Name: mechanostimulation of multicellular organisms through a high-throughput microfluidic compression system
Uploaded: 2022-12-23T13:15:00
Description: Watch this Scientific Journal Video about Mechanostimulation of Multicellular Organisms Through a High-Throughput Microfluidic Compression System at JoVE.com

This work was supported by the National Science Foundation (CMMI-1946456), the Air Force Office of Scientific Research (FA9550-18-1-0262), and the National Institute of Health (R01AG06100501A1; R21AR08105201A1).

1. Preparation of the silicon wafer mold
<ol>
	<li>Clean the silicon wafer (see Table of Materials) first with acetone and then with isopropyl alcohol (IPA).</li>
	<li>Place the silicon wafer on a 250 &#176;C hot plate for 30 min for dehydration bake (Figure 3A).</li>
	<li>Coat the silicon wafer with hexamethyldisilazane (HDMS) in a vapor prime oven (see Table of Materials) (process temperature: 150 &#176;C, process pressure: 2 Torr, proce...

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila-A+versatile+model+in+biology+&amp;+medicine.">Drosophila-A versatile model in biology &amp; medicine.</a> Materials Today. 14 (5), 190-195 (2011).</li><li>Konno, 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=State-of-the-art+technology+of+model+organisms+for+current+human+medicine.">State-of-the-art technology of model organisms for current human medicine.</a> Diagnostics. 10 (6), 392 (2020).</li><li>Morgan, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+limited+inheritance+in+Drosophila.">Sex limited inheritance in Drosophila.</a> Science. 32 (812), 120-122 (1910).</li><li>Wu, Q., Kumar, N., Velagala, V., Zartman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+to+reverse-engineer+multicellular+systems:+Case+studies+using+the+fruit+fly.">Tools to reverse-engineer multicellular systems: Case studies using the fruit fly.</a> Journal of Biological Engineering. 13 (1), 1-16 (2019).</li><li>Jayamohan, H., et al., Patrinos, G., 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=Chapter+11+-+Advances+in+Microfluidics+and+Lab-on-a-Chip+Technologies.">Chapter 11 - Advances in Microfluidics and Lab-on-a-Chip Technologies.</a> Molecular Diagnostics. , 197-217 (2017).</li><li>Scheler, O., Postek, W., Garstecki, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+developments+of+microfluidics+as+a+tool+for+biotechnology+and+microbiology.">Recent developments of microfluidics as a tool for biotechnology and microbiology.</a> Current Opinion in Biotechnology. 55, 60-67 (2019).</li><li>Mohammed, D., 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=Innovative+tools+for+mechanobiology:+Unraveling+outside-in+and+inside-out+mechanotransduction.">Innovative tools for mechanobiology: Unraveling outside-in and inside-out mechanotransduction.</a> Frontiers in Bioengineering and Biotechnology. 7, 162 (2019).</li><li>Shorr, A. Z., S&#246;nmez, U. M., Minden, J. S., LeDuc, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+mechanotransduction+in+Drosophila+embryos+with+mesofluidics.">High-throughput mechanotransduction in Drosophila embryos with mesofluidics.</a> Lab on a Chip. 19 (7), 1141-1152 (2019).</li><li>Kim, Y. 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=Mechanochemical+Actuators+of+Embryonic+Epithelial+Contractility.">Mechanochemical Actuators of Embryonic Epithelial Contractility.</a> Proceedings of the National Academy of Sciences. 40, 14366-14371 (2014).</li><li>Ashburner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila. A Laboratory Handbook. , (1989).</li><li>Qin, D., Xia, Y., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+lithography+for+micro-and+nanoscale+patterning.">Soft lithography for micro-and nanoscale patterning.</a> Nature Protocols. 5 (3), 491 (2010).</li><li>Lee, H., 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+new+fabrication+process+for+uniform+SU-8+thick+photoresist+structures+by+simultaneously+removing+edge+bead+and+air+bubbles.">A new fabrication process for uniform SU-8 thick photoresist structures by simultaneously removing edge bead and air bubbles.</a> Journal of Micromechanics and Microengineering. 21 (12), 125006 (2011).</li><li>Xia, Y., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+lithography.">Soft lithography.</a> Annual Review of Materials Science. 28 (1), 153-184 (1998).</li><li>Sonmez, U. M., Coyle, S., Taylor, R. E., LeDuc, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polycarbonate+heat+molding+for+soft+lithography.">Polycarbonate heat molding for soft lithography.</a> Small. 16 (16), 2000241 (2020).</li><li>Levario, T. J., Zhan, M., Lim, B., Shvartsman, S. Y., Lu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+trap+array+for+massively+parallel+imaging+of+Drosophila+embryos.">Microfluidic trap array for massively parallel imaging of Drosophila embryos.</a> Nature Protocols. 8 (4), 721-736 (2013).</li></ol>

The article describes the development of a microfluidic device to automatically align, immobilize, and precisely apply mechanical stimulation to hundreds of whole Drosophila embryos. The integration of the microfluidic system with a thin glass coverslip allowed for the imaging of embryos with high-resolution confocal microscopy during the stimulation. The microfluidic device also enabled the collection of the embryos right after the stimulation for running downstream biological assays. The design considerations,...

