Name: A Novel Platform for In Vitro Cellular Stretching and Imaging (Video) | JoVE Journal
Uploaded: 2026-03-10T13:45:00.000+00:00
Duration: 7 min 38 s
Description: Discover a low-cost, microscope-compatible cell stretching device for mechanobiology research using primary human airway smooth muscle cells. This platform enables live imaging of calcium dynamics and traction force microscopy under controlled mechanical strain.

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5 minutes

The authors thank Mr. Noah Joseph for his help in fabricating the molds. The authors thank Heitor Mourato and Glenn Thayer at the Boston University Scientific Instrument Facility for their help with machining the device. This work was supported by the National Science Foundation (NSF) Career grant #2047207 awarded to Harikrishnan Parameswaran. H.P. conceived the research. S.E.S. and S.R.P. developed the overall design of the device, and S.E.S. and H.P. designed the isotropic experiments. S.E.S conducted the isotropic molds experiments, analyzed the experimental data, and interpreted the results along with H.P. V.C. designed the uniaxial molds, optimized gel preparation and beading. B.M.G conducted the uniaxial gel experiments, analyzed the experimental data, and interpreted the results along with H.P. and V.C. S.E.S. and B.M.G prepared the figures. B.M.G, S.E.S, V.C. and H.P contributed to the drafting and editing of the manuscript. H.P approved the final version of the manuscript.

