Work is our laboratory is supported by grants from the National Institutes of Health.

Preparation
<ul>
<li> Coverslips should be autoclaved.</li>
<li> Sterile distilled or deionized water should be used to prepare solutions and for washing coverslips.</li>
<li> AC (40% w/v) and bis-AC (1% w/v) solutions are sterilized by 0.2 &mu;m filtration. Prepare 10% ammonium persulfate (APS; 100&mu;g/ml water) shortly before use and sterile filter. Replace the APS solution monthly.</li>
<li> Chemical reagents such as 3-APTMS, chloroform, glutaradehyde, NHS, and SurfaSil that cannot be autoclaved are kept in an assigned bottle used solely for the preparation of hydrogels.</li>
<li> For best results, hydrogels should be utilized within a couple of days after the overnight incubation with the appropriate ECM protein.</li>
<li> Prepare a 10% SurfaSil solution in chloroform (10 ml is usually enough for 20 top coverslips) in a 50 ml polypropylene Falcon tube prior to pouring of hydrogel. (Our lab usually siliconizes the top coverslips during the 0.5% glutaraldehyde incubation step). Add the coverslips to the Falcon tube and rock for at least 10 min. Decant the SurfaSil solution and air dry the coverslips on Kimwipes in the biological safety cabinet where the hydrogels will be prepared.</li>
<li> Prepare the heat-inactivated BSA solution as follows: a 20 mg/ml solution of fatty-acid free BSA in PBS is incubated in a 68&deg;C water bath for 30 min. The solution is then sterile filtered and stored at 4&deg;C.</li>
</ul>

