The overall goal of this method is to robustly and consistently generate engineered blood vessels of any size. This technique allows the reliable and effective engineering of blood vessels. The main advantage of this technique is that vessels of any size can be easily and reliably fabricated.
We first had the idea for this method when we were discussing how to engineer a tube of tissue and likened our method to constructing roadway tunnels, one segment at a time. To prepare the 3D printed inserts first add a thin layer of two, four, or six mL of uncured silicone to the bottoms of a 35, 60, or 100 millimeter Petri dish, respectively. To create posts for the 35 mm plates, pour PDMS into a different 100 mm plate to a seven mm height, and heat the PDMS on a hotplate at 60 degrees Celsius for two to three hours.
When the PDMS is cured, use a five mm biopsy punch to create cylindrical posts. Using a small amount of uncured PDMS to secure the cylinders to the center of each 35 mm plate. Place a 3D printed outer shell about 66.7 mm in diameter equidistant from the post in the 100 mm plates.
Before the PDMS at the bottom of the 60 and 100 mm plates is cured, place 10 and 20 mm diameter 3D printed posts centrally into each 60 and 100 mm plates respectively. Allow the dishes to cure in the open air on the 60 degree Celsius hotplate for two to three hours, followed by eighteen hours of polymer degassing. At the end of the degassing, sterilize the insides of all of the plates with 70%ethanol and cover each plate for 30 minutes.
Then carefully aspirate the ethanol from each plate to allow air drying. When the plates are dry transfer them to a biological safety cabinet next to their corresponding face up lids. and expose the materials to UV light for 30 minutes to complete the sterilization.
To prepare the Fibrin Hydrogel first add 0.5, 1.1 and 1.81 mL of growth medium supplemented with Fibrin gel and TGF Beta 1 to 35, 60 and 100 mm culture dishes respectively. Next, add 40 88.4, and 145 microliters of Thrombin to the 35, 60, and 100 mm plates respectively, and gently swirl each plate by hand. When the Thrombin is evenly distributed add 160, 354, and 580 microliters of Fibrinogen with a drop-wise circular motion to the Thrombin medium mixture in the 35, 60, and 100 mm plates, respectively, and gently swirl the plates to evenly distribute the Hydrogel.
Allow the Hydrogel to cure for 10 to 15 minutes at room temperature. Then trypsinize the smooth muscles cells of interest, expanded in 150 mm cell culture plates, and collect the detached cells by centrifugalization. Re-suspend the pellets in three ml of differentiation medium and use a two mL pipette to vigorously triturate the cells to break up any cell clumps.
After counting, split the cells into 2/10 10 to the 6th, 1/10 10 to the 7th, and 1.4 x 10 to the 7th cells per mL aliquots in 50 ml conical tubes, labeled according to their corresponding culture plates. Add differentiation medium to each tube to obtain final seeding volumes of two, four, and five mL for each 35, 60 and 100 mm plate respectively Then, carefully add the cell solutions drop-wise onto the prepared Hydrogel in each corresponding plate for expansion in the cell culture incubator. After two to four days the rings will have completely contracted in towards their posts.
So add 10, 20, or 35 mL of TGF Beta 1 to each 35, 60 and 100 mm ring, respectively. To assemble the vascular construct for the 35 mm vessel cut a two inch section from the top of a 50 mL of a polycarbonate conical tube And PDMS glue the top edge into a 35 mm plate to create a tall plate for ring stacking. For the 60 and 100 mm vessel tall ring stacking plates cut a 1.75 inch diameter polycarbonate tube into 2.5 inch sections length-wise to serve as the tall plate walls.
For the tall plate bottoms, cut a 0.125 inch thick polycarbonate sheet into two inch diameter circle pieces. Using acrylic solvent cement bind the polycarbonate tube sections to the circular cut pieces. 3D print five, ten, and 20 mm diameter posts with a 50 mm length.
Then add 10 mL of uncured silicone to each container, centrally placing each 3D printed post into each 35, 60 and 100 mm container before the PDMS is completely cured. And place the dishes on a 60 degree Celsius hotplate for two to three hours. When the PDMS is cured, sterilize the plates with 70%ethanol as just demonstrated, followed by exposure of the dried ethanol sterilized containers to UV light for 30 minutes.
Now, use two pairs of very fine forceps to carefully lift first one side, and then the other, of a tightly rolled smooth muscle Hydrogel ring from its post. Then carefully slide one side of the ring and then the other onto the tall post of the corresponding next sized larger container. Working circumferentially, with gentle gradual movements, slowly push the ring down the post, subsequently stacking additional tissue rings until the desired vessel length has been obtained.
When all of the rings have been stacked turn the plates so that the posts are parallel with the working surface, and gently add 40, 80, and 160 microliters of Thrombin to the outer surface of each 35, 60, and 100 mm vessel respectively while slowly rotating the plates. Next, add 40, 80, and 160 microliters of Fibrinogen to each 35, 60, and 100 mm construct respectively, as quickly and evenly as possible while briskly rotating the construct. The Thrombin and Fibrinogen will quickly set into a firm gel upon mixing.
Then add 20 mL of differentiation medium to each construct container and place the vessels into a 37 degree Celsius incubator until needed. Histological analysis reveals a high cellularity in all ringed sizes, with a small amount of residual Fibrin gel observed on the outer edges of the small rings. In the larger rings some Fibrin gel is interspersed with the cellular content, with indications of collagen production apparent in both the intermediate and large rings.
Tissue ring analysis for Alpha smooth muscle Actin and Tropomyosin expression reveals that all of the ring sizes are positive for both antibodies verifying that the smooth muscle phenotype was maintained. Tensile testing indicates a consistent trend of increasing strength correlating with an increasing ring and vessel size in all three sizes of rings. Cell seeding numbers for creating the various ring sizes also correlates with the seeding surface area.
These vessel constructs are also able to withstand flow for up to five minutes at flow rates from 100 to 417 ml per minute with only minor leaking observed where the vessels connect to the profusion system. Once mastered, the rings can be engineered in one hour if the technique is properly performed. While attempting this procedure it's important to remember to mix the Hydrogel components thoroughly to ensure setting of the gel and proper ring formation.
Following this procedure, other methods, like creating rings of other cell types, can be performed for engineering other types of tissues. After watching this video you should have a good understanding of how to use the ring stacking method to engineer vascular tissues of various sizes.
