Name: a lab-on-a-chip platform for stimulating osteocyte mechanotransduction and analyzing functional outcomes of bone remodeling
Uploaded: 2020-05-21T14:30:00
Description: Watch this Scientific Journal Video about A Lab-On-A-Chip Platform for Stimulating Osteocyte Mechanotransduction and Analyzing Functional Outcomes of Bone Remodeling at JoVE.com

This work was supported by the National Science Foundation under Grant Nos. (CBET 1060990 and EBMS 1700299). Also, this material is based upon work supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. (2018250692). Any opinions, findings, conclusions, or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation.

1. Chip mask preparation
NOTE: Steps 1.1 - 1.3 only need to be performed once upon initial receipt of the chip mask. They ensure the mask does not bow during use. The design of the microfluidic masks was previously described11,14. Masks were designed in-house and commercially fabricated using high resolution stereolithography (Figure 1A).
<ol>
	<li>Cover the top surface of the mask with ...

<ol>
	<li>Hemmatian, H., Bakker, A. D., Klein-Nulend, J., van Lenthe, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging,+Osteocytes,+and+Mechanotransduction.">Aging, Osteocytes, and Mechanotransduction.</a> Current Osteoporosis Reports. 15 (5), 401-411 (2017).</li><li>Bonewald, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+amazing+osteocyte.">The amazing osteocyte.</a> Journal of Bone and Mineral Research. 26 (2), 229-238 (2011).</li><li>Middleton, K., Al-Dujaili, S., Mei, X., Gunther, A., You, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+co-culture+platform+for+investigating+osteocyte-osteoclast+signalling+during+fluid+shear+stress+mechanostimulation.">Microfluidic co-culture platform for investigating osteocyte-osteoclast signalling during fluid shear stress mechanostimulation.</a> Journal of Biomechanics. 59, 35-42 (2017).</li><li>Kou, 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=A+multishear+microfluidic+device+for+quantitative+analysis+of+calcium+dynamics+in+osteoblasts.">A multishear microfluidic device for quantitative analysis of calcium dynamics in osteoblasts.</a> Biochemical and Biophysical Research Communications. 408 (2), 350-355 (2011).</li><li>Ma, H. 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W., Horton, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variable+osteogenic+performance+of+MC3T3-E1+subclones+impacts+their+utility+as+models+of+osteoblast+biology.">Variable osteogenic performance of MC3T3-E1 subclones impacts their utility as models of osteoblast biology.</a> Scientific Reports. 9 (1), 8299 (2019).</li><li>Beier, E. E., Holz, J. D., Sheu, T. J., Puzas, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevated+lifetime+lead+exposure+impedes+osteoclast+activity+and+produces+an+increase+in+bone+mass+in+adolescent+mice.">Elevated lifetime lead exposure impedes osteoclast activity and produces an increase in bone mass in adolescent mice.</a> Toxicological Sciences. 149 (2), 277-288 (2016).</li><li>Chaudhary, L. R., Hofmeister, A. M., Hruska, K. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+considerations+for+optimizing+bone+cell+culture+and+remodeling+in+a+lab-on-a-chip+platform.">Cellular considerations for optimizing bone cell culture and remodeling in a lab-on-a-chip platform.</a> Biotechniques. , (2019).</li><li>Truesdell, S. L., George, E. L., Seno, C. E., Saunders, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+printed+loading+device+for+inducing+cellular+mechanotransduction+via+matrix+deformation.">3D printed loading device for inducing cellular mechanotransduction via matrix deformation.