We gratefully acknowledge funding support by the NIH [grant numbers EB025765 and EB027062], DOD [award number W81XWH-18-1-0095], and the UCSF Health Innovation via Engineering (HIVE Fellowship). We gratefully acknowledge the Columbia University Stem Cell Core for their help and guidance with cell reprogramming.

All cell lines for this work were created and used in compliance with the institutional guidelines of Columbia University, NY, USA.
1. Bioreactor preparation
<ol>
	<li>Make bioreactor molds
	<ol>
		<li>Download a bioreactor CAD file from the Supplementary CAD File or create a custom own design.</li>
		<li>Generate a CNC toolpath from the 3D model using CAM software.</li>
		<li>Machine acetal molds using a CNC milling machine.</li>
	</ol>
	</li>
	<li>Fabricat...

<ol>
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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+the+neuromuscular+junction,+vitally+informative+for+understanding+function+and+the+molecular+mechanisms+of+congenital+myasthenic+syndromes.">Animal models of the neuromuscular junction, vitally informative for understanding function and the molecular mechanisms of congenital myasthenic syndromes.</a> International Journal of Molecular Sciences. 19 (5), 1326 (2018).</li><li>Jones, R. 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=Cellular+and+molecular+anatomy+of+the+human+neuromuscular+junction.">Cellular and molecular anatomy of the human neuromuscular junction.</a> Cell Reports. 21 (9), 2348-2356 (2017).</li><li>Takahashi, K., 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=Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors.">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131 (5), 861-872 (2007).</li><li>Rao, L., Qian, Y., Khodabukus, A., Ribar, T., Bursac, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+human+pluripotent+stem+cells+into+a+functional+skeletal+muscle+tissue.">Engineering human pluripotent stem cells into a functional skeletal muscle tissue.</a> Nature Communications. 9 (1), 126 (2018).</li><li>Madden, L., Juhas, M., Kraus, W. 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F., 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=Bioengineered+optogenetic+model+of+human+neuromuscular+junction.">Bioengineered optogenetic model of human neuromuscular junction.</a> Biomaterials. 276, 121033 (2021).</li><li>Vila, O. F., 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=Quantification+of+human+neuromuscular+function+through+optogenetics.">Quantification of human neuromuscular function through optogenetics.</a> Theranostics. 9 (5), 1232-1246 (2019).</li><li>Boyden, E. 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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=Functional+connectivity+under+optogenetic+control+allows+modeling+of+human+neuromuscular+disease.">Functional connectivity under optogenetic control allows modeling of human neuromuscular disease.</a> Cell Stem Cell. 18 (1), 134-143 (2016).</li><li>Santhanam, N., 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=Stem+cell+derived+phenotypic+human+neuromuscular+junction+model+for+dose+response+evaluation+of+therapeutics.">Stem cell derived phenotypic human neuromuscular junction model for dose response evaluation of therapeutics.</a> Biomaterials. 166, 64-78 (2018).</li><li>Phillips Ii, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epidemiology+of+myasthenia+gravis.">The epidemiology of myasthenia gravis.</a> Annals of the New York Academy of Sciences. 998 (1), 407-412 (2003).</li><li>Sanders, D. 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This system is an engineered 3D human tissue model that combines optogenetics and video processing to enable automated and unbiased evaluation of NMJ function. Using a standardized protocol, we have demonstrated the ability to measure changes in NMJ function during physiological development and characterize the damaging effects of pathologies such as neurotoxin exposure and myasthenia gravis patient sera.
Previous studies have reported the ability to model MG with optogenetic hPSC-derived moto...

