The overall goal of this procedure is to prepare ized liver grafts by using Decellularized liver matrix. This is accomplished by first cannulating a donor liver. The organ is then decellularized by perfusion with the series of detergent solutions.
Revascularization of the decellularized liver matrix is then carried out by introducing adult primary hepatocytes by perfusion. Finally, an in vitro profusion system is used to culture, the cellularize liver graft. Ultimately, results can be obtained that show hepatic function of the resized grafts in vitro through measurement of albumin urea and total bile acid secretion in the profusion medium.
The main advantage of this technique over existing midgets like scaffold fabrication using synthetic biomaterials and traditional tissue engineering techniques, is that, in my opinion, the presence of the tissue specific extracellular matrix composition. The perfusion decellularized scaffold has also the tissue specific micro architecture, such as the vascular structure. The presence of the vascular structure enables us to reintroduce the cells into the scaffold more uniformly.
It also enables us to perfuse it uniformly with nutrients to enhance cell survival. The deceleration part of the protocol will be demonstrated by Nima with the post-structural fellow in our laboratory. The re retailization part will be demonstrated by Gabriel Price, who's also a postdoctoral fellow.
Her work focuses on industrialization of these grafts To perform perfusion decellularization begin by harvesting a rat liver by portal vein cannulation. Using an 18 gauge catheter, leave the inferior and superior vena cava open. Keep the organ hydrated in phosphate buffered saline or PBS in a 10 centimeter petri dish.
Next, set up a perfusion system that consists of an eight liter reservoir, peristaltic pump, and a bubble trap. Fill the perfusion system with phosphate buffered saline or PBS and keep it running for 10 minutes. Then fill a 10 centimeter Petri dish with PBS and reduce the flow rate of the buffer to 1.2 milliliters per minute.
Carefully transfer the harvested liver into the Petri dish of PBS and allow it to perfuse overnight. The next day. Perfuse the liver with 0.01%sodium ESAL sulfate or SDS into distilled water for five minutes.
Then switch back to PBS for one hour. Repeat the SDS and PBS perfusion steps three more times, increasing the SDS perfusion Time to 10 15 and then finally 20 minutes. Then perfuse the liver with the following series 0.01%SDS for 24 hours.
0.1%SDS for 24 hours. 0.2%SDS for three hours, 0.5%SDS for three hours. Distilled water for 15 minutes, 1%Triton X 100 in distilled water for 30 minutes, which will remove any bound nucleic acids.
And finally, PBS for two hours, which will wash the decellularized liver matrix or DLM after the perfusion is complete if desired, resect all the lobes except the median lobe. Store the DLM in a clean and sealed Petri dish in PBS at four degrees Celsius until ready to use to sterilize the DLM. Flush it with sterile PBS containing 0.1%per acetic acid and 4%ethanol and incubate it for three hours at four degrees Celsius.
Next, wash the DLM with sterile PBS twice. Then with PBS containing 2%penicillin streptomycin, 10 micrograms per milliliter, gentamycin and 2.5 micrograms per milliliter. Amphotericin B.Store the decellularized liver in the same solution at four degrees Celsius until ready to use for ization experiments.
Two, to cellularize a decellularized liver matrix under a tissue culture hood. Begin with a perfusion system similar to the one used to decellularize the liver. Fill the perfusion system with 200 milliliters of culture medium, such as high glucose DMEM, 10%fetal bovine serum, 100 units per milliliter, penicillin, and 100 micrograms per milliliter.Streptomycin.
Place the DLM into the perfusion chamber and connect it to the perfusion system through the portal vein cannula while the pump is running at five milliliters per minute. To avoid formation of any air bubbles, allow the medium to perfuse the DLM for 30 minutes. Next, stop the flow in the perfusion system and slowly inject 50 million hepatocytes that have been isolated from an adult rat into the perfusion system through the bubble trap.
Restart the flow at 10 milliliters per minute and recirculate the medium for 10 minutes. Stop the flow of medium again. Inject an additional 50 million hepatocytes.
Then restart the flow again at 10 milliliters per minute. Repeat this procedure until a total of 200 million hepatocytes have been introduced into the DLM. Once all the cells have been injected into the DLM, collect the profuse eight into 50 milliliter centrifuge, tubes, and centrifuge at 800 RPM for five minutes.
Discard the supernatants and combine the pellets into a single tube. Determine the number of cells and their viability via trian blue exclusion. To establish the seeding efficiency to culture.
A cellularize liver graft. Set up a sterile perfusion system that includes an oxygenator. Fill the perfusion system with 50 milliliters of culture medium such as Williams E with 5%PBS, 0.5 units per milliliter of insulin.
20 nanograms per milliliter of EGF 14 nanograms per milliliter of glucagon. 7.5 micrograms per milliliter of hydrocortisone, 100 units per milliliter of penicillin and 100 micrograms per milliliter of streptomycin. Place the cellularize liver graft into the perfusion chamber and connect it to the perfusion system through the portal vein cannula while the pump is running at five milliliters per minute.
To avoid formation of any air bubbles, aseptically close the perfusion chamber and seal it tightly to avoid any leakage during the culturing. Incubate the perfusion system at 37 degrees Celsius with 10%carbon dioxide and increase the perfusion flow rate to 15 milliliters per minute. Connect the oxygenator to a 95%oxygen and 5%carbon dioxide gas mixture tank, and set the gas flow rate to 0.5 liters per minute.
This should achieve an oxygen partial pressure of approximately 400 millimeters of mercury. Continue the culture for up to 10 days with daily changes of culture.Medium. Sample the medium daily for albumin urea and total bile acid secretion to monitor the liver functions of the graft at the end of the culture period.
Sample the cellularize liver graft for molecular and histological analysis. The complete decellularization of a rat liver takes about 72 hours. Using the described protocol.
The resulting matrix retains 100%of the fibrillar collagen, 50%of the glyco amino glycans, and only 5%of the DNA of the native liver. The vascular structure of the matrix is preserved as evidenced by corrosion casting and scanning electron microscopy analysis. The presence of the vascular microarchitecture within the DLM facilitates its repopulation with cells with an efficiency of 96%and its subsequent perfusion.
For in vitro culture, the re liver graft may be cultured up to 10 days in vitro and displays proper liver functions as confirmed via albumin, urea, and total bile acid secretion. So after watching this video, you should have a good understanding of how to decellularize and ize the whole liver.