</a> Experimental Mechanics. 59 (8), 1223-1232 (2019).</li><li>George, E. L., Truesdell, S. L., Magyar, A. L., Saunders, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+mechanically+loaded+osteocytes+and+inflammation+on+bone+remodeling+in+a+bisphosphonate-induced+environment.">The effects of mechanically loaded osteocytes and inflammation on bone remodeling in a bisphosphonate-induced environment.</a> Bone. 127, 460-473 (2019).</li><li>George, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Quantifying the roles of stimulated osteocytes and inflammation in bone remodeling Doctor of Philosophy. , (2019).</li><li>York, S. L., Sethu, P., Saunders, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+osteocytic+microdamage+and+viability+quantification+using+a+microloading+platform.">In vitro osteocytic microdamage and viability quantification using a microloading platform.</a> Medical Engineering &amp; Physics. 38 (10), 1115-1122 (2016).</li><li>Hui, A. Y., Wang, G., Lin, B., Chan, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microwave+plasma+treatment+of+polymer+surface+for+irreversible+sealing+of+microfluidic+devices.">Microwave plasma treatment of polymer surface for irreversible sealing of microfluidic devices.</a> Lab Chip. 5 (10), 1173-1177 (2005).</li><li>Millare, B., 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=Dependence+of+the+quality+of+adhesion+between+poly(dimethylsiloxane)+and+glass+surfaces+on+the+conditions+of+treatment+with+oxygen+plasma.">Dependence of the quality of adhesion between poly(dimethylsiloxane) and glass surfaces on the conditions of treatment with oxygen plasma.</a> Langmuir. 24 (22), 13218-13224 (2008).</li><li>Lee, J. N., Park, C., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solvent+compatibility+of+poly(dimethylsiloxane)-based+microfluidic+devices.">Solvent compatibility of poly(dimethylsiloxane)-based microfluidic devices.</a> Analytical Chemistry. 75 (23), 6544-6554 (2003).</li><li>Regehr, K. 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=Biological+implications+of+polydimethylsiloxane-based+microfluidic+cell+culture.">Biological implications of polydimethylsiloxane-based microfluidic cell culture.</a> Lab Chip. 9 (15), 2132-2139 (2009).</li><li>Wang, Y., Burghardt, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uncured+PDMS+inhibits+myosin+in+vitro+motility+in+a+microfluidic+flow+cell.">Uncured PDMS inhibits myosin in vitro motility in a microfluidic flow cell.</a> Analytical Biochemistry. 563, 56-60 (2018).</li><li>Millet, L. J., Stewart, M. E., Sweedler, J. V., Nuzzo, R. G., Gillette, M. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+devices+for+culturing+primary+mammalian+neurons+at+low+densities.">Microfluidic devices for culturing primary mammalian neurons at low densities.</a> Lab Chip. 7 (8), 987-994 (2007).</li><li>Kilic, O., 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=Brain-on-a-chip+model+enables+analysis+of+human+neuronal+differentiation+and+chemotaxis.">Brain-on-a-chip model enables analysis of human neuronal differentiation and chemotaxis.</a> Lab Chip. 16 (21), 4152-4162 (2016).</li><li>Van Scoy, G. 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This article describes the foundations for fabricating a bone remodeling LOC platform for culturing osteocytes, osteoclasts, and osteoblasts. By altering the depth and size of the well within the chip, multiple configurations were developed for stimulating osteocytes with mechanical load and quantifying functional outcomes of bone remodeling (Figure 1B).
During chip assembly, optimizing the plasma oxidation protocol was critical for eliminating le...