The neuromuscular junction (NMJ) is the chemical synapse between motoneurons (MNs) and skeletal muscle cells (SkM) that allows for muscle contraction. Toxins, such as neurotoxin &#945;-bungarotoxin (BTX), or neuromuscular diseases (NMD) like myasthenia gravis (MG) can lead to degeneration of the NMJ and reductions in muscle control1. Bioengineered human tissue models better recapitulate the functional and physiological mechanisms of human NMJs and offer greater translational potential than animal models.
While animal models have advanced the understanding of the formation and function of the NMJ, there are significant differences between human and animal synapses that limit the translation of results to humans and make in vivo characterization of the NMJ challenging2,3,4. Studies have shown distinct physiological differences between mouse and human NMJs. Mice have larger NMJs and smaller active zone densities when compared to human NMJs4. Additionally, drug studies conducted in animal models do not always reflect the effects found in human clinical trials. Engineered human tissue models provide the opportunity to study the healthy development of the NMJ and the pathology of neuromuscular diseases and allow for drug screenings. Human induced pluripotent stem cells (hiPSCs)5 can be differentiated into a variety of cell types, including skeletal muscle cells6,7 and motoneurons8,9. hiPSCs can be generated easily from patient cells, allowing for better disease modeling10 and drug screening11,12 through patient-specific tissue models.
Two-dimensional (2D) monolayer co-cultures of SkMs and MNs lack the morphology, phenotype, organization, and functional behavior of physiological NMJs. NMJs randomly form in 2D culture, which inhibits the isolation of motor units for analysis, limits accurate functional measurements, and prevents their use for repeated, systematic experiments13. Three-dimensional (3D) tissue models of NMJs overcome many of these limitations, recapitulating the morphological and functional characteristics of physiological NMJs7,14,15,16,17. Using this model, the two tissue types are developed separately and then integrated by directing axonal growth, allowing for more organized NMJs to develop compared to 2D culture systems.
Our previous study demonstrated that combining optogenetics with tissue engineering can allow for accurate non-invasive stimulation and evaluation of NMJ function18,19. Through genetic engineering, light-sensitive proteins can be integrated into the genome of hiPSCs. Integrating channelrhodopsin-2 (ChR2), an ion channel that opens in response to blue light, into the membrane of excitable cells such as neurons allows for non-contact spatiotemporal control over cell activation20,21,22. hiPSCs carrying ChR2 can be differentiated into optogenetic motoneurons sensitive to blue light, removing the need for typical invasive electrodes that stimulate neurons and avoiding unwanted stimulation of the muscle cells by electrodes23. This system uses optogenetic motoneurons to stimulate contractions in non-optogenetic skeletal muscle cells. Combining video acquisition and controlled blue light illumination allows for the co-cultured tissues to be simultaneously stimulated and recorded for NMJ function.
MG is caused by autoantibodies targeting nicotinic acetylcholine receptors (AChR), which results in decreased NMJ function and muscle weakness24. It is diagnosed based on presented symptoms, electrodiagnosis, and detection of autoantibodies via serological blood tests. However, not all autoantibodies involved in MG have been identified, and some seronegative patients are diagnosed with MG but with no recognized antibodies25,26. Our system allows for repeated functional assessment of the NMJ before and after the addition of serum from MG patients, providing invaluable insight into the functional and biochemical changes caused by the MG antibodies18. Our protocol illustrates how to produce 3D in vitro models of functional human NMJ that can be used to diagnose and investigate NMJ pathologies and test potential therapeutics. We demonstrate the versatility of the system in two platforms, a microfluidic device, and a larger open-well bioreactor platform.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SkMDC</td>
