This work was supported by the Belgian National Foundation for Scientific Research (F.R.S.-FNRS) through &#8220;MIS Confocal Microscopy&#8221;, &#8220;Cr&#233;dit aux Chercheurs&#8221; grants and the &#8220;Nanomotility FRFC project&#8221; (no. 2.4622.11). T.G. doctoral fellowship is supported by the Foundation for Training in Industrial and Agricultural Research (FRIA). The authors gratefully acknowledge Sylvain Desprez for mechanical characterization and G&#233;raldine Circelli for confocal imaging.

1. Activating the Surface of Glass Coverslips
<ol>
	<li>Place glass circular coverslips (25 mm diameter) in a Petri dish and smear 0.1 M NaOH solution on it for 5 min&#160;(chemical fume hood recommended).</li>
	<li>Remove the NaOH solution and fully immerse coverslips with sterile ddH2O for 20 min&#160;while gently rocking on a rocking plate in a sterile culture hood.</li>
	<li>Drain sterile ddH2O and repeat the step 1.2.</li>
	<li>Remove the coverslips with sterile tweezers and place them in a new Petri dish with the activated face up.</li>
	<li>Dry coverslips under a steady flow of high-purity nitrogen gas.</li>
	<li>In a sterile culture hood, smear a thin layer of 3-(trimethoxysilyl)propyl acrylate (92%) on the activated side of the coverslip for 1&#160;hr.</li>
	<li>Extensively wash glass coverslips with 3 washes of sterile ddH2O and immerse them in sterile ddH2O in a new Petri dish.</li>
	<li>Tap the Petri dish with Parafilm and place it on a rocker plate under gentle agitation for 10 min.</li>
	<li>Remove the coverslips from ddH2O with sterile tweezers with fine tips and place them in a new Petri dish with the activated face up.</li>
	<li>Store at RT in a dry place with aluminum foil to avoid dust from sticking to the coverslips.</li>
</ol>
2. Preparation of Hydroxy-PAAm Hydrogels
<ol>
	<li>Prepare a weight of 65 mg of N-hydroxyethyl acrylamide (HEA) in a 1.5 ml&#160;Eppendorf tube. It is important to prepare a fresh HEA solution.</li>
	<li>Add 1 ml&#160;of 50 mM HEPES buffer to HEA and mix using a vortexer until the complete HEA dissolution.</li>
	<li>Add 400 &#181;l&#160;of 40% w/w in HEPES acrylamide solution and the required volume of 2% w/w in HEPES bis-acrylamide solution (see Table 1) to reach the desired hydrogel stiffness. Adjust with 50 mM HEPES to a final volume of 5 ml.</li>
	<li>Mix the solution using a vortexer and degas it in a vacuum chamber for 20 min&#160;in order to reduce oxygen concentration within the solution, which prevents hydroxy-PAAm polymerization.</li>
	<li>Under a sterile hood, filter the degased solution with a 0.2 &#181;m pore size filter in order to sterilize it.</li>
	<li>Activate circular glass coverslips (22 mm diameter) in a UV/ozone cleaner during 7 min.</li>
	<li>Prepare 100 &#181;l&#160;of 10% ammonium persulfate (APS) solution, that is 10 mg APS in 100 &#181;l&#160;ddH2O. It is important to prepare a fresh APS solution.</li>
	<li>Add 2.5 &#181;l&#160;of tetramethylenediamine (TEMED) and 25 &#181;l&#160;of APS solution to the sterilized hydroxy-PAAm solution (step 2.5) to initiate the polymerization. Mix the solution by 3 successive pipettings without introduction of bubbles, under sterile conditions.</li>
	<li>Under a sterile hood, place a 25 &#181;l&#160;drop of the hydroxy-PAAm solution on a 25 mm coverslip (available from step 1.9) and immediately place a 22 mm glass coverslip (prepared on step 2.5) on top of the droplet to squeeze the hydroxy-PAAm solution. Center the 22 mm glass coverslip with sterile tweezers and smooth out any bubbles.</li>
	<li>Allow hydroxy-PAAm hydrogels to polymerize at RT for 15 min. Invert manually the remaining hydroxy-PAAm solution in the Eppendorf tube to follow the completion of the polymerization process.</li>
	<li>Fully immerse coverslips with sterile ddH2O and carefully separate the 22 mm glass coverslips by introducing the edge of a razor blade between the 22 mm glass coverslips and the hydroxy-PAAm hydrogel layer.</li>
	<li>Wash hydroxy-PAAm hydrogels with sterile PBS (3 exchanges of PBS) and let the gels fully immersed in sterile PBS to maintain hydration.</li>
	<li>Store hydroxy-PAAm hydrogels in sterile PBS at 4 &#176;C for up to 3 days.</li>
</ol>
3. Polydimethylsiloxane (PDMS) Microstamp Fabrication