Primary human airway smooth muscle cells (ASMCs) used in this study were obtained from ATCC (Virginia), and donor information was de-identified in accordance with NIH guidelines. This study was approved by the Biosafety Committee of Northeastern University prior to experimentation.
NOTE: For instructions regarding the fabrication of the device as well as device dimensions, please see the CAD file available at our GitHub repository: https://github.com/Breathe-Lab/A-Novel-Platform-for-In-Vitro-Cellular-Stretching-and-Imaging.
1. Design of 3D printed mold
<ol>
	<li>Isotropic mold dimensions
	<ol>
		<li>Outer diameter of the mold is 37 mm.</li>
		<li>Outer height of the mold is 4.1 mm.</li>
		<li>Outer wall thickness is 2.1 mm.</li>
		<li>Base thickness of the mold is 2 mm.</li>
		<li>Attachment holes are located 2 mm from the edge.</li>
		<li>The inner cell culture well area has a diameter of 20 mm and a depth of 1.9 mm. 
		NOTE: The completed PDMS mold should have a cell culture area thickness of 200 &#181;m.</li>
	</ol>
	</li>
	<li>Uniaxial mold dimensions
	<ol>
		<li>Outer dimensions of the mold are 41 mm x 12 mm x 4 mm (L x W x H).</li>
		<li>Attachment holes are 3 mm from the width edge and 4 mm from the length edge with a post depth of 1 mm.</li>
		<li>Outer wall thickness is 1.5 mm on the width edge and 2 mm on the length edge.</li>
		<li>The inner cell culture well dimensions are 26 mm x 5.5 mm x 1 mm (L x W x H).</li>
		<li>Base thickness is 2 mm. 
		&#8203;NOTE: Molds were designed in SolidWorks, 3D printed using a FormLabs liquid grey resin system, and fabricated at Northeastern University &#39;s makerspace.</li>
	</ol>
	</li>
	<li>Instructions only for the initial use of the mold
	<ol>
		<li>Sand down the rough edges of the mold print.</li>
		<li>Coat the negative mold with clear nail polish to facilitate subsequent removal of the PDMS mold after curing. Allow the mold to air-dry for 24 h.</li>
	</ol>
	</li>
</ol>
2. Fabrication of optically clear, elastic gels with tunable substrate stiffness for cell culture
NOTE: Wear appropriate PPE (gloves, lab coat, eye protection) when handling PDMS and NuSil.
<ol>
	<li>Add Sylgard 184 base and curing agent in a 10:1 ratio by weight into a plastic dish.</li>
	<li>Mix vigorously with tongue depressor (or similar tool) for 2-3 min.</li>
	<li>Degas the mixture for 1 h.</li>
	<li>Pour the PDMS mixture into the custom negative mold (Figure 2A,B).
	<ol>
		<li>Add 1.3 g to the isotropic mold.</li>
		<li>Add 0.5 g for the uniaxial mold. 
		&#8203;NOTE: Take care to spread the gel evenly across the mold.</li>
	</ol>
	</li>
	<li>Degas the mold for 1 h.</li>
	<li>Allow the gel in the mold to settle on a flat surface at room temperature for 2 h.</li>
	<li>Cure at 60 &#176;C overnight.</li>
	<li>Tumble mix parts A and B of NuSil Gel-8100 in equal proportions (1:1) for 15 min.
	<ol>
		<li>Mix with Sylgard 184 crosslinker at 0.36% of total volume to produce a substrate with a stiffness of 13 kPa 32.</li>
		<li>Add no crosslinker to produce a substrate with a stiffness of 0.3 kPa.</li>
	</ol>
	</li>
	<li>Degas the mixture for 30 min.</li>
	<li>Carefully remove the PDMS gel from the custom mold. Place the gel onto a 40 mm coverslip. 
	NOTE: Take care not to tear the gels as they are fragile.</li>
	<li>Pour the NuSil mixture onto the negative area of the PDMS gel.
	<ol>
		<li>Isotropic gel: 100 &#181;L.</li>
		<li>Uniaxial gel: 50 &#181;L.</li>
	</ol>
	</li>
	<li>Coat the respective PDMS gels with the NuSil mixture using a spin coater for 50 s at 7 x g to attain a uniform substrate thickness of 100 &#181;m.</li>
	<li>Allow the gel to settle on a flat level surface at room temperature for 2 h before being cured at 60 &#176;C overnight. 
	NOTE: Please take care to ensure all curing steps are performed on a level surface. An uneven gel will not allow the full region of interest to remain in the imaging plane of the microscope. If imaging reveals focus variation across the field of view, the gel may have cured on an unlevel surface and should be remade.</li>
	<li>Store the completed cell culture dishes in covered petri dishes until use (Figure 2D-E).</li>
</ol>
3. Matrix protein coating
<ol>
	<li>Add 500 &#181;L of poly-L-lysine (PLL, 500 &#181;g/mL) to each substrate.</li>
	<li>Incubate at room temperature for 1 h.</li>
	<li>Wash substrates 3 &#215; with 1 mL of PBS, followed by 3 &#215; with 1 mL of 10 mM HEPES buffer (pH 8.0).</li>
	<li>Add 500 &#181;L of mPEG-SVA (50 mg/mL) to each substrate.</li>
	<li>Incubate at room temperature for 1 h.</li>
	<li>Wash substrates 3 &#215; with 1 mL of PBS.</li>
	<li>Replace PBS with 500 &#181;L of PLPP (14.5 mg/mL).</li>
	<li>Using the PRIMO micropatterning system with Leonardo software, project the predefined pattern onto the substrate surface with UV light for 30 s.</li>
	<li>Wash substrates 3 &#215; with 1 mL of PBS and place under UV for 15 min for sterilization.</li>
	<li>Add 500 &#181;L of 0.1% fluorescein conjugated gelatin to each substrate.</li>
	<li>Incubate at room temperature for 1 h.</li>
	<li>Wash 3 &#215; with 1 mL of PBS. 
	NOTE: To visualize patterns, incubate with 0.1% fluorescein-conjugated gelatin for 1 h at room temperature.</li>
	<li>Proceed with imaging.</li>
</ol>
4. Human Airway smooth muscle cell culture in elastic dishes
NOTE: Perform all cell culture procedures in a biosafety hood using standard aseptic technique.
<ol>
	<li>Obtain primary human airway smooth muscle cells (ASMCs) from ATCC (Virginia) and ensure donor information is de-identified in accordance with NIH guidelines. Confirm Institutional Biosafety Committee approval prior to experimentation.</li>
	<li>Use cells from three healthy human donors of different sexes and ages with no history of asthma/ disease. Example: one female (age 18) and two males (ages 34 and 59).</li>
	<li>Culture cells under standard conditions (37 &#176;C, 5% CO &#8322;) and use cells up to passage 6 for all experiments.</li>
	<li>Grow cells in the following complete growth medium:
	<ol>
		<li>DMEM/F12 (500 mL)</li>
		<li>10% fetal bovine serum (FBS) (50 mL)</li>
		<li>1&#215; penicillin/streptomycin (5 mL)</li>
		<li>1&#215; MEM nonessential amino acids (5 mL)</li>
		<li>Amphotericin B (0.5 mL)</li>
	</ol>
	</li>
	<li>Switch cells to serum-free medium at least 24 h before measurements. Prepare serum-free medium with:
	<ol>
		<li>Ham &#39;s F-12 medium (500 mL)</li>
		<li>1 &#215; penicillin/streptomycin (5 mL)</li>
		<li>Amphotericin B (50 &#181;g/L)</li>