			<td>Cook Myosite</td>
			<td>P01059-14M</td>
			<td></td>
		</tr>
		<tr>
			<td>Media and Supplements</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>ThermoFisher Scientific</td>
			<td>12634-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin solution</td>
			<td>Millipore Sigma</td>
			<td>A9576-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>G-5 Supplement (100X)</td>
			<td>ThermoFisher Scientific</td>
			<td>17503-012</td>
			<td></td>
		</tr>
		<tr>
			<td>Geneticin Selective Antibiotic (G418 Sulfate) (50 mg/mL)</td>
			<td>ThermoFisher Scientific</td>
			<td>10131-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin, Recombinant Human</td>
			<td>Millipore Sigma</td>
			<td>91077C-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354277</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR Plus</td>
			<td>Stem Cell Technologies</td>
			<td>100-0276</td>
			<td></td>
		</tr>
		<tr>
			<td>MyoTonic Growth Media Kit</td>
			<td>Cook Myosite</td>
			<td>MK-4444</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2 Supplement</td>
			<td>ThermoFisher Scientific</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>NBactiv4 500 mL</td>
			<td>BrainBits LLC</td>
			<td>Nb4-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>ThermoFisher Scientific</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal-A Medium</td>
			<td>ThermoFisher Scientific</td>
			<td>A13710-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Sigma Aldrich</td>
			<td>P2443</td>
			<td></td>
		</tr>
		<tr>
			<td>ReLeSR</td>
			<td>Stem Cell Technologies</td>
			<td>5872</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasticware</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>30 mm cage cube system</td>
			<td>ThorLabs</td>
			<td>CM1-DCH, CP33, ER1-P4 and ER2-P4</td>
			<td></td>
		</tr>
		<tr>
			<td>37 &#181;m Reversible Strainer, large</td>
			<td>Stem Cell Technologies</td>
			<td>27250</td>
			<td></td>
		</tr>
		<tr>
			<td>546 nm short-pass excitation filter</td>
			<td>Semrock</td>
			<td>FF01-546/SP-25</td>
			<td></td>
		</tr>
		<tr>
			<td>573 nm dichroic mirror</td>
			<td>Semrock</td>
			<td>FF573-Di01&#8211;25x36</td>
			<td></td>
		</tr>
		<tr>
			<td>594 nm long- pass emission filter</td>
			<td>Semrock</td>
			<td>BLP01-594R-25</td>
			<td></td>
		</tr>
		<tr>
			<td>594 nm long-pass excitation filter</td>
			<td>Semrock</td>
			<td>BLP01-594R-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Blue (470nm) Rebel LED on a SinkPAD-II 10mm Square Base - 65 lm @ 700mA</td>
			<td>LuxeonStarLEDs</td>
			<td>SP-05-B4</td>
			<td></td>
		</tr>
		<tr>
			<td>Carclo 29.8&#176; Frosted 10 mm Circular Beam Optic - Integrated Legs</td>
			<td>LuxeonStarLEDs</td>
			<td>10413</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 60 mm Ultra-Low Attachment Culture Dish</td>
			<td>Corning</td>
			<td>3261</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat sink</td>
			<td>LuxeonStarLEDs</td>
			<td>LPD-19-10B</td>
			<td></td>
		</tr>
		<tr>
			<td>Optics</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pluriStrainer 400 &#181;m, 25 pack, sterile</td>
			<td>PluriSelect</td>
			<td>43-50400-03</td>
			<td></td>
		</tr>
		<tr>
			<td>pluriStrainer 500 &#181;m, 25 pack, sterile</td>
			<td>PluriSelect</td>
			<td>43-50500-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Red (627nm) Rebel LED on a SinkPAD-II 10mm Square Base - 65 lm @ 700mA</td>
			<td>LuxeonStarLEDs</td>
			<td>SP-05-R5</td>
			<td></td>
		</tr>
		<tr>
			<td>ring-actuated iris diaphragm</td>
			<td>ThorLabs</td>
			<td>SM1D12D</td>
			<td></td>
		</tr>
		<tr>
			<td>T-Cube LED drivers</td>
			<td>ThorLabs</td>
			<td>LEDD1B, KPS101</td>
			<td></td>
		</tr>
		<tr>
			<td>Molds</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Female Threaded Hex Standoffs,&#160; 3 1/2&#34; 10-32, Partially Threaded 1/2&#34;</td>
			<td>McMaster</td>
			<td>91920A046</td>
			<td></td>
		</tr>
		<tr>
			<td>Low-Profile&#160;C-Clamp</td>
			<td>McMaster</td>
			<td>1705A12</td>
			<td></td>
		</tr>
		<tr>
			<td>Growth Factors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 3&#8242;,5&#8242;-cyclic monophosphate</td>
			<td>Millipore Sigma</td>