Bone is a highly dynamic tissue that requires intricate coordination among the three major cell types: osteocytes, osteoblasts, and osteoclasts. Multicellular interactions among these cells are responsible for the bone loss that occurs during paralysis and long-term immobility and for the bone formation that occurs in response to growth and exercise. Osteocytes, the most abundant bone cell type, are highly sensitive to mechanical stimuli applied to the bone. Mechanical stimulation alters osteocyte metabolic activity and leads to an increase in key signaling molecules1,2. Through this process, known as mechanotransduction, osteocytes can directly coordinate the activities of osteoblasts (bone forming cells) and osteoclasts (bone resorbing cells). Maintaining bone homeostasis requires a tight regulation between bone formation and bone resorption rates; however, disruptions in this process can result in disease states such as osteoporosis or osteopetrosis.
The complexity of interactions between these three cell types lends itself well to investigation utilizing microfluidic and lab-on-a-chip (LOC) technologies. To that end, our lab has recently established proof of concept of a LOC platform for analyzing bone resorption and formation (functional outcomes) in the bone remodeling process. The platform can be used for the study of cellular interactions, altered loading environments, and investigational drug screening. In recent years, various microfluidic devices have been developed for investigating the molecular signaling pathways that regulate bone remodeling; however, many of these systems quantify remodeling through indirect markers that are indicative of functional activity3,4,5,6,7. An advantage of our system is that it can be used for direct quantification of functional outcomes. Bone remodeling is a long-term process. As such, direct quantification of bone resorption and formation requires a culturing system that can be maintained for a minimum of several weeks to months8,9,10,11. Thus, when developing the LOC platform, we established long-term culturing protocols necessary for formation and resorption and have maintained cells within the system for up to seven weeks11. Additionally, we incorporated appropriate culturing substrates for both cell types into the platform; osteoclasts were cultured directly on bone, and osteoblasts, which are known to be plastic adherent, were cultured on polystyrene discs. Further, we addressed issues concerning sterility, long-term cytotoxicity and chip disassembly for remodeling analysis11,12.
The LOC platform can also be used to induce osteocyte mechanotransduction through matrix deformation. A 3D printed mechanical loading device was developed to pair with the LOC and apply a static out of plane distention to stretch the cells13. To accommodate this mechanical load, the depth of the well within the LOC was increased. This small scale, simple mechanical loading device can be easily produced by labs with limited engineering experience, and we have previously shared drawings of the 3D printed components13. In the current work, we demonstrate some of the novel techniques necessary for the successful use of the LOC. Specifically, we demonstrate chip fabrication, cell seeding on functional substrates, mechanical loading and chip disassembly for remodeling quantification. We believe that the explanation of these techniques benefit from a visual format.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acrylic sheet</td>
			<td>Optix</td>
			<td>--</td>
			<td>3.175 mm thick</td>
		</tr>
		<tr>
			<td>Angled dispensing tips</td>
			<td>Jensen Global</td>
			<td>JG18-0.5X-90</td>
			<td>Remove plastic connector prior to use</td>
		</tr>
		<tr>
			<td>Biopsy punch</td>
			<td>Robbins Instruments</td>
			<td>RBP-10</td>
			<td>1 mm diameter</td>
		</tr>
		<tr>
			<td>Bone wafers</td>
			<td>Boneslices.com</td>
			<td>0.4 mm thick</td>
			<td>Bovine cortical bone</td>
		</tr>
		<tr>