		<li>1&#215; MEM nonessential amino acids (5 mL)</li>
	</ol>
	</li>
	<li>UV-sterilize stretchable cell culture dishes for 15 min.</li>
	<li>Incubate sterilized dishes with 0.1% gelatin for 1 h at room temperature prior to seeding (Figure 2F).
	<ol>
		<li>For Patterned Substrates:
		<ol>
			<li>Seed cells in elastic cell culture wells at a density of 104&#160;cells/cm2.</li>
			<li>Incubate in 10% serum medium for 10 min to promote attachment to patterned areas.</li>
			<li>Wash wells with PBS to remove unattached cells.</li>
			<li>Fill wells with 10% serum medium and incubate for 24 h.</li>
			<li>Replace with serum-free medium and incubate for 24 h before measurements.</li>
		</ol>
		</li>
		<li>For Non-Patterned Substrates:
		<ol>
			<li>Seed cells at the desired density: 102&#160;cells/cm2&#160;for isolated cells. 103&#160;cells/cm2&#160;for sparse cells. 104cells/cm2&#160;for confluent cells.</li>
			<li>Incubate in 10% serum medium for 24 h.</li>
			<li>Replace with serum-free medium and incubate for 24 h before measurements.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
5. Fluorescent calcium imaging and analysis
<ol>
	<li>Serum-starve airway ASMCs for 24 h.</li>
	<li>Prepare FLIPR Ca2+&#160;6 dye according to manufacturer &#39;s instructions.</li>
	<li>Mix FLIPR Ca2+&#160;6 dye with serum-free medium at a 1:1 ratio.</li>
	<li>Incubate cells with the FLIPR Ca &#178; &#8314; 6 solution at 37 &#176;C and 5% CO &#8322; for 2 h.</li>
</ol>
6. Imaging Setup
<ol>
	<li>Use a Leica DMi8 inverted microscope equipped with:
	<ol>
		<li>Leica DFC6000 camera</li>
		<li>Leica LED8 light engine</li>
		<li>5&#215;/0.12 dry objective</li>
	</ol>
	</li>
	<li>Set excitation at 480/40 nm and emission at 527/50 nm.</li>
	<li>Acquire 16-bit images at a frame rate of 2 Hz during stretch experiments.</li>
</ol>
7. Data analysis
<ol>
	<li>Import image sequences into Fiji (ImageJ) 33.</li>
	<li>Manually define regions of interest (ROIs) in the cytoplasm of each cell.</li>
	<li>Extract the mean grayscale intensity values for each ROI over time.</li>
	<li>Process fluorescence intensity data using a custom MATLAB script. 
	NOTE: Fluorescence intensity is proportional to cytosolic [Ca2+].</li>
</ol>
8. Traction force measurements and analysis
NOTE: All codes, including those for image analysis, traction force microscopy, and motor control, are available at our GitHub repository:&#160;<a href="https://github.com/Breathe-Lab/A-Novel-Platform-for-In-Vitro-Cellular-Stretching-and-Imaging">https://github.com/Breathe-Lab/A-Novel-Platform-for-In-Vitro-Cellular-Stretching-and-Imaging</a>
<ol>
	<li>Coat PDMS-NuSil gel substrates with a layer of fluorescent beads to serve as fiducial markers for traction force microscopy.</li>
	<li>Prepare a 0.025% solution of 1 &#181;m diameter red fluorescent carboxylate-modified microspheres in HBSS by diluting to a 1:4000 ratio.</li>
	<li>Vortex the bead solution for 10 s.</li>
	<li>Add the bead solution to the gel.
	<ol>
		<li>880 &#181;L to isotropic gel.</li>
		<li>420 &#181;L to uniaxial gel.</li>
	</ol>
	</li>
	<li>Incubate at room temperature for 1 h to allow the beads to adhere.</li>
	<li>After 1 h, carefully remove the solution. 
	NOTE: Pour off the bead solution gently on soft gels. Improper removal prevents proper bead distribution.</li>
	<li>Gently blot any residual solution with a Q-tip or Kimwipe.</li>
	<li>Image the beads with a Leica DMi8 microscope equipped with a 10x/0.32 dry objective.</li>
	<li>Capture images in the pre-stretch condition and continuously throughout the stretching protocol. 
	NOTE: Perform all imaging at the center of the substrate where strain uniformity is highest. If needed, refocus the microscope at the stretched position before capturing images.</li>
	<li>Analyze the images using a custom MATLAB code based on Fourier traction force microscopy to calculate displacement vectors and corresponding planar strain fields 34.</li>
	<li>Represent data as the average of N trials.</li>
</ol>
9. Calibration of the cell stretching device
<ol>
	<li>Add 500 &#181;L of H&#8322;O to the well to simulate the weight of cell culture media and keep the protein pattern moist.</li>
	<li>For protocol regarding creating specific protein patterns, please see section 3 titled &#34;Matrix Protein Coating &#34;.</li>
	<li>Image fluorescent patterns every 400 &#181;m steps of motor displacement, ramping up to a maximum of 2800 &#181;m, and then returning to zero in reverse 400 &#181;m increments.</li>
	<li>Characterize the relationship between linear displacement from the stepper motor and isotropic deformation of the stretchable cell culture dish at both macroscopic and regional scales by measuring the changes in area and eccentricity of fluorescent protein-patterned letters spelling &#34;BREATHE &#34; on the elastic substrate during stretching. Use a rectangular bounding box encompassing all letters to calculate the area and eccentricity of the entire word.</li>
	<li>Apply image segmentation in MATLAB to quantify these parameters for each individual letter.</li>
	<li>Define zero displacement by the unstretched dimensions of the cell culture dish.</li>
	<li>Repeat each experiment across two to three independently prepared stretchable cell culture wells to ensure reproducibility.</li>
</ol>
10. Data presentation and statistical testing
<ol>
	<li>For stretcher calibration experiments, define trials as the number of cycles.</li>
	<li>Show each trial separately instead of combining them into a single curve to demonstrate system repeatability.</li>
	<li>Indicate error bars as standard deviation.</li>
	<li>Use a one-way ANOVA to test for significant differences in datasets with three or more groups influenced by one independent factor.</li>
	<li>Use two-way ANOVAs followed by post hoc pairwise comparisons to test for significant differences in datasets influenced by two independent factors.</li>
	<li>Apply the t-test for pairwise comparisons when data are normally distributed.</li>
	<li>Apply the Mann-Whitney rank sum test to compare median values when data are not normally distributed.</li>
	<li>Report the specific tests used, the number of samples, and the corresponding P-values, along with the results.</li>
	<li>Use a P-value threshold of 0.05 to determine statistically significant differences between datasets.</li>
	<li>Perform statistical tests in SigmaStat.</li>
	<li>Plot figures in GraphPad Prism and MATLAB.</li>
</ol>

