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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<li>Moraes, C., Sun, Y., Simmons, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microfabricated+platforms+for+mechanically+dynamic+cell+culture%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Microfabricated platforms for mechanically dynamic cell culture.</a> Journal of Visualized Experiments. (46), e224(2010).</li>
<li>Sim, W. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+pneumatic+micro+cell+chip+for+the+differentiation+of+human+mesenchymal+stem+cells+under+mechanical+stimulation%5BTitle%5D)+AND+%22Lab+on+a+Chip%22%5BJournal%5D)">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/pubmed?term=((Valve-based+microfluidic+compression+platform%3A+single+axon+injury+and+regrowth%5BTitle%5D)+AND+%22Lab+on+a+Chip%22%5BJournal%5D)">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/pubmed?term=((Advanced+microfluidic+device+designed+for+cyclic+compression+of+single+adherent+cells%5BTitle%5D)+AND+%22Frontiers+in+Bioengineering+and+Biotechnology%22%5BJournal%5D)">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/pubmed?term=((Interconnectable+dynamic+compression+bioreactors+for+combinatorial+screening+of+cell+mechanobiology+in+three+dimensions%5BTitle%5D)+AND+%22ACS+Applied+Materials+%26+Interfaces%22%5BJournal%5D)">Interconnectable dynamic compression bioreactors for combinatorial screening of cell mechanobiology in three dimensions.</a> ACS Applied Materials & Interfaces. 10 (16), 13293-13303 (2018).</li>
<li>Erickson, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+tunable%2C+three-dimensional+in+Vitro+culture+model+of+growth+plate+cartilage+using+alginate+hydrogel+acaffolds%5BTitle%5D)+AND+%22Tissue+Engineering+Part+A%22%5BJournal%5D)">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/pubmed?term=((Three-dimensional+confocal+microscopy+indentation+method+for+hydrogel+elasticity+measurement%5BTitle%5D)+AND+%22Langmuir%22%5BJournal%5D)">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/pubmed?term=((Mechanical+characterization+of+bulk+Sylgard+184+for+microfluidics+and+microengineering%5BTitle%5D)+AND+%22Journal+of+Micromechanics+and+Microengineering%22%5BJournal%5D)">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 protocol are 1) fabricating PDMS layer with pneumatic microchannels (Layer 1) without any air bubbles since air bubbles in Layer 1 may damage pneumatic microchannels while the backing transparency film is peeled off, 2) maintaining a constant temperature (e.g., 80 &#176;C) for curing PDMS balloons (Layer 2) because the elasticity of PDMS is known to depend on the curing temperature36, 3) aligning the alginate gel constructs with the PDMS balloons, and 4) using fresh amino-silanized glass plate (Glass plate 2) for bonding the alginate gel columns on Glass plate 2 within two days after the salinization treatment.
The major limitation of this protocol is that it is relatively labor intensive to fabricate the device because the process involves photolithography and multiple steps of soft lithography. Additionally, the performance of microfluidic cell compression devices fabricated based on our protocol needs to be evaluated whenever different types of hydrogels and cells are used. This is because any differences in the mechanical properties of hydrogels and cells will affect device performance.
Although our microfluidic cell compression device is for applying dynamic compression to chondrocytes, its performance was evaluated by imaging statically compressed alginate gels and cells. This is because it was hard to image gels and cells under dynamic compression with the conventional confocal microscopy. We compared static (14 kPa, 1 h) and dynamic compression (14 kPa, 1 Hz, 1 h) in terms of the permanent deformation of alginate gel and found that the permanent deformation of the gel under the dynamic compression was negligible compared to the static compression (see Supplementary Figure S3).
One advantage of our method is that it can be used for other cell types which need 3D culture environment. The resultant compression of the device can be modulated depending on applications by changing the diameter and thickness of the PDMS balloons and/or the pressure in the balloon. It is also possible to modify the elasticity of the PDMS balloon by adjusting the mixing ratio between the prepolymer and the curing agent. Cells in this device can be imaged in real time using light/fluorescence microscopy, and the device can be rapidly disassembled for cell harvest to enable downstream analysis. Another advantage is the ability to generate five distinct mechanical stress levels with five technical replicates per each stress level using a single device. Combining replication and a dose-response analysis ensures a high degree of rigor and reproducibility in the results.

