We thank Drs. Christopher Moraes and Stephen A. Morin for their support for device design and fabrication. This study was supported by Bioengineering for Human Health grant from the University of Nebraska-Lincoln (UNL) and the University of Nebraska Medical Center (UNMC), and grant AR070242 from the NIH/NIAMS. We thank Janice A. Taylor and James R. Talaska of the Advanced Microscopy Core Facility at the University of Nebraska Medical Center for providing assistance with confocal microscopy.

NOTE: Wear personal protective equipment (PPE) such as gloves and lab coat for every step in this protocol.
1. Master mold fabrication
NOTE: Perform step 1.1 - 1.3 in a fume hood.
<ol>
	<li>Glass treatment 
	NOTE: Wear a face shield, gloves, and a lab coat for step 1.1.
	<ol>
		<li>Make Piranha solution (60 mL) by mixing sulfuric acid (H2SO4) and hydrogen peroxide (H2O2) with a volume ratio of 3:1. 
		CAUTION: Do not use Piranha solution and acetone in the same fume hood due to the explosion hazard.</li>
		<li>Place a glass plate (50.8 mm &#215; 76.2 mm &#215; 1.2 mm) in Piranha solution for 30 min at 40 &#176;C.</li>
		<li>Rinse the glass plate with deionized water (diH2O).</li>
		<li>Place the glass plate in acetone at room temperature for 10 min.</li>
		<li>Rinse the glass plate with isopropanol and dry it with nitrogen (N2) gas.</li>
		<li>Bake the glass plate at 200 &#176;C for 20 min on a hot plate. All subsequent baking steps use a hot plate.</li>
	</ol>
	</li>
	<li>SU-8 seed layer formation on the glass plate
	<ol>
		<li>Spread SU-8 5 on the glass plate with a disposable pipette to cover around 2/3 of the plate&#8217;s surface area.</li>
		<li>Spin the glass plate with SU-8 5 photoresists at 500 rpm for 35 s (initial spinning cycle) and then 2,500 rpm for 40 s (final spinning cycle) using a spin coater. All subsequent spin coating steps include the same initial spinning cycle.</li>
		<li>Bake the SU-8 5 coated glass plate at 65 &#176;C for 2 min and then at 95 &#176;C for 5 min.</li>
		<li>Expose the SU-8 5 coated glass plate to UV light (60 mW/cm2, distance between UV lamp and photomask is 20 cm, total amount of UV light energy = 60 mJ/cm2) for 1 s. 
		NOTE: The UV exposure time should be adjusted according to the power of a used UV light.</li>
		<li>Bake the SU-8 5 coated glass plate at 65 &#176;C for 2 min and at 95 &#176;C for 5 min.</li>
		<li>Place the baked glass plate in the SU-8 developer for 2 min.</li>
		<li>Bake the SU-8 5 coated glass at 180 &#176;C for 20 min.</li>
	</ol>
	</li>
	<li>SU-8 channel pattern fabrication using photolithography (Figure 2a Step 1-3)
	<ol>
		<li>Pour SU-8 100 on the SU-8 5 seeded glass plate to cover around 2/3 of the plate&#8217;s surface area.</li>
		<li>Spin the glass plate with SU-8 100 at 3,000 rpm for 38 s (&#8776; 90 &#956;m thick).</li>
		<li>Bake the SU-8 100 coated glass plate at 65 &#176;C for 10 min and then at 95 &#176;C for 30 min. If SU-8 100 is still sticky after this procedure, bake the glass plate for a longer time at 95 &#176;C until SU-8 100 becomes non-sticky.</li>
		<li>Place a high-resolution microchannel photomask (25,400 dpi, see Supplementary Figure 1) on the SU-8 100 coated glass plate and expose the photomask covered glass plate to UV light for 4 s (total amount of UV light energy = 240 mJ/cm2). 
		NOTE: The UV exposure time should be adjusted according to the power of a used UV light.</li>
		<li>Remove the photomask from the glass plate and bake the glass plate at 65 &#176;C for 2 min and then 95 &#176;C for 20 min.</li>
		<li>Keep the baked glass plate in a container, which is wrapped with aluminum foil to block any light, for overnight curing.</li>
		<li>Develop SU-8 100 channel patterns on the glass plate in the SU-8 developer for 15 min.</li>
		<li>Wash the SU-8 patterned glass plate (master mold) with isopropyl alcohol and dry it with N2 gas. If white particles remain during this process, repeat step 7 for 5 min.</li>
	</ol>
	</li>
</ol>
