A Novel Platform for In Vitro Cellular Stretching and Imaging

Summary

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.

Abstract

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'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's ease of operation is facilitated by its stepper motor-driven design, which supports the generation of programmable cyclic waveforms. We demonstrate the device'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.

Introduction

Cells experience mechanical forces from their surrounding environment that play a critical role in regulating many aspects of cellular function, including determining cell fate^1,2, intercellular communication^3,4, and healthy growth^2,5. Additionally, changes in these mechanical forces can lead to increased disease progression and onset of disease, including extracellular matrix (ECM) remodeling⁶ and tumor growth^7,8,9,10. 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 muscles^11,12. 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 forces^{4,16,17,18,19,20}. 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 tissues^21,22. 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 process^26,27.

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. 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.

Protocol

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

1. Isotropic mold dimensions
   1. Outer diameter of the mold is 37 mm.
   2. Outer height of the mold is 4.1 mm.
   3. Outer wall thickness is 2.1 mm.
   4. Base thickness of the mold is 2 mm.
   5. Attachment holes are located 2 mm from the edge.
   6. 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 µm.
2. Uniaxial mold dimensions
   1. Outer dimensions of the mold are 41 mm x 12 mm x 4 mm (L x W x H).
   2. Attachment holes are 3 mm from the width edge and 4 mm from the length edge with a post depth of 1 mm.
   3. Outer wall thickness is 1.5 mm on the width edge and 2 mm on the length edge.
   4. The inner cell culture well dimensions are 26 mm x 5.5 mm x 1 mm (L x W x H).
   5. Base thickness is 2 mm.
      ​NOTE: Molds were designed in SolidWorks, 3D printed using a FormLabs liquid grey resin system, and fabricated at Northeastern University 's makerspace.
3. Instructions only for the initial use of the mold
   1. Sand down the rough edges of the mold print.
   2. 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.

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.

1. Add Sylgard 184 base and curing agent in a 10:1 ratio by weight into a plastic dish.
2. Mix vigorously with tongue depressor (or similar tool) for 2-3 min.
3. Degas the mixture for 1 h.
4. Pour the PDMS mixture into the custom negative mold (Figure 2A,B).
   1. Add 1.3 g to the isotropic mold.
   2. Add 0.5 g for the uniaxial mold.
      ​NOTE: Take care to spread the gel evenly across the mold.
5. Degas the mold for 1 h.
6. Allow the gel in the mold to settle on a flat surface at room temperature for 2 h.
7. Cure at 60 °C overnight.
8. Tumble mix parts A and B of NuSil Gel-8100 in equal proportions (1:1) for 15 min.
   1. Mix with Sylgard 184 crosslinker at 0.36% of total volume to produce a substrate with a stiffness of 13 kPa ³².
   2. Add no crosslinker to produce a substrate with a stiffness of 0.3 kPa.
9. Degas the mixture for 30 min.
10. 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.
11. Pour the NuSil mixture onto the negative area of the PDMS gel.
    1. Isotropic gel: 100 µL.
    2. Uniaxial gel: 50 µL.
12. 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 µm.
13. Allow the gel to settle on a flat level surface at room temperature for 2 h before being cured at 60 °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.
14. Store the completed cell culture dishes in covered petri dishes until use (Figure 2D-E).

3. Matrix protein coating

1. Add 500 µL of poly-L-lysine (PLL, 500 µg/mL) to each substrate.
2. Incubate at room temperature for 1 h.
3. Wash substrates 3 × with 1 mL of PBS, followed by 3 × with 1 mL of 10 mM HEPES buffer (pH 8.0).
4. Add 500 µL of mPEG-SVA (50 mg/mL) to each substrate.
5. Incubate at room temperature for 1 h.
6. Wash substrates 3 × with 1 mL of PBS.
7. Replace PBS with 500 µL of PLPP (14.5 mg/mL).
8. Using the PRIMO micropatterning system with Leonardo software, project the predefined pattern onto the substrate surface with UV light for 30 s.
9. Wash substrates 3 × with 1 mL of PBS and place under UV for 15 min for sterilization.
10. Add 500 µL of 0.1% fluorescein conjugated gelatin to each substrate.
11. Incubate at room temperature for 1 h.
12. Wash 3 × with 1 mL of PBS.
    NOTE: To visualize patterns, incubate with 0.1% fluorescein-conjugated gelatin for 1 h at room temperature.
13. Proceed with imaging.