NOTE: The fabrication of a silicon master is required prior to start the PDMS microstamp fabrication. This microfabrication of a silicon master can be done by lithographic techniques, which requires specialized equipment and training. Collaborations with a nanofabrication facility are encouraged to fabricate the silicon master. Alternatively, contact a company that fabricates custom-made microstructured silicon masters on demand. It is important to note that the fabrication of the silicon master needs only to be done once. Indeed, microstructured silicon masters can be used indefinitely to produce elastomeric stamps.
<ol>
	<li>Mix PDMS and the curing agent in a 10:1 ratio in a plastic beaker and mix thoroughly using a pipette for 10 min.</li>
	<li>Degas the PDMS mixture under vacuum to remove air bubbles that were formed during the step 3.1.</li>
	<li>Place the microstructured silicon master in a Petri dish and cast a 10-mm thick layer of degassed PDMS mixture on it without forming bubbles.</li>
	<li>Let cure the PDMS for 2 hr at 60 &#176;C in an oven.</li>
	<li>In a dust-free environment, peel-off the PDMS layer and excise 1 cm2 microstamps with a scalpel.</li>
	<li>Using forceps, place PDMS microstamps pattern-up in a Petri dish.</li>
</ol>
4. Micropatterning Hydroxy-PAAm Hydrogels
<ol>
	<li>Place PDMS microstamps in an ethanol/water (50/50) solution and sonicate for 15 min.</li>
	<li>Dry the stamps with a stream of nitrogen flow and place them pattern-up in a UV/Ozone cleaner (&#955; &#60; 200 nm) for 7 min.</li>
	<li>Under a sterile hood, place a 150-&#181;l&#160;drop of a desired protein solution (e.g., 100 &#181;g/ml&#160;laminin in PBS or 25 &#181;g/ml&#160;fibronectin in PBS) onto the microstructured surface of a 1-cm2 PDMS stamp.</li>
	<li>Optional: Modify the concentration of the protein solution to modulate the cell-ligand density.</li>
	<li>Spread the protein solution across the stamp surface by moving it with a sterile tip of a pipette toward each corner of the stamp.</li>
	<li>Leave the protein solution to adsorb on the PDMS stamp for 60 min&#160;under a sterile hood. Turn off lamps to avoid protein damage.</li>
	<li>Under a sterile hood, transfer hydroxy-PAAm coated coverslips (available from step 2.13) into a Petri dish.</li>
	<li>Remove excess PBS from the surface of hydroxy-PAAm substrates with a low nitrogen stream under sterile conditions. Stop the procedure as soon as no evidence of standing water on the gel surface is observed. The gel should not be dried thoroughly at this stage.</li>
	<li>Dry carefully the structured surface of the PDMS stamp with a steady flow of high-purity nitrogen gas.</li>
	<li>Grasp the protein-coated stamp with dressing tissue forceps and place the structured surface in contact with the dried hydrogel surface. Apply brief pressure points with the tip of tweezers on the top of the PDMS stamp to ensure a good contact between stamp microfeatures and the hydrogel surface.</li>
	<li>Leave the PDMS stamp on the hydrogel surface for 1 hr at RT.</li>
	<li>Gently remove PDMS stamps from hydroxy-PAAm hydrogels with dressing tissue forceps and follow the step 4.1 to clean the stamp.</li>
	<li>Wash extensively the stamped hydroxy-PAAm hydrogels by 3 exchanges of PBS (pH = 7.4) in sterile conditions for 10 min&#160;per exchange.</li>
	<li>Optional: Additional micropatterns of other ECM proteins can be added to the hydroxy-PAAm surface by following steps 4.5 to 4.11.</li>
	<li>Passivate non-printed zones with a sterile solution of BSA at 5 mg/ml&#160;in PBS during one night at 4 &#176;C under a gentle agitation on a rocking plate.</li>
	<li>Wash extensively by 3 exchanges of PBS (pH = 7.4) in sterile conditions for 10 min&#160;per exchange. At this stage, stamped hydroxy-PAAm hydrogels can be stored at 4 &#176;C up to one week.</li>
</ol>
5. Cell Deposition on Micropatterned hydroxy-PAAm Hydrogels
<ol>
	<li>Incubate coverslips in cell media for 30-45 min prior to plating cells.</li>
	<li>Wash adherent cells cultured in a 75 cm2 culture flask with sterile PBS at 37 &#176;C and detach it with 3 ml&#160;of trypsin-EDTA or accutase for 10 min.</li>
	<li>Transfer the desired amount of pre-warmed complete growth medium appropriate for your cell line into the flask containing the detached cells and centrifuge the cell suspension for 3 min&#160;at 650 x g.</li>
	<li>Remove the supernatant with a micropipette and resuspend the cells in complete culture medium at 15-20,000 cells/ml.</li>
	<li>Add 4 ml&#160;of the cell solution to a micropatterned coverslip (obtained from Step 5.1) and place the cell-covered coverslip in a culture incubator at 37 &#176;C and 5% humidity for 1-2 hr.</li>
	<li>Aspirate gently unattached cells and replace culture medium. Return the attached cells to the incubator and let them spread fully (3-6 hr, depending on the cell type).</li>
</ol>