2. Pneumatic actuation unit
<ol>
	<li>Microchannel layer (Layer 1) 
	NOTE: Perform step 2.1.1 - 2.1.2 in a fume hood.
	<ol>
		<li>Drop 200 &#956;L of (Tridecafluoro-1,1,2,2-Tetrahydrooctyl)-1-Trichlorosilane on a coverslip and place it in a vacuum chamber with the master mold.</li>
		<li>Apply vacuum for 2 min in the chamber and wait for 6 h (or overnight) for silanization of the mold.</li>
		<li>Mix PDMS with a weight ratio of 10:1 (prepolymer:curing agent) for 5 min. 
		NOTE: All subsequent PDMS casting step contains the same prepolymer and curing agent weight ratio (10:1).</li>
		<li>Pour PDMS on the master mold and degas PDMS in a vacuum chamber for 30 min.</li>
		<li>Sandwich PDMS with a piece of transparency film.</li>
		<li>Clamp the above sandwiched assembly with a glass plate, foam pads and plexiglass plates (Figure 2a Step 4).</li>
		<li>Cure PDMS in an oven at 80 &#176;C for 6 h.</li>
		<li>Isolate the PDMS layer (Layer 1) with the transparency film from the sandwiched structure (Figure 2a Step 5).</li>
		<li>Activate surfaces of Layer 1 and a clean glass plate (Glass plate 1; 50.8 mm &#215; 76.2 mm &#215; 1.2 mm) using a plasma cleaner for 1 min. 
		NOTE: Plasma cleaning time may vary based on the power of a used plasma cleaner.</li>
		<li>Bond Layer 1 onto Glass plate 1 and place them in the oven at 80 &#176;C for 30 min.</li>
		<li>Remove the transparency film from Layer 1.</li>
	</ol>
	</li>
	<li>Thin PDMS membrane (Layer 2)
	<ol>
		<li>Spin coat uncured PDMS on a transparency film at 1,000 rpm for 1 min to obtain a 60 &#956;m thick PDMS layer.</li>
		<li>Partially cure spin coated PDMS (Layer 2) in the oven at 80 &#176;C for 20-30 min.</li>
		<li>Activate Layer 1 on Glass plate 1 and Layer 2 using the plasma cleaner for 1 min. 
		NOTE: Plasma cleaning time may vary based on the power of a used plasma cleaner.</li>
		<li>Bond the Layer 2 onto Layer 1 and place them in the oven at 80 &#176;C overnight.</li>
	</ol>
	</li>
	<li>Tubing block
	<ol>
		<li>Place metal tubes vertically on a petri dish.</li>
		<li>Gently pour PDMS in the dish to submerge about 3/4 of the metal tubes.</li>
		<li>Cure PDMS in the oven at 60 &#176;C for 6 h (or overnight).</li>
		<li>Cut a piece of PDMS block containing one metal tube.</li>
		<li>Punch a hole on Layer 2 for the inlet.</li>
		<li>Activate the PDMS block and Layer 2 using the plasma cleaner for 1 min.</li>
		<li>Attach the PDMS block onto the inlet part of Layer 2 and place the entire actuation unit in the oven at 80 &#176;C for overnight.</li>
	</ol>
	</li>
</ol>
3. Alginate-chondrocyte (or bead) constructs
<ol>
	<li>Amino-silanized glass plate
	<ol>
		<li>Cut a glass plate (50.8 mm &#215; 76.2 mm &#215; 1.2 mm) into two half-sized glass plates (50.8 mm &#215; 38.1 mm &#215; 1.2 mm) using a diamond scriber.</li>
		<li>Place the glass plates in 0.2 M hydrogen chloride (HCl) and gently shake (e.g., 55 rpm) them overnight.</li>
		<li>Rinse the glass plates with diH2O.</li>
		<li>Shake the glass plates in 0.1 M sodium hydroxide (NaOH) for 1 h at 55 rpm and rinse them with diH2O.</li>
		<li>Shake the glass plates in 1% (v/v) 3-aminopropyltrimethoxysilane (APTES) for 1 h at 55 rpm and rinse them with diH2O.</li>
		<li>Dry the amino-silanized glass plates in a fume hood overnight.</li>
	</ol>
	</li>
	<li>Agarose gel mold for alginate gel constructs
	<ol>
		<li>Mix 5% (w/v) agarose and 200 mM calcium chloride (CaCl2) in diH2O.</li>
		<li>Boil the agarose gel solution with a microwave oven (or a hot plate). Boiling time varies based on the volume of the agarose gel solution and the power of the microwave oven (or the temperature of the hot plate).</li>
		<li>Pour the boiled agarose gel solution onto an aluminum mold (see Supplementary Figure S2), and sandwich it with a glass plate.</li>
		<li>Wait for 5 min and unmold the solidified agarose gel from the aluminum mold.</li>
	</ol>
	</li>
	<li>Growth plate chondrocyte harvest
	<ol>
		<li>Isolate growth plates from the hind limbs of neonatal mice.</li>
