The overall goal of this procedure is to engineer biological based vascular grafts using a pulsatile bioreactor system. This is accomplished by preparing a biodegradable scaffold from PGA mesh where the cells will grow row and assembling them in a bioreactor chamber. The scaffolds are then seated with smooth muscle cells and a lid with air and feeding tube seals the bioreactor chamber.
The bioreactor is then connected to a flow system to provide pulsatile flow to vessels in the bioreactor. In the reactor, the cells grow into vessels that pass immunochemical, biochemical and mechanical tests. The implications of this technique extend towards therapy of coronary artery disease because this technique is a promi promising approach to regenerate implantable small diameter vascular grafts for arterial bypass.
Although this method can provide insight into vascular engineering, it can be applied to other organ systems such are under cyclic physiological forces. Generally, individual new to this method will struggle. You will require a lot of practice to fully master the skills.
We first had the idea for this approach when we realized the importance of applying physiological mechanical stimulation to regenerate engineering constructs. Visual demonstration of this method is critical as the bioreactor sing process are difficult to learn because there are many steps and can be acquired only through a detailed demonstration. To begin, use distilled water to clean 30 centimeter long sections of three millimeter inner diameter silicone tubing and allow it to air dry.
Use scissors to cut out a 1.1 centimeter by eight centimeter section of PGA mesh for each piece of tubing using a hemostat, so the PGA mesh to the tubing with Dexon 6.0 suture tie three surgical knots and follow with single stitches with hemostat, the secured mesh makes a PGA scaffold. Wash the scaffolds by first dipping them into one molar sodium hydroxide for one to two minutes, and then three dips into distilled water for two minutes per dip. Pat them dry between washes and allow them to air dry in a hood for 15 minutes after the third wash.
Once dried, suture one centimeter Dacron cuffs onto each end of the mesh with three stitches. Leave a two to three millimeter tubing overlap and do not puncture the tubing. Be sure to leave about 15 centimeters of free suture for later use in this protocol.
All the bioreactor components and tools are auto claved beforehand. The day before initiating the bioreactor culture, the bioreactor must be partially assembled submerge the bioreactor components the scaffolds, so mesh silicone tubing, the surgical instruments and a thin wire longer than the length of the bioreactor in 70%ethanol bath for at least 30 minutes While submerged. Use the wire to pull the silicone tubing through sidearms of the reactor.
Now emerge the bioreactor and attach the PGA scaffolds inside by fastening the Dacron cuffs over the flared glass lips. Use the excess suture to tie down each cuff. Next, using connectors, attach the silicone tubing on one side of the scaffold with one pair of bioreactor arms and attach the tubing on the other side of the scaffold to the other two arms.
When the connectors are installed, pull them out of the sidearms and flush the bioreactor out with ethanol. Leave the reactor soaking for 10 minutes on each side. Set the bioreactor on some Petri dishes and flush it out with tissue culture.
Grade water. Using a pipette, be sure to also flush out the mesh and tubing. Now place a sterile stir bar in the reactor for future use and dry the apparatus overnight in hood with blower on and UV off from this point forward.
Do not lean over the reactor as this could lead to contamination During setup, all the connectors must be wiped clean with alcohol. Before attachments are made, many of the joints are then reinforced with paraform, which must be soaked in 70%ethanol before it is applied. So have plenty of both.
At the ready as a precaution, place a sterile Petri dish over each bioreactor opening to protect the PGA scaffold inside from contaminants. Now begin the setup by attaching the injection port to the third unused arm of bioreactor. Next, connect the IV bag with the flow system.
With the walrus tubing set, tie off the blue end as close to the Y junction as possible and clamp the tube. Attach the red tubing to far end of the IV bag. Then attach the white tube to one side of flow system.
Insert a three-way stop cock into flow system. Attach one end of the pressure transducer tube to the stop cock and attach other end to middle opening of the IV bag. Now to partially fill the flow system and check for leaks, add 350 milliliters of PBS and 1%fungi zone to the IV bag.