Procedure
<ol>
<li> Place a layer of Parafilm on the bottom half of a 150-mm Petri dish.</li>
<li> Place up to 9 25-mm coverslips on top of the Parafilm and cover them with 1 ml 0.1 M NaOH. Incubate for 3 min and then aspirate with a vacuum line.</li>
<li> Working in a chemical hood, place 0.5 ml 3-APTMS on each coverslip. Incubate for 3 min and then aspirate the APTMS. If you wait too long, a foam will form. </li>
<li> Rinse the coverslips once with 20 ml deionized water in the same dish. Remove the coverslips from the dish using curved forceps and transfer them, with their treated-side facing up, to a new 150-mm dish. Wash the coverslips with deionized water three times, on the rocker, for 10 min each wash. If you fail to remove all the APTMS, it will react with the glutaraldehyde in the next step and leave a white cloudy precipitate (Figure 1).</li>
<li> Thaw the glutaraldehyde (~10 min before use).</li>
<li> Using curved forceps, transfer the coverslips to a clean dish layered with Parafilm and aspirate any remaining liquid. Use a vacuum line or Kimwipe to blot the remaining water as needed.</li>
<li> Cover each coverslip completely with 0.5 ml of 0.5% glutaraldehyde in sterile deionized water and incubate for 30 min in a chemical hood. Aspirate the glutaraldehyde. Rinse and wash the coverslips as in step 4. Dry the coverslips completely. [You can stop here and leave the coverslips for several weeks in a dry area]. </li>
<li> When you are ready to prepare the hydrogels, use curved forceps to transfer the coverslips, reactive side up, to a sheet of Parafilm that has been taped onto the surface of the biological safety cabinet. Make sure that the coverslips are flat on the Parafilm surface.</li>
<li> Prepare a saturated NHS solution in toluene. (Dissolve a small amount of NHS in enough toluene for the particular experiment. Keep adding NHS bit by bit until the NHS no longer dissolves. The saturated solution is usually cloudy and pink.)</li>
<li> Next, prepare the acrylamide, bis-acrylamide, water and APS to reach the desired acrylamide percentage. Add the reagents in microcentrifuge tubes as outlined below to a total volume of 0.8ml.</li>
<li> Then, one aliquot at a time, add the NHS and TEMED to the 0.8 ml AC solution, vortex briefly and IMMEDIATELY pour 3-5 gels using 140 &mu;l per coverslip in the biological safety hood. Whenever possible, all steps from this point should be performed in a biological safety cabinet. [Note: different size coverslips can be utilized based upon need. If 18-mm coverslips are used, use ~33 &mu;l AC solution per coverslip and an 18-mm top siliconized coverslip.] 
 <table border="1">
 <tbody>
 <tr>
 <td>&nbsp;</td>
 <td colspan="4">Bis-AC (%)</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>0.3 (stiff)</td>
 <td>0.15</td>
 <td>0.06</td>
 <td>0.03 (soft)</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&mu;l</td>
 <td>&mu;l</td>
 <td>&mu;l</td>
 <td>&mu;l</td>
 </tr>
 <tr>
 <td>Water</td>
 <td>402</td>
 <td>522</td>
 <td>594</td>
 <td>618</td>
 </tr>
 <tr>
 <td>AC</td>
 <td>150</td>
 <td>150</td>
 <td>150</td>
 <td>150</td>
 </tr>
 <tr>
 <td>Bis-AC</td>
 <td>240</td>
 <td>120</td>
 <td>48</td>
 <td>24</td>
 </tr>
 <tr>
 <td>APS</td>
 <td>8</td>
 <td>8</td>
 <td>8</td>
 <td>8</td>
 </tr>
 <tr>
 <td>TEMED</td>
 <td>1</td>
 <td>1</td>
 <td>1</td>
 <td>1</td>
 </tr>
 <tr>
 <td>NHS</td>
 <td>228</td>
 <td>228</td>
 <td>228</td>
 <td>228</td>
 </tr>
 </tbody>
 </table>
 </li>
<li> Quickly place the siliconized 25-mm coverslip on top of each gel before it begins to polymerize. Addition of the top coverslip should allow the AC to completely cover the bottom coverslip. Incubate this "sandwich" at room temperature until the AC polymerizes. (Check the residual AC solution in the microcentrifuge tube to determine when polymerization has occurred. Usually a few minutes for stiff gels and a little longer for soft ones will suffice.). </li>
<li> Carefully pick up the sandwich (sterile gloves on!) and slide off the top coverslip until it overhangs the polymerized gel. You can then pry it off the gel. If you wait too long before removing the top coverslip, the gel will rip as the coverslip is removed.</li>
<li> Discard the top coverslip. Place the bottom gel-coverslips (hereafter called the hydrogel) into 6-well plates with 2 ml PBS/well. PBS can be added before or after the gel-coverslip to each well. Wash the hydrogels with PBS three times, on a rocker, 5 min per wash. </li>
<li> Repeat steps 11-14 until the required number of hydrogels have been prepared.</li>
<li> Cover each hydrogel with 2 ml of a fibronectin solution (3 &mu;g/ml in PBS) or other ECM protein (see Klein et al.13 for details.). The ECM protein becomes covalently bound to the hydrogel during overnight incubation at 4&deg;C. </li>
<li> Aspirate the ECM solution and block unreacted NHS with 1mg/ml heat-inactivated fatty-acid free BSA in serum-free media for at least 30 min at 37&deg;C in a cell culture incubator. Rinse the hydrogels once with sterile PBS or cell culture medium.</li>
<li> Plate cells in the appropriate culture medium containing FBS. The number of cells seeded on the hydrogel should be determined by the user based upon the degree of cell spreading and confluency required for experimentation. Approximately 105 cells is usually sufficient for western blotting and qPCR analysis.</li>
<li> Following the incubation period, cell protein or mRNA can be extracted. The coverslips are carefully removed from the wells using curved forceps and placed (cell-side down) on top of 100 &mu;l droplets of lysis buffer which have been spaced out (2-3cm) along a sheet of Parafilm on the lab bench. (We use standard SDS sample buffer and TRIzol, respectively, to prepare samples for western blotting and to isolate RNA for qPCR.) Incubate the cells with the lysis buffer for exactly one minute. Remove the coverslips and transfer the lysis buffer to a microcentrifuge tube. Alternatively for RNA extraction only, the user can transfer each hydrogel to a new 6-well plate and add 1ml of TRIzol/well. Incubate for 3 minutes and remove TRIzol solution for storage in a microcentrifuge tube.</li>
</ol>

Additional information on procedures such as immunofluorescence, BrdU staining, transfection, etc. for cells seeded on hydrogels is described in Klein et al. 200713.