		<li>Place the growth plates in 1 mL of 0.25% collagenase for 3 h in an incubator at 37 &#176;C and 8% carbon dioxide (CO2), to remove extracellular matrix (ECM).</li>
		<li>Centrifuge the digested sample for 5 min at 125 x g to make a chondrocyte pellet and remove supernatant from the sample. Here, 1 x g is the acceleration of gravity.</li>
		<li>Resuspend the chondrocyte pellet in 1 mL of Dulbecco&#39;s modified Eagle&#39;s medium (DMEM).</li>
		<li>Count the number of chondrocytes in DMEM using a cell counter.</li>
		<li>Centrifuge the chondrocytes in DMEM for 5 min at 125 x g to make a chondrocyte pellet again and remove supernatant from the sample.</li>
		<li>Optionally, resuspend the chondrocyte pellet in the chondrocyte culture media (CCM)34 containing 2 &#956;M calcein AM, and incubate the sample at 37 &#176;C for 30 min. Repeat step 3.3.6 and move to step 3.3.8.</li>
		<li>Resuspend the chondrocyte pellet in a desired volume of CCM and keep them in the incubator at 37 &#176;C and 8% CO2 before use.</li>
	</ol>
	</li>
	<li>Alginate-chondrocyte (or bead) constructs (Figure 2c)
	<ol>
		<li>Mix 1.5% (w/v) alginate in phosphate-buffered saline (PBS) with 5 mg/mL of sulfo-NHS, 10 mg/mL of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC).</li>
		<li>Add 8 x 106 chondrocytes into the 1 mL of alginate gel solution [or Add 3 &#956;L of 1 &#956;m-diameter fluorescent beads (542/612 nm) in 1 mL of alginate gel solution (0.3% (v/v))].</li>
		<li>Place 150 &#956;L of the alginate-chondrocyte (or bead) solution on the amino-silanized glass plate fabricated in Step 3.1.</li>
		<li>Cover the alginate gel solution with the agarose gel mold for 3 min.</li>
		<li>Remove excessive alginate gel solution overflowing from the agarose gel mold with a razor blade and remove the agarose gel mold. Then, cylindrical alginate-chondrocyte (or bead) constructs are obtained on the amino-silanized glass plate.</li>
		<li>Place the alginate-chondrocyte constructs in cross-linking solution (50 mM CaCl2 / 140 mM NaCl in diH2O) for 1 min for further polymerization.</li>
	</ol>
	</li>
</ol>
4. Device assembly (Figure 2d)
NOTE: PDMS spacers and 3D printed clamps need to be prepared separately.
<ol>
	<li>Locate four 1 mm thick PDMS spacers on the four corners of the Layer 2 of the actuation unit.</li>
	<li>Place 700 &#956;L of CCM to cover the air chambers of Layer 2.</li>
	<li>Place the alginate-chondrocyte (or bead) constructs on Layer 2 while carefully aligning the constructs with the air chambers.</li>
	<li>Clamp the device with 3D printed clamps (Figure 1c).</li>
</ol>
5. Actuation of the device
<ol>
	<li>Connect the outlet of an air pump (see Table of Materials) with the inlet of a solenoid valve with a silicon tubing.</li>
	<li>Connect the outlet of the solenoid valve with the inlet of the assembled device with a silicon tubing.</li>
	<li>Connect the solenoid valve with a function generator.</li>
	<li>Manipulate the solenoid valve with a square wave (e.g., 1 Hz) generated by the function generator.</li>
	<li>Turn on the air pump to actuate the device pneumatically.</li>
</ol>
6. Imaging of chondrocytes in the device
NOTE: To obtain a good image quality, image chondrocytes (or fluorescent beads) in alginate gel through Glass plate 2 because expanded PDMS balloons and air chambers can distort optical images. If an inverted microscope is used for imaging, the device needs to be setup so that Glass plate 2 faces downward.
<ol>
	<li>Prepare the device with chondrocytes (or fluorescent beads) as shown in previous sections.</li>
	<li>Take z-stack images of chondrocytes (or fluorescent beads) with a confocal microscope before and under compression, respectively. Choose a z-step size based on the depth of field of a used confocal imaging system.</li>
	<li>The height of chondrocytes (or an alginate-bead construct) can be measured with automatic image processing method shown in previous literatures26,35.</li>
</ol>

