To begin, rinse glutaraldehyde cross-linked gelatin coated cover slips in PBS containing calcium magnesium about five times on an orbital shaker. During the first three rinses, lift the cover slips using a sterile tweezer to allow thorough rinsing of the glutaraldehyde. Next, replace the culture medium of mouse RECs with fresh medium supplemented with sterile, filtered ascorbic acid.
After 15 days of treatment, rinse the cells with calcium magnesium free PBS. Incubate the cells in 250 microliters of warm decellularization buffer for two to three minutes. Confirm the decellularization of the cells under a phase contrast microscope.
Gently pipette out the buffer without disturbing the REC secreted subendothelial matrix. Then add 0.5 milliliters of PBS into each well. After removing the PBS, add 200 microliters of RNase free DNase one.
Incubate the mixture at 37 degrees Celsius for 30 minutes to remove all cellular debris. Next, view the Macroscale fibrous subendothelial matrix under a phase contrast microscope. Then use a confocal microscope to view the finer nanoscale matrix fibers at 100x magnification.
Subendothelial matrix aggregates were observed on the glass cover slips following decellularization of 10 day or 15 day cultures of human or mouse RECs. Confocal microscopy of the decellularized matrices showed a dense nano fibrillar collagen-IV and fibronectin matrix.