<ol>
	<li>Grevesse, T., Versaevel, M., Circelli, G., Desprez, S., Gabriele, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+route+to+functionalize+polyacrylamide+gels+for+the+independent+tuning+of+mechanotransduction+cues.">A simple route to functionalize polyacrylamide gels for the independent tuning of mechanotransduction cues.</a> Lab Chip. 13 (5), 777-780 (2013).</li><li>Gabriele, S., Benoliel, A. M., Bongrand, P., Th&#233;odoly, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+investigation+reveals+distinct+roles+for+actin+cytoskeleton+and+myosin+II+activity+in+capillary+leukocyte+trafficking.">Microfluidic investigation reveals distinct roles for actin cytoskeleton and myosin II activity in capillary leukocyte trafficking.</a> Biophys. J. 96 (10), 4308-4318 (2009).</li><li>Atmanli, A., Domian, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+aligned+functional+myocardial+tissue+through+microcontact+printing.">Generation of aligned functional myocardial tissue through microcontact printing.</a> J. Vis. Exp. (73), e50288 (2013).</li><li>Gabriele, S., Versaevel, M., Preira, P., Th&#233;odoly, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+microfluidic+method+to+select,+isolate,+and+manipulate+single-cells+in+mechanical+and+biochemical+assays.">A simple microfluidic method to select, isolate, and manipulate single-cells in mechanical and biochemical assays.</a> Lab Chip. 10 (11), 1459-1467 (2010).</li><li>Le Berre, M., Aubertin, J., Piel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+control+of+nuclear+confinement+identifies+a+threshold+deformation+leading+to+lamina+rupture+and+induction+of+specific+genes.">Fine control of nuclear confinement identifies a threshold deformation leading to lamina rupture and induction of specific genes.</a> Integr. Biol. 4 (11), 1406-1414 (2012).</li><li>Rundqvist, 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=High+fidelity+functional+patterns+of+an+extracellular+matrix+protein+by+electron+beam-based+inactivation.">High fidelity functional patterns of an extracellular matrix protein by electron beam-based inactivation.</a> J. Am. Chem. Soc. 129 (59), 59-67 (2006).</li><li>Sorribas, H., Padeste, C., Tiefenauer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photolithographic+generation+of+proteins+micropatterns+for+neuron+culture+applications.">Photolithographic generation of proteins micropatterns for neuron culture applications.</a> Biomaterials. 23 (3), 893-900 (2002).</li><li>Holden, M. A., Cremer, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+activated+patterning+of+dye-labeled+molecules+on+surfaces.">Light activated patterning of dye-labeled molecules on surfaces.</a> J. Am. Chem. Soc. 125 (27), 8074-8075 (2003).</li><li>Cheng, Q., Li, S., Komvopoulos, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma-assisted+surface+chemical+patterning+for+single-cell+culture.">Plasma-assisted surface chemical patterning for single-cell culture.</a> Biomaterials. 30 (25), 4203-4210 (2009).</li><li>Kumar, A., 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=Features+of+gold+having+micrometer+to+centimeter+dimensions+can+be+formed+through+a+combination+of+stamping+with+an+elastomeric+stamp+and+an+alkanethiol+"ink"+followed+by+chemical+etching.">Features of gold having micrometer to centimeter dimensions can be formed through a combination of stamping with an elastomeric stamp and an alkanethiol "ink" followed by chemical etching.</a> Appl. Phys. Lett. 63 (14), 2002-2004 (1993).</li><li>Tseng, Q., 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=New+micropatterning+method+of+soft+substrates+reveals+that+different+tumorigenic+signals+can+promote+or+reduce+cell+contraction+levels.">New micropatterning method of soft substrates reveals that different tumorigenic signals can promote or reduce cell contraction levels.</a> Lab Chip. 11, 2231-2240 (2011).</li><li>Palchesko, R. N., Zhang, L., Sun, Y., Feinberg, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+Polydimethylsiloxane+Substrates+with+Tunable+Elastic+Modulus+to+Study+Cell+Mechanobiology+in+Muscle+and+Nerve.">Development of Polydimethylsiloxane Substrates with Tunable Elastic Modulus to Study Cell Mechanobiology in Muscle and Nerve.</a> PLoS ONE. 7, e51499 (2012).</li><li>Herrick, W. G., Nguyen, T. V., Sleiman, M., McRae, S., Emrick, T. S., Peyton, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PEG-Phosphorylcholine+hydrogels+as+tunable+and+versatile+platforms+for+mechanobiology.">PEG-Phosphorylcholine hydrogels as tunable and versatile platforms for mechanobiology.</a> Biomacromolecules. 14, 2294-2304 (2013).</li><li>Versaevel, M., Grevesse, T., Riaz, M., Lantoine, J., Gabriele, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropatterning+hydroxy-PAAm+hydrogels+and+sylgard+184+silicone+elastomers+with+tunable+elastic+moduli.">Micropatterning hydroxy-PAAm hydrogels and sylgard 184 silicone elastomers with tunable elastic moduli.</a> Methods in Cell Biology. 121, 33-48 (2014).</li><li>Kandow, C. E., Georges, P. C., Janmey, P. A., Beningo, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyacrylamide+hydrogels+for+cell+mechanics:+steps+towards+optimization+and+alternative+uses.">Polyacrylamide hydrogels for cell mechanics: steps towards optimization and alternative uses.</a> Methods Cell Biol. 83, 29-46 (2007).</li><li>Hemphill, M. A., 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+possible+role+for+integrin+signaling+in+diffuse+axonal+injury.">A possible role for integrin signaling in diffuse axonal injury.</a> PLoS ONE. 6 (7), e22899 (2011).</li><li>Damljanovic, V., Lagerholm, B. C., Jacobson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bulk+and+micropatterned+conjugation+of+extracellular+matrix+proteins+to+characterized+polyacrylamide+substrates+for+cell+mechanotransduction+assays.">Bulk and micropatterned conjugation of extracellular matrix proteins to characterized polyacrylamide substrates for cell mechanotransduction assays.</a> BioTechniques. 39 (6), 847-851 (2005).</li><li>Hynd, M. R., Frampton, J. P., Dowell-Mesfin, N., Turner, J. N., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+cell+growth+on+protein-functionalized+hydrogel+surfaces.">Directed cell growth on protein-functionalized hydrogel surfaces.</a> Journal of Neuroscience Methods. 162, 255-263 (2007).</li><li>Tseng, Q., 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=new+micropatterning+method+of+soft+substrates+reveals+that+different+tumorigenic+signals+can+promote+or+reduce+cell+contraction+levels.">new micropatterning method of soft substrates reveals that different tumorigenic signals can promote or reduce cell contraction levels.</a> Lab Chip. 11 (13), 2231-2240 (2011).</li><li>Versaevel, M., Grevesse, T., Gabriele, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+coordination+between+cell+and+nuclear+shape+within+micropatterned+endothelial+cells.">Spatial coordination between cell and nuclear shape within micropatterned endothelial cells.</a> Nat. Commun. 3, 671 (2012).</li><li>Versaevel, M., Grevesse, T., Riaz, M., Gabriele, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Confinement:+Putting+the+squeeze+on+the+nucleus.">Cell Confinement: Putting the squeeze on the nucleus.</a> Soft Matter. 9, 6665-6676 (2013).</li><li>Trappman, B., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+cells+sense+extracellular+matrix+stiffness:+a+material's+perspective.">How cells sense extracellular matrix stiffness: a material's perspective.</a> Current Opinion in Biotechnology. 24, 1-6 (2013).</li></ol>