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Y., 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+pneumatic+micro+cell+chip+for+the+differentiation+of+human+mesenchymal+stem+cells+under+mechanical+stimulation.">A pneumatic micro cell chip for the differentiation of human mesenchymal stem cells under mechanical stimulation.</a> Lab on a Chip. 7 (12), 1775-1782 (2007).</li><li>Hosmane, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Valve-based+microfluidic+compression+platform:+single+axon+injury+and+regrowth.">Valve-based microfluidic compression platform: single axon injury and regrowth.</a> Lab on a Chip. 11 (22), 3888-3895 (2011).</li><li>Ho, K. K. Y., Wang, Y. L., Wu, J., Liu, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+microfluidic+device+designed+for+cyclic+compression+of+single+adherent+cells.">Advanced microfluidic device designed for cyclic compression of single adherent cells.</a> Frontiers in Bioengineering and Biotechnology. 6 (148), (2018).</li><li>Seo, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interconnectable+dynamic+compression+bioreactors+for+combinatorial+screening+of+cell+mechanobiology+in+three+dimensions.">Interconnectable dynamic compression bioreactors for combinatorial screening of cell mechanobiology in three dimensions.</a> ACS Applied Materials &amp; Interfaces. 10 (16), 13293-13303 (2018).</li><li>Erickson, A. 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=A+tunable,+three-dimensional+in+Vitro+culture+model+of+growth+plate+cartilage+using+alginate+hydrogel+acaffolds.">A tunable, three-dimensional in Vitro culture model of growth plate cartilage using alginate hydrogel acaffolds.</a> Tissue Engineering Part A. 24 (1-2), 94-105 (2018).</li><li>Lee, D., Rahman, M. M., Zhou, Y., Ryu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+confocal+microscopy+indentation+method+for+hydrogel+elasticity+measurement.">Three-dimensional confocal microscopy indentation method for hydrogel elasticity measurement.</a> Langmuir. 31 (35), 9684-9693 (2015).</li><li>Johnston, I. D., McCluskey, D. K., Tan, C. K. L., Tracey, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+characterization+of+bulk+Sylgard+184+for+microfluidics+and+microengineering.">Mechanical characterization of bulk Sylgard 184 for microfluidics and microengineering.</a> Journal of Micromechanics and Microengineering. 24 (3), 035017 (2014).</li></ol>