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<li>Atcha, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+low-cost+mechanical+stretching+device+for+uniaxial+strain+of+cells%3A+a+platform+for+pedagogy+in+mechanobiology%5BTitle%5D)+AND+%22J+Biomech+Eng%22%5BJournal%5D)">A low-cost mechanical stretching device for uniaxial strain of cells: a platform for pedagogy in mechanobiology.</a> J Biomech Eng. 140, 0810051-0810059 (2018).</li>
<li>Tschumperlin, D. J., Margulies, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Equibiaxial+deformation-induced+injury+of+alveolar+epithelial+cells+in+vitro%5BTitle%5D)+AND+%22Am+J+Physiol%22%5BJournal%5D)">Equibiaxial deformation-induced injury of alveolar epithelial cells in vitro.</a> Am J Physiol. 275, L1173-L1183 (1998).</li>
<li>Meza, D., Abejar, L., Rubenstein, D. A., Yin, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+shearing-stretching+device+that+can+apply+physiological+fluid+shear+stress+and+cyclic+stretch+concurrently+to+endothelial+cells%5BTitle%5D)+AND+%22J+Biomech+Eng%22%5BJournal%5D)">A shearing-stretching device that can apply physiological fluid shear stress and cyclic stretch concurrently to endothelial cells.</a> J Biomech Eng. 138, 4032550(2016).</li>
<li>Ursekar, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Design+and+construction+of+an+equibiaxial+cell+stretching+system+that+is+improved+for+biochemical+analysis%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Design and construction of an equibiaxial cell stretching system that is improved for biochemical analysis.</a> PLoS One. 9, e90665(2014).</li>
<li>Pelham, R. J., Wang, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+locomotion+and+focal+adhesions+are+regulated+by+substrate+flexibility%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Cell locomotion and focal adhesions are regulated by substrate flexibility.</a> Proc Natl Acad Sci U S A. 94, 13661-13665 (1997).</li>
<li>Cell stretching bioreactor FAQs. , Flexcell International. 
 <a target="_blank" href="https://www.flexcellint.com/resources/faq">https://www.flexcellint.com/resources/faq</a> (2024).</li>
<li>Bossé, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+strain+on+airway+smooth+muscle+during+a+deep+inspiration+to+total+lung+capacity%5BTitle%5D)+AND+%22J+Eng+Sci+Med+Diagn+Ther%22%5BJournal%5D)">The strain on airway smooth muscle during a deep inspiration to total lung capacity.</a> J Eng Sci Med Diagn Ther. 2, 010802(2019).</li>
<li>Yoshie, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Traction+force+screening+enabled+by+compliant+PDMS+elastomers%5BTitle%5D)+AND+%22Biophys+J%22%5BJournal%5D)">Traction force screening enabled by compliant PDMS elastomers.</a> Biophys J. 114, 2194-2199 (2018).</li>
<li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fiji%3A+an+open+source+platform+for+biological+image+analysis%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Fiji: an open source platform for biological image analysis.</a> Nat Methods. 9 (7), 676-682 (2012).</li>
<li>Butler, J. P., Toli ć-Nørrelykke, I. M., Fabry, B., Fredberg, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Traction+fields%2C+moments%2C+and+strain+energy+that+cells+exert+on+their+surroundings%5BTitle%5D)+AND+%22Am+J+Physiol+Cell+Physiol%22%5BJournal%5D)">Traction fields, moments, and strain energy that cells exert on their surroundings.</a> Am J Physiol Cell Physiol. 282, C595-C605 (2002).</li>
<li>Dessalles, C. A., Leclech, C., Castagnino, A., Barakat, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integration+of+substrate-+and+flow-derived+stresses+in+endothelial+cell+mechanobiology%5BTitle%5D)+AND+%22Commun+Biol%22%5BJournal%5D)">Integration of substrate- and flow-derived stresses in endothelial cell mechanobiology.</a> Commun Biol. 4, 764(2021).</li>
<li>Ji, G., Barsotti, R. J., Feldman, M. E., Kotlikoff, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stretch-induced+calcium+release+in+smooth+muscle%5BTitle%5D)+AND+%22J+Gen+Physiol%22%5BJournal%5D)">Stretch-induced calcium release in smooth muscle.</a> J Gen Physiol. 119, 533-543 (2002).</li>
<li>Davis, M. J., Meininger, G. A., Zawieja, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stretch-induced+increases+in+intracellular+calcium+of+isolated+vascular+smooth+muscle+cells%5BTitle%5D)+AND+%22Am+J+Physiol%22%5BJournal%5D)">Stretch-induced increases in intracellular calcium of isolated vascular smooth muscle cells.</a> Am J Physiol. 263, H1292-H1299 (1992).</li>
<li>Naruse, K., Sokabe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Involvement+of+stretch-activated+ion+channels+in+Ca2%2B+mobilization+to+mechanical+stretch+in+endothelial+cells%5BTitle%5D)+AND+%22Am+J+Physiol%22%5BJournal%5D)">Involvement of stretch-activated ion channels in Ca2+ mobilization to mechanical stretch in endothelial cells.</a> Am J Physiol. 264, C1037-C1044 (1993).</li>
<li>Arold, S. P., Bartol ák-Suki, E., Suki, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Variable+stretch+pattern+enhances+surfactant+secretion+in+alveolar+type+II+cells+in+culture%5BTitle%5D)+AND+%22Am+J+Physiol+Lung+Cell+Mol+Physiol%22%5BJournal%5D)">Variable stretch pattern enhances surfactant secretion in alveolar type II cells in culture.</a> Am J Physiol Lung Cell Mol Physiol. 296, L574-L581 (2009).</li>
<li>Cell stretching bioreactors. , Flexcell International. 
 <a target="_blank" href="https://www.flexcellint.com">https://www.flexcellint.com</a> (2024).</li>
<li>Mitrossilis, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+response+to+stiffness+exhibits+muscle-like+behavior%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Single-cell response to stiffness exhibits muscle-like behavior.</a> Proc Natl Acad Sci U S A. 106, 18243-18248 (2009).</li>
<li>Parameswaran, H., Lutchen, K. R., Suki, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+computational+model+of+the+response+of+adherent+cells+to+stretch+and+changes+in+substrate+stiffness%5BTitle%5D)+AND+%22J+Appl+Physiol%22%5BJournal%5D)">A computational model of the response of adherent cells to stretch and changes in substrate stiffness.</a> J Appl Physiol. 116, 825-834 (2014).</li>
</ol>