Living systems constantly experience and respond to various mechanical inputs throughout their lifetimes1. Mechanotransduction has been linked to many diseases, including developmental disorders, muscle and bone loss, and neuropathologies through signaling pathways directly or indirectly affected by the mechanical environment2. However, the genes and proteins that are regulated by mechanical stimulation3 in the mechanosensitive signaling pathways4 remain largely unknown5, preventing the elucidation of the mechanical regulation mechanisms and the identification of molecular targets for diseases associated with pathological mechanotransduction6,7. One limiting factor in projecting mechanobiology studies onto the related physiological processes is using individual cells with conventional culture dishes instead of intact multicellular organisms. Model organisms, such as Drosophila melanogaster (fruit fly), have contributed greatly to understanding the genes, signaling pathways, and proteins involved in animal development8,9,10. Nevertheless, using Drosophila and other multicellular model organisms in mechanobiology research has been hindered by challenges with experimental tools. Conventional techniques for preparing, sorting, imaging, or applying various stimuli require mostly manual manipulation; these approaches are time-consuming, require expertise, introduce variability, and limit the experimental design and sample size11. Recent microtechnological advancements are a great resource for enabling novel biological assays with very high throughput and highly controlled experimental parameters12,13,14.
This article describes the development of an enhanced microfluidic device to align, immobilize, and precisely apply mechanical stimulation in the form of uniaxial compression to hundreds of whole Drosophila embryos15&#160;(Figure 1). Integration of the microfluidic system with a glass coverslip allowed high-resolution confocal imaging of the samples during the stimulation. The microfluidic device also enabled fast collection of the embryos after the stimulation for running -omics assays (Figure 2). Explanations of the design considerations of this device, as well as the fabrication using soft lithography and experimental characterization, are described herein. Since making a silicon wafer mold of such a device requires a uniform coating of thick photoresist (thickness &#62;200 &#181;m) over large areas with high aspect ratio (AR) trenches (AR &#62;5), this method considerably modified the traditional photolithographic mold fabrication protocol. In this way, this method facilitated the handling, adhesion, coating, patterning, and development of the photoresist. Additionally, potential pitfalls and their solutions are discussed. Lastly, the versatility of this design and fabrication strategy was demonstrated using other multicellular systems such as Drosophila egg chambers and brain organoids16.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 mL tri-cornered perforated plastic beakers with 60 mm Petri dishes</td>
			<td>Fisher</td>
			<td>14-955-111B</td>
			<td>Perferate with air holes</td>
		</tr>
		<tr>
			<td>100 mm P B &#60;100&#62; 0-100 500um SSP Test Grade Si Wafer</td>
			<td>University Wafer</td>
			<td>452</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy punches</td>
			<td>Ted Pella</td>
			<td>15110</td>
			<td></td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td></td>
			<td></td>
			<td>Not brand specific</td>
		</tr>
		<tr>
			<td>Blunt needle set</td>
			<td>CML Supply</td>
			<td>901</td>
			<td></td>
		</tr>
		<tr>
			<td>Contact Mask Aligner</td>
			<td>Quintel</td>
			<td>Q4000 MA</td>
			<td></td>
		</tr>
		<tr>
			<td>Cutting mat</td>
			<td>Dahle</td>
			<td>Vantage 10670</td>
			<td>size: 24&#34; x 36&#34;</td>
		</tr>
		<tr>
			<td>Developer</td>
			<td>Kayaku Advance Materials</td>
			<td>SU-8 2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Direct Write Lithographer</td>
			<td>Heidelberg</td>
			<td>MLA100</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td></td>
			<td></td>
			<td>Any commericailly availble dissecting microscope with transmitted light</td>
		</tr>
		<tr>
			<td>Glass petri dish</td>
			<td>Fisher</td>
			<td>FB0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slide</td>
			<td>Warner Instruments</td>
			<td>64-0710&#160; (CS-24/60)</td>
			<td></td>
		</tr>
		<tr>
			<td>HMDS Vapor Prime Oven</td>
			<td>Yes Engineering</td>
			<td>YES-3TA</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td></td>
			<td></td>
			<td>Not brand specific</td>
		</tr>
		<tr>
			<td>Oven</td>
			<td>Labnet</td>
			<td>I5110A</td>
			<td></td>
		</tr>
		<tr>
			<td>Paintbrush</td>
			<td></td>
			<td></td>
			<td>Not brand specific</td>
		</tr>
		<tr>
			<td>PDMS</td>
			<td>Dow Corning</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr>
			<td>Photoresist</td>
			<td>MicroChem</td>
			<td>SU-8 2100</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-32G</td>
			<td></td>
		</tr>
		<tr>
			<td>Portable pressure source</td>
			<td>hygger Quietest</td>
			<td>HGD946</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure gauge</td>
			<td>Cole-Parmer</td>
			<td>EW-68950-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin Coater</td>
			<td>Laurell</td>
			<td>WS-650-8B</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloro(1H,1H,2H,2H-perfluorooctyl)silane (PFOCTS)</td>
			<td>Sigma-Aldrich</td>
			<td>448931-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X 100</td>
			<td>Fisher</td>
			<td>AAA16046AP</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing</td>
			<td>Saint-Gobain</td>
			<td>02-587-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic Cleaner</td>
			<td>Cole-Parmer</td>
			<td>UX-08895-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum Pump</td>
			<td>Cole-Parmer</td>
			<td>EW-07164-87</td>
			<td></td>
		</tr>
	</tbody>
</table>