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&#039;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 diameters are 1.2, 1.4, 1.6, 1.8 and 2.0 mm, respectively. The performance of the device was quantified with confocal microscopy with static compression conditions and image processing. Static compression was employed for the microscopic imaging because the z-stack imaging with the confocal microscopy takes a few minutes, so it is too slow for quantitative imaging during the dynamic compression.
Figure 3a shows the 5 &#215; 5 arrays of alginate hydrogel columns (diameter: ~0.8 mm, height: ~1 mm) cast on Glass plate 2. These gel constructs were imaged by adding fluorescent beads in the gel. Figure 3b shows an example case that the gel column was compressed by 33.8% in height by the largest PDMS balloon. The resultant compressive strain of the gel constructs increased by approximately 5% per 0.2 mm increment in the PDMS balloon diameter as shown in Figure 3c.
Compressive deformation of chondrocytes was determined by imaging the cells in a 613 &#956;m &#215; 613 &#956;m &#215; 40&#8211;55 &#956;m (x &#215; y &#215; z) volume near the gel construct center as shown in Figure 4a. Figure 1d shows an example image of a chondrocyte that was compressed by 16% by the largest PDMS balloon. Figure 4b shows the distribution of the measured cell compression strain values, and overall cells were compressed more by larger PDMS balloons. Therefore, the amount of alginate gel and chondrocyte compression were controlled by the diameter of PDMS balloons (Figure 3 and Figure 4) with a constant pressure of 14 kPa.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59676/59676fig01.jpg" /> 
Figure 1. Microfluidic chondrocyte compression device. 
(a) Schematic of the assembled device. A 5 &#215; 5 array of alginate&#8211;chondrocyte constructs are aligned on PDMS balloons with 5 different diameters (D = 1.2, 1.4, 1.6, 1.8 and 2.0 mm), where D is the diameter of PDMS balloon (or air chamber). (b) Schematic of the device operation. The device is actuated by pneumatic pressure which expands PDMS balloons. (c) Image of an actual device (coin diameter = 19 mm). (d) Vertical cross-sections of a chondrocyte before (left) and under (right) compression on the largest PDMS balloon (D = 2.0 mm) (cell compressive strain, &#949;cell = |cell height change/initial cell height| x&#160;100 = 16%). This figure is reproduced from 26.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59676/59676fig02v2.jpg" /> 
Figure 2. Detailed steps of microfluidic chondrocyte compression device fabrication. 
(a) Photolithography for generating a SU-8 master mold and following soft lithography for creating PDMS layer with pneumatic microchannels (Layer 1). (b) Thin PDMS membrane (Layer 2) on a transparency film generated by spin coating. (c) Cylindrical alginate gel casting method on glass (Glass plate 2). (d) Assembly of the microfluidic chondrocyte compression device. This figure is reproduced from 26. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59676/59676fig02v2large.jpg" target="_blank">Please click here to download a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59676/59676fig03.jpg" /> 
Figure 3. Measurement of alginate gel deformation under static compression. 
(a) 5 x&#160;5 arrays of cylindrical alginate gel constructs (diameter: ~800 &#956;m, height: ~1 mm). (b) Alginate gel compressed by the largest PDMS balloon (D = 2.0 mm). The compressive strain of the&#160;alginate gel is 33.8%. (c) Compressive strain of alginate gel (&#949;gel) increases around 5% per 0.2 mm increment of PDMS balloon diameter (D). Error bar: standard deviation. Red line: linear fitting line This figure is reproduced from 26.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59676/59676fig04.jpg" /> 
Figure 4. Measurement of chondrocyte deformation under static compression. 
(a) A z-stack image [613 &#956;m &#215; 613 &#956;m &#215; 40&#8211;55 &#956;m (x &#215; y &#215; z)] was obtained in the middle of the gel construct, 300&#8211;400 &#956;m from the gel bottom. (b) Different magnitudes of chondrocyte compressive strain (&#949;cell) resulted as a function of the PDMS balloon diameter (D). <img alt="Icon" src="/files/ftp_upload/59676/star.jpg" />: mean values. <img alt="Icon" src="/files/ftp_upload/59676/diamond.jpg" />: each data points. Top (or bottom) and middle lines of the box are the standard deviation and median value, respectively. This figure is reproduced from 26.
Figure S1. Microchannel photomask design for step 1.3.4 (unit = mm). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59676/59676supfig01.jpg" target="_blank">Please click here to download this figure.</a>
Figure S2. Aluminum mold design for step 3.2.3 (unit = mm). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59676/59676supfig02.jpg" target="_blank">Please click here to download this figure.</a>
Figure S3. Permanent deformation of the alginate gel (1.5%, w/v) under 1 h-long dynamic (1 Hz) and static compression. This figure is reproduced from 26. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59676/59676supfig03.jpg" target="_blank">Please click here to download this figure.</a>

