A Novel Platform for In Vitro Cellular Stretching and Imaging

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.