Representative Results
Thorough washing of the coverslips following addition of APTMS is an important step in producing "reactive" coverslips. If one fails to remove the APTMS completely, it will react with the glutaraldehyde in the following step and produce a white cloudy precipitate as seen in Figure 1A. Figure 1B shows a properly washed and dried coverslip. If the precipitate develops, the whole procedure must be restarted from the beginning as the coverslip is no longer useable.
Following hydrogel formation and coating with ECM proteins overnight, cells can be seeded the following day. As Figure 2 shows, there is a distinct difference between cell spreading on stiff versus soft hydrogels. As can be seen by phalloidin staining in mouse embryonic fibroblasts (MEFs), cells spread to a greater extent on stiff as compared to soft hydrogels. Indeed, most cells attaching to a soft hydrogel will remain compact and attach less efficiently.
Although only MEF morphology is shown in Figure 2, the difference in cell spreading is consistent across several other cell lines tested11-12,14.
Secrets to Success
<ol>
<li> Once the procedure is started, it is very important to keep the coverslips with the "reactive" side up and keep in mind which side has been coated.</li>
<li> Gloves should be worn at all time during the procedure to provide a working environment that is as sterile as possible.</li>
<li> Most of the initial steps are performed between a chemical fume hood and the lab bench. All steps subsequent to removing excess glutaraldehyde from the coverslips should be executed under a biological safety cabinet.</li>
<li> Due to differences in cell spreading (Figure 2), we recommend seeding twice the number of cells on soft hydrogels as compared to stiff hydrogels.</li>
<li> The AC polymerization process is extremely quick. For first time users, one can decrease the amount of APS or TEMED in the solution to prolong the polymerization process. Do not try to prepare more than a few coverslips at one time.</li>
<li> In cell cycle studies where synchronization in G0 is required, we usually serum-starve cells in plastic culture dishes, trypsinize the cells and reseed them on hydrogels of different compliances in the presence of mitogens (FBS and/or growth factors). This procedure ensures that the starting population of G0-synchronized cells is identical in all samples. However, for many signal transduction studies, the required mitogen stimulation period may not be long enough to allow for attachment and spreading of cells. In this case, we serum-starve the cells on the hydrogels and then directly stimulate them with mitogen.</li>
</ol>
 <img src="/files/ftp_upload/2089/2089fig1a.jpg" alt="Figure 1a" /> 
 Figure 1A. Improperly washed coverslip. Following addition of APTMS, coverslip was washed for 1-2 minutes before addition of the glutaraldehyde solution. A precipitate forms on the coverslip that has not been washed according to the steps outlined in Procedure.
 <img src="/files/ftp_upload/2089/2089fig1b.jpg" alt="Figure 1b" /> 
Figure 1B. Properly washed coverslip. Following addition of APTMS, coverslip was washed for three times 10 minutes each before addition of the glutaraldehyde solution. No precipitate forms on the coverslip.
 <img src="/files/ftp_upload/2089/2089fig2.jpg" alt="Figure 2" /> 
Figure 2. Morphology of cells on hydrogels of varying stiffness. Established MEFs were seeded on fibronectin-coated hydrogels of high or low stiffness for 9 hours. Following the incubation period, the cells were fixed, permeabilized and stained with FITC-phalloidin which binds to f-actin. MEFs on high stiffness gels exhibit stress fibers and are well spread compared to those seeded on low stiffness hydrogels. Scale bar= 50&mu;m.
 <img src="/files/ftp_upload/2089/2089fig3.jpg" alt="Figure 3" /> 
Figure 3. Representative quantitative PCR result of mouse cyclin D1 mRNA levels. Serum starved mouse embryonic fibroblasts were plated onto high (3% acrylamide) or low (0.3% acrylamide) stiffness hydrogels and stimulated with 10% FBS for 9 hrs. Following RNA extraction, real-time quantitative PCR analysis was performed for cyclin D1 mRNA levels (normalized to 18S RNA). G0 represents cyclin D1 mRNA from quiescent cells. Cyclin D1 mRNA levels increase significantly on stiff hydrogels but not on soft hydrogels. Data are mean +/- SD of duplicate PCR reactions.

