This method can help answer key questions in the field of biomaterials, such as how cell matrix interactions affect sulfate. The main advantage of this technique is that it generates the first material that accurately reflects the native kidney cortex biochemical micro environments. Line a tissue culture hood with an under pad.
Place a one liter beaker containing a stir bar, a 150 millimeter sterile tissue culture dish, and the whole kidney into the hood. Fill the beaker with 500 milliliters of 1%SDS solution. Place the kidney in the sterile tissue culture dish.
Remove all perirenal fat by lightly shaving around the renal capsule with a scalpel. Then, make a shallow eight to 10 centimeter incision across the superior end of the kidney, to break open the renal capsule without damaging the underlying cortex tissue. Use two hemostat clamps to remove the renal capsule by peeling it away from the cortex tissue.
Bisect the kidney along the coronal plane by using the scalpel along the lateral side of the kidney. Isolate cortex tissue from both halves by carving out the medullar region with the scalpel. Then, dice the cortex tissue into 0.5 centimeter cubed pieces, and remove any large, visible vessels.
To isolate extracellular matrix from the kidney, place the diced cortex tissue into the beaker containing SDS solution. Cover the beaker with autoclaved aluminum foil and place it on a stir plate at approximately 400 RPM, outside of the tissue culture hood. After 24 hours of stirring, bring the beaker into a tissue culture hood.
Add a 40 micron sterile cell strainer made with nylon mesh. And then, fill a separate 1000 milliliter beaker with 200 milliliters of bleach, and place it in the tissue culture hood. Dispose of the SDS solution through the cell strainer, into the beaker containing bleach.
Pipet out any remaining SDS solution, until only decellularized tissue and the cell strainer remain in the beaker. Leave the cell strainer in the beaker, and add 500 milliliters of fresh SDS solution. Cover the beaker with the same aluminum foil, and place onto a stir plate at the same speed as before.
After decellularization and washing, transfer the decellularized tissue, referred to as kidney ECM from this point on, into a 30 milliliter self-standing conical tube. And fill it with cell culture grade water, until all of the tissue is submerged. First, use a tissue homogenizer to homogenize the kidney ECM in the conical tube for two minutes, until an opaque solution with no visible pieces of ECM is obtained.
Then, submerge the conical tube containing the kidney ECM in liquid nitrogen until boiling surrounding the tube no longer persists. Store the kidney ECM at minus four degrees Celsius overnight. To lyophilize the frozen decellularized tissue, slightly loosen the conical tube cap to allow for gas exchange, and place the tube into a lyophilization machine.
Lyophilize the kidney ECM for three days, or until it resembles a fine, white powder. To chemically digest and solubilize the gel, add carefully weighed porcine gastric pepsin, and 0.01 normal hydrochloric acid to a scintillation vile containing a stir bar. Stir at approximately 500 RPM, until all the pepsin has dissolved.
Then, transfer the lyophilized kidney ECM to the scintillation vile and leave the solution on a stir plate at approximately 500 RPM for three days to make the stock kidney ECM hydrogel. In a tissue culture hood, pipet the required volumes of cell culture media, one normal sodium hydroxide and M199 supplement, into a sterile 30 milliliter self-standing conical tube. Mix the neutralizing reagent solution with a micro spatula.
Use a sterile one milliliter syringe to transfer the appropriate volume of stock kidney ECM hydrogel to the neutralizing reagent solution. Use a micro spatula to gently mix the solution until a homogenous in color, hydrogel solution is obtained. Avoid introducing air bubbles by stirring slowly and gently.
To incorporate cells into the kidney ECM hydrogel, resuspend three hundred thousand cells in 10 microliters of medium for each hydrogel. Pipet 10 microliters of cell suspension into the final kidney ECM gel. Stir the solution with a micro spatula until the cells are evenly distributed.
Use a one milliliter syringe to fill the desired cell culture device with the kidney ECM hydrogel. Allow the gel to set at 37 degrees Celsius for one hour before transferring or plating the cells onto the kidney ECM in the cell culture device. Analysis of decellularized cortex tissue by mass spectrometry, revealed the presence of proteins associated with the basil lamina, with Collagen-IV and Collagen-I being the most highly represented.
Collagen-IV, A1 and A2 chains, ubiquitous in all basement membranes were conserved. Collagen-IV, A3 and A5 chains, present only in basement membranes of the glomerulus, were also detected. Common iso forms of Laminins and Collagen-I were also detected.
Human kidney peritubular microvascular endothelial cells, or HKMECs, cultured on Collagen-I, kidney ECM, and a 1:1 mixture gel, showed differences in phenotype. HKMECs cultured on Collagen-I, displayed uniform CD31 expression, seen in red. While HKMECs cultured on the two gels containing kidney ECM, displayed reduced CD31 expression in uneven distributions.
Matrix type did not appear to affect VWF expression, shown in green. While attempting this procedure, it is important to remember to fully homogenize the decellularized kidney cortex tissue. Furthermore, when mixing the gel with neutralizing reagents, avoid the introduction of air bubbles.
Micro physiological systems allow for the study of organ function in a controlled laboratory setting. The creation of such systems requires the implementation of cells, matrices, and stimuli. The kECM hydrogel fabricated here, will allow us to study such systems that recapitulate the kidney micro environments.