			<td>Bovine calf serum</td>
			<td>Hyclone</td>
			<td>SH30072</td>
			<td></td>
		</tr>
		<tr>
			<td>Calipers</td>
			<td>Global Industrial</td>
			<td>T9F534164</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell spatula</td>
			<td>TPP</td>
			<td>99010</td>
			<td></td>
		</tr>
		<tr>
			<td>Chip mask</td>
			<td>ProtoLabs</td>
			<td>Custom-designed</td>
			<td>Print material: Accura SL 5530</td>
		</tr>
		<tr>
			<td>Cork borer</td>
			<td>Fisher Scientific</td>
			<td>07-865-10B</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton tipped applicator</td>
			<td>Puritan</td>
			<td>806-WCL</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture dish (100 mm)</td>
			<td>Corning</td>
			<td>430591</td>
			<td>Sterile, Non-tissue culture treated</td>
		</tr>
		<tr>
			<td>Culture dish (150 mm)</td>
			<td>Corning</td>
			<td>430597</td>
			<td>Sterile, Non-tissue culture treated</td>
		</tr>
		<tr>
			<td>Double sided tape</td>
			<td>3M Company</td>
			<td>Scotch 237</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Hyclone</td>
			<td>SH30910</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fisher Scientific</td>
			<td>22-327379</td>
			<td></td>
		</tr>
		<tr>
			<td>Leveling box</td>
			<td>Custom-made</td>
			<td>--</td>
			<td>3D printed</td>
		</tr>
		<tr>
			<td>Masking tape</td>
			<td>3M Company</td>
			<td>Scoth 2600</td>
			<td></td>
		</tr>
		<tr>
			<td>MC3T3-E1 preosteoblasts</td>
			<td>ATCC</td>
			<td>CRL-2593</td>
			<td>Subclone 4</td>
		</tr>
		<tr>
			<td>Mechanical loading device</td>
			<td>Custom-made</td>
			<td>--</td>
			<td>3D printed</td>
		</tr>
		<tr>
			<td>Minimum essential alpha medium</td>
			<td>Gibco</td>
			<td>12571-063</td>
			<td></td>
		</tr>
		<tr>
			<td>MLO-Y4 osteocytes</td>
			<td>--</td>
			<td>--</td>
			<td>Gift from Dr. Lynda Bonewald</td>
		</tr>
		<tr>
			<td>Packaging tape</td>
			<td>Duck Brand</td>
			<td>--</td>
			<td>Standard packaging tape</td>
		</tr>
		<tr>
			<td>Paraffin film</td>
			<td>Bemis Parafilm</td>
			<td>PM999</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Invitrogen</td>
			<td>p4333</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-001</td>
			<td>Expanded plasma cleaner</td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane kit</td>
			<td>Dow Corning</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene coverslips</td>
			<td>Nunc Thermanox</td>
			<td>174942</td>
			<td>Sterile, tissue culture treated</td>
		</tr>
		<tr>
			<td>Oven</td>
			<td>Quincy Lab</td>
			<td>12-180</td>
			<td></td>
		</tr>
		<tr>
			<td>RAW264.7 preosteoclasts</td>
			<td>ATCC</td>
			<td>TIB-71</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>BD Medical</td>
			<td>372611</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone tubing</td>
			<td>Saint-Gobain Tygon</td>
			<td>ABW00001</td>
			<td>ID: 1/32&#34; (0.79 mm), OD: 3/32&#34; (2.38 mm)</td>
		</tr>
		<tr>
			<td>SolidWorks software</td>
			<td>Dassault Syst&#232;mes</td>
			<td>--</td>
			<td>Used to generate 3D printed models and perform FEA</td>
		</tr>
		<tr>
			<td>Spray adhesive</td>
			<td>Loctite</td>
			<td>2323879</td>
			<td>Multi-purpose adhesive</td>
		</tr>
		<tr>
			<td>Syringe (5 ml)</td>
			<td>BD Medical</td>
			<td>309646</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Harvard Apparatus</td>
			<td>70-2213</td>
			<td>Pump 11 Pico Plus</td>
		</tr>
		<tr>
			<td>Tapered laboratory spatula</td>
			<td>Fisher Scientific</td>
			<td>21-401-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Two-part expoxy</td>
			<td>Loctite</td>
			<td>1395391</td>
			<td>5 minute quick set</td>
		</tr>
		<tr>
			<td>Type I collagen</td>
			<td>Corning</td>
			<td>354236</td>
			<td>Rat tail collagen</td>
		</tr>
		<tr>
			<td>Vacuum desiccator</td>
			<td>Bel-Art</td>
			<td>F42010-0000</td>
			<td></td>
		</tr>
		<tr>
			<td>Waterproof sealant</td>
			<td>Gorilla</td>
			<td>8090001</td>
			<td>100% silicone sealant</td>
		</tr>
	</tbody>
</table>