4. Human Airway smooth muscle cell culture in elastic dishes

NOTE: Perform all cell culture procedures in a biosafety hood using standard aseptic technique.

1. 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.
2. 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).
3. Culture cells under standard conditions (37 °C, 5% CO ₂) and use cells up to passage 6 for all experiments.
4. Grow cells in the following complete growth medium:
   1. DMEM/F12 (500 mL)
   2. 10% fetal bovine serum (FBS) (50 mL)
   3. 1× penicillin/streptomycin (5 mL)
   4. 1× MEM nonessential amino acids (5 mL)
   5. Amphotericin B (0.5 mL)
5. Switch cells to serum-free medium at least 24 h before measurements. Prepare serum-free medium with:
   1. Ham 's F-12 medium (500 mL)
   2. 1 × penicillin/streptomycin (5 mL)
   3. Amphotericin B (50 µg/L)
   4. 1× MEM nonessential amino acids (5 mL)
6. UV-sterilize stretchable cell culture dishes for 15 min.
7. Incubate sterilized dishes with 0.1% gelatin for 1 h at room temperature prior to seeding (Figure 2F).
   1. For Patterned Substrates:
      1. Seed cells in elastic cell culture wells at a density of 10⁴ cells/cm².
      2. Incubate in 10% serum medium for 10 min to promote attachment to patterned areas.
      3. Wash wells with PBS to remove unattached cells.
      4. Fill wells with 10% serum medium and incubate for 24 h.
      5. Replace with serum-free medium and incubate for 24 h before measurements.
   2. For Non-Patterned Substrates:
      1. Seed cells at the desired density: 10² cells/cm² for isolated cells. 10³ cells/cm² for sparse cells. 10⁴cells/cm² for confluent cells.
      2. Incubate in 10% serum medium for 24 h.
      3. Replace with serum-free medium and incubate for 24 h before measurements.

5. Fluorescent calcium imaging and analysis

1. Serum-starve airway ASMCs for 24 h.
2. Prepare FLIPR Ca²⁺ 6 dye according to manufacturer 's instructions.
3. Mix FLIPR Ca²⁺ 6 dye with serum-free medium at a 1:1 ratio.
4. Incubate cells with the FLIPR Ca ² ⁺ 6 solution at 37 °C and 5% CO ₂ for 2 h.

6. Imaging Setup

1. Use a Leica DMi8 inverted microscope equipped with:
   1. Leica DFC6000 camera
   2. Leica LED8 light engine
   3. 5×/0.12 dry objective
2. Set excitation at 480/40 nm and emission at 527/50 nm.
3. Acquire 16-bit images at a frame rate of 2 Hz during stretch experiments.

7. Data analysis

1. Import image sequences into Fiji (ImageJ) ³³.
2. Manually define regions of interest (ROIs) in the cytoplasm of each cell.
3. Extract the mean grayscale intensity values for each ROI over time.
4. Process fluorescence intensity data using a custom MATLAB script.
   NOTE: Fluorescence intensity is proportional to cytosolic [Ca²⁺].

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: https://github.com/Breathe-Lab/A-Novel-Platform-for-In-Vitro-Cellular-Stretching-and-Imaging

1. Coat PDMS-NuSil gel substrates with a layer of fluorescent beads to serve as fiducial markers for traction force microscopy.
2. Prepare a 0.025% solution of 1 µm diameter red fluorescent carboxylate-modified microspheres in HBSS by diluting to a 1:4000 ratio.
3. Vortex the bead solution for 10 s.
4. Add the bead solution to the gel.
   1. 880 µL to isotropic gel.
   2. 420 µL to uniaxial gel.
5. Incubate at room temperature for 1 h to allow the beads to adhere.
6. After 1 h, carefully remove the solution.
   NOTE: Pour off the bead solution gently on soft gels. Improper removal prevents proper bead distribution.
7. Gently blot any residual solution with a Q-tip or Kimwipe.
8. Image the beads with a Leica DMi8 microscope equipped with a 10x/0.32 dry objective.
9. 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.
10. Analyze the images using a custom MATLAB code based on Fourier traction force microscopy to calculate displacement vectors and corresponding planar strain fields ³⁴.
11. Represent data as the average of N trials.

