This method explains a detailed procedure for saphenous vein tissue engineering. We also demonstrate preparation of decellularization setups and bioreactor for recellularization using simple apparatus. The main advantage of this technique is that a simple blood sample is required for recellularization, thus eliminating painful bone marrow collection and time-consuming cell culture procedures.
For Triton-X 100 perfusion and washing, prepare decellularization setup one. To a two liter chamber, tape all three pieces of tube A.Tape one tube to the wall where the edge touches the bottom of the chamber for the detergent's inlet. Tape the other two tubes to the opposite wall with a distance of five to 10 centimeters in between, depending on length of the vein.
At least five centimeters of these tubes should also be taped to the chamber's floor. Connect the detergent's inlet tube to tube B using the male and female lure connectors. Then connect tube B to tube F, and tube F to tube C.Connect tube C to the vein inlet.
Now, place tube F into the cassette of the peristaltic pump. Take another tube C and connect one end to the vein's outlet. Place the other end into a glass jar equipped with a bottom hose outlet and positioned 45 centimeters above the chamber, and tape it to secure.
This serves as the detergent outlet. Then, push one end of tube G into the hose outlet of the glass jar, and place the other end into the vein chamber. Next, prepare decellularization setup two for TnBP perfusion as detailed in the text protocol.
Also prepare decellularization setup three for DNA's perfusion as detailed in the text, and place it at 37 degrees Celsius. To prepare the recellularization bioreactor, insert tube H into one port of a four port cap to serve as the vein's outlet. Into the second port, insert tube I to serve as the media inlet.
And into the third port, insert tube J for the vein's inlet. Next, insert the other end of tube H into the fourth port to serve as the media outlet. Connect the reducing connectors to the inner ends of tubes H and J.Bend the other end of tube J into a U shape and tie it with a suture.
Now, place a 60 milliliter tube with a flat base into a 250 milliliter glass. Next, connect one end of tube K to the vein's inlet and connect the other end to tube E.Connect tube E to a second tube K, and then to the media inlet. Then place the prepared cap setup into the tube present in the bottle.
Finally, sterilize the bioreactor by autoclaving. Perform two freeze-thaw cycles on a human saphenous vein as detailed in the text protocol before cutting a biopsy two millimeters in length with scissors. Fix the biopsy in formaldehyde for 24 to 48 hours at room temperature.
Tie each end of the vein to the barbs of a male and female lure connector with a suture. Connect the vein to decellularization setup one and fill the chamber with one liter of solution two. Now, perfuse for 15 minutes at 35 milliliters per minute and empty the contents of the setup.
Then, add one liter of solution four and perfuse for four hours at 35 milliliters per minute. Empty the contents, add 500 milliliters of solution two, perfuse for five minutes, and again empty the contents. Disconnect the vein from perfusion setup one, and connect it to perfusion setup two.
Next, add one liter of solution five, turn on the stirrer, and perfuse for four hours at 35 milliliters per minute. Disconnect the vein from perfusion setup two, and wash the vein's outside twice with 20 milliliters of ultrapure water. Then use a syringe to wash the vein's inside twice with 20 milliliters of ultrapure water for five to 10 minutes.
After perfusing with perfusion setup three as described in the text protocol, connect the vein to perfusion setup one. Fill one liter of solution two and perfuse overnight at 35 milliliters per minute. Repeat the perfusion process four times for complete removal of nuclear material.
Following perfusion, empty the setup, fill one liter of solution two, and perfuse for 24 hours at 35 milliliters per minute. After 24 hours, empty the setup again and fill with one liter of ultrapure water. Perfuse for another 24 hours at 35 milliliters per minute.
Finally, repeat the perfusion using the same conditions but with solution seven. After verification of decellularization as described in the text protocol, sterilize the vein by placing it in a 50 milliliter tube containing 50 milliliters of solution eight. Agitate at 80 rotations per minute in a shaker for one hour at 37 degrees Celsius.
Working in a laminar flow cabinet to maintain sterility, now use sterile forceps to transfer the sterile vein to a new 50 milliliter tube containing 50 milliliters of sterile solution seven, containing 500 microliters of anti anti. Agitate the vein as before for 12 hours, changing solution seven at least twice in between. Using sterile forceps, transfer the vein to a new 50 milliliter tube, add 50 milliliters of endothelial media, and agitate for 11 hours.
Now, connect the vein by tying it to the bioreactor's vein inlet and outlet with sterile suture and forceps, and tighten the cap setup to the 250 milliliter bottle. Fill the bioreactor with the same endothelial medium. Add heparin at 50 units per milliliter and perfuse for one hour at two milliliters per minute in the incubator.
Collect 15 to 25 milliliters of fresh blood from the donor in heparin-coated vacutainer tubes and dilute it one to one with steen solution. Later, add endocrine gland derived vascular endothelial growth factor, basic fibro blast growth factor, and a subtle salicylic acid. After emptying the endothelial medium from the bioreactor, add the blood into it and perfuse for 48 hours at two milliliters per minute in a 37 degrees Celsius incubator with five percent carbon dioxide.
Approximately 12 hours later, work in the laminar flow cabinet to remove a drop of blood from the bioreactor and measure glucose using a blood glucose monitoring device. If the level is less than three millimoles per liter, add glucose to bring it within the range of seven to nine millimoles per liter. After 48 hours back in the incubator, drain the blood from the bioreactor in the laminar flow cabinet.
Add 30 milliliters of sterile solution seven, perfuse for five minutes, and drain the solution. Repeat this process until the blood red color is lost. Finally, add 30 to 45 milliliters of endothelial medium and perfuse for 96 hours in the incubator at two milliliters per minute.
Disconnect the recellularized vein from the bioreactor in the laminar flow cabinet, cut five millimeter edges of the vein using scissors, and discard. Take a biopsy as before, place in formaldehyde, and perform verification of recellularization as described in the text protocol. The gross morphology of a normal vein looks bright red in color.
The red color is lost in progressive decellularization cycles, and after five cycles of treatment, looks pale and white. The vein recellularized by perfusion of peripheral blood and endothelial medium again looked bright red in color. Shown here is a representative Haemotoxylin and Eosin staining image of normal vein showing the presence of many blue nuclei.
However, blue nuclei were not seen in the Haemotoxylin and Eosin staining of the decellularized vein. In the recellularized vein perfused with blood and endothelial medium, the Haemotoxylin and Eosin staining showed the presence of cell attachment at the lumen. After watching this video, you should have a good understanding of how to perform decellularization of saphenous vein by detergents and recellularization with peripheral blood and endothelial medium.
This technique of decellularization using perfusion of Triton-X 100, TnBP, and DNA's under pressure was successful and reproducible in the removal of nuclei from human saphenous veins. Though this technique takes 20 days to complete, storing the decellularized vein as an after-shelf product will decrease the procedure time to eight days. Recellularization of veins using the recipient's peripheral blood may be considered clinically relevant.
Vein's recellularized with similar protocol showed promise in clinical transplantation by providing a functional blood supply after operation. With slight modifications, this protocol can be used for any vein type, and as a personalized graft in clinics for all vascular surgeries.
