The overall goal of this procedure is to observe and analyze the effect of cell matrix interactions on fiber realignment and matrix remodeling activities in a fibrin gel system. This is accomplished by first polymerizing, a three dimensional fibrin gel containing microbeads onto cover glass contained within an environmentally controlled bioreactor. The second step is to prepare a highly concentrated cell suspension that is used to position clusters of fibroblasts or X explants into geometric patterns on the surface of the gel.
Next, the sample is imaged every 15 minutes using differential interference contrast microscopy over the course of 48 hours. The final step is to measure the extent of fibrin reorganization and remodeling in the form of strain from different regions of the gel using digital image correlation. Ultimately, this explant system allows one to arrange the cells into simple geometric patterns, which makes it easier to visualize and probe the mechanisms underlying cell driven matrix remodeling and fiber realignment.
This method can answer key questions in the field of mechanical biology, such as what are the effects of very spatial and temporal mechanical cues on cell behavior and the resulting changes these generate in the surrounding tissue matrix. Demonstrate in this procedure will be Arabic de Jesus, a graduate student from my lab Gently dissolve fibrinogen into prewarm saline at 37 degrees Celsius overnight to create a 33.3 milligram per milliliter stock solution. Prepare a stencil on perfil to lay out the location of each x explant following the desired geometry space.
Each dot approximately one to two millimeters apart. This distance corresponds to an ideal spacing for generating fiber alignment between explants. Next, thoroughly clean all components of the bioreactor using 70%ethanol and place the cleaned components under the UV of a tissue culture hood for two to three hours prior to mentation.
After the components of the bioreactor have been sterilized, attach the stencil underneath the area of the 60 millimeter diameter cover glass where the sample will be prepared using tape. Next, prepare the microbead suspension, followed by the fibrinogen and thrombin. Stock solutions combine 0.017 milliliters of the Microbead stock solution and 0.149 milliliters of DMEM into a micro centrifuge tube to achieve a concentration of 10 million beads per milliliter.
Then sonicate the suspension for 10 minutes to disperse the beads and homogenize the solution. Add 0.22 milliliters of the fibrinogen stock solution to 0.44 milliliters of 20 millimolar heaps buffer, and 0.1667 milliliters of the microbead solution in a 15 milliliter centrifuge tube. Gently mix the components and then place the tube on ice in a separate 15 milliliter centrifuge tube.
Mixed together. 32.8 microliters of thrombin dissolved in saline at 25 units per milliliter. 131 microliters of 20 millimolar heaps buffer, and 2.46 microliters of two molar calcium chloride.
Next carefully mix the prepared thrombin and fibrinogen solutions by pipetting up and down five to 10 times until the solution is evenly distributed. Try to minimize introducing bubbles to the mixture by not fully discharging the pipette while mixing. Pipette the mixed solution into eight millimeters square PDMS molds on cover glass as soon as possible, allow the gel to polymerize at room temperature.
The addition of thrombin will cost the solution to gel quickly in around 30 seconds following polymerization, seal the bioreactor and prepare the cell explants to begin harvest 70 to 90%confluent human dermal fibroblast cells from a T 75 flask using trypsin and spin them down at 200 times G for five minutes. Transfer the bioreactor to a biosafety cabinet and carefully remove the lid following aseptic conditions. Then aspirate off the media and resuspend the cell pellet to a final concentration of 20 million cells per milliliter in DMEM with 10%FBS.
Next, create explants by loading cells into a micro pipette and injecting 0.3 microliters of the cell suspension into the polymerized fibrin gel. Following the pattern on the stencil, each explan should contain approximately 6, 000 cells. Once the explants have been positioned on the gel seal the bioreactor.
Next, insert the heating blocks and connect the thermocouples to the temperature controller. Set the temperature controller to 37 degrees Celsius and incubate the gels for one hour to allow the cells to settle and attach to the fibrin matrix. Then at approximately five milliliters of DMEM that has been conditioned for two to three hours with 5%carbon dioxide and supplemented with 10%fetal bovine serum 1%penicillin streptomycin 0.1%amphotericin B and 10 micrograms per milliliter, aprotinin directly into the bioreactor chamber.
Then reseal the bioreactor. Use a syringe to deliver the additional five milliliters of carbon dioxide conditioned medium via the barbed fitting on the inlet port to completely fill the bioreactor carefully remove any bubbles that form set up a syringe pump with a 10 milliliter or 30 milliliter syringe filled with additional 5%carbon dioxide conditioned medium. Connect the syringe directly to the inlet port on the bioreactor lid with lure lock fitted sterile tubing.
Set the perfusion rate on the syringe pump to 0.01 milliliters per minute. Then connect modified pieces of tubing to both outlet ports and place the ends into a 100 milliliter beaker. To collect waste, use a lab jack to set the heights of the inlet and outlet feeds so that a pressure differential does not develop in the bioreactor supply.
Fresh 5%carbon dioxide conditioned medium to the bioreactor throughout the experiment. In order to maintain pH supply nutrients and remove waste products, a 10 milliliter syringe filled with medium will last about 16 hours. Position the 20 XDIC objective under the view port of the microscope and ensure that the polarizer analyzer and prism are all in place.
Then open the imaging software. Focus the objective on the area between the three x explan and save the X, Y, and Z coordinates of this location in the imaging software. In order to image the entire area between and around the x explan, select the option to acquire a large image and specify the size of the area.
This will allow the acquisition of multiple images around the specified area and create a large tiled image, set the light intensity and exposure time to the lowest values possible. This will help to avoid cell death caused by phototoxicity while still providing sufficient resolution to discriminate between cells, microbeads and fibrin fibers. Note that in this particular experiment, the fibrin fibers are not visible due to the high density of light scattering beads in the gel, reduce the bead density or gel thickness in order to observe fibrin fibers.
Next, begin strain tracking. By first creating or downloading custom MATLAB code, then register images using microbeads throughout the gel as reference points. Registering the image will ensure that the images obtained are taken at the exact same location each time.
Fiber realignment was observed between explan by capturing tiled images every 15 minutes. Over a 48 hour period, individual regions were extracted from the tiled images and analyzed with the strain tracking algorithm. In order to measure local strains from microbead displacement contour plots show the distribution of maximum principle strain in the area of fiber realignment corresponding to the strap.
Once mastered, this technique can be completed in approximately three To four hours if performed properly. Following this procedure, other methods like fluorescent labeling of collagen and cytoskeletal proteins as well as quantification of cell migration can be performed in order to answer additional questions like what mechano biological pathways are involved in tissue remodeling, and how are these processes modulated by various biochemicals.