None of the authors has any competing interests to declare.

In this protocol, we describe the design, development, validation, and implementation of a novel microscope compatible cell stretcher that can mimic isotropic and uniaxial strains while simultaneously imaging live cells. This device is compact and can fit on a Leica DMi8 microscope stage, confining all moving parts to a single plane to allow for live imaging while maintaining focus on the substrate and underlying cells. This was done utilizing angled arms inspired by the geometry of a two-dimensional Hoberman sphere. Thi...

Cells experience mechanical forces from their surrounding environment that play a critical role in regulating many aspects of cellular function, including determining cell fate1,2, intercellular communication3,4, and healthy growth2,5. Additionally, changes in these mechanical forces can lead to increased disease progression and onset of disease, including extracellular matrix (ECM) remodeling6 and tumor growth7,8,9,10. Cells can sense and respond to these mechanical cues, which include changes in the stiffness of the ECM4, as well as cyclic strain in organs such as the heart, lungs, and muscles11,12. In vitro studies have demonstrated that ECM stiffness and composition can influence the rigidity of the cell cytoskeleton, both at rest and following exposure to contractile agonists13. Numerous in vitro and in situ studies have examined how transient, static, and cyclic strains affect cells and tissues, showing that responses vary based on strain type, rate, and amplitude14. For example, recent studies found that a temporary stretch reduces the stiffness of adherent cells in proportion to the applied strain, wherein a sustained increase in strain results in an initial rapid increase in stiffness, followed by a gradual relaxation15. Additionally, calcium signaling, the primary molecular regulator of force generation, is impacted by stretch and other mechanical forces4,16,17,18,19,20. Studying how stiffness and strain influence mechanobiological pathways requires modulating both factors while monitoring cellular responses. Therefore, we developed a novel microscope-compatible cell stretcher capable of inducing either isotropic or uniaxial stretch, with independent control over substrate stiffness, matrix protein coating, and a range of programmable waveforms.
In the past, researchers have similarly created devices to impose specific motions and mechanical stretches on cell cultures and tissues21,22. The simplest case, uniaxial motion, is a mechanical stretch imposed on a single dimension. For example, some devices have been developed to impose mechanical stretch and strain on tissue strips23 and collagen fibers24, or to align cellular orientation and induce cellular migration25. Isotropic stretch refers to a type of stretching in which an equal force is applied to a gel or substrate from all directions, resulting in uniform strain across the material. This stretch simulates the even force distribution that cells experience in certain physiological environments, such as within the alveoli of the lungs, where cells are subjected to radial expansion and contraction during the breathing process26,27.
Existing stretcher devices have several limitations that constrain their use for mechanobiology-related research. Many designs use substrate stiffness values exceeding the typical physiological range to withstand mechanical strain and support the weight of cell culture media28. However, the use of such non-physiological stiffness may also affect cell behavior, potentially limiting the relevance of the model to natural tissue environments29. Additionally, some stretcher designs obstruct the objective or light path, preventing imaging, while others use membranes too thick for clear observation28. Even when stretchers are compatible with microscopes and use thin substrates, issues like membrane sagging and focus loss are common. Commercial platforms such as Flexcell use vacuum-based deformation to stretch flexible membranes. While this approach can generate stable, homogeneous strain fields, it is not optimized for live-cell microscopy, and microscope-compatible configurations require expensive additional accessories.&#160;Additionally, these platforms can achieve a maximum strain of only 21.8%30, whereas deep inspirations can produce strains exceeding 30%, with even higher strains observed in diseases like asthma and COPD31.
Therefore, we designed a novel cell stretching device that enables precise material control of substrate stiffness and ECM composition, while allowing for variable isotropic and uniaxial strains to be applied to adherent cells. This platform is well-suited for researchers investigating mechanobiological questions who require live imaging during mechanical stimulation with substrates with physiologically relevant stiffness. The device uses a mechanically driven approach inspired by the geometry of a two-dimensional Hoberman sphere, which expands and contracts through scissor-like joints (Figure 1, Supplementary Figure 1). A single stepper motor drives 16 radially arranged arms through a linkage mechanism, producing coordinated expansion and contraction of eight attachment posts. This configuration generates homogeneous in-plane strain by applying equal radial displacement to all posts simultaneously. The unique design and compact footprint of the device facilitate live cell imaging with an inverted fluorescence microscope during in-plane isotropic and uniaxial stretch, at a fraction of the cost of commercial alternatives. The linear stepper motor is controlled with a custom Arduino code, enabling programmable cyclic waveforms with adjustable periods and amplitudes. In this study, we demonstrate that the device delivers precise and reproducible strains ranging from less than 1% to 15%, with a geometric capacity exceeding 200%. Additionally, we quantify the effects of mechanical stretch on intracellular calcium dynamics and cell traction forces of primary human airway smooth muscle cells.

