged resting membrane potentials and intrinsic electrical responses. To further characterize the sensitivity of this approach, dose-dependent effects of intoxication on synaptic activity were measured 20 hr after addition of BoNT/A. Intoxication with 0.005 pM BoNT/A resulted in a significant decrement in mEPSCs, with a median inhibitory concentration (IC50) of 0.013 pM. Comparisons of median doses indicate that functional measurements of synaptic inhibition are faster, more specific and more sensitive than SNARE cleavage assays or the mouse lethality assay. These data validate the use of synaptically coupled, stem cell-derived neurons for the highly specific and sensitive detection of CNTs.

A custom protocol is described to differentiate mouse ES cells into defined populations of highly pure neurons exhibiting functioning synapses and emergent network behavior. Electrophysiological analysis demonstrates the loss of synaptic transmission following exposure to botulinum neurotoxin serotypes /A-/G and tetanus neurotoxin.

Funktionale Bewertung der biologischen Neurotoxine in Networked Cultures für Stammzell abgeleitete Zentralnervensystem Neuronen

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, ...

Forschung

Neurowissenschaften

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions in newborn infants. While mosquitos are the main route of viral transmission, it has also been shown to spread via sexual contact and vertical mother-to-fetus transmission. In this latter case of transmission, due to the unique viral tropism of ZIKV, the virus is believed to predominantly target the neural progenitor cells (NPCs) of the developing brain.
Here a method for modeling ZIKV infection, and the resulting microcephaly, that occur when human cerebral organoids are exposed to live ZIKV is described. The organoids display high levels of virus within their neural progenitor population, and exhibit severe cell death and microcephaly over time. This three-dimensional cerebral organoid model allows researchers to conduct species-matched experiments to observe and potentially intervene with ZIKV infection of the developing human brain. The model provides improved relevance over standard two-dimensional methods, and contains human-specific cellular architecture and protein expression that are not possible in animal models.

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

This protocol describes a technique used to model Zika virus infection of the developing human brain. Using wildtype or engineered stem cell lines, researchers may use this technique to uncover the various mechanisms or treatments that may affect early brain infection and resulting microcephaly in Zika virus-infected embryos.

Modellierung der Zika-Virus-Infektion des entwickelnden menschlichen Gehirns In Vitro mit Stammzellen abgeleitet zerebrale Organellen

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

Einfache Erstellung einer High-Yield-Kultur der induzierten Neuronen aus Menschenhaut Erwachsenen Fibroblasten

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

Method for High Speed Stretch Injury of Human Induced Pluripotent Stem Cell-derived Neurons in a 96-well Format

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven therapies for TBI have been developed. This paper presents a novel method for pre-clinical neurotrauma research intended to complement existing pre-clinical models. It introduces human pathophysiology through the use of human induced pluripotent stem cell-derived neurons (hiPSCNs). It achieves loading pulse duration similar to the loading durations of clinical closed head impact injury. It employs a 96-well format that facilitates high throughput experiments and makes efficient use of expensive cells and culture reagents. Silicone membranes are first treated to remove neurotoxic uncured polymer and then bonded to commercial 96-well plate bodies to create stretchable 96-well plates. A custom-built device is used to indent some or all of the well bottoms from beneath, inducing equibiaxial mechanical strain that mechanically injures cells in culture in the wells. The relationship between indentation depth and mechanical strain is determined empirically using high speed videography of well bottoms during indentation. Cells, including hiPSCNs, can be cultured on these silicone membranes using modified versions of conventional cell culture protocols. Fluorescent microscopic images of cell cultures are acquired and analyzed after injury in a semi-automated fashion to quantify the level of injury in each well. The model presented is optimized for hiPSCNs but could in theory be applied to other cell types.

Here we present a method for a human in vitro model of stretch injury in a 96-well format on a timescale relevant to impact trauma. This includes methods for fabricating stretchable plates, quantifying the mechanical insult, culturing and injuring cells, imaging, and high content analysis to quantify injury.

Methode für High-Speed Strecke Verletzungen der menschlichen induzierten pluripotenten Stammzellen abgeleitet Neuronen in einem 96-Well-Format

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven ...