Using a 60 milliliter syringe before or after connecting the to the flow system, adjust the stopcock to control the flow direction of PBS and squeeze the bag to flush the system. This step can be done either prior to or post cell seating. Now cells can be seated in bioreactor.
In this example, resus suspend 8 million SM cells, which is about one confluent T 75 in 1.25 milliliters of medium and drip them uniformly onto the PGA scaffold from Dacron junction to Dacron junction. After adding the cells, wipe the rim of bioreactor clean with alcohol. Avoid hovering over the reactor.
Next, carefully assemble the silicone stopper lid. Make sure not to expose the bottom of the lid as it is removed from the autoclave bag or during any of the following attachment steps. First, attach the injection port to feeding tube at the male connector.
Second, attach PTFE 0.20 micron filters to each of the three airports. Third para, film the injection port. Insert the assembled silicone stopper lid into glass bioreactor.
Make sure that the feeding tube does not touch the seated PGA scaffolds. Then seal with paraform. Now connect the bioreactor to the flow system and place the bioreactor and flow system inside a 37 degrees Celsius incubator.
For 25 to 30 minutes, the reactor must be set on its side and rotated every five minutes. Next, while still in the incubator, fill the bioreactor chamber with 400 milliliters of four to 10 culture media using a pump through a feeding tube. Now incubate the seeded scaffolds without any pulsatile pumping for six to seven days at 37 degrees Celsius with 5%carbon dioxide.
There is no need for medium change or vitamin C supplementation during this time before turning on the pump, inspect the tubing for leaks or kinks. Then adjust the speed rate of the pump to maintain a pressure range between negative 30 and 270 millimeters of mercury. Monitor and maintain this pressure daily throughout culture.
Medium changes and supplementation of ascorbic acid should be done twice a week while the scaffold grows with pulsatile pumping to feed fresh medium first, attach the injection port and PTFE filter onto the feeding lids for both medium change and medium waste disposal purposes. Next, place the feeding tube firmly in the dual directional pump and insert one end to feeding port of the bioreactor and the other end to the feeding lid. Now slowly start pumping out 200 milliliters of medium.
Gradually the pump speed can be increased. Then attach a new feeding tube and slowly start pumping 200 milliliters of fresh medium. To add ascorbic acid to the reactor, remove 25 milliliters of medium using a sterile 30 milliliter syringe.
Add five milliliters of sterile ascorbic acid solution and then return the 25 milliliters. Medium engineered vessels will appear to be opaque and achieve a wall thickness of approximately 250 microns after eight week culture. Under pulsatile conditions, hemat, toin and eosin staining of engineered vessels shows tissue morphology changes with time and with pulsing.
The luminal side of the vessels is indicated with an L masson's tri CHRO stain. For collagen shows the four week pulsed vessel have more collagen than its non pulses counterpart white arrows point to remaining PGA fragments in the vessels by 12 weeks. Immunochemical analysis shows the expression of the early smooth muscle marker alpha actin the intermediate smooth muscle marker cal poin one and the late smooth muscle marker myosin heavy chain Once mastered, this technique can be done in seven to eight hours if it is performed properly.
While attempting this procedure, it is important to remember to perform everything as cleanly and sterly as possible following this procedure. Other methods like modification of the lumen of the engineered vessels can be performed in order to answer additional questions like the importance of luminal surface modification in the maintenance of engineered vessels during in vivo studies. After its development.
This technique paved the way for researchers in the field of tissue engineering, especially vascular engineering, to explore potential means to treat coronary artery diseases in animal models. After watching this video, you should have a good understanding of how to achieve implantable engineer vascular grafts through a dynamic postal culture. Don't forget that working with sodium hydroxide can be dangerous, and precautions such as eye protection and lab coat should always be used while performing this procedure.
