The overall goal of this procedure is to create 3D tissue constructs from aggregated cells and cell derived matrix that can be used for functional analysis. This is accomplished by first milling a polycarbonate mold to create round bottomed annular wells. Next poly dimethyl Sloane or PDMS is cast onto the polycarbonate mold to create a template.
Then the PDMS template is used to form aros wells. The final step is to seed cells into aros wells to allow them to aggregate and form cohesive tissue rings. Ultimately, this method can be used to achieve cellular self-assembly and tissue engineering using different cell types, and enables analysis of the contribution of cells and cell derived extracellular matrix to tissue structure and function.
The main advantage of this technique over other tissue engineering approaches is that we can generate tissue constructs entirely from cells and the extracellular matrix that they produce more rapidly than previously reported, and in a format which is conducive to mechanical analysis, Begin by milling a half inch thick piece of polycarbonate to create 15 round bottomed annular wells with a center post diameter of two millimeters. Clean the polycarbonate mold to remove any plastic debris generated from the milling process and let the mold dry. Next mix PDMS at a 10 to one ratio of base curing agent Degas the solution to remove all air bubbles and then pour it into the polycarbonate mold.
Then degas the mixture on the mold again to remove any remaining air bubbles and cure the PDMS in an oven at 60 degrees Celsius for four hours while the PDMS is curing dissolve. 2%AROS in DMEM once cured carefully remove the PDMS template by slowly peeling it away from the polycarbonate, then wash the PDMS with soap and water and autoclave it along with the DMEM AROS solution. Now place the autoclave PDMS template on a level surface and fill it with molten aros by first pipetting aros into each of the center post molds, and then by pipetting aros into the space around them.
Allow the aros to solidify for about 15 minutes. Then invert the mold to release the aros from the PDMS. Cut the excess aros from around each of the wells, and then place one aros well into each well of a 12 well plate.Now.
Add cell culture media around the outside of the aros well without covering the top of the well. Then place the plates into the incubator to allow them to equilibrate while the cells are prepared. At 90%confluence trypsin, rat aortic smooth muscle cells and resus.
Suspend the cells at a concentration of five times 10 to the six cells per milliliter. Then pipette 100 microliters of the resuspended cell suspension into each of the equilibrated aros wells. Using a circular motion to apply the cells to each of the wells.
Place the plates in the incubator to sit undisturbed for 24 hours. Exchange the medium every two days by aspirating the media from around the well and then refilling each well until the aros mold is completely submerged. And at the conclusion of the culture, fill a small Petri dish with PBS.
Remove each ring from its aros mold by sliding it over the top of the center post. Place the rings in the Petri dishes with PBS Center, a ring under the digital imaging system. Use edge detection software to measure the thickness of the ring in four positions, top, bottom, left, and right around its circumference.
To perform uni axial tensile testing. Mount a ring sample on two thin wire grips. Extend the grips until a five milli Newton tear load is applied to the ring.
Then enter the cross-sectional area calculated from the thickness measurements and record the gauge length. Pre cycle the ring eight times between the tear load and 50 kilo pascal. Stress at a rate of 10 millimeters per minute after the eighth pre cycle pulled to failure at 10 millimeters per minute.
For each ring, measure the ultimate tensile stress and failure strain and calculate the maximum tangent modulus from the acquired data. In addition to functional in vitro studies, these living cell rings can also fuse to form tissue tubes. Use surgical scissors to cut the ends of silicone tubes at an angle to create beveled edges and then autoclave the silicone tubes while the tubes are being autoclaved.
Fill a Petri dish with media transfer sterilized custom silicone tube holders to a biosafety cabinet and place in an empty Petri dish. Next, remove the rings from the aros wells and place them in the Petri dish filled with media. Place the silicone tubes in the same petri dish to wet them before use.
Use forceps to place the tip of a silicone tube into the center of a ring and gently slide the ring onto the silicone tubing. Slide the rings into contact with one another by gently pushing them successively in both directions along the tube. Once the rings have been mounted, align silicone tubes within the polycarbonate holders and screw the two parts of the holder together.
Add 55 milliliters of media to the Petri dish. Exchange the media every three days for the duration of culture to remove the tissue tubes. First, fill a Petri dish with PBS.
Then release the silicone tubes from the polycarbonate holder and use forceps to slide the tissue tubes off the silicone tube and into the dish. When performed correctly, the cells settle in the bottoms of the aros wells and contract within 24 hours after seating to generate a tissue ring around the center post in the Agarro s well, this is a representative plot of a stress strain curve obtained from a uni axial tensile test of a two millimeter tissue ring generated from rat smooth muscle cells. The ultimate tensile strength, the maximum tensile modulus, and the failure strain values are then used to compare the mechanical properties of tissue ring samples grown under different culture parameters.
The mechanical properties of tissue rings can be controlled by altering culture parameters, including cell seating number, the length of culture, the culture medium, and the cell type used. In addition to rat smooth muscle cells. Human primary, smooth muscle cells form tissue rings with similar tensile strength.
Human mesenchymal stem cells also aggregate to form rings, but they were not strong enough for mechanical testing. Our system enables fabrication of engineered tissue constructs entirely from cells simply by seeding cells into our custom aros wells. Once the initial polycarbonate mold has been machined, we can make an unlimited number of PDMS templates that then can be used to make the molds so no additional equipment is required.
The system is versatile in that we've used a number of different cell types in the system to make tissue rings, and so this system could be used potentially for both tissue engineering applications as well as in vitro studies of the contributions of cells and extracellular matrix to tissue structure and function.