No conflicts of interest declared.

Many in vitro observations in modern cell biology have been performed on rigid glass coverslips, often coated with a thin layer of ECM proteins or synthetic peptides containing the RGD sequence. However, such basic culture substrates do not recapitulate the whole physicochemical complexity of the ECM and thus do not provide an accurate model for studying cellular mechanotransduction processes. To tackle this problem, we propose a simple alternative to functionalize two-dimensional hydrogels with any desired amount and nature of ECM proteins. This method allows independent control of important mechanotransduction cues, such as matrix stiffness, cellular morphology and confinement or cell-ligand density. In addition, hydroxy-PAAm hydrogels overcome one of the greatest limitations of current functionalization methods, which is the inability to immobilize and to control the spatial distribution of more than one ECM protein. Due to the high affinity of hydroxy-PAAm hydrogels for biomolecules, one can control the spatial distribution of various ECM proteins on the same surface, regardless the matrix stiffness, by using sequential microprintings, which do not require any supplementary equipment.
We propose a novel procedure based on the incorporation of hydroxyl groups within acrylamide hydrogels to surmount their intrinsically non-adhesive properties with minimal requirements in cost or expertise. In comparison with existing techniques for patterning proteins on hydrogels, the hydroxy-PAAm method avoids the use of harsh toxic chemicals (e.g., hydrazine hydrate), expensive photoreactive crosslinkers (e.g., Sulfo-Sanpah) and the dependence on UV lamp power and positioning to functionalize soft hydrogels. Hydroxy-PAAm hydrogels remain stable and active for several weeks, can be mass produced, easily microprinted and their high affinity for biomolecules allows to investigate synergistic effects of conjugated ECM proteins on cellular functions. In addition, hydroxy-PAAm hydrogels can be used to quantitatively investigate the amount of contractile forces exerted by cells on their surrounding microenvironment. Indeed, we have shown previously1,20 that fluorescent beads can be easily embedded in hydroxy-PAAm hydrogels, in order to use cell traction force microscopy (TFM) to measure the displacement field and estimate the resulting traction field exerted by eukaryotic cells plated on two-dimensional micropatterns.
As many other microprinting-based methods, the main limitation of hydroxy-PAAm hydrogels concerns the spatial resolution of protein microfeatures. On&#160;stiff hydrogels (E &#62; 10 kPa), the spatial resolution is around a micrometer and mainly determined by the resolution of the stamp, depending on the lithography technique used to fabricate the silicon mold. On softer hydrogels, the spatial resolution becomes partly determined by the surface deformation during the protein transfer, which becomes the principal limitation to reach micrometer spatial resolution. The approach presented here is currently limited to two-dimensional substrates. However this method is highly scalable and further studies are currently underway in our laboratory to extend this method to three-dimensional structures.
We hope that hydroxy-PAAm hydrogels may help to decipher key mechanisms of complex cellular and tissue processes that are intimately related to the physicochemical properties of the cell microenvironment21,22, including embryonic development, inflammatory responses, wound healing, neurite outgrowth, adult tissue homeostasis, and the pathogenesis of diseases such as fibrosis and cancer.