<table><tbody><tr><td>&amp;nbsp;Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium/F12</td><td>Fisher Scientific</td><td>12634010</td><td>500 mL&amp;nbsp;</td></tr><tr><td>(4-benzoylbenzyl) trimethylammonium chloride (PLPP)</td><td>Alv&amp;eacute;ole</td><td>N/A</td><td>14.5mg/mL</td></tr><tr><td>0.1% fluorescein conjugated gelatin&amp;nbsp;</td><td>Invitrogen</td><td>G13187</td><td></td></tr><tr><td>0.1% Gelatin Solution</td><td>Sigma-Aldrich</td><td>ES-006</td><td>500 mL&amp;nbsp;</td></tr><tr><td>1 &amp;mu;m diameter red fluorescent carboxylate-modified microspheres</td><td>FluoSpheres</td><td>F8816</td><td></td></tr><tr><td>1&amp;times; MEM nonessential amino acid solution</td><td>Sigma-Aldrich</td><td>M7145</td><td>100 mL</td></tr><tr><td>1&amp;times; penicillin/streptomycin</td><td>Fisher Scientific</td><td>15-140-122</td><td>100 mL</td></tr><tr><td>10% fetal bovine serum</td><td>Thermo-Scientific</td><td>A5256701</td><td>500 mL&amp;nbsp;</td></tr><tr><td>15 mL tube</td><td>Fisher Scientific</td><td>14-959-53A</td><td></td></tr><tr><td>1x glutamine&amp;nbsp;</td><td>Fisher Scientific</td><td>MT10010CM</td><td></td></tr><tr><td>1x Insulin-Transferrin-Selenium Growth Supplement</td><td>Corning Life Sciences</td><td>25-800-CR</td><td></td></tr><tr><td>amphotericin B</td><td>Sigma-Aldrich</td><td>A2942</td><td>25 &amp;mu;g/liter</td></tr><tr><td>Ball Bearings</td><td>McMaster-Carr&amp;nbsp;</td><td>N/A</td><td></td></tr><tr><td>Calcium Chloride Dihydrate</td><td>Sigma-Aldrich</td><td>223506</td><td></td></tr><tr><td>Carriage Rail</td><td>McMaster-Carr&amp;nbsp;</td><td>N/A</td><td></td></tr><tr><td>FLIPR Ca&lt;sup&gt;2+&lt;/sup&gt; 6</td><td>Molecular Devices</td><td>R8190</td><td></td></tr><tr><td>GraphPad Prism</td><td>Dotmatics</td><td>N/A</td><td></td></tr><tr><td>Ham&amp;rsquo;s F-12 medium&amp;nbsp;</td><td>Sigma-Aldrich</td><td>N8641</td><td>500 mL&amp;nbsp;</td></tr><tr><td>Hanks&#039; Balanced Salt Solution&amp;nbsp;</td><td>Thermo-Scientific</td><td>14025092</td><td>500 mL&amp;nbsp;</td></tr><tr><td>HEPES buffer&amp;nbsp;</td><td>Thermo-Scientific</td><td>15630080</td><td>100 mL</td></tr><tr><td>Leonardo Software</td><td>Alv&amp;eacute;ole</td><td>N/A</td><td></td></tr><tr><td>Matlab</td><td>Mathworks</td><td>N/A</td><td></td></tr><tr><td>methoxy polyethylene glycol&amp;ndash;Succinimidyl Valerate (mPEG-SVA)</td><td>Laysan Bio Inc</td><td>N/A</td><td>50mg/mL</td></tr><tr><td>Nusil Gel-8100</td><td>Nusil&amp;nbsp;</td><td>N/A</td><td></td></tr><tr><td>Petri Dishes</td><td>Thermo-Scientific</td><td>263991</td><td>90 mm&amp;nbsp;</td></tr><tr><td>phosphate-buffered saline&amp;nbsp;</td><td>Sigma-Aldrich</td><td>10010023</td><td>500 mL&amp;nbsp;</td></tr><tr><td>poly-L-lysine (PLL)</td><td>Sigma-Aldrich</td><td>P8920</td><td>500&amp;mu;g/mL</td></tr><tr><td>Primary human airway smooth muscle cells&amp;nbsp;</td><td>ATCC</td><td>PCS-130-011</td><td></td></tr><tr><td>PRIMO optical module&amp;nbsp;</td><td>Alv&amp;eacute;ole</td><td>N/A</td><td></td></tr><tr><td>Screws</td><td>McMaster-Carr&amp;nbsp;</td><td>N/A</td><td></td></tr><tr><td>SigmaStat</td><td>Systat Software</td><td>N/A</td><td></td></tr><tr><td>Spin Coater</td><td>Laurell</td><td>Model WS-650MZ-23NPPB</td><td></td></tr><tr><td>Stretcher and Stage Attachment Components&amp;nbsp;</td><td>Boston University&#039;s Scientific Instrument Facility</td><td>N/A</td><td>Stainless Steel</td></tr><tr><td>Sylgard 184 base Elastomer and Curing agent kit</td><td>Dow</td><td>H047J8Q026</td><td></td></tr><tr><td>T-25 Tissue Culture Flask - Vent Cap, Sterile</td><td>CellTreat</td><td>229331</td><td></td></tr><tr><td>Tongue depressor&amp;nbsp;</td><td>Thermo-Scientific</td><td>22-363-154</td><td></td></tr></tbody></table>

