This protocol allows the fibroblast build and organize their own environment under defined mechanical conditions. These results in anisotropic tissues with stiffness and matrix compositions comparable to the cell's natural environment. This method allows to compare up to 48 conditions in parallel.
It is flexible with respect to the used cell type and culture conditions. By using only a few well-defined components, it is reproducible between laboratories. By applying pro-fibrotic factors or antifibrotic drugs, this protocol can be used to study the underlying processes and therapies of fibrotic diseases of any kind.
Demonstrating the procedure will be Alisa Degrave, a PhD student from our laboratory, and myself. To begin with, thaw the cryo-preserved cardiac fibroblasts in a water bath at 37 degrees Celsius for approximately two minutes until the vial is left with only a small amount of ice. Next, transfer the cell suspension dropwise using a two milliliter serological pipette into an appropriate sterile centrifuge tube containing 10 milliliters of fibroblast growth medium.
For optimal cell retrieval, rinse the cryo-vial with one milliliter of FGM and transfer it to the centrifuge tube. Gently resuspend the cells and transfer into a cell culture flask. Replenish FGM every other day for five days or until the cells have reached 80%confluency.
After aspirating the medium from the cultured cells, wash cells with six milliliters of PBS and aspirate, then add six milliliters of the cell dissociation reagent to the cells and incubate until the cell detachment is visible. Neutralize enzymatic activity by adding six to 12 milliliters of FGM to the dislodged cells in the cell association reagent. Gently pipette up and down four to eight times using a 10 milliliter serological pipette to ensure a single-cell suspension and transfer the cells into a fresh 50 milliliter collection tube.
Verify the yield with the help of an automated cell counter as per the manufacturer's instructions and pellet down the cells. After centrifugation, aspirate the supernatant, flick the tube to dislodge the pellet and resuspend the cells in FGM. Next, strain the cell suspension through a 40 micrometer mesh cell strainer, then using an automated cell counter, assess cell number and viability by electric current exclusion to ensure reliable cell numbers before proceeding with engineered connective tissues or ECT preparation.
To prepare for ECT, adjust the cell suspension to the 8.9 million cells per milliliter at 20 to 25 degrees Celsius by adding FGM and keep the cells on ice. Transfer all the other tubes containing the individual components of ECT hydrogel mixture onto ice and pre-chill an empty 50 milliliter centrifuge tube to prepare the mixture. Start preparing the ECT hydrogel mixture by adding components described in the text to the pre-chilled 50 milliliter centrifuge tube avoiding air bubble formation.
Firstly, pipette the acid soluble collagen type I hydrogel into a serological pipette with a wide bore tip. Adjust the salt content of the collagen solution by adding the DMEM while gently swirling the tube for proper mixing. To neutralize the pH, add 0.2 molar sodium hydroxide while swirling the tube and confirm the neutralization by observing the red color of the phenol red indicator.
Then add the cell suspension dropwise while gently swirling the tube. Mix the suspension by gently piping up and down only once using a serological pipette with a wide bore tip to avoid bubble formation and minimize the shear stress. Gently swirl the tube 10 times for thorough mixing.
Keep the centrifuge tube containing ECT hydrogel mixture on ice throughout the casting process. Wet a one milliliter pipette tip in ECT hydrogel mixture, then distribute 180 microliters of hydrogel mixture evenly into each mold of the 48-well casting plate avoiding excessive shear forces that may affect the integrity of the collagen matrix assembly and ensuring that the entire plate finishes in 15 to 20 minutes. Ensure that a complete loop forms within the mold as discontinuous distribution of ECT mixture will prevent a complete ECT ring formation.
Avoid pipetting into the inner well and forming bubbles during pipetting to ensure a uniform ECT hydrogel casting for homogeneous and functional tissue formation. Carefully place the 48-well casting plate inside the cell culture incubator to reconstitute the ECT hydrogel mixture for 15 to 30 minutes. After incubation, it appears gel-like and opaque.
Add 600 microliters of 37 degrees Celsius warm FGM per well along the wall gently to avoid ECT detachment from the bottom. Replace the medium every day with 500 microliters of FGM until analysis. At the desired time points, use a stereo microscope to record microscopic images of the top and side views of the ECT.
Use an image processing program to perform a line scan analysis. Set a scale and use the straight line tool to trace and measure the ECT diameters at a minimum of six positions per arm in each imaging plane. Image the 48-well casting plate under a recording device with an integrated area scan camera placed at a fixed distance equipped with a high-resolution monochrome image sensor and a near-UV light source.
Perform automated detection of the poles'tips containing fluorescent dye for maximizing the contrast. Measure the distance between the poles from daily records using an automated analysis by running the recorded images on software to detect high-contrast bright pixels on a dark background or an image processing program. Remove the ECT from retaining poles and insert a transferring hook and ECT loop.
Then transfer the ECT onto two hooks clamped to the stationary arm and the transducer arm of an extensional dynamic mechanical riometer equipped with a 37 degree Celsius tempered organ bath filled with PBS. Apply uniaxial tension at a constant linear rate by setting the riometer at approximately 1%of the initial distance between the hooks per second. A constant stretching rate of 0.03 millimeters per second can be used with typical ECT dimensions.
Tear the force of the transducer and initiate the stretch. Keep recording until the point of ECT rupture after normalizing the measured force by cross-sectional area and plot the stress-strain curve determining different biomechanical parameters. Just before the tissue starts micro fracturing, the upper limit of the elastic region corresponds to the yield point and its strain is a measure of tissue elasticity.
The plastic region is in between the yield point and the failure point. The failure point corresponds to a sudden drop in the stress due to rupture of the tissue, defining the ultimate strain which is a measure of tissue extensibility. The third measuring point corresponds to the maximum strength defined by the highest stress that the tissue can bear without breaking during the stretch.
The resilience and toughness given by the area under the curve corresponds to the energy absorbed by the tissue up to the yield point and failure point respectively. For each obtained curve, the slope of the linear part of the elastic region corresponds to Young's modulus, also known as elastic modulus. It is a mechanical property that measures the stiffness of the tissue.
Using this protocol, ECT compaction and contraction under control conditions and in the presence of FCS ensued a few hours after casting and notably increased up to day five. When ECT was treated with the actin polymerization inhibitor Latrunculin A, the ECT compaction was reduced as indicated by the significantly higher cross-section area compared to control. The contraction of the tissues was assessed during the five days of culture.
In the absence of Latrunculin A, the contraction gradually increased up to day five reaching about 40%contraction. However, Latrunculin A presence affected the tissue contraction resulting in only about 20%maximum contraction. Actin polymerization inhibition led to a significant reduction of about 50%in the tissue stiffness over the control.
These results demonstrate that the actin cytoskeletal integrity is essential for ECT compaction, contraction, and stiffening. It is important to select a reliable, high-quality collagen solution and ensure that a single-cell suspension with high viability is used to reconstitute engineered connective tissues.