Many aspects of the local cellular microenvironment (e.g., rigidity, pore size, nature of proteins, or cell-ligand density) provide a coordinate set of regulatory cues that control cellular processes such as motility, cell proliferation, differentiation, and gene expression. Modifications of the physicochemical properties of the extracellular environment can be perceived by cells and cause different physiological consequences, including deformation of cellular polarization, migration, and differentiation. It remains unclear, however, how cells translate ECM modifications into cellular biochemical signals. It is therefore of major importance to engineer controlled in vitro microenvironments that can reproduce the interactions between cells and their microenvironment for studying mechanotransduction pathways. To address this problem, we have recently introduced a novel method1, called hydroxy-PAAm hydrogels, to easily generate two-dimensional soft matrices that permit to independently control important mechanotransduction cues: matrix stiffness, cell geometry and confinement, nature of the protein and cell-ligand density.
ECM directs cellular processes via gradients in morphogens (chemotaxis), adhesive proteins (haptotaxis), and stiffness (durotaxis). Over the last few decades, advanced in vitro platforms have been developed to isolate these extracellular cues in order to tease out how cells are able to translate biochemical and biophysical features into physiological processes2-5. Electron-beam6, photolithography7, photochemical immobilization8, or plasma-assisted techniques9 have been developed to direct the growth of living cells on micropatterned substrates. Although these techniques have yielded important results, most of them do not allow discrimination between the individual influence of different cues on cell behavior and they require technical facilities that few laboratories can afford. Among these techniques, microcontact printing (&#181;CP), has emerged as a robust and accessible method to create cell-adhesive micro-islands10. More recently, extensive efforts11-14 have been made to develop &#181;CP on hydrogels with tunable rigidities in order to reproduce the wide range of rigidities observed in living tissues. Among these works, polyacrylamide (PAAm) has become popular15 and is already one of the most commonly used polymer-based matrices for cell biomechanics assays.
PAAm surfaces are commonly functionalized with the heterobifunctional cross-linker N-sulfosuccinimidyl-6-[4&#39;-azido-2&#39;-nitrophenylamido] (sulfo-SANPAH) and ECM proteins are linked to the surface by UV activation of the sulfo-SANPAH nitrophenyl azide groups16. Another technique consists in coupling hydrazine to proteins that have been severely oxidized with periodate17. Hynd and coworkers introduced a technique for patterning biomimetic hydrogel surfaces with protein and peptides that requires photopolymerization in presence of an acroyl-streptavidin monomer18. More recently, Tseng et al. have reported a new micropatterning method19 based on deep UV exposure of PAAm through an optical quartz mask that requires to incubate activated PA gels with 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) water solutions prior to add the protein. Despite the ability of these techniques to create homogeneous and reproducible proteins micropatterns, most of them suffer major limitations: long synthesis processes (e.g., dialysis, lyophilization, etc), expensive chemical compounds (e.g., hyaluronic acid, sulfo-SANPAH) or deep UV irradiation. In addition, these techniques do not allow independent modulation of substrate stiffness, micropattern geometry, ECM protein nature, and cell-ligand density.
Taking these limitations into account, we have developed a novel and simple acrylamide-based approach that allows immobilization of a variety of proteins and biomolecules on soft hydrogels and permits independent tuning of mechanotransduction cues in order to decipher their role on cellular functions. Instead of treating PAAm hydrogels with harsh chemical compounds, we introduce a commercial acrylamide monomer with hydroxyl groups during PAAm polymerization. This simple operation overcomes the intrinsic anti-adhesive property of PAAm hydrogels without any other technical requirements.
The presence of hydroxyl groups leads to a high affinity of hydroxy-PAAm hydrogels for proteins and biomolecules that form hydrogen-bonding interactions. In combination with &#181;CP, hydroxy-PAAm hydrogels enable a rapid generation of two-dimensional culture platform with an independent control on matrix rigidity, type of ECM proteins, cell-ligand density and confined adhesiveness, which are envisioned to be a powerful platform for studying mechanotransduction.
The purpose of this protocol is to provide the necessary information for easily making hydroxy-PAAm hydrogels without any expertise in material sciences. The ultimate goal is to provide a means for researchers to ask physiologically relevant questions at the cellular and tissue levels that may lead to a better understanding of mechanotransduction pathways involved in pathophysiological mechanisms.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">UV/Ozone Photoreactor</td>
			<td style="width:71px;">Ultra-Violet Products</td>
			<td style="width:119px;">Model PR-100</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Rocking plate</td>
			<td style="width:71px;">IKAcWerke</td>
			<td style="width:119px;">Model KS 130 Basic</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Vortexer</td>
			<td style="width:71px;">Scientific Industries</td>
			<td style="width:119px;">Model Vortex Genie2</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">Vacuum degassing chamber</td>
			<td style="width:71px;">Applied Vacuum Engineering</td>
			<td style="width:119px;">DP- 8-KIT</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Parafilm</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">P7793-1EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Stainless steel forceps with fine tip</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z225304-1EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Dressing tissue forceps</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">F4392-1EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Petri dishes in polystyrene</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">P5731-500EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Aluminium foil, thickness 0.5&#160;mm</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">266574-3.4G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Isopore membrane filter (0,2 &#181;m pore size)</td>
			<td style="width:71px;">Millipore</td>
			<td style="width:119px;">GTTP Filter code</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Round glass coverslip (22 mm diameter)</td>
			<td style="width:71px;">Neuvitro</td>
			<td style="width:119px;">GG-22</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Round glass coverslip (25 mm diameter)</td>
			<td style="width:71px;">Neuvitro</td>
			<td style="width:119px;">GG-25</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Variable volume micropipette</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z114820</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Protein microcentrifuge tubes</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z666505-100EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Scalpel handles</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">S2896-1EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Scalpel blades</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">S2771-100EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Cell culture flasks (75 cm2)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">CLS430641</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Ultrasonic bath tray, solid (stainless steel)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z613983-1EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;"></td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Name of the Reagent</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalogue Number</td>
			<td style="width:167px;">Comments</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Polydimethylsiloxane&#160;</td>
			<td style="width:71px;">Dow Corning</td>
			<td style="width:119px;">Sylgard 184 silicone elastomer kit</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Acrylamide (powder)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">A3553</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">N,N&#8217;-Methylenebis(acrylamide)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">146072</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">N-Hydroxyethylacrylamide</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">697931</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">N,N,N&#8217;,N&#8217;-Tetramethylethylenediamine</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">T9281</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Amonium PerSulfate (APS)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">A3678</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">3-(Trimetoxysilyl)propyle acrylate</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">1805</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Human Plasma Fibronectin</td>
			<td style="width:71px;">Millipore</td>
			<td style="width:119px;">FC010</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Laminin from EHS</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">L2020</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Sodium hydroxyde</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">221465-25G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">Double-distilled water (ddH2O)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Endothelial cell growth medium</td>
			<td style="width:71px;">Cells Applications</td>
			<td style="width:119px;">211K-500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Human Umbilical Vein Endothelial Cells (HUVEC)</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">C-003-5C</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Accutase</td>
			<td style="width:71px;">PAA laboratories</td>
			<td style="width:119px;">L11-007</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">HEPES buffer solution 1M in H20</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">83264-500ML-F</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Antibiotics-antimycotics</td>
			<td style="width:71px;">PAA laboratories</td>
			<td style="width:119px;">P11-002</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Phosphate Buffer Saline solution</td>
			<td style="width:71px;">PAA laboratories</td>
			<td style="width:119px;">H15-002</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Alexa Fluor 488 Phaloidin</td>
			<td style="width:71px;">Molecular Probes</td>
			<td style="width:119px;">A12379</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-vinculin antibody produced in mouse</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">V9131</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Goat anti-mouse antibody-tetramethylrhodamine</td>
			<td style="width:71px;">Molecular Probes</td>
			<td style="width:119px;">T-2762</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-Fibronectin</td>
			<td rowspan="2" style="width:71px;">Sigma-Aldrich</td>
			<td rowspan="2" style="width:119px;">F3648</td>
			<td rowspan="2" style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">(rabbit)</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Streptavidin&#160;</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">41469</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-Laminin antibody</td>
			<td rowspan="2" style="width:71px;">Sigma-Aldrich</td>
			<td rowspan="2" style="width:119px;">L9393</td>
			<td rowspan="2" style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">(rabbit)</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-rabbit IgG-FITC</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">F7512</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Trypsin-EDTA solution</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">T3924-100ML</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Absolute ethanol</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">459844-2.5L</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