			<td>A9501-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR 99021, 10 mg</td>
			<td>Tocris</td>
			<td>4423/10</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPT 10 mg</td>
			<td>R&#38;D Systems</td>
			<td>2634/10</td>
			<td></td>
		</tr>
		<tr>
			<td>Human CNTF, research grade, 5 &#181;g</td>
			<td>Miltenyl Biotec</td>
			<td>130-096-336</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Vitronectin Protein, CF</td>
			<td>R&#38;D Systems</td>
			<td>2349-VN-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Vitronectin Protein, CF</td>
			<td>R&#38;D Systems</td>
			<td>2349-VN-100</td>
			<td></td>
		</tr>
		<tr>
			<td>IGF1 Recombinant Human Protein</td>
			<td>ThermoFisher Scientific</td>
			<td>PHG0078</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin mouse protein, natural</td>
			<td>ThermoFisher Scientific</td>
			<td>23017015</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human Agrin Protein</td>
			<td>R&#38;D Systems</td>
			<td>6624-AG-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human GDNF Protein, CF 50ug</td>
			<td>R&#38;D Systems</td>
			<td>212-GD-050/CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human Neurotrophin 3&#160;100 ug</td>
			<td>Cell Sciences</td>
			<td>CRN500D</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human Neurotrophin-4</td>
			<td>Cell Sciences</td>
			<td>CRN501B</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human Sonic Hedgehog/Shh (C24II) N-Terminus</td>
			<td>R&#38;D Systems</td>
			<td>1845-SH-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human/Murine/Rat BDNF 50 ug</td>
			<td>Peprotech</td>
			<td>450-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Retinoic Acid, 50 mg</td>
			<td>Millipore Sigma</td>
			<td>R2625-50</td>
			<td></td>
		</tr>
		<tr>
			<td>SAG Smoothened Agonist</td>
			<td>Millipore Sigma</td>
			<td>566660</td>
			<td></td>
		</tr>
		<tr>
			<td>SB431542 10 mg</td>
			<td>Stem Cell Technologies</td>
			<td>72234</td>
			<td></td>
		</tr>
		<tr>
			<td>StemMACS LDN-193189</td>
			<td>Miltenyl Biotec</td>
			<td>130-103-925</td>
			<td></td>
		</tr>
		<tr>
			<td>Vitronectin from human plasma</td>
			<td>Millipore Sigma</td>
			<td>V8379-50UG</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632 dihydrochloride</td>
			<td>Tocris</td>
			<td>1254</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-actinin mAb (Mouse IgG1)</td>
			<td>Abcam</td>
			<td>ab9465</td>
			<td></td>
		</tr>
		<tr>
			<td>Choline Acetyltransferase (ChAT) (Goat)</td>
			<td>Millipore</td>
			<td>AB144P</td>
			<td></td>
		</tr>
		<tr>
			<td>Desmin mAb (Mouse IgG1)</td>
			<td>Dako</td>
			<td>M076029-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Myosin Heavy Chain (MHC) (Mouse IgG2b)</td>
			<td>DSHB</td>
			<td>MF20</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Arduino Uno R3</td>
			<td>Arduino</td>
			<td>A000066</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated stage</td>
			<td>Applied scientific instrumentation</td>
			<td>MS- 2000 XYZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Expanded plasma cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-001&#160;(115V)</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen Countess Automated Cell Counter</td>
			<td>Marshal Scientific</td>
			<td>I-CACC</td>
			<td></td>
		</tr>
		<tr>
			<td>IX-81 Inverted fluorescence microscope</td>
			<td>Olympus</td>
			<td>IX-ILL100LH</td>
			<td></td>
		</tr>
		<tr>
			<td>Series Stage Top Incubator System</td>
			<td>Tokai Hit STX</td>
			<td>TOKAI-HIT-STXG</td>
			<td></td>
		</tr>
		<tr>
			<td>Zyla 4.2 sCOMS Camera</td>
			<td>Andor Technology</td>
			<td>ZYLA-4.2P-CL10</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Arduino Software (IDE)</td>
			<td>Arduino</td>
			<td>IDE 1.8.19</td>
			<td></td>
		</tr>
		<tr>
			<td>Mastercam</td>
			<td>Mastercam</td>
			<td>Mastercam for Solidworks</td>
			<td></td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>Matlab</td>
			<td>R2021b</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS elements</td>
			<td>Nikon</td>
			<td>Basic Research</td>
			<td></td>
		</tr>
		<tr>
			<td>Solidworks 3D CAD</td>
			<td>Solidworks</td>
			<td>Solidworks Standard</td>
			<td></td>
		</tr>
	</tbody>
</table>

