The overall goal of this procedure is to create fibrin based constructs that can be used to study cell and collagen reorganization. This is accomplished by first isolating human venous saphenous cells from donor tissue and expanding them in culture. The second step is to glue Velcro strips onto flexible membranes.
At the bottom of six well plates to create a square space at the center, which will hold the construct. Next, a mixture of fibrinogen thrombin cells and fluorescently labeled beads are added to the space in between the Velcro strips and is allowed to polymerize to form a gel. The final step is to condition the cells and induce oriented matrix formation by applying either a uniaxial cyclical strain to the construct or by releasing static strain.
By trimming the fibrin gel from the Velcro posts in a single direction. Ultimately, results can be obtained that describe changes in cellular and collagen reorganization by monitoring the constructs over time using confocal microscopy. This method can help answer key questions in the collagen remodeling field relating to the speed of collagen formation and reorientation by different cell types.
To begin, acquire human donor tissue from the venous saana magna in accordance with guidelines for the secondary use of material. Then isolate the human venous saphenous cells or H VSCs from the tissue by following the protocol by Schnell et al, and store them in liquid nitrogen. When needed, expand cells by placing one vial containing 250, 000 quick thaw H VSCs into a T 75 flask with 15 milliliters of prewarm growth.
Medium consisting of 440 milliliters of advanced DMEM 50 milliliters of fetal bovine serum. Five milliliters of 100 X pen strep and five milliliters of 200 millimolar L glut max. Change the growth media every two to three days for approximately two weeks until the cells are confluent.
Once confluent, harvest and split the cells up to passage.Nine. Using trypsin until there is an adequate number of cells for the planned experiment. Approximately 1.5 million cells are required for each construct.
When an adequate number of cells have been grown, harvest them at 90%co fluency and then suspend the cells in growth media at 15 million cells per milliliter. Place them on ice until they are needed. Prepare silicone glue by mixing the elastomer and curing agent in a 10 to one ratio.
Use this glue to attach seven millimeter by three millimeter strips caught from the soft side of Velcro to the flexible membrane of flex cell plates. To form a cross shape with an open center, dry the silicone glue overnight in an oven at 60 degrees Celsius to ensure a hardening of the glue. Then sterilize the plate by adding five milliliters of 70%ethanol to each well and incubating for 30 minutes at room temperature.
Rinse the wells three times with sterile PBS. Then remove the PBS from the wells and the Velcro strips. Place the plate uncovered under the UV lamp of a tissue culture hood for 30 minutes to sterilize the surfaces.
Once finished, cover the plate and keep it sterile until use. Next, prepare tissue engineering medium by adding 130 milligrams of allic acid, two phosphate to 500 milliliters of growth medium during the first seven days of culture. Also, add one milligram per milliliter epsilon aminocaproic acid to prevent the fibrin from degrading, dissolve fibrinogen to a concentration of 10 milligrams of actual protein per milliliter of tissue engineering Medium supplemented with aminocaproic acid by mixing gently.
Then sterile. Filter the fibrinogen solution through a 0.22 micron syringe filter and store it on ice until use. Next, prepare the thrombin solution by diluting stock thrombin to a final concentration of 10 international units per milliliter in tissue engineering medium supplemented with aminocaproic acid and store it on ice until use.
Next place, 100 microliters of the cell suspension prepared earlier into a micro centrifuge tube for each tissue construct that will be made. Centrifuge the cells at 350 times G for seven minutes, and then discard the supernatant, resuspend the cells in 50 microliters of the thrombin solution and add four microliters, a blue fluorescent polystyrene microspheres to be used as internal reference markers. Next, draw up 50 microliters of the fibrinogen solution into a pipette tip.
Increase the volume of the pipette to 100 microliters and then pipette the 50 microliters solution of fibrinogen into the centrifuge tube. With the thrombin and cell mixture, gently mix the thrombin and fibrinogen together by drawing up the 100 microliter mixture to prevent bubble formation. Once the solution is thoroughly mixed, pipette the gel mixture into and in between the Velcro strips we're quickly as the typical gelian time for the fibrin gels, once mixed is on the order of 20 seconds, incubate the constructs for 30 minutes at 37 degrees Celsius in a humidified 5%carbon dioxide buffered incubator to allow them to gel.
Then add six milliliters of tissue engineering medium with aminocaproic acid to each well, and place the plate back into the incubator. Replace the media every two to three days for the first seven days. After one week, remove the aminocaproic acid supplement from the media and continue to culture the gels with the tissue engineering media.
Still exchanging it every two to three days over the first five days in culture, allow the constructs to compact and reorganize under static strain forming collagen fibers, bi axially due to the fiber constraints to induce collagen reorganization in a single direction under static conditions. First, culture the cells for one additional week at day 12, cut the tissue construct loose from two Velcro strips in a single direction. This will cause strain in the opposite direction as the fibrin compacts.
Alternatively, for cyclic loading, place the plates on top of a set of loading posts in a flex cell base plate, equipped with a gasket on day six, the loading posts will support the flexible membrane of the wells and allow the base of the posts to be pulled down to create strain in a single direction. Next, set up the software protocol to apply a cyclic strain to the construct. The program shown here is for intermittent strain resembling a sine wave, straining the construct from zero to 5%strain at one hertz for periods of three hours, alternating with three hours of rest.
With the program prepared, turn on the vacuum pump and start the program. The controller will regulate the vacuum strength in the system, which pulls down on the membranes around the support posts to apply cyclic strain to the constructs in accordance with the program. Run the program for one week taking care to exchange the media every two to three days after one week of conditioning induce collagen reorganization by turning the rectangular post by 90 degrees to change the direction of strain for three days.
To visualize active real-time collagen remodeling, add cell tracker orange. Add a concentration of four micromolar to stain the cytoplasm and CNA 35 OG 4 88. Add a concentration of one micromolar to visualize the collagen fibers.
These probes will not interfere with cell viability or collagen formation. Then incubate the cells for one hour in an incubator at 37 degrees Celsius. Following incubation, remove the culture plate from the incubator and transport it to a confocal laser scanning microscope to visualize cells and collagen.
Take images of the gels at the surface and up to approximately 60 microns into the scaffold in order to view the changes in organization at these levels over time. Then return the plate to the mechanical conditioning setup inside the incubator. Repeat this procedure every few days to track the progress of cellular reorganization in response to the stimuli.
The fluorescent microspheres added to the fibrinogen mixture prior to polymerization become entrapped in the fibrin network and work well as reference markers to ensure visualization of the same area at different time points when cultured under static conditions and attach to all four posts. No real fiber or cellular orientation can be seen three days after trimming two of the fiber post connections. The uni axial strain caused by contraction has aligned the collagen fibers shown in green.
Human venous saphenous cells that have been cyclically strained in a single direction for one week are shown here at the surface of the construct and 60 microns into the gel. When the direction of strain was rotated 90 degrees, the cellular response after three days was found to be quicker at the surface than in the core of the tissue. Once mastered engineering, these FI based tissue constructs can be performed in around two hours.