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.

The shallow-well configuration can be used for analyzing functional activity of osteoblasts and osteoclasts. Bone formation via osteoblasts and resorption via osteoclasts requires culturing times on the order of several weeks to months. Bone formation from MC3T3-E1 pre-osteoblasts was quantified using alizarin red and von Kossa stains11,15. At day 49, the average surface area stained with alizarin red was 10.7% &#177; 2.2% (mean &...

Watch this Scientific Journal Video about A Lab-On-A-Chip Platform for Stimulating Osteocyte Mechanotransduction and Analyzing Functional Outcomes of Bone Remodeling at JoVE.com

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

Here we present protocols for culturing bone cells within an adaptable lab-on-a-chip platform. These techniques can be used to further our understanding of the mechanisms that regulate mechanically-induced bone remodeling. Our system allows for long-term culturing of bone cells, which enables direct quantification of bone formation and resorption in response to altered mechanical loading environments.Use of these foundational techniques can lead to discoveries that extend to a number of bone-related issues such as osteoporosis, fracture healing, and periprosthetic osteolysis. To create the well and microchannel layer, combine the PDMS prepolymer and curing agent at a ratio of 10 to one in a plastic cup and mix vigorously, then degas the polymer for 30 minutes. Slowly pour the mixture over the appropriate pre-leveled mask.Let it sit for 30 minutes and bake it at 45 degrees Celsius for 18 hours. Loosen the edges of the PDMS with a tapered laboratory spatula and carefully peel it away from the mask, then cut out individual chips with a scalpel and a 3D-printed template and surface clean them with packaging tape. To make the PDMS membrane layer, repeat the previous steps for mixing, pouring, and baking the PDMS.Remove the PDMS sheet from the leveling box and cut individual membranes that match the dimensions of the well and microchannel layer. Use calipers to measure the membrane thickness at the center and discard any membranes that are outside of the desired thickness, then carefully clean the membranes with packaging tape and place them on a piece of paraffin film. To make the PDMS lid layer, repeat the previous steps for mixing, pouring, and baking the PDMS.After cutting individual lids to size, punch access holes through the PDMS with a one millimeter biopsy punch and then surface clean them with packaging tape. Activate the surfaces of layers one and two with a plasma cleaner for 30 seconds on a medium radio frequency power setting, then align the access holes in layer one with the microchannels in layer two and firmly press the two layers together. For the deep-well design, repeat this process with layers two and three using double-sided tape to attach the paraffin film of layer three to a flat surface during plasma treatment.Trim off excess material from the PDMS membrane and carefully peel away the sheet of paraffin film from the bottom of the chip. Bake the chip at 65 degrees Celsius for 10 minutes to increase bond strength between the PDMS layers, then insert angle dispensing tips into the access holes in the lid and secure them with a two-part epoxy using a micropipette tip to apply the epoxy around each tip. After the epoxy has fully cured, surface clean the chip with 70%ethanol and transfer it to a biosafety cabinet.Connect a five milliliter syringe to the dispensing tips with sterile silicone tubing and fill the entire chip with 70%ethanol for at least 30 seconds. Remove the ethanol from the chip and sterilize it with UV light overnight. On the next day, wash the chip three times with distilled water, fill it with water, and remove the tubing from the dispensing tips.Incubate the chip for at least 48 hours at 37 degrees Celsius. Place a compression spring around the shaft of the platen and insert it into the central hole on the bottom of the base, then attach the dial block to the bottom of the base with four self-tapping screws. Place a second compression spring around the central screw.Insert the screw into the hexagonal-shaped hole in the bottom of the dial and screw the assembly into the threaded hole in the center of the dial block. Screw four male-female standoffs into the bottom of the base. Remove slack from the device by turning the dial counterclockwise until the top of the platen is below the top of the base, then slowly turn the dial clockwise until the top of the platen is level with the top of the base.Use a five milliliter syringe to remove the water from the deep-well chip and coat the bottom of the well with 200 microliters of type I collagen in acetic acid for an hour. Rinse the chip three times using DPBS with calcium and magnesium and place it into the chip holder. Seed the chip with MLO-Y4 osteocytes in MEM alpha medium supplemented with calf serum, FBS, and penicillin/streptomycin.Remove the tubing from the dispensing tips, place the chip into a deep-well culture dish, and incubate the cells at 37 degrees Celsius and five percent carbon dioxide for 72 hours. After the incubation, attach sterile tubing to dispensing tips and use a five milliliter syringe to remove the spent culture medium from the chip, then slowly refill the chip with fresh medium. Place the chip holder into the rectangular inset on the top of the loading device base, feed the tubing through the slots located on the lid, and secure the lid to the base.Apply load to the cells by turning the dial clockwise until the desired platen displacement is reached, then place the loading device into an empty P1000 micropipette tip box and incubate the cells with the load for 15 minutes. After the incubation, remove the load from the cells by turning the dial counterclockwise to the original starting position, then remove the lid from the device, place the chip holder into the deep-well culture plate, and incubate the cells for a 90-minute post load recovery period. Use a five milliliter syringe to remove the conditioned medium from the chip and remove the chip from the chip holder, then use a tapered spatula to break the bond between the PDMS lid and well layer.The cells are now ready to be analyzed. The shallow-well configuration can be used for analyzing functional activity of osteoblasts and osteoclasts. Bone formation from preosteoblasts was quantified using alizarin red and von Kossa stains.Bone resorption from osteoclasts was quantified using toluidine blue staining and scanning electron microscopy was used to verify the presence of resorption pits. The deep-well chip configuration was used to induce osteocyte mechanotransduction by stretching the cells via a static out of plane distension. Images of osteocyte seeded in chips demonstrate that the 48-hour incubation of the chip with distilled water is critical for maintaining cell viability in typical morphology.During bouts of loading, osteocytes were exposed to a strain gradient induced on the PDMS membrane. The relationship between average equivalent strain and platen displacement for values between one and two millimeters was determined and a representative heat map of the induced strain was generated. Following loading, cell viability was analyzed with lactate dehydrogenase staining and the annexin V and dead cell assay.Lactate dehydrogenase stain of stretched osteocytes showed a lighter staining near the outer edge of the well, which corresponds to the location of higher strain values. Our platform provides a high degree of versatility and can be used to investigate a variety of factors that regulate bone remodeling such as load-induced mechanotransduction, inflammation, and drug effects. The techniques presented here provide a foundation for developing a true bone organ on a chip that could greatly enhance our understanding of the multicellular intricacies that regulate bone health.