The authors have nothing to disclose.

To test the effects of compressive stress on growth plate chondrocytes, we developed the microfluidic chondrocyte compression device (Figure 1) to apply various levels of compressive stress to the chondrocytes in the alginate hydrogel scaffold for 3D culture in high throughput ways. To assist other researchers to adopt our device or to develop similar devices, we provided details of the device fabrication steps in this protocol article.
The crucial steps in this p...

Micro-engineered platforms are valuable tools for studying the molecular, cellular, and tissue level biology because they enable dynamic control of both the physical and chemical microenvironments1,2,3,4,5,6,7,8. Thus, multiple hypotheses can be simultaneously tested in a tightly controlled manner. In the case of growth plate cartilage, there are increasing evidences of an important role of compressive stress in modulating bone growth through action on the growth plate cartilage9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25. However, the mechanism of action of compressive stress &#8211; in particular, how stress guides the formation of chondrocyte columns in the growth plate &#8211; is poorly understood.
The goal of this protocol is to create a pneumatically actuating microfluidic chondrocyte compression device26 to elucidate mechanisms of mechanobiology in growth plate chondrocytes (Figure 1a-c). The device consists of two parts: the pneumatic actuation unit and the alginate gel construct. The microfluidic pneumatic actuation unit is fabricated using polydimethylsiloxane (PDMS) based on the photo- and soft-lithography. This unit contains a 5 x 5 array of thin PDMS membrane balloons which can be inflated differently based on their diameters. The alginate gel construct consists of the chondrocytes embedded in a 5 x&#160;5 array of alginate gel cylinders, and the entire alginate-chondrocyte constructs are assembled with the actuation unit. The alginate gel constructs are compressed by the pneumatically inflated PDMS balloons (Figure 1b). The microfluidic device can generate five different levels of compressive stress simultaneously in a single platform based on differences in the PDMS balloon diameter. Thus, a high-throughput test of chondrocyte mechanobiology under multiple compression conditions is possible.
The microfluidic device described in this protocol has many advantages over the conventional compression device such as external fixators14,21,23 and macroscopic compression devices16,19,27,28 for studying chondrocyte mechanobiology: 1) The microfluidic device is cost effective because it consumes smaller volume of samples than the macroscopic compression device, 2) The microfluidic device is time effective because it can test multiple compression conditions simultaneously, 3) The microfluidic device can combine mechanical and chemical stimuli by forming a concentration gradient of chemicals based on the limited mixing in microchannels, and 4) Various microscopy techniques (time-lapse microscopy and fluorescence confocal microscopy) can be applied with the microfluidic device made of transparent PDMS.
We adopted and modified the method of Moraes et al.7,29 to create different compressive stress levels in a single device to enable high-throughput mechanobiology studies of chondrocyte compression. Our approach is appropriate for cells (e.g., chondrocytes) which need three-dimensional (3D) culture environment and for biological assays after compressing cells. Although some microfluidic cell compression devices can compress cells cultured on two-dimensional (2D) substrates30,31,32, they cannot be used for chondrocytes because 2D cultured chondrocytes dedifferentiate. There are microfluidic platforms for compressing 3D cultured cells in photopolymerized hydrogels7,33, but they are limited in isolating cells after compression experiments because isolating cells from photopolymerized hydrogel is not easy. Additionally, the effects of ultraviolet (UV) exposure and photo crosslinking initiators on cells may need to be evaluated. In contrast, our method allows rapid isolation of cells after compression experiments for post biological assays because alginate hydrogels can be depolymerized quickly by calcium chelators. The detailed device fabrication and characterization methods are described in this protocol. A brief procedure for fabricating the microfluidic chondrocyte compression device is shown in Figure 2.