<ol>
	<li>Beattie, D., Xu, C., Vito, R., Glagov, S., Whang, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+analysis+of+heterogeneous,+atherosclerotic+human+aorta.">Mechanical analysis of heterogeneous, atherosclerotic human aorta.</a> J Biomech Eng. 120, 602-607 (1998).</li><li>Bernini, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arterial+stiffness,+intima-media+thickness+and+carotid+artery+fibrosis+in+patients+with+primary+aldosteronism.">Arterial stiffness, intima-media thickness and carotid artery fibrosis in patients with primary aldosteronism.</a> J Hypertens. 26, 2399-2405 (2008).</li><li>Boonyasirinant, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aortic+stiffness+is+increased+in+hypertrophic+cardiomyopathy+with+myocardial+fibrosis:+novel+insights+in+vascular+function+from+magnetic+resonance+imaging.">Aortic stiffness is increased in hypertrophic cardiomyopathy with myocardial fibrosis: novel insights in vascular function from magnetic resonance imaging.</a> J Am Coll Cardiol. 54, 255-2562 (2009).</li><li>Discher, D. E., Janmey, P., Wang, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+cells+feel+and+respond+to+the+stiffness+of+their+substrate.">Tissue cells feel and respond to the stiffness of their substrate.</a> Science. 310, 1139-1143 (2005).</li><li>Duprez, D. A., Cohn, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arterial+stiffness+as+a+risk+factor+for+coronary+atherosclerosis.">Arterial stiffness as a risk factor for coronary atherosclerosis.</a> Curr Atheroscler Rep. 9, 139-144 (2007).</li><li>Lee, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prediction+of+mechanical+properties+of+human+atherosclerotic+tissue+by+high-frequency+intravascular+ultrasound+imaging.+An+in+vitro+study.">Prediction of mechanical properties of human atherosclerotic tissue by high-frequency intravascular ultrasound imaging. An in vitro study.</a> Arterioscler Thromb. 12, 1-5 (1992).</li><li>Levental, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+crosslinking+forces+tumor+progression+by+enhancing+integrin+signaling.">Matrix crosslinking forces tumor progression by enhancing integrin signaling.</a> Cell. 139, 891-906 (2009).</li><li>Paszek, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tensional+homeostasis+and+the+malignant+phenotype.">Tensional homeostasis and the malignant phenotype.</a> Cancer Cell. 8, 241-254 (2005).</li><li>Samani, A., Zubovits, J., Plewes, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastic+moduli+of+normal+and+pathological+human+breast+tissues:+an+inversion-technique-based+investigation+of+169+samples.">Elastic moduli of normal and pathological human breast tissues: an inversion-technique-based investigation of 169 samples.</a> Phys Med Biol. 52, 1565-1576 (2007).</li><li>Wells, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+matrix+stiffness+in+regulating+cell+behavior.">The role of matrix stiffness in regulating cell behavior.</a> Hepatology. 47, 1394-1400 (2008).</li><li>Pelham, R. J., Wang, Y. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+locomotion+and+focal+adhesions+are+regulated+by+substrate+flexibility.">Cell locomotion and focal adhesions are regulated by substrate flexibility.</a> Proc. Natl. Acad Sci USA. 94, 13661-13665 (1997).</li><li>Klein, E. A., Yung, Y., Castagnino, P., Kothapalli, D., Assoian, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+adhesion,+cellular+tension,+and+cell+cycle+control.">Cell adhesion, cellular tension, and cell cycle control.</a> Methods Enzymol. 426, 155-175 (2007).</li><li>Klein, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-cycle+control+by+physiological+matrix+elasticity+and+in+vivo+tissue+stiffening.">Cell-cycle control by physiological matrix elasticity and in vivo tissue stiffening.</a> Current Biology. 19, 1511-1518 (2009).</li></ol>

No conflicts of interest declared.

A crucial element of the hydrogel polymerization process is to avoid air bubble formation which will allow cells to bind to the glass coverslip rather than the ECM-coated hydrogel itself. This can be prevented by carefully pipetting the polymerization solution after vortexing and visually making sure that no air bubbles have become trapped in the gel. We always recommend preparing additional "reactive" coverslips and hydrogels to ensure having enough for experimentation.
Particular attention ...