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 ...

a-microfluidic-platform-for-stimulating-chondrocytes-with-dynamic

Nanyang Technological University

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JoVE Journal

Bioengineering

6.5K Views.  University of Nebraska Medical Center. Dit pneumatisch geactiveerde microfluïde compressieapparaat kan worden gebruikt om mechanobiologie te bestuderen in 3D hydrogelculturen van groeiplaatchondrocyten. Het microfluïde apparaat is kosten- en tijdeffectief omdat het tegelijkertijd meerdere grootheden van een compressie kan genereren op kleine hoeveelheden monsters, en verschillende microscopische beeldvormingen kunnen met het apparaat worden toegepast. Onze methode kan ook worden toegepast op de studie van mechanobiologie van and...

Video: A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

Chondrogenesis from stem cells requires fine tuning the culture conditions. Here, we present a magnetic approach for condensing cells, an essential step to initiate chondrogenesis. In addition, we show that dynamic maturation in a bioreactor applies mechanical stimulation to the cellular constructs and enhances cartilaginous extracellular matrix production.

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

Real-time Visualization and Analysis of Chondrocyte Injury Due to Mechanical Loading in Fully Intact Murine Cartilage Explants

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread chondrocyte death, potentially leading to irreversible breakdown of the joint and the onset of osteoarthritis. Additionally, maintenance of chondrocyte viability is important in osteochondral graft procedures for optimal surgical outcomes. We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine synovial joints after application of controlled mechanical loads or impacts. This method can be used in comparative studies to investigate the effects of different mechanical loading regimens, different environmental conditions or genetic manipulations, as well as different stages of cartilage degeneration on short- and/or long-term vulnerability of in situ articular chondrocytes. The goal of the protocol introduced in the manuscript is to assess the spatial extent of cell injury/death on the articular surface of murine synovial joints. Importantly, this method enables testing on fully intact cartilage without compromising native boundary conditions. Moreover, it allows for real-time visualization of vitally stained articular chondrocytes and single image-based analysis of cell injury induced by application of controlled static and impact loading regimens. Our representative results demonstrate that in healthy cartilage explants, the spatial extent of cell injury depends sensitively on load magnitude and impact intensity. Our method can be easily adapted to investigate the effects of different mechanical loading regimens, different environmental conditions or different genetic manipulations on the mechanical vulnerability of in situ articular chondrocytes.

We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine joints after application of controlled mechanical loads or impacts. This method can be used to investigate how osteoarthritis, genetic factors and/or different loading regimens affect the vulnerability of in situ chondrocytes.

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread ...