<table>
	<tbody>
		<tr>
			<td>(3-Aminopropyl)triethoxysilane (ATPES)</td>
			<td>Sigma-Aldrich</td>
			<td>741442-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>(Tridecafluoro-1, 1, 2, 2-Tetrahydrooctyl)-1-Trichlorosilane</td>
			<td>United Chemical Technologies</td>
			<td>T2492-KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylic sheet</td>
			<td>McMaster-Carr</td>
			<td>8560K354</td>
			<td></td>
		</tr>
		<tr>
			<td>Air pump</td>
			<td>Schwarzer Precision</td>
			<td>SP 500 EC-LC4.5V DC</td>
			<td>We used the model purchased in 2015. The internal design and performance of air pump (SP 500 EC-LC) changed in early 2016. Also, air pump performance has changed in the course of time. Thus, air pressure generated by an SP 500 EC-LC air pump should be calibrated before use.</td>
		</tr>
		<tr>
			<td>Alginate powder</td>
			<td>FMC Corporation</td>
			<td>Pronova UP MVG</td>
			<td></td>
		</tr>
		<tr>
			<td>Barb Straight Connectors (Metal tube)</td>
			<td>Pneumadyne</td>
			<td>EB40-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcein AM</td>
			<td>Invitrogen</td>
			<td>C3100MP</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td>11960-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Dyed red aqueous fluorescent particles</td>
			<td>Thermo Fisher Scientific</td>
			<td>R0100</td>
			<td></td>
		</tr>
		<tr>
			<td>EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride)</td>
			<td>Thermo Fisher Scientific</td>
			<td>22980</td>
			<td></td>
		</tr>
		<tr>
			<td>Foam pad</td>
			<td>GRAINGER</td>
			<td>Item # 5GCE8</td>
			<td></td>
		</tr>
		<tr>
			<td>Function / Arbitrary Waveform Generator</td>
			<td>Keysight Technologies</td>
			<td>33210A</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Fisher Chemical</td>
			<td>A144-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide</td>
			<td>Fisher BioReagents</td>
			<td>BP2633500</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl alcohol</td>
			<td>BDH1174-4LP</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>22-267-013</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS)</td>
			<td>Dow Corning</td>
			<td>184 SIL ELAST KIT 0.5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Power supply</td>
			<td>Keysight Technologies</td>
			<td>E3630A</td>
			<td></td>
		</tr>
		<tr>
			<td>SeaKem LE Agarose</td>
			<td>Lonza</td>
			<td>50004</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Fisher Chemical</td>
			<td>S318-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Solenoid manifold</td>
			<td>Pneumadyne</td>
			<td>MSV10-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Solenoid valve</td>
			<td>Pneumadyne</td>
			<td>S10MM-30-12-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin coater</td>
			<td>Laurell Technologies</td>
			<td>WS-650Mz-23NPPB</td>
			<td></td>
		</tr>
		<tr>
			<td>SU8 Developer</td>
			<td>MicroChem Corp.</td>
			<td>Y020100 4000L1PE</td>
			<td></td>
		</tr>
		<tr>
			<td>SU8-100</td>
			<td>MicroChem Corp.</td>
			<td>Y131273 0500L1GL</td>
			<td></td>
		</tr>
		<tr>
			<td>SU8-5</td>
			<td>MicroChem Corp.</td>
			<td>Y131252 0500L1GL</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfo-NHS (N-hydroxysulfosuccinimide)</td>
			<td>Thermo Fisher Scientific</td>
			<td>24510</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfuric acid</td>
			<td>EMD Millipore</td>
			<td>MSX12445</td>
			<td></td>
		</tr>
	</tbody>
</table>

a microfluidic platform for stimulating chondrocytes with dynamic compression

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth plate cartilage architecture and results in growth modulation of long bones of children. To determine the role of compressive stress in bone growth, we created a microfluidic device actuated by pneumatic pressure, to dynamically (or statically) compress growth plate chondrocytes embedded in alginate hydrogel cylinders. In this article, we describe detailed methods for fabricating and characterizing this device. The advantages of our protocol are: 1) Five different magnitudes of compressive stress can be generated on five technical replicates in a single platform, 2) It is easy to visualize cell morphology via a conventional light microscope, 3) Cells can be rapidly isolated from the device after compression to facilitate downstream assays, and 4) The platform can be applied to study mechanobiology of any cell type that can grow in hydrogels.