<table><tbody><tr><td>Glutaraldehyde, 70%</td><td>Sigma-Aldrich</td><td>G7776</td><td>Store at -20&amp;#x00B0;C</td></tr><tr><td>3-APTMS (3-Aminopropyltrimethosysilane 97%)</td><td>Sigma-Aldrich</td><td>281778</td><td>Store at room temperature</td></tr><tr><td>SurfaSil Siliconizing Fluid</td><td>Thermo Fisher Scientific, Inc.</td><td>42800</td><td>Store at room temperature</td></tr><tr><td>NHS (N-hydroxysucinimide Ester)</td><td>Sigma-Aldrich</td><td>A-8060</td><td>Store at 4&amp;#x00B0;C Replace monthly</td></tr><tr><td>Albumin, bovine serum, essentially fatty acid free</td><td>Sigma-Aldrich</td><td>A6003-100G</td><td>Store at 4&amp;#x00B0;C</td></tr><tr><td>Coverslips (25mm)</td><td>Fisher Scientific</td><td>12-545-86 25 Cir 1D</td><td></td></tr><tr><td>Coverslips (18mm)</td><td>Fisher Scientific</td><td>12-545-84 18 Cir 1D</td><td></td></tr></tbody></table>

studying the effects of matrix stiffness on cellular function using acrylamide-based hydrogels

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and cardiovascular disease1-11. Traditional cell biological approaches to studying cellular function involve culturing cells on a rigid substratum (plastic dishes or glass coverslips) which cannot account for the effect of an elastic ECM or the variations in ECM stiffness between tissues. To model in vivo tissue compliance conditions in vitro, we and others use ECM-coated hydrogels. In our laboratory, the hydrogels are based on polyacrylamide which can mimic the range of tissue compliances seen biologically12. "Reactive" cover slips are generated by incubation with NaOH followed by addition of 3-APTMS. Glutaraldehyde is used to cross-link the 3-APTMS and the polyacrylamide gel. A solution of acrylamide (AC), bis-acrylamide (Bis-AC) and ammonium persulfate is used for the polymerization of the hydrogel. N-hydroxysuccinimide (NHS) is incorporated into the AC solution to crosslink ECM protein to the hydrogel. Following polymerization of the hydrogel, the gel surface is coated with an ECM protein of choice such as fibronectin, vitronectin, collagen, etc.
The stiffness of a hydrogel can be determined by rheology or atomic force microscopy (AFM) and adjusted by varying the percentage of AC and/or bis-AC in the solution12. In this manner, substratum stiffness can be matched to the stiffness of biological tissues which can also be quantified using rheology or AFM. Cells can then be seeded on these hydrogels and cultured based upon the experimental conditions required. Imaging of the cells and their recovery for molecular analysis is straightforward. For this article, we define soft substrata as those having elastic moduli (E) &lt;3000 Pascal and stiff substrata/tissues as those with E &gt;20,000 Pascal.

Watch this Scientific Journal Video about Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels at JoVE.com

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

studying-effects-matrix-stiffness-on-cellular-function-using

Research

JoVE Journal

Biology

22.0K Views.  University of Pennsylvania. The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.

Video: Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Preparation of Complaint Matrices for Quantifying Cellular Contraction

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling. Focal adhesions are macromolecular assemblies that couple the contractile F-actin cytoskeleton to the ECM. This connection allows for the transmission of intracellular mechanical forces across the cell membrane to the underlying substrate. Recent work has shown the mechanical properties of the ECM regulate focal adhesion and F-actin morphology as well as numerous physiological processes, including cell differentiation, division, proliferation and migration. Thus, the use of cell culture substrates has become an increasingly prevalent method to precisely control and modulate ECM mechanical properties.
To quantify traction forces at focal adhesions in an adherent cell, compliant substrates are used in conjunction with high-resolution imaging and computational techniques in a method termed traction force microscopy (TFM). This technique relies on measurements of the local magnitude and direction of substrate deformations induced by cellular contraction. In combination with high-resolution fluorescence microscopy of fluorescently tagged proteins, it is possible to correlate cytoskeletal organization and remodeling with traction forces.
Here we present a detailed experimental protocol for the preparation of two-dimensional, compliant matrices for the purpose of creating a cell culture substrate with a well-characterized, tunable mechanical stiffness, which is suitable for measuring cellular contraction. These protocols include the fabrication of polyacrylamide hydrogels, coating of ECM proteins on such gels, plating cells on gels, and high-resolution confocal microscopy using a perfusion chamber. Additionally, we provide a representative sample of data demonstrating location and magnitude of cellular forces using cited TFM protocols.