9. Calibration of the cell stretching device

1. Add 500 µL of H₂O to the well to simulate the weight of cell culture media and keep the protein pattern moist.
2. For protocol regarding creating specific protein patterns, please see section 3 titled "Matrix Protein Coating ".
3. Image fluorescent patterns every 400 µm steps of motor displacement, ramping up to a maximum of 2800 µm, and then returning to zero in reverse 400 µm increments.
4. 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 "BREATHE " on the elastic substrate during stretching. Use a rectangular bounding box encompassing all letters to calculate the area and eccentricity of the entire word.
5. Apply image segmentation in MATLAB to quantify these parameters for each individual letter.
6. Define zero displacement by the unstretched dimensions of the cell culture dish.
7. Repeat each experiment across two to three independently prepared stretchable cell culture wells to ensure reproducibility.

10. Data presentation and statistical testing

1. For stretcher calibration experiments, define trials as the number of cycles.
2. Show each trial separately instead of combining them into a single curve to demonstrate system repeatability.
3. Indicate error bars as standard deviation.
4. Use a one-way ANOVA to test for significant differences in datasets with three or more groups influenced by one independent factor.
5. Use two-way ANOVAs followed by post hoc pairwise comparisons to test for significant differences in datasets influenced by two independent factors.
6. Apply the t-test for pairwise comparisons when data are normally distributed.
7. Apply the Mann-Whitney rank sum test to compare median values when data are not normally distributed.
8. Report the specific tests used, the number of samples, and the corresponding P-values, along with the results.
9. Use a P-value threshold of 0.05 to determine statistically significant differences between datasets.
10. Perform statistical tests in SigmaStat.
11. Plot figures in GraphPad Prism and MATLAB.

Results

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...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbecco’s modified Eagle’s medium/F12	Fisher Scientific	12634010	500 mL
(4-benzoylbenzyl) trimethylammonium chloride (PLPP)	Alvéole	N/A	14.5mg/mL
0.1% fluorescein conjugated gelatin	Invitrogen	G13187
0.1% Gelatin Solution	Sigma-Aldrich	ES-006	500 mL
1 μm diameter red fluorescent carboxylate-modified microspheres	FluoSpheres	F8816
1× MEM nonessential amino acid solution	Sigma-Aldrich	M7145	100 mL
1× penicillin/streptomycin	Fisher Scientific	15-140-122	100 mL
10% fetal bovine serum	Thermo-Scientific	A5256701	500 mL
15 mL tube	Fisher Scientific	14-959-53A
1x glutamine	Fisher Scientific	MT10010CM
1x Insulin-Transferrin-Selenium Growth Supplement	Corning Life Sciences	25-800-CR
amphotericin B	Sigma-Aldrich	A2942	25 μg/liter
Ball Bearings	McMaster-Carr	N/A
Calcium Chloride Dihydrate	Sigma-Aldrich	223506
Carriage Rail	McMaster-Carr	N/A
FLIPR Ca2+ 6	Molecular Devices	R8190
GraphPad Prism	Dotmatics	N/A
Ham’s F-12 medium	Sigma-Aldrich	N8641	500 mL
Hanks' Balanced Salt Solution	Thermo-Scientific	14025092	500 mL
HEPES buffer	Thermo-Scientific	15630080	100 mL
Leonardo Software	Alvéole	N/A
Matlab	Mathworks	N/A
methoxy polyethylene glycol–Succinimidyl Valerate (mPEG-SVA)	Laysan Bio Inc	N/A	50mg/mL
Nusil Gel-8100	Nusil	N/A
Petri Dishes	Thermo-Scientific	263991	90 mm
phosphate-buffered saline	Sigma-Aldrich	10010023	500 mL
poly-L-lysine (PLL)	Sigma-Aldrich	P8920	500μg/mL
Primary human airway smooth muscle cells	ATCC	PCS-130-011
PRIMO optical module	Alvéole	N/A
Screws	McMaster-Carr	N/A
SigmaStat	Systat Software	N/A
Spin Coater	Laurell	Model WS-650MZ-23NPPB
Stretcher and Stage Attachment Components	Boston University's Scientific Instrument Facility	N/A	Stainless Steel
Sylgard 184 base Elastomer and Curing agent kit	Dow	H047J8Q026
T-25 Tissue Culture Flask - Vent Cap, Sterile	CellTreat	229331
Tongue depressor	Thermo-Scientific	22-363-154