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.

Figure 1A presents the co-polymerization of acrylamide (AAm) and bisacrylamide (bis-AAm) with N-hydroxyethylacrylamide (HEA) monomers containing a primary hydroxyl formed by random radical polymerization a hydrophilic network of polyacrylamide with embedded hydroxyl groups (hydroxy-PAAm). In this protocol, a weight 65 mg of HEA must be diluted in a volume of 1 ml&#160;of HEPES. Knowing that the density of HEA is roughly equal to one, we assume that we obtain a working volume of 1,065 &#181;l&#160;(HEA+HEPES). As presented in Table 1, the total addition of 1,065 &#181;l&#160;to 400 &#181;l&#160;of AAm and 50 &#181;l&#160;of bisAAm leads to a volume of 1,515 &#181;l. Therefore a volume of 3,485 &#181;l&#160;of HEPES is needed to adjust the solution to a final volume of 5 ml. As indicated in the step 2.6, the surface of the 22 mm glass coverslip must be activated during 7 min&#160;in a UV/Ozone in order to remove it without causing any damages to the hydrogel surface. Indeed, the glass surface becomes more hydrophilic after the UV/Ozone treatment and can be therefore easily removed by immersing the whole system in water. In addition, the UV/Ozone treatment prevents chemical contamination of the 22 mm coverslip, which is in direct contact with the hydrogel surface. This technique permits to obtain a flat circular polyacrylamide surface (22 mm in diameter) bound to a glass coverslip (25 mm in diameter).
As presented in&#160;Figure 1B, micropatterns of proteins can be created on the surface of hydroxy-PAAm hydrogels by microcontact printing. In this experimental procedure, UV/Ozone exposures permit to reduce temporarily the intrinsic hydrophobicity of the PDMS stamps by forming silanol groups at the surface. Indeed, the 185 nm line produces ozone from molecular oxygen while the 254 nm line converts the ozone to atomic oxygen. This reactive species attacks the siloxane backbone of PDMS to form oxygen rich SiOx silica-like layer and Si-OH surface groups. The oxidized PDMS surface is known to recover its hydrophobicity in just hours after exposure to air due to the migration of low molecular weight uncrosslinked polymeric chains from the bulk phase to the surface. This strategy helps the spreading of the protein solution on the surface of the PDMS stamp.
One of the key advantages of this method is to independently modulate matrix stiffness (Figure&#160;2A), micropattern geometry (Figure&#160;2B), cell-ligand density (Figure&#160;2C), and protein nature (Figures&#160;3A and 3B). As shown in Figure 2A, hydroxy-PAAm hydrogels show a linear dependence of elastic moduli on cross-linker concentration from few kPa to several dozen kPa, allowing fine reproduction of the rigidity of the cellular microenvironment. Figure 2B presents epifluorescent images of laminin (LM) rectangular micropatterns deposited on hydroxy-PAAm hydrogels of three different stiffnesses (2.5, 15, and 40 kPa), demonstrating clearly the independent tuning of micropattern geometry and matrix stiffness. Figure 2C shows that the cell-ligand density can be modulated by varying the concentration of the protein solution used to incubate PDMS stamps.&#160;Figure 3 presents a fluorescence image of collagen lines stamped across laminin and fibronectin lines by using sequential microcontact printings.
It is interesting for the readers to note that various sizes and shapes of micropatterns have been stamped efficiently, regardless the hydrogel stiffness. We have reproduced successfully microfeatures of proteins with sharp edges (e.g., triangles and stars) and straight (e.g., lines), or curved (e.g., circles) shapes, demonstrating that there is no major limitation to the pattern complexity. For single cell experiment, the micropattern surface area was between 400 and 2,500 &#181;m2, the lower value corresponding to the limit of single cell viability. As many other microprinting-based methods, the main limitation of the presented method concerns its spatial resolution. On stiff hydrogels (E &#62; 10 kPa), the spatial resolution is around the micrometer and mainly determined by the resolution of the stamp, whereas on &#34;soft&#34;&#160;hydrogels, the limit of resolution becomes partly determined by the surface deformation that may occur during the protein transfer.
The toxicity of hydroxy-PAAm hydrogels was investigated by quantifying the viability of primary endothelial cells plated for 1, 2, and 3 days in culture on homogeneously coated and microprinted soft hydroxy-PAAm hydrogels (Figure 4A). About 80% of HUVECs plated on micropatterned soft hydroxy-PAAm hydrogels maintained their viability for three days, demonstrating the biocompatibility of hydroxy-PAAm hydrogels for cell culture. Interestingly, similar viability was obtained in our laboratory with primary cortical neuron cells. In addition, the primary endothelial cells proliferate more on &#8220;stiff&#8221; (500 kPa) compared to &#8220;soft&#8221; (25 kPa) substrates (Figure 4B), indicating that this method provides an appropriate microenvironment for studying cellular proliferation across a wide range of rigidities.