a-lab-on-chip-platform-for-stimulating-osteocyte-mechanotransduction

Research

JoVE Journal

Bioengineering

Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata are separated from their axon terminals due to skeletal growth of the enlarging organism (Weiss 1941; Gray, Hukkanen et al. 1992). This mechanotransduction induces a secondary mode of neuronal growth capable of accommodating continual elongation of the axon (Bray 1984; Heidemann and Buxbaum 1994; Heidemann, Lamoureux et al. 1995; Pfister, Iwata et al. 2004).
Axon Stretch Growth (ASG) is conceivably a central factor in the maturation of short embryonic processes into the long nerves and white matter tracts characteristic of the adult nervous system. To study ASG in vitro, we engineered bioreactors to apply tension to the short axonal processes of neuronal cultures (Loverde, Ozoka et al. 2011). Here, we detail the methods we use to prepare bioreactors and conduct ASG. First, within each stretching lane of the bioreactor, neurons are plated upon a micro-manipulated towing substrate. Next, neurons regenerate their axonal processes, via growth cone extension, onto a stationary substrate. Finally, stretch growth is performed by towing the plated cell bodies away from the axon terminals adhered to the stationary substrate; recapitulating skeletal growth after growth cone extension.
Previous work has shown that ASG of embryonic rat dorsal root ganglia neurons are capable of unprecedented growth rates up to 10mm/day, reaching lengths of up to 10cm; while concurrently resulting in increased axonal diameters (Smith, Wolf et al. 2001; Pfister, Iwata et al. 2004; Pfister, Bonislawski et al. 2006; Pfister, Iwata et al. 2006; Smith 2009). This is in dramatic contrast to regenerative growth cone extension (in absence of mechanical stimuli) where growth rates average 1mm/day with successful regeneration limited to lengths of less than 3cm (Fu and Gordon 1997; Pfister, Gordon et al. 2011). Accordingly, further study of ASG may help to reveal dysregulated growth mechanisms that limit regeneration in the absence of mechanical stimuli.

A unique tissue engineering method was developed to elongate numerous nerve fibers in culture by recapitulating axon stretch growth; a form of nervous system growth whereby nerves elongate in conjunction with growth of the enlarging body.

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata ...

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, each offering its own advantages and shortcomings5,6. Most prominently, due to the microscale device dimensions, a high modulus is required to facilitate implantation into living tissue. Conversely, the stiffness of the device should match the surrounding tissue to minimize induced local strain7-9. Therefore, we recently developed a new class of bio-inspired materials to meet these requirements by responding to environmental stimuli with a change in mechanical properties10-14. Specifically, our poly(vinyl acetate)-based nanocomposite (PVAc-NC) displays a reduction in stiffness when exposed to water and elevated temperatures (e.g. body temperature). Unfortunately, few methods exist to quantify the stiffness of materials in vivo15, and mechanical testing outside of the physiological environment often requires large samples inappropriate for implantation. Further, stimuli-responsive materials may quickly recover their initial stiffness after explantation. Therefore, we have developed a method by which the mechanical properties of implanted microsamples can be measured ex vivo, with simulated physiological conditions maintained using moisture and temperature control13,16,17. 
To this end, a custom microtensile tester was designed to accommodate microscale samples13,17 with widely-varying Young's moduli (range of 10 MPa to 5 GPa). As our interests are in the application of PVAc-NC as a biologically-adaptable neural probe substrate, a tool capable of mechanical characterization of samples at the microscale was necessary. This tool was adapted to provide humidity and temperature control, which minimized sample drying and cooling17. As a result, the mechanical characteristics of the explanted sample closely reflect those of the sample just prior to explantation.
The overall goal of this method is to quantitatively assess the in vivo mechanical properties, specifically the Young's modulus, of stimuli-responsive, mechanically-adaptive polymer-based materials. This is accomplished by first establishing the environmental conditions that will minimize a change in sample mechanical properties after explantation without contributing to a reduction in stiffness independent of that resulting from implantation. Samples are then prepared for implantation, handling, and testing (Figure 1A). Each sample is implanted into the cerebral cortex of rats, which is represented here as an explanted rat brain, for a specified duration (Figure 1B). At this point, the sample is explanted and immediately loaded into the microtensile tester, and then subjected to tensile testing (Figure 1C). Subsequent data analysis provides insight into the mechanical behavior of these innovative materials in the environment of the cerebral cortex.

