The overall goal of this procedure is to repopulate Acellular three-dimensional rodent kidney extracellular matrix scaffolds with human renal epithelial cells. This is accomplished by first decellularizing, a haw rat kidney with a series of detergent solutions perfused through the renal artery over a period of 26 hours. The kidney is then sterilized and the acellular renal extracellular matrix scaffolds are injected with 40 million human renal cortical tubular epithelial or RCTE cells under continuous antegrade arterial perfusion within a custom designed Biore reactor system.
Ultimately, the ability of the RCTE cells to repopulate the kidney scaffold can be assessed by ER and perfusion assay and immunohistochemical analysis. This 3D cell culture technique allows investigators to repopulate whole organ extracellular matrix. Scaffold cells are deposited within the peritubular space within the extracellular matrix to allow investigation and binding factors that influence cell growth, differentiation and maturation.
The main advantage of this technique over alternative methods like the fabrication of synthetic polymer scaffolds, is that the renal extracellular matrix facilitates the adhesion, proliferation, and maturation of the perfused cells within an intricate network of natural structural proteins, growth factors and other biological components. Begin the decellularization procedure by connecting the renal artery catheter of each thawed kidney to the end of the perfusion circuit tubing downstream from the pump, ensuring that no air bubbles are entrapped in the catheter. If air bubbles are present in the catheter, use a 200 microliter micro pipette to draw the bubbles out and fill the catheter with fluid.
Suspend the kidney along the inner wall of an empty five liter beaker so that the renal artery not kinked or coiled, and adjust the pump drive to five milliliters per minute. Then press start after confirming that each kidney is perfusing by observing the dripping of solutions from the bottom of the organ plus each kidney with the appropriate solutions as designated in the flow chart at the end of the perfusion store the decellularized kidneys in PBS in a 50 milliliter conical tube at four degrees celsius or a maximum of two. We after assembling the perfusion circuit, remove the red screw cap on the bioreactor head and pipette 50 milliliters of a 0.1%per acetic acid, 4%ethanol solution into the medium reservoir through the opening.
Using a large pump cartridge, connect the peristaltic pump tubing segment to the pump head. Then adjust the flow rate to five milliliters per minute. Press start and allow the circuit to prime when the liquid fills the profusion line and reaches the remaining open port of the three-way stop cock.
Use a male lure plug to plug the port. Allow the circuit to fully prime until no air is observed in the perfusion circuit tubing or on the inside of the inlet. Lure acceptor expel bubbles from the perfusion circuit by temporarily increasing the flow rate of the pump.
When the system is fully primed, stop the pump drive and you sterilize six inch forceps. To carefully plug the female lure end of the renal artery catheter into the male inlet, lure acceptor on the inner surface of the bioreactor head, making sure that the connection is tight and that no air is left in the catheter. Allow the kidney to gently hang from the renal artery catheter so that the renal artery does not twist or kink.
Then tighten the metallic clamp holding the bioreactor head and body together so that the bioreactor reservoir is tightly sealed and close the screw cap. Now sterilize the kidneys with a room temperature perfusion of the per acetic acid ethanol solution at five milliliters per minute. After one hour, stop the pump and open the red screw cap.
Using a sterile pest, your pipette carefully aspirate all of the solution from the bioreactor reservoir. Then pipette 50 milliliters of fresh PBS into the bioreactor reservoir. Close the screw cap and start the pump for three sequential one hour perfusions at room temperature at five milliliters per minute.
After the final PBS rinse, aspirate the saline from the reservoir and add 50 milliliters of medium to the reservoir with a screw cap closed. Transfer the bioreactor with the attached perfusion circuit to a large capacity incubator at 37 degrees Celsius and 5%carbon dioxide. Then connect the bioreactor perfusion circuit to the pump and perfuse the kidneys at four milliliters per minute for at least one hour prior to seeding.
To cellularize, the kidneys collect a sufficient number of culture flasks for the desired seating concentration. Next, after aspirating the culture medium from each flask, use 10 milliliters of the appropriate cell dissociating enzyme to lift the renal cortical tubular epithelial cells from the flask at 37 degrees Celsius. After 10 minutes, begin checking the flasks on a face contrast microscope every two minutes until fold detachment is observed.
Then after taking a small aliquot of cells for counting, dilute the associated cell suspension in an equal volume of premed medium and centrifuge the cells resus. Suspend the pellet with an appropriate volume of culture medium to obtain a final concentration of two times 10 to the seventh cells per milliliter. Transfer two milliliters of the seating suspension, a 35 millimeter culture dish, and then draw the two milliliter volume into a sterile five milliliter syringe.
Transfer the perfusion bioreactor to a biological safety cabinet and turn the stop cock valve to close the flow to the seating port. Remove the male lure slip, plug from the stop cock and connect the syringe. Then quickly transfer the perfusion circuit back to the incubator and use the large pump cartridge to secure the peristaltic pump tubing segment to the pump head to seat the cells.
Close the stop cock valve port pointing toward the pump and slowly inject the cells into the kidney scaffold. Then turn the stopcock valve to close the flow from the syringe and start the pump at 25 milliliters per minute. After 15 minutes, lower the pump flow rate to four milliliters per minute, replacing the medium the following day and every two days thereafter.
To assess the viability and proliferation of the cells aseptically exchange the culture medium with 10 milliliters of zarin working solution for a four milliliter per minute perfusion of the kidney scaffolds after exactly one hour, collect the conditioned and partially reduced zarin solution and perform another four milliliter per minute perfusion. With 100 milliliters of fresh culture, medium kidneys sequentially profused with water and dilute detergent solutions became progressively more transparent over a 26 hour period by the final detergent perfusion, the kidneys vascular network and in particular, the inter lober vessels are prominently displayed in the decellularized scaffold. The entire organ is cleared of native cells leaving behind the intact basement membrane network of glomeruli tubules and collecting ducts and the extracellular matrix of decellularized blood vessels.
In addition to the larger vessels, the microvascular basement membranes within the glomeruli retain their structural integrity as well. RCTE cells seeded into the kidney scaffold as just demonstrated home primarily to the cortical regions of the kidney scaffold where they preferentially repopulate the pereg glomerular tubules. Few cells embed within the glomeruli at day one post seeding, and by day seven, the glomeruli are virtually devoid of cells while the majority of these cells occupy the cortical regions of the renal extracellular matrix.
After seven days of antegrade perfusion culture, many RCTE line tubules are present in the outer medullary and papillary tubules and collecting ducks after RCTE cell repopulation and one week of perfusion culture. However, the transparency observed following decellularization is lost and the ized kidney appears opaque and closer in appearance to its native state. Following this procedure, other methods like or immunochemical analysis of conditioned culture media can be performed to answer additional questions like, how do the metabolic or phenotypic states of these cells change within the renal extracellular matrix?
The ability to develop extracellular scaffolds and to add back donor sales to the matrix allows researchers to delineate growth and maturation in three dimensions within natural renal exo matrix scaffolds. The ultimate goal is to use this technology as a tool to better understand the mechanisms of renal repair and regeneration.