To decouple the effect of substrate stiffness and spreading area on mechanotransduction, primary endothelial cells (HUVECs) were plated on rectangular FN-coated micropatterns (constant area of 1,200 &#181;m2) deposited on hydroxy-PAAm hydrogels of three different stiffnesses (2.5, 8.5, and 25 kPa). Then, HUVECs were stained for actin filaments, vinculin and DNA by following a standard immunocytochemistry procedure, as used for mammalian cells plated on glass substrates. On the softest substrates, HUVECs exhibit low actin fiber density and a rounded nucleus, whereas on stiffer substrates, actin fibers are straighter and thicker and the nucleus deformed (Figure 5). Changes in substrate stiffness at constant spreading area modulate tension and distribution of actin stress fibers, which results in the remodeling of the nucleus. This observation suggests a mechanical coupling between matrix stiffness, cytoskeleton, and nucleus.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Final AAm/bisAAm ratio</td>
			<td>AAm 40% (&#181;l)</td>
			<td>Weight of HEA to dissolve in 1 ml HEPES (mg)</td>
			<td>bis AAm (2%) 
			(&#181;l)</td>
			<td>HEPES (&#181;l)</td>
			<td>Young&#8217;s modulus 
			(103 Pa)</td>
		</tr>
		<tr>
			<td>0.2/50 
			0.5/50 
			1/50 
			2/50 
			3/50</td>
			<td>400 
			400 
			400 
			400 
			400</td>
			<td>65 
			65 
			65 
			65 
			65</td>
			<td>50 
			125 
			250 
			500 
			750</td>
			<td>3,485 
			3,410 
			3,285 
			3,035 
			2,785</td>
			<td>~1.4 
			~&#160;3.6 
			~&#160;8.7 
			~17.2 
			~&#160;25</td>
		</tr>
	</tbody>
</table>
Table 1.&#160;Preparation of hydroxy-PAAm hydrogels with various rigidities. Working solutions to prepare hydroxy-PAAm hydrogels with final stiffness ranging from 1.4 to 25 kPa.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51010/51010fig1highres.jpg" width="500" /> 
Figure 1. (A)&#160;Acrylamide (AAm, in black), N,N&#8217;-methylenebisacrylamide (bis-AAm, in blue) and N-hydroxyethylacrylamide (HEA, in red) were mixed together to form hydroxy-PAAm hydrogels. (B) Schematic representation of the different steps of the micropatterning procedure on hydroxy-PAAm hydrogels. A solution of protein is first incubated during 1&#160;hr on the structure face of a microstamp (step #1). The hydrogel surface is gently dried with a nitrogen flow (step #2). The microstamp is then placed in formal contact with the hydroxy-PAAm surface during 1&#160;hr (step #3). After successive washes, the microprinted surface is passivated with a sterile BSA solution deposited O/N at 4 &#176;C (step #5) to block non-printed areas. Finally, the microprinted hydroxy-PAAm surface is washed several times with sterile PBS and is ready for cell seeding (step #6). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51010/51010fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51010/51010fig2highres.jpg" width="500" /> 
Figure 2. (A)&#160;The evolution of the stiffness of hydroxy-PAAm hydrogels is linearly correlated with the amount of bis-AAm cross-linker (the red line is a linear regression with R2=0,995). (B) Fluorescence images of rectangular laminin micro-features stamped on hydroxy-PAAm hydrogels of different stiffnesses (E = 2.5, 15, and 40 kPa). Scale bars correspond to (A) 65 &#181;m, (B) 80 &#181;m, and (C) 50 &#181;m. (C) Parallel lines of 15 &#181;m width form a LM gradient on a 25 kPa hydroxy-PAAm hydrogel, as indicated by the evolution of the fluorescence intensity profile. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51010/51010fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51010/51010fig3highres.jpg" width="500" /> 
Figure 3. (A)&#160;Immunofluorescence image of Fibronectin (in red) and Laminin (in green) stripes crossed at 90&#176;. (B) Multilabeling of Fibronectin (in red), Laminin (in green) and collagen (in blue) stripes microprinted subsequently on a 25 kPa hydrogel PAAm substrate. Scale bars correspond to 30 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51010/51010fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51010/51010fig4highres.jpg" width="500" /> 
Figure 4. (A)&#160;The quantification of cell viability demonstrated excellent cell survival at 24, 48, and 72 hr&#160;after being plated on homogeneously-coated and micropatterned hydroxy-PAAm surfaces. The number indicated at the bottom of each bar corresponds to the total number of cells counted for the viability assay. (B) Quantification of HUVECs proliferation on 25 and 500 kPa homogeneously-coated substrates after 1 (white bars) and 7 (hashed bars) days of culture. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51010/51010fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" fo:content-width="5in" src="/files/ftp_upload/51010/51010fig5highres.jpg" width="500" /> 
Figure 5.&#160;Fluorescent images of immunostained primary endothelial cells cultured on rectangular FN-coated micropatterns, which were deposited on hydroxy-PAAm hydrogels of 2.5 kPa, 8.5 kPa, and 25 kPa (from left to right). The actin stress fibers (in green) were stained with Alexa Fluor 488 phalloidin, focal adhesions (in red) were stained with a mouse polyclonal primary antibody and labeled with a red-fluorescent IgG secondary antibody and the DNA was stained in blue with DAPI. The scale bars correspond to 17 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51010/51010fig5highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Preparation of Hydroxy-PAAm Hydrogels for Decoupling the Effects of Mechanotransduction Cues at JoVE.com