In this video, we demonstrate the experimental techniques used to fabricate compliant, extracellular matrix (ECM) coated substrates suitable for cell culture, and which are amenable to traction force microscopy and observing effects of ECM stiffness on cell behavior.

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling.  Focal adhesions are ...

Bioengineering

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 ...

Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a matrix that is elastic and much softer (0.1 &#8211; 100 kPa). Since such stiffness mismatch greatly affects cell responses, there is a strong interest in developing hydrogel materials that span a wide range of stiffness to serve as cell substrates. Polyacrylamide gels, which are inexpensive and cover the stiffness range of all soft tissues in the body, are the hydrogel of choice for many research groups. However, polyacrylamide gel preparation is lengthy, tedious, and only suitable for small batches. Here, we describe an assay which by utilizing a permanent flexible plastic film as a structural support for the gels, enables the preparation of polyacrylamide gels in a multiwell plate format. The technique is faster, more efficient, and less costly than current methods and permits the preparation of gels of custom sizes not otherwise available. As it doesn&#8217;t require any specialized equipment, the method could be easily adopted by any research laboratory and would be particularly useful in research focused on understanding stiffness-dependent cell responses.

Here, a method that enables quick, efficient, and inexpensive preparation of polyacrylamide gels in a multiwell plate format is described. The method does not require any specialized equipment and could be easily adopted by any research laboratory. It would be particularly useful in research focused on understanding stiffness-dependent cell responses.

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a ...

An Optimized O9-1/Hydrogel System for Studying Mechanical Signals in Neural Crest Cells

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to various organs and tissues. Tissue stiffness produces mechanical force, a physical cue that plays a critical role in NCC differentiation; however, the mechanism remains unclear. The method described here provides detailed information for the optimized generation of polyacrylamide hydrogels of varying stiffness, the accurate measurement of such stiffness, and the evaluation of the impact of mechanical signals in O9-1 cells, a NCC line that mimics in vivo NCCs.
Hydrogel stiffness was measured using atomic force microscopy (AFM) and indicated different stiffness levels accordingly. O9-1 NCCs cultured on hydrogels of varying stiffness showed different cell morphology and gene expression of stress fibers, which indicated varying biological effects caused by mechanical signal changes. Moreover, this established that varying the hydrogel stiffness resulted in an efficient in vitro system to manipulate mechanical signaling by altering gel stiffness and analyzing the molecular and genetic regulation in NCCs. O9-1 NCCs can differentiate into a wide range of cell types under the influence of the corresponding differentiation media, and it is convenient to manipulate chemical signals in vitro. Therefore, this in vitro system is a powerful tool to study the role of mechanical signaling in NCCs and its interaction with chemical signals, which will help researchers better understand the molecular and genetic mechanisms of neural crest development and diseases.

Detailed step-by-step protocols are described here for studying mechanical signals in vitro using multipotent O9-1 neural crest cells and polyacrylamide hydrogels of varying stiffness.

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to ...

Developmental Biology

A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, function, and fate in general. Although increasingly more adherent cell types&#39; responses to matrix stiffness have been characterized, how adherent cells&#39; susceptibility to bacterial infection depends on matrix stiffness is largely unknown, as is the effect of bacterial infection on the biomechanics of host cells. We hypothesize that the susceptibility of host endothelial cells to a bacterial infection depends on the stiffness of the matrix on which these cells reside, and that the infection of the host cells with bacteria will change their biomechanics. To test these two hypotheses, endothelial cells were used as model hosts and Listeria monocytogenes as a model pathogen. By developing a novel multi-well format assay, we show that the effect of matrix stiffness on infection of endothelial cells by L. monocytogenes can be quantitatively assessed through flow cytometry and immunostaining followed by microscopy. In addition, using traction force microscopy, the effect of L. monocytogenes infection on host endothelial cell biomechanics can be studied. The proposed method allows for the analysis of the effect of tissue-relevant mechanics on bacterial infection of adherent cells, which is a critical step towards understanding the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria. This method is also applicable to a wide variety of other types of studies on cell biomechanics and response to substrate stiffness where it is important to be able to perform many replicates in parallel in each experiment.