Use of Pre-Assembled Plastic Microfluidic Chips for Compartmentalizing Primary Murine Neurons

Microfabricated methods to compartmentalize neurons have become essential tools for many neuroscientists. This protocol describes the use of a commercially available pre-assembled plastic chip for compartmentalizing cultured primary rat hippocampal neurons. These plastic chips, contained within the footprint of a standard microscope slide, are compatible with high-resolution, live, and fluorescence imaging. This protocol demonstrates how to retrograde label neurons via isolated axons using a modified rabies virus encoding a fluorescent protein, create isolated microenvironments within one compartment, and perform axotomy and immunocytochemistry on-chip. Neurons are cultured for &gt;3 weeks within the plastic chips, illustrating the compatibility of these chips for long-term neuronal cultures.

This protocol describes the use of plastic chips to culture and compartmentalize primary murine neurons. These chips are preassembled, user-friendly, and compatible with high-resolution, live, and fluorescence imaging. This protocol describes how to plate rat hippocampal neurons within these chips and perform fluidic isolation, axotomy and immunostaining.

Einsatz von vormontierten Kunststoff mikrofluidischen Chips für Abschottung primäre Murine Neuronen

Microfabricated methods to compartmentalize neurons have become essential tools for many neuroscientists. This protocol describes the use of a ...

Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model system to explore disease mechanisms and carry out high content screening (HCS) to interrogate compound libraries or identify gene mutation phenotypes. However, with human cells the transition from NPC to functional, mature neuron requires several weeks. Synapses typically start to form after 3 weeks of differentiation in monolayer culture, and several neuron-specific proteins, for example the later expressing pan-neuronal marker NeuN, or the layer 5/6 cerebral cortical neuron marker CTIP2, begin to express around 4-5 weeks post-differentiation. This lengthy differentiation time can be incompatible with optimal culture conditions used for small volume, multi-well HCS platforms. Among the many challenges are the need for well-adhered, uniformly distributed cells with minimal cell clustering, and culture procedures that foster long-term viability and functional synapse maturation. One approach is to differentiate neurons in a large volume format, then replate them at a later time point in HCS-compatible multi-wells. Some main challenges when using this replating approach concern reproducibility and cell viability, due to the stressful disruption of the dendritic and axonal network. Here we demonstrate a detailed and reliable procedure for enzymatically resuspending human induced pluripotent stem cell (hiPSC)-derived neurons after their differentiation for 4-8 weeks in a large-volume format, transferring them to 384-well microtiter plates, and culturing them for a further 1-3 weeks with excellent cell survival. This replating of human neurons not only allows the study of synapse assembly and maturation within two weeks from replating, but also enables studies of neurite regeneration and growth cone characteristics. We provide examples of scalable assays for neuritogenesis and synaptogenesis using a 384-well platform.

This protocol describes a detailed procedure for resuspending and culturing human stem cell derived neurons that were previously differentiated from neural progenitors in vitro for multiple weeks. The procedure facilitates imaging-based assays of neurites, synapses, and late-expressing neuronal markers in a format compatible with light microscopy and high-content screening.

Post-Differenzierung Replating von Human Pluripotent Stem Cell abgeleiteten Neuronen für High-Content Screening von Neuritogenese und Synapse Reifung

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model ...

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.

Migration, Chemo-Attraktion und Co-Culture-Assays für menschliche Stammzell-abgeleitete Endothelzellen und GABAerge Neuronen

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have ...

Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human neurons, which may contribute to neurodegeneration in various disease states. Although it is challenging to examine mitochondrial dynamics in live human nerves, such paradigms are critical for studying the role of mitochondria in neurodegeneration. Described here is a protocol for analyzing mitochondrial transport and mitochondrial morphology in forebrain neuron axons derived from human induced pluripotent stem cells (iPSCs). The iPSCs are differentiated into telencephalic glutamatergic neurons using well-established methods. Mitochondria of the neurons are stained with MitoTracker CMXRos, and mitochondrial movement within the axons are captured using a live-cell imaging microscope equipped with an incubator for cell culture. Time-lapse images are analyzed using software with &#34;MultiKymograph&#34;, &#34;Bioformat importer&#34;, and &#34;Macros&#34; plugins. Kymographs of mitochondrial transport are generated, and average mitochondrial velocity in the anterograde and retrograde directions is read from the kymograph. Regarding mitochondrial morphology analysis, mitochondrial length, area, and aspect ratio are obtained using the ImageJ. In summary, this protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology to facilitate studies of neurodegenerative diseases.