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

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 ...

preparation-hydroxy-paam-hydrogels-for-decoupling-effects

Nanyang Technological University

Research

JoVE Journal

Bioengineering

13.2K Views.  Université de Mons. 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.

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

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

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 ...

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific molecular mechanisms are identified, new therapeutic strategies can be developed to treat abnormalities in specific behaviors caused by degenerative diseases or aging of the nervous system. The marine snail Aplysia californica is well suited for the investigations of cellular and molecular basis of behavior because neural circuitry underlying a specific behavior could be easily determined and the individual components of the circuitry could be easily manipulated. These advantages of Aplysia have led to several fundamental discoveries of neurobiology of learning and memory. Here we describe a preparation of the Aplysia nervous system for the electrophysiological and molecular analyses of individual neurons. Briefly, ganglion dissected from the nervous system is exposed to protease to remove the ganglion sheath such that neurons are exposed but retain neuronal activity as in the intact animal. This preparation is used to carry out electrophysiological measurements of single or multiple neurons. Importantly, following the recording using a simple methodology, the neurons could be isolated directly from the ganglia for gene expression analysis. These protocols were used to carry out simultaneous electrophysiological recordings from L7 and R15 neurons, study their response to acetylcholine and quantitating expression of CREB1 gene in isolated single L7, L11, R15, and R2 neurons of Aplysia.

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

Marine snail Aplysia californica has been widely used as a neurobiology model for the studies on cellular and molecular basis of behavior. Here a methodology is described for exploring the nervous system of Aplysia for the electrophysiological and molecular analyses of single neurons of identified neural circuitry.

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific ...

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and functionalize polymer chains with compounds that are able to improve biocompatibility, mechanical properties, or cell viability allows the design of novel materials to meet new challenges in the biomedical field. With the polymer functionalization strategies, click chemistry is a powerful tool to improve cell-compatibility and drug delivery properties of polymeric devices. Similarly, the fundamental need of biomedicine to use sterile tools to avoid potential adverse-side effects, such as toxicity or contamination of the biological environment, gives rise to increasing interest in the microwave-assisted strategy.
The combination of click chemistry and the microwave-assisted method is suitable to produce biocompatible hydrogels with desired functionalities and improved performances in biomedical applications. This work aims to synthesize RGD-functionalized hydrogels. RGD (arginylglycylaspartic acid) is a tripeptide that can mimic cell adhesion proteins and bind to cell-surface receptors, creating a hospitable microenvironment for cells within the 3D polymeric network of the hydrogels. RGD functionalization occurs through Huisgen 1,3-dipolar cycloaddition. Some PAA carboxyl groups are modified with an alkyne moiety, whereas RGD is functionalized with azido acid as the terminal residue of the peptide sequence. Finally, both products are used in a copper catalyzed click reaction to permanently link the peptide to PAA. This modified polymer is used with carbomer, agarose and polyethylene glycol (PEG) to synthesize a hydrogel matrix. The 3D structure is formed due to an esterification reaction involving carboxyl groups from PAA and carbomer and hydroxyl groups from agarose and PEG through microwave-assisted polycondensation. The efficiency of the gelation mechanism ensures a high degree of RGD functionalization. In addition, the procedure to load therapeutic compounds or biological tools within this functionalized network is very simple and reproducible.

We present a protocol for the synthesis of RGD-functionalized hydrogels as devices for cell and drug delivery. The procedure involves copper catalyzed alkyne-azide cycloaddition (CuAAC) between alkyne-modified polyacrylic acid (PAA) and a RGD-azide derivative. The hydrogels are formed using microwave-assisted polycondensation and their physicochemical properties are investigated.

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. One way to address this challenge is to develop new materials that enable measurement of cell function within the material. Here, a method is presented for measurement of cellular matrix metalloproteinase (MMP) activity in 3D hydrogels in a 96-well format. In this system, a poly(ethylene glycol) (PEG) hydrogel is functionalized with a fluorogenic MMP cleavable sensor. Cellular MMP activity is proportional to fluorescence intensity and can be measured with a standard microplate reader. Miniaturization of this assay to a 96-well format reduced the time required for experimental set up by 50% and reagent usage by 80% per condition as compared to the previous 24-well version of the assay. This assay is also compatible with other measurements of cellular function. For example, a metabolic activity assay is demonstrated here, which can be conducted simultaneously with MMP activity measurements within the same hydrogel. The assay is demonstrated with human melanoma cells encapsulated across a range of cell seeding densities to determine the appropriate encapsulation density for the working range of the assay. After 24 h of cell encapsulation, MMP and metabolic activity readouts were proportional to cell seeding density. While the assay is demonstrated here with one fluorogenic degradable substrate, the assay and methodology could be adapted for a wide variety of hydrogel systems and other fluorescent sensors. Such an assay provides a practical, efficient and easily accessible 3D culturing platform for a wide variety of applications.

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

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).