A Lab-On-A-Chip Platform for Stimulating Osteocyte Mechanotransduction and Analyzing Functional Outcomes of Bone Remodeling

Bone remodeling is a tightly regulated process that is required for skeletal growth and repair as well as adapting to changes in the mechanical environment. During this process, mechanosensitive osteocytes regulate the opposing responses between the catabolic osteoclasts and anabolic osteoblasts. To better understand the highly intricate signaling pathways that regulate this process, our lab has developed a foundationary lab-on-a-chip (LOC) platform for analyzing functional outcomes (formation and resorption) of bone remodeling within a small scale system. As bone remodeling is a lengthy process that occurs on the order of weeks to months, we developed long-term cell culturing protocols within the system. Osteoblasts and osteoclasts were grown on functional activity substrates within the LOC and maintained for up to seven weeks. Afterward, chips were disassembled to allow for the quantification of bone formation and resorption. Additionally, we have designed a 3D printed mechanical loading device that pairs with the LOC platform and can be used to induce osteocyte mechanotransduction by deforming the cellular matrix. We have optimized cell culturing protocols for osteocytes, osteoblasts, and osteoclasts within the LOC platform and have addressed concerns of sterility and cytotoxicity. Here, we present the protocols for fabricating and sterilizing the LOC, seeding cells on functional substrates, inducing mechanical load, and disassembling the LOC to quantify endpoint results. We believe that these techniques lay the groundwork for developing a true organ-on-a-chip for bone remodeling.

Here, we present protocols for analyzing bone remodeling within a lab-on-a-chip platform. A 3D printed mechanical loading device can be paired with the platform to induce osteocyte mechanostransduction by deforming the cellular matrix. The platform can also be used to quantify bone remodeling functional outcomes from osteoclasts and osteoblasts (resorption/formation).

Bone remodeling is a tightly regulated process that is required for skeletal growth and repair as well as adapting to changes in the mechanical ...

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not regenerate very efficiently in vivo; and as a result, osteoarthritis leads to irreversible cartilage loss and is accompanied by chronic pain and immobility 1,2. Cartilage tissue engineering offers promising potential to regenerate and restore tissue function. This technology typically involves seeding chondrocytes into natural or synthetic scaffolds and culturing the resulting 3D construct in a balanced medium over a period of time with a goal of engineering a biochemically and biomechanically mature tissue that can be transplanted into a defect site in vivo 3-6. Achieving an optimal condition for chondrocyte growth and matrix deposition is essential for the success of cartilage tissue engineering. 
 In the native joint cavity, cartilage at the articular surface of the bone is bathed in synovial fluid. This clear and viscous fluid provides nutrients to the avascular articular cartilage and contains growth factors, cytokines and enzymes that are important for chondrocyte metabolism 7,8. Furthermore, synovial fluid facilitates low-friction movement between cartilaginous surfaces mainly through secreting two key components, hyaluronan and lubricin 9 10. In contrast, tissue engineered cartilage is most often cultured in artificial media. While these media are likely able to provide more defined conditions for studying chondrocyte metabolism, synovial fluid most accurately reflects the natural environment of which articular chondrocytes reside in.
 Indeed, synovial fluid has the advantage of being easy to obtain and store, and can often be regularly replenished by the body. Several groups have supplemented the culture medium with synovial fluid in growing human, bovine, rabbit and dog chondrocytes, but mostly used only low levels of synovial fluid (below 20&#37;) 11-25. While chicken, horse and human chondrocytes have been cultured in the medium with higher percentage of synovial fluid, these culture systems were two-dimensional 26-28. Here we present our method of culturing human articular chondrocytes in a 3D system with a high percentage of synovial fluid (up to 100&#37;) over a period of 21 days. In doing so, we overcame a major hurdle presented by the high viscosity of the synovial fluid. This system provides the possibility of studying human chondrocytes in synovial fluid in a 3D setting, which can be further combined with two other important factors (oxygen tension and mechanical loading) 29,30 that constitute the natural environment for cartilage to mimic the natural milieu for cartilage growth. Furthermore, This system may also be used for assaying synovial fluid activity on chondrocytes and provide a platform for developing cartilage regeneration technologies and therapeutic options for arthritis.