We have developed a multi-well format polyacrylamide-based assay for probing the effect of extracellular matrix stiffness on bacterial infection of adherent cells. This assay is compatible with flow cytometry, immunostaining, and traction force microscopy, allowing for quantitative measurements of the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria.

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, ...

Immunology and Infection

Experimental and Data Analysis Workflow for Soft Matter Nanoindentation

Nanoindentation refers to a class of experimental techniques where a micrometric force probe is used to quantify the local mechanical properties of soft biomaterials and cells. This approach has gained a central role in the fields of mechanobiology, biomaterials design&#160;and tissue engineering, to obtain a proper mechanical characterization of soft materials with a resolution comparable to the size of single cells (&#956;m). The most popular strategy to acquire such experimental data is to employ an atomic force microscope (AFM); while this instrument offers an unprecedented resolution in force (down to pN) and space (sub-nm), its usability is often limited by its complexity that prevents routine measurements of integral indicators of mechanical properties, such as Young&#39;s Modulus (E). A new generation of nanoindenters, such as those based on optical fiber sensing technology, has recently gained popularity for its ease of integration while allowing to apply sub-nN forces with &#181;m spatial resolution, therefore being suitable to probe local mechanical properties of hydrogels and cells.
In this protocol, a step-by-step guide detailing the experimental procedure to acquire nanoindentation data on hydrogels and cells using a commercially available ferrule-top optical fiber sensing nanoindenter is presented. Whereas some steps are specific to the instrument used herein, the proposed protocol can be taken as a guide for other nanoindentation devices, granted some steps are adapted according to the manufacturer&#39;s guidelines. Further, a new open-source Python software equipped with a user-friendly graphical user interface for the analysis of nanoindentation data is presented, which allows for screening of incorrectly acquired curves, data filtering, computation of the contact point through different numerical procedures, the conventional computation of E, as well as a more advanced analysis particularly suited for single-cell nanoindentation data.

The protocol presents a complete workflow for soft material nanoindentation experiments, including hydrogels and cells. First, the experimental steps to acquire force spectroscopy data are detailed; then, the analysis of such data is detailed through a newly developed open-source Python software, which is free to download from GitHub.

Nanoindentation refers to a class of experimental techniques where a micrometric force probe is used to quantify the local mechanical properties of soft ...

Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes and therapeutic responses. Accounting for matrix effects earlier in the drug development pipeline is expected to increase the likelihood of clinical success of novel therapeutics. Biomaterial-based strategies recapitulating specific tissue microenvironments in 3D cell culture exist but integrating these with the 2D culture methods primarily used for drug screening has been challenging. Thus, the protocol presented here details the development of methods for 3D culture within miniaturized biomaterial matrices in a multi-well plate format to facilitate integration with existing drug screening pipelines and conventional assays for cell viability. Since the matrix features critical for preserving clinically relevant phenotypes in cultured cells are expected to be highly tissue- and disease-specific, combinatorial screening of matrix parameters will be necessary to identify appropriate conditions for specific applications. The methods described here use a miniaturized culture format to assess cancer cell responses to orthogonal variation of matrix mechanics and ligand presentation. Specifically, this study demonstrates the use of this platform to investigate the effects of matrix parameters on the responses of patient-derived glioblastoma (GBM) cells to chemotherapy.

The present protocol describes an experimental platform to assess the effects of mechanical and biochemical cues on chemotherapeutic responses of patient-derived glioblastoma cells in 3D matrix-mimetic cultures using a custom-made UV illumination device facilitating high-throughput photocrosslinking of hydrogels with tunable mechanical features.

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes ...