engineering and characterization of an optogenetic model of the human neuromuscular junction

Many neuromuscular diseases, such as myasthenia gravis (MG), are associated with dysfunction of the neuromuscular junction (NMJ), which is difficult to characterize in animal models due to physiological differences between animals and humans. Tissue engineering offers opportunities to provide in vitro models of functional human NMJs that can be used to diagnose and investigate NMJ pathologies and test potential therapeutics. By incorporating optogenetic proteins into induced pluripotent stem cells (iPSCs), we generated neurons that can be stimulated with specific wavelengths of light. If the NMJ is healthy and functional, a neurochemical signal from the motoneuron results in muscle contraction. Through the integration of optogenetics and microfabrication with tissue engineering, we established an unbiased and automated methodology for characterizing NMJ function using video analysis. A standardized protocol was developed for NMJ formation, optical stimulation with simultaneous video recording, and video analysis of tissue contractility. Stimulation of optogenetic motoneurons by light to induce skeletal muscle contractions recapitulates human NMJ physiology and allows for repeated functional measurements of NMJ over time and in response to various inputs. We demonstrate this platform&#39;s ability to show functional improvements in neuromuscular connectivity over time and characterize the damaging effects of patient MG antibodies or neurotoxins on NMJ function.

Neuromuscular junctions were generated by co-culturing optogenetic hiPSC-derived motoneurons with non-optogenetic skeletal muscle tissue. Human primary skeletal myoblasts (SkM) were seeded into the platforms and differentiated into multinucleated myotubes using the 2-week protocol. The optogenetic motoneurons were differentiated separately, but in parallel with the myotube differentiation, and then seeded into the platform (Figure 1). The tissues began contracting in response to blue light s...

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Engineering and Characterization of an Optogenetic Model of the Human Neuromuscular Junction

We describe a reproducible, automated, and unbiased imaging system for characterizing neuromuscular junction function using human engineered skeletal muscle tissue and optogenetic motoneurons. This system allows for the functional quantification of neuromuscular connectivity over time and detects diminished neuromuscular function caused by neurotoxins and myasthenia gravis patient serum.

Many neuromuscular diseases, such as myasthenia gravis (MG), are associated with dysfunction of the neuromuscular junction (NMJ), which is difficult to ...