A 3D system of culturing human articular chondrocytes in high levels of synovial fluid is described. Synovial fluid reflects the most natural microenvironment for articular cartilage, and can be easily obtained and stored. This system thus can be used for studying cartilage regeneration and for screening therapeutics for treating arthritis.

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not ...

Biology

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy this deficiency, several medical interventions have been developed. One such method is to resurface the damaged area with tissue-engineered cartilage; however, the engineered tissue typically lacks the biochemical properties and durability of native cartilage, questioning its long-term survivability. This limits the application of cartilage tissue engineering to the repair of small focal defects, relying on the surrounding tissue to protect the implanted material. To improve the properties of the developed tissue, mechanical stimulation is a popular method utilized to enhance the synthesis of cartilaginous extracellular matrix as well as the resultant mechanical properties of the engineered tissue. Mechanical stimulation applies forces to the tissue constructs analogous to those experienced in vivo. This is based on the premise that the mechanical environment, in part, regulates the development and maintenance of native tissue1,2. The most commonly applied form of mechanical stimulation in cartilage tissue engineering is dynamic compression at physiologic strains of approximately 5-20% at a frequency of 1 Hz1,3. Several studies have investigated the effects of dynamic compression and have shown it to have a positive effect on chondrocyte metabolism and biosynthesis, ultimately affecting the functional properties of the developed tissue4-8. In this paper, we illustrate the method to mechanically stimulate chondrocyte-agarose hydrogel constructs under dynamic compression and analyze changes in biosynthesis through biochemical and radioisotope assays. This method can also be readily modified to assess any potentially induced changes in cellular response as a result of mechanical stimuli.

The biosynthesis of cartilaginous extracellular matrix by chondrocytes can be affected by application of mechanical stimuli. This method describes the technique of applying dynamic compressive strains to chondrocytes encapsulated in 3D constructs and the evaluation of induced changes in chondrocyte metabolism.

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy ...

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 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 ...

Creation of a Knee Joint-on-a-Chip for Modeling Joint Diseases and Testing Drugs

The high prevalence of debilitating joint diseases like osteoarthritis (OA) poses a high socioeconomic burden. Currently, the available drugs that target joint disorders are mostly palliative. The unmet need for effective disease-modifying OA drugs (DMOADs) has been primarily caused by the absence of appropriate models for studying the disease mechanisms and testing potential DMOADs. Herein, we describe the establishment of a miniature synovial joint-mimicking microphysiological system (miniJoint) comprising adipose, fibrous, and osteochondral tissue components derived from human mesenchymal stem cells (MSCs). To obtain the three-dimensional (3D) microtissues, MSCs were encapsulated in photocrosslinkable methacrylated gelatin before or following differentiation. The cell-laden tissue constructs were then integrated into a 3D-printed bioreactor, forming the miniJoint. Separate flows of osteogenic, fibrogenic, and adipogenic media were introduced to maintain the respective tissue phenotypes. A commonly shared stream was perfused through the cartilage, synovial, and adipose tissues to enable tissue crosstalk. This flow pattern allows the induction of perturbations in one or more of the tissue components for mechanistic studies. Furthermore, potential DMOADs can be tested via either &#34;systemic administration&#34; through all the medium streams or &#34;intraarticular administration&#34; by adding the drugs to only the shared &#34;synovial fluid&#34;-simulating flow. Thus, the miniJoint can serve as a versatile in vitro platform for efficiently studying disease mechanisms and testing drugs in personalized medicine.

We provide detailed methods for generating four types of tissues from human mesenchymal stem cells, which are used to recapitulate the cartilage, bone, fat pad, and synovium in the human knee joint. These four tissues are integrated into a customized bioreactor and connected through microfluidics, thus generating a knee joint-on-a-chip.

The high prevalence of debilitating joint diseases like osteoarthritis (OA) poses a high socioeconomic burden. Currently, the available drugs that target ...

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

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