Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

The overall goal of this procedure is to engineer tubular constructs for vascular tissue regeneration. This is accomplished by first combining type one collagen and smooth muscle cells in cylindrical geometry with the use of a molding technique. The second step is to mature the construct in a specifically designed static bioreactor.

Next, the mature constructs are carefully harvested and endothelial cells are seated on the lumen of the construct using a rotating wall bioreactor actor. Ultimately, histology, metabolic assays and mechanical tests are performed to evaluate the properties of these tubular constructs. The originality of this technique is that it uses just one step to combine cells and collagen to yield tissue engineer construct with targeted mechanical properties without any additional physical or chemical treatment.

Moreover, the construct can be easily manipulated thanks to specifically design grips, which allow for their manipulation and end links. This method can provide an insight in the interplay between cells and extracellular matrix during growth and remodeling for vascular tissue regeneration. In addition, it can be applied for a number of targeted tissues, such as tendons, skin grafts, cardiac patch, and nervous system.

To begin fabricate and assemble the static bioreactor as described in the accompanying text protocol included are details on reservoir and gauze, grip fabrication, mandrel cap, and the housing mold assembly and sterilization techniques. Next, expand porcine aortic smooth muscle cells in a 175 square centimeter tissue culture flask until they are 90%confluent. Using standard culture techniques, harvest the cells and then resuspend them at 4 million cells per milliliter in complete culture medium.

Next, combine one part buffer solution, two parts sterile collagen solution at four grams per liter and one part cell suspension. In this order, measure the pH of the mixture and ensure it is between pH 7.0 and 7.4. Then gently pour the cell collagen mixture into the assembled housing mold complex.

Make sure there are no air bubbles in the mixture. Let the collagen gel sit at room temperature for one hour in a sterile environment. After one hour, remove the housing tube and carefully transfer the construct into the reservoir containing 35 milliliters of prewarm culture medium.

Incubate the construct in a vertical position for one or two weeks of static maturation. Change the culture medium every two days by aspirating the old medium from the lower septum port and refilling the reservoir with an equivalent amount of fresh culture.Medium. If desired, collect one milliliter of both the old and fresh culture medium at each media exchange for biochemical analysis every hour for the first 12 hours and every 24 hours thereafter.

Remove the construct from the culture medium under sterile conditions and place it orthogonally in the path of a scanning laser interferometer to measure the construct's thickness following one to two weeks of static maturation. Transfer the bioreactor into a cell culture hood and gently remove the construct from its mandrel. Once free, place it into a 100 millimeter diameter Petri dish containing 40 milliliters of fresh culture medium.

Next, set up a micro mechanical tester equipped with a five to 10 Newton load cell and grips that extend into a bath of PBS kept at 37 degrees Celsius. Remove the construct from its Petri dish and connect it to specialized gripping devices for longitudinal tests and designed to connect to the gauze portion of the construct without crushing the gel. Use Teflon tape to prevent any slipping of the gripping devices during testing.

Then mount the construct onto the micro mechanical tester. Next, perform a fatigue test on the longitudinal direction by first stretching the construct to its initial gauge length and holding it there for 10 minutes. Then apply 30 cycles of a 10%cyclic strain at a rate of 5%per second.

Increase the strain by incremental steps of 10%cyclic strain until the sample fails to perform tests in the circumferential direction. First section the specimen into 10 millimeter long ring constructs. Then mount the rings onto stainless steel bars used in place of the grips.

As with the longitudinal direction tests, first, stretch the construct to its initial gauge length and hold it there for 10 minutes. Next, apply 30 cycles of a 10%cyclic strain at a rate of 5%per second. Once the construct has matured and it has been removed from its mandrel, fix the construct that both ends to the reservoir and place it in the reservoir assembly.

As shown here. Fill the exterior of the reservoir with 30 milliliters of culture.Medium. Next, fill 75%of the luminal volume of the construct with a mixture of proteins, and then close both ends of the construct.

To avoid any leakage of the protein solution, place the bioreactor inside an incubator and rotate the bioreactor at 4.02 times 10 to the negative five GForce for one hour to coat the luminal surface with proteins while the lumin is being coated. Harvest human umbilical vein and tial cells cultured with standard techniques when they're 90%confluent Resus suspend the cells and supplemented M1 99 culture medium. Next, see the human umbilical vein and urothelial cells into the lumen of the construct.

With the density of 1000 cells per square centimeter, close the upper extremities of the construct to avoid any leakage of the cell solution. Incubate the constructs hosted into the rotating wall bioreactor culture for two days at a constant rotation of 4.02 times 10 to the negative 5G force. During static maturation of the vessel constructs, the external diameter of the cellular rise structures rapidly reduced.

As shown here, the diameter of the constructs reduced by 60%of their initial value in day one of static culture. By day seven, the constructs had reduced almost 85%Both the longitudinal and circumferential strain cycles of the constructs followed viscoelastic properties typical of collagen based constructs. Overall, the constructs were stronger in the longitudinal direction compared with the circumferential direction at the same strain range, Massen tricone staining performed on the endothelial constructs showed a highly homogenous endothelium.

Smooth muscle cells exhibited spindle like shaped morphology and appeared homogenously dispersed through the wall while the endothelial cells appeared well spread on the luminal side. After his development. This technique paved the way for researchers in biomechanics and regenerative medicine to explore the mechanical behavior of engineered and native tissues.

After watching this video, you should have now a good understanding of the how vascular constructs can be rapidly engineered ma and finally become strong enough to be handled and used for mechanical conditioning in a dynamic ator. We hope that our efforts and results have convinced you about the high potential of cells in themodel of extracellular matrix for the development of biological functional tissues.