mechanostimulation of multicellular organisms through a high-throughput microfluidic compression system

During embryogenesis, coordinated cell movement generates mechanical forces that regulate gene expression and activity. To study this process, tools such as aspiration or coverslip compression have been used to mechanically stimulate whole embryos. These approaches limit experimental design as they are imprecise, require manual handling, and can process only a couple of embryos simultaneously. Microfluidic systems have great potential for automating such experimental tasks while increasing throughput and precision. This article describes a microfluidic system developed to precisely compress whole Drosophila melanogaster (fruit fly) embryos. This system features microchannels with pneumatically actuated deformable sidewalls and enables embryo alignment, immobilization, compression, and post-stimulation collection. By parallelizing these microchannels into seven lanes, steady or dynamic compression patterns can be applied to hundreds of Drosophila embryos simultaneously. Fabricating this system on a glass coverslip facilitates the simultaneous mechanical stimulation and imaging of samples with high-resolution microscopes. Moreover, the utilization of biocompatible materials, like PDMS, and the ability to flow fluid through the system make this device capable of long-term experiments with media-dependent samples. This approach also eliminates the requirement for manual mounting which mechanically stresses samples. Furthermore, the ability to quickly collect samples from the microchannels enables post-stimulation analyses, including -omics assays which require large sample numbers unattainable using traditional mechanical stimulation approaches. The geometry of this system is readily scalable to different biological systems, enabling numerous fields to benefit from the functional features described herein including high sample throughput, mechanical stimulation or immobilization, and automated alignment.

The microfluidic system is divided into two sub-compartments separated by deformable PDMS sidewalls. The first compartment is the liquid system where Drosophila embryos are introduced, automatically aligned, lined up, and compressed. The second compartment is a gas system where the gas pressure at either side of the compression channels is controlled via dead-end microchannels to precisely control the effective width of the compression channels. The microfluidic device is sealed with a glass slide at th...

Watch this Scientific Journal Video about Mechanostimulation of Multicellular Organisms Through a High-Throughput Microfluidic Compression System at JoVE.com

Mechanostimulation of Multicellular Organisms Through a High-Throughput Microfluidic Compression System

The present protocol describes the design, fabrication, and characterization of a microfluidic system capable of aligning, immobilizing, and precisely compressing hundreds of Drosophila melanogaster embryos with minimal user intervention. This system enables high-resolution imaging and recovery of samples for post-stimulation analysis and can be scaled to accommodate other multicellular biological systems.

During embryogenesis, coordinated cell movement generates mechanical forces that regulate gene expression and activity. To study this process, tools such ...