Impaired mitochondrial transport and morphology are involved in various neurodegenerative diseases. The presented protocol uses induced pluripotent stem cell-derived forebrain neurons to assess mitochondrial transport and morphology in hereditary spastic paraplegia. This protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology, which will facilitate the study of neurodegenerative disease.

Analyse von mitochondrialem Transport und Morphologie bei humaninduzierten Pluripotenten Stammzell-abgeleiteten Neuronen bei erblicher spastischer Paraplegie

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human ...

Subretinal Transplantation of Human Embryonic Stem Cell-Derived Retinal Tissue in a Feline Large Animal Model

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and Leber Congenital Amaurosis (LCA) cause progressive and debilitating vision loss. There is an unmet need for therapies that can restore vision once photoreceptors have been lost. Transplantation of human pluripotent stem cell (hPSC)-derived retinal tissue (organoids) into the subretinal space of an eye with advanced RD brings retinal tissue sheets with thousands of healthy mutation-free photoreceptors and has a potential to treat most/all blinding diseases associated with photoreceptor degeneration with one approved protocol. Transplantation of fetal retinal tissue into the subretinal space of animal models and people with advanced RD has been developed successfully but cannot be used as a routine therapy due to ethical concerns and limited tissue supply. Large eye inherited retinal degeneration (IRD) animal models are valuable for developing vision restoration therapies utilizing advanced surgical approaches to transplant retinal cells/tissue into the subretinal space. The similarities in globe size, and photoreceptor distribution (e.g., presence of macula-like region area centralis) and availability of IRD models closely recapitulating human IRD would facilitate rapid translation of a promising therapy to the clinic. Presented here is a surgical technique of transplanting hPSC-derived retinal tissue into the subretinal space of a large animal model allowing assessment of this promising approach in animal models.

Presented here is a surgical technique for transplanting human pluripotent stem cell (hPSC)-derived retinal tissue into the subretinal space of a large animal model.

Subretinale Transplantation von menschlichem embryonalem Stammzellgewebe in einem katzenartigen Großtiermodell

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and ...

Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) provide a powerful tool for modeling Amyotrophic Lateral Sclerosis (ALS) pathophysiology in vitro. Multi-electrode array (MEA) recordings are a means to record electrical field potentials from large populations of neurons and analyze network activity over time. It was previously demonstrated that the presence of hiPSC-A that are differentiated using techniques to promote a spinal cord astrocyte phenotype improved maturation and electrophysiological activity of regionally specific spinal cord hiPSC-motor neurons (MN) when compared to those cultured without hiPSC-A or in the presence of rodent astrocytes. Described here is a method to co-culture spinal cord hiPSC-A with hiPSC-MN and record electrophysiological activity using MEA recordings. While the differentiation protocols described here are particular to astrocytes and neurons that are regionally specific to the spinal cord, the co-culturing platform can be applied to astrocytes and neurons differentiated with techniques specific to other fates, including cortical hiPSC-A and hiPSC-N. These protocols aim to provide an electrophysiological assay to inform about glia-neuron interactions and provide a platform for testing drugs with therapeutic potential in ALS.

We describe a method for differentiating spinal cord human induced pluripotent-derived astrocytes and neurons and their co-culture for electrophysiological recording.

Etablierung einer elektrophysiologischen Plattform zur Modellierung von ALS mit regionalspezifischen humanen pluripotenten Stammzell-abgeleiteten Astrozyten und Neuronen

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) provide a powerful tool for modeling Amyotrophic Lateral Sclerosis (ALS) ...