a novel platform for in vitro cellular stretching and imaging

A Novel Platform for In Vitro Cellular Stretching and Imaging

Cells respond to mechanical cues from their environment, such as changes in extracellular matrix (ECM) stiffness and cyclic strain, which regulate cellular processes including cell fate determination, intercellular communication, and development. Alterations in these forces contribute to or drive disease progression in conditions like asthma, hypertension, and cancer. While existing in-vitro stretching devices can impose uniaxial or isotropic strains, they often use non-physiological stiffnesses, limit live imaging, or cannot achieve high strain amplitudes relevant to physiological and pathological conditions. Here, we present a compact, microscope-compatible stretcher device that applies controlled isotropic or uniaxial strain to adherent cells on elastomeric culture dishes. These dishes feature a tunable Young&#39;s modulus and are compatible with a variety of matrix protein coatings for cell adhesion, allowing independent modulation of substrate stiffness and ECM composition. Importantly, the device&#39;s ease of operation is facilitated by its stepper motor-driven design, which supports the generation of programmable cyclic waveforms. We demonstrate the device&#39;s capability by quantifying intracellular calcium dynamics and cell traction forces in primary human airway smooth muscle cells under mechanical stretch. This platform provides a versatile tool for investigating the effect of mechanical cues on cellular function in both healthy and disease-relevant contexts.

The cell stretcher induces macroscopic area changes that reflect localized deformations. To assess isotropic behavior, we measured the area changes of a fluorescent gelatin protein pattern forming the word "BREATHE" on a stretchable cell culture well, along with its individual letters, on an isotropic substrate. Our results demonstrated that both the word and the individual letters exhibited equivalent changes in area (Figure 3A,B). In Figure 3B...

Here, we present a novel device that applies isotropic and uniaxial strain waveforms to cells while supporting culture on substrates with tunable stiffness and customizable protein matrices. This simple, economical platform enables researchers to investigate how mechanical cues regulate cellular function in both fundamental studies and disease-relevant contexts.

Cells respond to mechanical cues from their environment, such as changes in extracellular matrix (ECM) stiffness and cyclic strain, which regulate ...