This protocol explains how to fabricate and use microfluidics technologies to leverage the experimental benefits in sample throughput, automated handling, and the ability to precisely apply mechanical stress while simultaneously visualizing samples. This technique minimizes the manual handling of multicellular biological organisms, such as embryos and organoids, for their immobilization, alignment, and live imaging. It also enables the application of mechanical compression to them for mechanobiology studies.Fabrication of molds with high aspect ratio features for this microfluidic chip can be challenging with conventional microfabrication techniques. The tips explained in this video article can overcome these challenges. To begin, clean the silicon wafer first with acetone, and then with isopropyl alcohol.Place the silicon wafer on a 250-degree Celsius hot plate for 30 minutes. for dehydration bake. Coat the silicon wafer with hexamethyldisiloxane in a vapor prime oven.Place a bottle of SU-8 2100 Photoresist in a 60-degrees Celsius oven for 15 minutes to reduce its viscosity. Pour one milliliter of the heated Photoresist for each inch of the silicon wafer placed on the hot plate until the Photoresist covers most of the surface. Apply pre-spin first at 250 rotations per minute for 30 seconds, and then at 350 rotations per minute for another 30 seconds, both with 100 RPM per second acceleration.Next, apply a spin first at 500 rotations per minute for 15 seconds with 100 RPM per second acceleration, and then at 1, 450 rotations per minute for 30 seconds with 300 RPM per second acceleration. Remove the edge bead with a clean room swab, and spray acetone on the wafer to remove imperfections and promote uniform coating. Expose the silicone wafer to 350 millijoule per centimeter square UV light through the photo mask.Using the contact mask aligner, apply post-exposure bake to the silicon wafer, and let it cool down to room temperature. Place the beaker inside another larger beaker, and fill the larger beaker with a fresh developer solution. Place a metal mesh on the beaker.Place the silicon wafer on the metal mesh upside down. Leave the silicon wafer submerged in the developer for 30 minutes with the stirrer turned on. Transfer the silicon wafer into an ultrasonic bath sonicator filled with the fresh developer for one hour at 40 kilohertz.Then take the wafer out from the beaker, and wash it with a fresh developer solution. Prepare the pre-cured PDMS solution by mixing the PDMS base with the curing agent at a 10-to-one ratio. Degas the mixture by placing it into a centrifuge for 500 g for five minutes at room temperature.Pour the pre-cured PDMS on a silicon wafer, and degas it again. Finally, place the uncured PDMS into 60-degrees Celsius oven for curing. Use a scalpel to cut the borders of the cured PDMS region corresponding to the microfluidic chip geometry.Punch the inlet and outlet holes on PDMS using a biopsy punch, or a needle with a blunt tip. Place the PDMS on the glass slide with its patterned surface facing toward the glass slide after plasma treatment to seal the microchannels via covalent bonding. Allow Oregon-R adult flies to lay eggs on apple juice agar plates, and collect the plates at the desired developmental time after egg laying for the given experiment.Flood the agar with embryo egg wash, and gently agitate the embryos with a paintbrush to dislodge them from the agar. Transfer the embryos to a 50%bleach solution for 90 seconds, stirring occasionally. Strain the embryos through a tissue sieve, and thoroughly wash away the bleach solution with water.Transfer the embryos to a 90-millimeter glass Petri dish with enough embryo egg wash to fully cover the embryos. Examine the embryos with transillumination on a dissecting microscope, and select embryos of the desired developmental stage for loading into the microfluidic device. Prime all seven embryo microchannels by filling them with 0.4 micrometers of filtered IPA through the main embryo inlet port.Replace the IPA with 0.4 micrometers of filtered deionized water. Then replace the DI water with embryo egg wash solution. Collect approximately 100 pre-selected embryos from the glass Petri dish using a glass pipette.Next, pipette the embryos into the embryo inlet port, and apply approximately three PSI negative pressure to the gas inlet using a portable vacuum pump to open up the embryo microchannels. Then tilt the microfluidic chip downward for the embryos to automatically align and settle into the embryo microchannels. If the embryo microchannel inlets get clogged by multiple embryos entering simultaneously, tilt the microfluidic chip upward, and then down again to clear the clogging.Based on the required throughput, introduce as many as 300 embryos into the embryo microchannels. Once the embryo loading is completed, remove the vacuum to immobilize the embryos. Then tilt the micro fluidic chip back to the horizontal position.Connect a portable positive pressure source with a pressure gauge to the gas inlet to apply three PSI compression. If live imaging experiments will be conducted on the mechanically stimulated embryos, place the microfluidic chip on a standard microscope stage glass slide holder with the gas inlet connected to the pressure source. Examine the embryos under a fluorescent microscope inside the microfluidic channels.Once the compression experiment is completed, the embryos can be collected for downstream analysis. To do this, first, apply the vacuum to the gas inlet to free the embryos. Then tilt the microfluidic chip upward for the embryos to move downward toward the embryo introduction port.Collect the embryos from the microfluidic chip using a glass pipette. The functionality of the microfluidic device was experimentally determined by loading Drosophila embryos into the compression channels and applying positive pressure to the gas channels. The embryos do not experience significant compression under vacuum, or in neutral pressure states.They are compressed when positive pressure is applied. Measurements of the decreasing width of the embryos under a microscope demonstrate how gas pressure can be used to obtain a target compression level. Microfluidic devices made in this way allow for chemical stimulation of immobilized samples.The stimulation allows for high spatiotemporal imaging. A fundamental question in developmental biology is how do mechanical forces regulate protein and gene expression during development? This technology allows us to apply mechanical stimulation to a large number of embryos at once, so that we can isolate sufficient amounts of protein and RNA to perform comparative proteomics, or transcriptomics experiments.This will enable researchers to learn more about the proteins and genes that are sensitive to mechanical forces.

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