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

A method is discussed by which the in vivo mechanical behavior of stimuli-responsive materials is monitored as a function of time. Samples are tested ex vivo using a microtensile tester with environmental controls to simulate the physiological environment. This work further promotes understanding the in vivo behavior of our material.

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, ...

Preparation of Hydroxy-PAAm Hydrogels for Decoupling the Effects of Mechanotransduction Cues

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their extracellular matrix (ECM) environment. Eukaryotic cells constantly sense their local microenvironment through surface mechanosensors to transduce physical changes of ECM into biochemical signals, and integrate these signals to achieve specific changes in gene expression. Interestingly, physicochemical and mechanical parameters of the ECM can couple with each other to regulate cell fate. Therefore, a key to understanding mechanotransduction is to decouple the relative contribution of ECM cues on cellular functions.
Here we present a detailed experimental protocol to rapidly and easily generate biologically relevant hydrogels for the independent tuning of mechanotransduction cues in vitro. We chemically modified polyacrylamide hydrogels (PAAm) to surmount their intrinsically non-adhesive properties by incorporating hydroxyl-functionalized acrylamide monomers during the polymerization. We obtained a novel PAAm hydrogel, called hydroxy-PAAm, which permits immobilization of any desired nature of ECM proteins. The combination of hydroxy-PAAm hydrogels with microcontact printing allows to independently control the morphology of single-cells, the matrix stiffness, the nature and the density of ECM proteins. We provide a simple and rapid method that can be set up in every biology lab to study in vitro cell mechanotransduction processes. We validate this novel two-dimensional platform by conducting experiments on endothelial cells that demonstrate a mechanical coupling between ECM stiffness and the nucleus.

We present a new polyacrylamide hydrogel, called hydroxy-PAAm, that allows a direct binding of ECM proteins with minimal cost or expertise. The combination of hydroxy-PAAm hydrogels with microcontact printing facilitates independent control of many cues of the natural cell microenvironment for studying cellular mechanostransduction.

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their ...

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

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 ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

Rodent Behavioral Testing to Assess Functional Deficits Caused by Microelectrode Implantation in the Rat Motor Cortex

Medical devices implanted in the brain hold tremendous potential. As part of a Brain Machine Interface (BMI) system, intracortical microelectrodes demonstrate the ability to record action potentials from individual or small groups of neurons. Such recorded signals have successfully been used to allow patients to interface with or control&#160;computers, robotic limbs, and their own limbs. However, previous animal studies have shown that a microelectrode implantation in the brain not only damages the surrounding tissue but can also result in functional deficits. Here, we discuss a series of behavioral tests to quantify potential motor impairments following the implantation of intracortical microelectrodes into the motor cortex of a rat. The methods for open field grid, ladder crossing, and grip strength testing provide valuable information regarding the potential complications resulting from a microelectrode implantation. The results of the behavioral testing are correlated with endpoint histology, providing additional information on the pathological outcomes and impacts of this procedure on the adjacent tissue.

We have shown that a microelectrode implantation in the motor cortex of rats causes immediate and lasting motor deficits. The methods proposed herein outline a microelectrode implantation surgery and three rodent behavioral tasks to elucidate potential changes in the fine or gross motor function due to implantation-caused damage to the motor cortex.

Medical devices implanted in the brain hold tremendous potential. As part of a Brain Machine Interface (BMI) system, intracortical microelectrodes ...

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 ...