This protocol describes how to engineer functional 3D in vitro human neuromuscular junctions that can be used to investigate neuromuscular pathologies and test potential therapeutics. This technique uses simultaneous optogenetic stimulation and video processing. It's quantified changes in neuromuscular junction function during development and due to external factors, such as neurotoxins and serum from myasthenia gravis patients.The steps involving the muscle cell and motor neuron seeding into the bioreactor can be challenging at first. Keeping the collagen gel mixture on ice can help extend the time available to seed all of the cells before the gel solidifies. Begin by mixing a 10-to-1 base to curing agent mixture of polydimethylsiloxane, or PDMS, and place the mixture into a vacuum chamber.Close all the valves and turn on the vacuum to degas the mixture for at least 30 minutes until no air bubbles remain. Pour the mixture into molds and degas the molds in the vacuum chamber for 1 hour, then close the molds with the top half of the mold in the correct orientation. Place a hexagonal steel rod over the center and clamp on both sides.Next, refill the top with PDMS and cure the molds in a 65-degree Celsius oven for at least 4 hours. After the molds have cooled down to room temperature, remove the platforms from the molds. Meanwhile, so glass cover slips in 1%non-ionic surfactant polyol for 30 minutes, ensuring that the cover slips do not stack and are all properly coated, then rinse them well with distilled water and dry them overnight at 65-degrees Celsius.Treat the glass cover slips and PDMS platform using a plasma cleaner on high with 6 liters per minute of oxygen for 1 to 2 minutes. Once treated, bond the two together by pressing the PDMS down onto the cover slip for at least 30 seconds. Prepare a 4-to-1 mix of 3 milligrams per milliliter collagen 1 and solubilized basement membrane matrix, then resuspend the myoblasts in this freshly prepared collagen mixture.Next, add 15 microliters of this cell-collagen suspension to each muscle chamber of the bioreactor, making sure to spread the suspension across both pillars using the pipette tip. Allow the cell gel mixture to polymerize at 37 degrees Celsius for 30 minutes, then fill each bioreactor well with 450 microliters of myotonic growth media. On day 11 of motoneuron differentiation, transfer the cells and media to a 50-milliliter tube and allow the neurospheres to settle to the bottom for 5 minutes.Aspirate the supernatant and resuspend the cells in 15 milliliters of motoneuron suspension culture medium supplemented with 20 nanograms per milliliter of brain-derived neurotrophic factor and 10 nanograms per milliliter of glial cell-derived neurotrophic factor. On day 14 of the differentiation protocols, seed the motoneurons into the platform. Prepare a 4-to-1 gel mixture of 2 milligrams per milliliter collagen 1 and solubilized basement membrane matrix.Use a 400-nanometer cell strainer to select large neurospheres and resuspend them in the prepared gel mixture. Carefully aspirate media from the reservoir and the neurosphere well, then add 15 microliters of the gel mixture into the neurosphere channel. Load a 10-microliter pipette with 10 microliters of gel and pick one neurosphere.Deposit the neurosphere into the neurosphere channel, ensuring that it is in the chamber, then slowly raise the pipette while releasing the remaining gel once the neurosphere is deposited. Allow the gel to polymerize for 30 minutes at 37 degrees Celsius, then add 450 microliters of NbActiv4 supplemented with 20 nanograms per milliliter of brain-derived neurotrophic factor and 10 nanograms per milliliter of glial cell-derived neurotrophic factor to the reservoirs. For imaging, use an inverted fluorescent microscope with a scientific complementary metal-oxide semiconductor camera software.Set the Binning to 2-by-2, Exposure to 20 milliseconds, Rolling shutter on, Readout Rate to 540 megahertz, dynamic range to 12-bit and Gain 1 and Sensor Mode to Overlap. Use a 2x objective on the microscope to image the microtissues and attach a live-cell chamber to the microscope stage. Select the region of interest that contains the innervated skeletal tissues to minimize the file size and processing time, then place a 594 nanometer long-pass emission filter between the sample and the imaging objective to filter out blue light pulses from the camera.Next, place a rectangular 4-well plate containing four bioreactors into the live-cell chamber, then click live view and center and focus the image with the desired ROI. Download the custom macro code from the GitHub folder to control the stage position, the Arduino board, and video acquisition, then set the output movie as day underscore tissue group underscore tissue name underscore experiment dot ND2. Run the macro code with the desired X and Y coordinates set on the stage and acquire a fast time lapse with 1700 frames at 50 frames per second.After imaging, replace the media and return the samples to the incubator. Allow at least 24 hours between image acquisition sessions to avoid tissue fatigue. For movie processing, download the files from the GitHub folder and use the custom MATLAB code to process the videos in batch analysis.Open recursive OS analysis script to have all the acquired videos in the same folder. Adjust the pre-analysis parameters and run the recursiveOSAnalysis script to analyze the movies through parallel processing. Adjust the post-analysis parameters like baseline time, peak threshold, min-min prominence, and min-min width as needed.Finally, generate video and graph output by running recursiveOSMovie to generate a video file for each tissue with its respective contractility trace. This protocol was tested using two systems with different scales, a microfluidic device yielding 1-millimeter-long skeletal muscle tissues comprising approximately 20, 000 myoblasts and the open-well bioreactor system resulting in 4-millimeter-long muscle tissues comprising approximately 450 myoblasts. An optical setup was built to allow for controlled stimulation of motoneurons with blue light and the NMJ function was elevated using a custom microscope macro script paired with a custom Arduino code.This code includes tunable adjustable parameters and determines if a contraction is triggered based on the time between a light pulse and the start of the contractility peak. The system captured improvement of NMJ function in tissues showing an increase in triggered responses one week after tissue fabrication, followed by a stable function for an additional week. Alpha-bungarotoxin stopped all triggered and spontaneous contractions resulting in a complete disruption of NMJ function, demonstrating that light stimulation of the tissues requires a functional NMJ.Analysis of the tissues treated with 20%myasthenia gravis serum for 48 hours showed decreased function compared to control tissues treated with serum from healthy donors. 48 hours after removing and washing out the serum, the engineered NMJs showed recovery of function. Make sure all bioreactors are in the oven for equal time to avoid stiffness variability between batches.Also, be sure to verify motor neuron seeding using the microscope. Following this procedure, metabalomics, proteomics, and CRISPR screening can be performed to gather additional information and to better understand the mechanistic changes caused by diseased serum.

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JoVE Journal

Bioengineering

2.2K vistas.  Columbia University. Este protocolo describe cómo diseñar uniones neuromusculares humanas 3D in vitro funcionales que se pueden usar para investigar patologías neuromusculares y probar posibles terapias. Esta técnica utiliza la estimulación optogenética simultánea y el procesamiento de video. Se cuantifican los cambios en la función de la unión neuromuscular durante el desarrollo y debido a factores externos, como las neurotoxinas y el suero de pacientes con miastenia gravis.Los pasos que involucr...