Name: engineering and characterization of an optogenetic model of the human neuromuscular junction
Uploaded: 2022-04-14T13:00:00
Description: Watch this Scientific Journal Video about Engineering and Characterization of an Optogenetic Model of the Human Neuromuscular Junction at JoVE.com

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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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...

Watch this Scientific Journal Video about Engineering and Characterization of an Optogenetic Model of the Human Neuromuscular Junction at JoVE.com

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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Research

JoVE Journal

Bioengineering

Tissue Engineering of the Intestine in a Murine Model

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to preoperative weights within 40 days.1 In humans, massive small bowel resection can result in short bowel syndrome, a functional malabsorptive state that confers significant morbidity, mortality, and healthcare costs including parenteral nutrition dependence, liver failure and cirrhosis, and the need for multivisceral organ transplantation.2 In this paper, we describe and document our protocol for creating tissue-engineered intestine in a mouse model with a multicellular organoid units-on-scaffold approach. Organoid units are multicellular aggregates derived from the intestine that contain both mucosal and mesenchymal elements,3 the relationship between which preserves the intestinal stem cell niche.4 In ongoing and future research, the transition of our technique into the mouse will allow for investigation of the processes involved during TESI formation by utilizing the transgenic tools available in this species.5The availability of immunocompromised mouse strains will also permit us to apply the technique to human intestinal tissue and optimize the formation of human TESI as a mouse xenograft before its transition into humans. Our method employs good manufacturing practice (GMP) reagents and materials that have already been approved for use in human patients, and therefore offers a significant advantage over approaches that rely upon decellularized animal tissues. The ultimate goal of this method is its translation to humans as a regenerative medicine therapeutic strategy for short bowel syndrome.

This article and the accompanying video present our protocol for generating tissue-engineered intestine in the mouse, using an organoid units-on-scaffold approach.

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow

Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using affinity chromatography.&#160;While affinity chromatography using porous protein A agarose beads packed in columns is arguably the most common method of laboratory scale isolation of antibodies and recombinant proteins expressing Fc fragments of IgG, it can be a time consuming and expensive process.&#160;Time and financial constraints are especially daunting in small basic science labs that must recover hundreds of micrograms to milligram quantities of protein from dilute solutions, yet lack access to high pressure liquid delivery systems and/or personnel with expertise in bioseparations. Moreover, product quantification and characterization may also excessively lengthen processing time over several workdays and inflate expenses (consumables, wages, etc.).&#160;Therefore, a fast, inexpensive, yet effective protocol is needed for laboratory scale isolation and characterization of antibodies and other proteins possessing an Fc fragment.&#160;To this end, we have devised a protocol that can be completed by limited-experience technical staff in less than 9 hr (roughly one workday) and as quickly as 4 hr, as opposed to traditional methods that demand 20+ work hours. Most required equipment is readily available in standard biomedical science, biochemistry, and (bio)chemical engineering labs, and all reagents are commercially available.&#160;To demonstrate this protocol, representative results are presented in which chimeric&#160;murine galectin-1 fused to human Fc (Gal-1hFc) from cell culture supernatant was isolated using a protein A membrane adsorber.&#160;Purified Gal-1hFc was quantified using an expedited Western blotting analysis procedure and characterized using flow cytometry. The streamlined workflow can be modified for other Fc-expressing proteins, such as antibodies, and/or altered to incorporate alternative quantification and characterization methods.

Compared with traditional affinity chromatography using protein A agarose bead-packed columns, protein A membrane adsorbers can significantly speed laboratory-scale isolation of antibodies and other Fc fragment-expressing proteins.&#160;Appropriate analysis and quantification methods can further accelerate protein processing, allowing isolation/characterization to be completed in one workday, instead of 20+ work hours.

Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular processes. There is a need for culturing and measuring systems that incorporate programmed illumination and stimulation of optogenetic systems. We present a protocol for building and using a continuous culturing apparatus to illuminate microbial cells with programmed doses of light, and automatically acquire and analyze images of cells in the effluent. The operation of this apparatus as a chemostat allows the growth rate and the cellular environment to be tightly controlled. The effluent of the continuous cell culture is regularly sampled and the cells are imaged by multi-channel microscopy. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses in the fluorescence intensity and cellular morphology of cells sampled from the culture effluent are measured over multiple days without user input. We demonstrate the utility of this culturing apparatus by dynamically inducing protein production in a strain of Saccharomyces cerevisiae engineered with an optogenetic system that activates transcription.

We designed a continuous culturing apparatus for use with optogenetic systems to illuminate cultures of microbes and regularly image cells in the effluent with an inverted microscope. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses to illumination can be measured over several days.

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically been studied in vivo in animal models showing characteristic patterns of cell migration. However, in vivo models have several limitations, including intercellular mediators that are difficult to access and analyze, as well as the inability to directly analyze human neutrophils. Because of these limitations, there is a need for an in vitro platform that studies swarming with human neutrophils and provides easy access to the molecular signals generated during swarming. Here, a multistep microstamping process is used to generate a bioparticle microarray that stimulates swarming by mimicking an in vivo infection. The bioparticle microarray induces neutrophils to swarm in a controlled and stable manner. On the microarray, neutrophils increase in speed and form stable swarms around bioparticle clusters. Additionally, supernatant generated by the neutrophils was analyzed and 16 proteins were discovered to have been differentially expressed over the course of swarming. This in vitro swarming platform facilitates direct analysis of neutrophil migration and protein release in a reproducible, spatially controlled manner.

This protocol generates bioparticle microarrays that provide spatially controlled neutrophil swarming. It provides easy access to the mediators that neutrophils release during migration and allows for quantitative imaging analysis.

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically ...

Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, regardless of the method used. Toward this goal, optogenetic gene expression systems have gained much attention over the past decade for spatiotemporal control of protein levels in mammalian cells. However, most existing circuits controlling light-induced gene expression vary in architecture, are expressed from plasmids, and utilize variable optogenetic equipment, creating a need to explore characterization and standardization of optogenetic components in stable cell lines. Here, the study provides an experimental pipeline of reliable gene circuit construction, integration, and characterization for controlling light-inducible gene expression in mammalian cells, using a negative feedback optogenetic circuit as a case example. The protocols also illustrate how standardizing optogenetic equipment and light regimes can reliably reveal gene circuit features such as gene expression noise and protein expression magnitude. Lastly, this paper may be of use for laboratories unfamiliar with optogenetics who wish to adopt such technology. The pipeline described here should apply for other optogenetic circuits in mammalian cells, allowing for more reliable, detailed characterization and control of gene expression at the transcriptional, proteomic, and ultimately phenotypic level in mammalian cells.

Reliably controlling light-responsive mammalian cells requires the standardization of optogenetic methods. Toward this goal, this study outlines a pipeline of gene circuit construction, cell engineering, optogenetic equipment operation, and verification assays to standardize the study of light-induced gene expression using a negative-feedback optogenetic gene circuit as a case study.

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, ...