This article shows detailed steps of the microfluidic chondrocyte compression device fabrication (Figure 2). The device contains a 5 x&#160;5 arrays of cylindrical alginate-chondrocyte constructs, and these constructs can be compressed with five different magnitudes of compression (Figure 1, Figure 3 and Figure 4). The height of the pneumatic microchannel is around 90 &#956;m, and the PDMS balloon diame...

Watch this Scientific Journal Video about A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression at JoVE.com

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

This article provides detailed methods for fabricating and characterizing a pneumatically actuating microfluidic device for chondrocyte compression.

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth ...

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6.5K Views.  University of Nebraska Medical Center. This article provides detailed methods for fabricating and characterizing a pneumatically actuating microfluidic device for chondrocyte compression.

Video: A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher neutrophil counts in the blood are usually an indicator of ongoing infections, while low neutrophil counts are a warning sign for higher risks for infections. To accomplish their functions, neutrophils also have to be able to move effectively from the blood where they spend most of their life, into tissues, where infections occur. Consequently, any defects in the ability of neutrophils to migrate can increase the risks for infections, even when neutrophils are present in appropriate numbers in the blood. However, measuring neutrophil migration ability in the clinic is a challenging task, which is time consuming, requires large volume of blood, and expert knowledge. To address these limitations, we designed a robust microfluidic assays for neutrophil migration, which requires a single droplet of unprocessed blood, circumvents the need for neutrophil separation, and is easy to quantify on a simple microscope. In this assay, neutrophils migrate directly from the blood droplet, through small channels, towards the source of chemoattractant. To prevent the granular flow of red blood cells through the same channels, we implemented mechanical filters with right angle turns that selectively block the advance of red blood cells. We validated the assay by comparing neutrophil migration from blood droplets collected from finger prick and venous blood. We also compared these whole blood (WB) sources with neutrophil migration from samples of purified neutrophils and found consistent speed and directionality between the three sources. This microfluidic platform will enable the study of human neutrophil migration in the clinic and the research setting to help advance our understanding of neutrophil functions in health and disease.

This protocol details an assay designed to measure human neutrophil chemotaxis from one droplet of whole blood with robust reproducibility. This approach circumvents the need for neutrophil separation and requires only a few minutes of assay preparation time. The microfluidic chip enables the repeated measure of neutrophil chemotaxis over time in infants or small mammals, where sample volume is limited.

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher ...

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments,&#160;as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier,&#160;more quantifiable extracellular relay of signals to the cells. 
For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by their natural environment, root hair morphology and function are difficult to study and often excluded from plant research. In recent years, microfluidic platforms have offered a way to visualize root systems at high resolution without disturbing the roots during transfer to an imaging system. The microfluidic platform presented here builds on previous plant-on-a-chip research by incorporating a two-layer device to confine the Arabidopsis thaliana main root to the same optical plane as the root hairs. This design enables the quantification of root hairs on a cellular and organelle level and also prevents z-axis drifting during the addition of experimental treatments. We describe how to store the devices in a contained and hydrated environment, without the need for fluidic pumps, while maintaining a gnotobiotic environment for the seedling. After the optical imaging experiment, the device may be disassembled and used as a substrate for atomic force or scanning electron microscopy while keeping fine root structures intact.

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

This article demonstrates how to culture Arabidopsis thaliana seedlings in a two-layer microfluidic platform that confines the main root and root hairs to a single optical plane. This platform can be used for real-time optical imaging of fine root morphology as well as for high-resolution imaging by other means.

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by ...

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved useful as a convenient live imaging platform providing capabilities for precise control of experimental conditions in real time. In this article, we demonstrate live imaging of individual worms employing WormSpa, a previously-published custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes. To illustrate the capability, we performed proof-of-principle experiments in which worms were infected in the device with pathogenic bacteria, and the dynamics of expression of immune response genes and egg laying were monitored continuously in individual animals. The simple design and operation of this device make it suitable for users with no previous experience with microfluidic-based experiments. We propose that this approach will be useful for many researchers interested in longitudinal observations of biological processes under well-defined conditions.

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In this article, we demonstrate live imaging of individual worms employing a custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes.

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...