Phototunable Bioprinted Hydrogels to Probe Cellular Responses to ECM Stiffening

3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture platforms and dynamically triggering changes, such as increasing microenvironmental stiffness, enables researchers to model changes in the extracellular matrix (ECM) that occur during fibrotic disease progression. Herein, a method is presented for 3D bioprinting a phototunable hydrogel biomaterial capable of two sequential polymerization reactions within a gelatin support bath. The technique of Freeform Reversible Embedding of Suspended Hydrogels (FRESH) bioprinting was adapted by adjusting the pH of the support bath to facilitate a Michael addition reaction. First, the bioink containing poly(ethylene glycol)-alpha methacrylate (PEG&#945;MA) was reacted off-stoichiometry with a cell-degradable crosslinker to form soft hydrogels. These soft hydrogels were later exposed to photoinitator and light to induce the homopolymerization of unreacted groups and stiffen the hydrogel. This protocol covers hydrogel synthesis, 3D bioprinting, photostiffening, and endpoint characterizations to assess fibroblast activation within 3D structures. The method presented here enables researchers to 3D bioprint a variety of materials that undergo pH-catalyzed polymerization reactions and could be implemented to engineer various models of tissue homeostasis, disease, and repair.

Author Spotlight: Understanding Chronic Lung Diseases Using 3D Printed Phototunable Hydrogels 

This article describes how to 3D bioprint phototunable hydrogels to study extracellular matrix stiffening and fibroblast activation.

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture ...

Preparation of DNA-crosslinked Polyacrylamide Hydrogels

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, the effect of tissue elasticity on cell function is a major area of mechanobiology research because tissue stiffness modulates with disease, development, and injury. Static tissue-mimicking materials, or materials that cannot alter stiffness once cells are plated, are predominately used to investigate the effects of tissue stiffness on cell functions. While information gathered from static studies is valuable, these studies are not indicative of the dynamic nature of the cellular microenvironment in vivo. To better address the effects of dynamic stiffness on cell function, we developed a DNA-crosslinked polyacrylamide hydrogel system (DNA gels). Unlike other dynamic substrates, DNA gels have the ability to decrease or increase in stiffness after fabrication without stimuli. DNA gels consist of DNA crosslinks that are polymerized into a polyacrylamide backbone. Adding and removing crosslinks via delivery of single-stranded DNA allows temporal, spatial, and reversible control of gel elasticity. We have shown in previous reports that dynamic modulation of DNA gel elasticity influences fibroblast and neuron behavior. In this report and video, we provide a schematic that describes the DNA gel crosslinking mechanisms and step-by-step instructions on the preparation DNA gels.

Our laboratory has developed DNA-crosslinked polyacrylamide hydrogels, a dynamic hydrogel system, to better understand the effects of modulating tissue stiffness on cell function. Here, we provide schematics, descriptions, and protocols to prepare these hydrogels.

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, ...

Studying Tissue Rigidity and Shear Stress Effects on Endothelial Cells

In Vitro Model Integrating Substrate Stiffness and Flow to Study Endothelial Cell Responses

We present an innovative in vitro model aimed at investigating the combined effects of tissue rigidity and shear stress on endothelial cell (EC) function, which are crucial for understanding vascular health and the onset of diseases such as atherosclerosis. Traditionally, studies have explored the impacts of shear stress and substrate stiffness on ECs, independently. However, this integrated system combines these factors to provide a more precise simulation of the mechanical environment of the vasculature. The objective is to examine EC mechanotransduction across various tissue stiffness levels and flow conditions using human ECs. We detail the protocol for synthesizing gelatin methacrylate (GelMA) hydrogels with tunable stiffness and seeding them with ECs to achieve confluency. Additionally, we describe the design and assembly of a cost-effective flow chamber, supplemented by computational fluid dynamics simulations, to generate physiological flow conditions characterized by laminar flow and appropriate shear stress levels. The protocol also incorporates fluorescence labeling for confocal microscopy, enabling the assessment of EC responses to both tissue compliance and flow conditions. By subjecting cultured ECs to multiple integrated mechanical stimuli, this model enables comprehensive investigations into how factors such as hypertension and aging may affect EC function and EC-mediated vascular diseases. The insights gained from these investigations will be instrumental in elucidating the mechanisms underlying vascular diseases and in developing effective treatment strategies.

We synthesized and characterized a tunable gelatin-based substrate for culturing vascular endothelial cells (ECs) under relevant vascular flow conditions. This biomimetic surface replicates both physiological and pathological conditions, enabling the study of mechanical forces on EC behavior and advancing our understanding of vascular health and disease mechanisms.

We present an innovative in vitro model aimed at investigating the combined effects of tissue rigidity and shear stress on endothelial cell (EC) function, ...