Our goal was to develop a low cost hoberman inspired microscope compatible cell stretching device to investigate how mechanical stretch influences cellular function. This device allows researchers to study life cell responses to mechanical stimuli, mimicking physiological systems such as a beating heart or breathing lungs. To begin where appropriate personal protective equipment.Mix silgard 184 base and curing agent in a 10 to one ratio in a plastic dish. With a tongue depressor mix the components vigorously for two to three minutes. Then place the dish in a degassing chamber for one hour.Next, take the prepared 3D printed molds and add 1.3 grams of the PDMS mixture into the isotropic mold. Then add 0.5 grams of the PDMS mixture to the uni axial mold. Spread the gel evenly across the mold and degas the filled mold for one hour.Allow the gel in the mold to settle on a flat surface at room temperature for two hours. Then cure the mold in an oven at 60 degrees celsius overnight. Next, mix parts A and B of NuSil gel 8100 in equal proportions.Mix the combined gel with silgard 184 crosslinker at 0.36%of the total volume to produce a substrate stiffness of 13 kilopascal. To produce a soft substrate with a stiffness of 0.3 kilopascal, skip adding the crosslinker, then tumble mix the solution for 15 minutes. Place the NuSil mixture in a degassing chamber and degas for 30 minutes.Now carefully remove the PDMS gel from the custom mold and place it onto a 40 millimeter cover slip without tearing it. Next place the PDMS gel in the spin coder. Pipette 100 microliters of the NuSil mixture onto the negative area of the isotropic PDMS gel.Then pipette 50 microliters of the NuSil mixture onto the negative area of the uni axial PDMS gel. After that code at seven G for 50 seconds. To attain a thickness of 100 micrometers.Allow the coated gel to settle on a flat level surface at room temperature for two hours. Place the gel in an oven and cure at 60 degrees Celsius overnight. Now seed human airway, smooth muscle cells in elastic cell culture wells and incubate under standard conditions.Afterwards, mix cells with the FLIPR Calcium six solution for imaging. For traction force measurement, coat the PDMS NuSil gel substrates with a layer of fluorescent beads to serve as fiducial markers. Prepare a 0.025%solution of one micrometer diameter red fluorescent carboxylate modified microspheres in HBSS.Using a vortex mixer vortex the bead solution for 10 seconds. Then pipette 880 microliters of the bead solution to the isotropic gel. Add 420 microliters of the bead solution to the uni axial gel.After incubating the gel at room temperature for one hour, carefully remove the bead solution to preserve bead distribution. Then use a cotton swab or laboratory wipe to gently blot any residual solution from the gel surface. Next, use an inverted microscope equipped with a camera, light engine, and a 10 x dry objective.Set the excitation to 475 by 40 nanometers and the emission to 555 by 50 nanometers. Acquire 16 bit images at a frame rate of two hertz during stretch experiments. Now place isotropic gel on the stretcher and then image the fluorescent beads using an inverted microscope equipped with a 10 x dry objective Capture images in the pre-stretch condition.Continuously capture images throughout the stretching protocol at the center of the substrate. Refocus the microscope at the stretched position if necessary before capturing images. Analyze the images using custom MATLAB code based on furier traction force microscopy to calculate displacement vectors and corresponding planar strain fields.Represent the data as the average of end trials. Applying a 240 micrometer displacement caused fluorescent beads to shift radially outward from the center. Traction force microscopy revealed an isotropic displacement field with vectors radiating outward from the center and increasing in magnitude with distance.The isotropic displacement field generated a homogeneous planar strain field. Increasing the displacement to 1000 micrometers resulted in displacement vectors that increased in magnitude while maintaining radial orientation. The corresponding strain field at 1000 micrometers increased in magnitude while remaining radially symmetric.In uni axial gels subjected to a 250 micrometer displacement, displacement vectors were predominantly aligned along a single axis, consistent with uni axial strain. The strain waveforms closely mirrored the programmed motor displacements and remained synchronized across three repeated cycles. Strain values at maximum displacement remained consistent across three independent samples.Applying a transient displacement up to 5, 555 micrometers produced strains ranging from greater than 0.1%to 12%A transient 0.5%strain induced an increase in intracellular calcium, evident as higher fluorescence intensity after stretch. Using our stretcher, researchers can conduct live cell calcium imaging, traction force microscopy, and quantify cellular mechanical responses to isotropic or uni axial stretch. A critical step is to ensure that gels cure on a level surface as uneven thickness can shift the substrate out of focus and reduce optical clarity during live imaging.Future studies can investigate stretch induced cytoskeletal reorientation, pathological strain conditions relevant to asthma or lung injury, and across diverse cell types.

a-novel-platform-for-in-vitro-cellular-stretching-and-imaging

Research

JoVE Journal

Bioengineering

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

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

A novel imaging protocol was developed using a custom motor-driven mechanical actuator to allow the measurement of real time responses to mechanical strain in live cells. Relevant to mechanobiology, the system can apply strains up to 20% while allowing near real-time imaging with confocal or atomic force microscopy.

Live Cell Imaging during Mechanical Stretch

Using a previously designed device to apply mechanical strain to adherent cells, this paper describes a redesigned substratum geometry and a customized apparatus for high-resolution single-cell imaging of strained cells with a 100x oil immersion objective.

High-resolution Imaging of Nuclear Dynamics in Live Cells under Uniaxial Tensile Strain

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

This work presents a flexible protocol for utilizing fluorescently labeled elastomeric contractible surfaces (FLECS) Technology in microwell format for simplified, hands-off quantification of single-cell contractile forces based on visualized displacements of fluorescent protein micropatterns.

Simplified, High-throughput Analysis of Single-cell Contractility using Micropatterned Elastomers

Here, we present a versatile equibiaxial stretching device that is pneumatically actuated and compatible with high-resolution microscopy.

Equibiaxial Stretching Device for High Magnification Live-Cell Confocal Fluorescence Microscopy

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment.  Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the 'larva chip' for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Discover a low-cost, microscope-compatible cell stretching device for mechanobiology research using primary human airway smooth muscle cells. This platform enables live imaging of calcium dynamics and traction force microscopy under controlled mechanical strain.

A Novel Platform for In Vitro Cellular Stretching and Imaging

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