The overall goal of this procedure is to rapidly isolate collagen type one from a dermal sample. This is accomplished by first slicing the dermal sample into small pieces. In the second step, the non collagenous solubilized proteins and polysaccharides are removed by rapid agitation of the dermal sample in sodium acetate.
Next, the sample is rinsed with water to remove all traces of the sodium acetate. In the final step, the collagen type one is extracted from the dermal sample using citrate buffer centrifuge to remove any remaining solids from the sample and then filtered using a disposable centrifugation device to concentrate the final product. Ultimately, collagen type one can be further polymerized from this concentrate.
The main advantage of this technique over existing methods is that our technique reduces the isolation time of collagen. Type one from dermal samples from over one week to under three hours. Begin by rinsing a 25 to 50 gram dermal sample in ice cold distilled water, and then use depilatory cream to remove any wool, fur or hair.
Next, use a single edge razor blade to scrape the sample clean of connective tissue and fat, and rinse the sample in more ice cold distilled water. Then use the razor to slice the skin sample into one by one centimeter pieces. To remove the non collagenous solubilized material, first weigh out five grams of sample per 50 milliliter conical tube and add 30 milliliters of ice cold, 0.5 molar sodium acetate to each sample.
Then using a 50 milliliter tube adapter, mix the tubes in a benchtop homogenizer at the six meters per second setting. After one minute, discard the supernatant and mix the samples again in more ice cold sodium acetate for a total of seven wash cycles after the last wash, rinse the sample and ice cold distilled water and mix the samples. One more time to remove the residual sodium acetate.
Then use a spatula to compress the samples against the side of each tube to remove the excess liquid and transfer each sample to fresh individual 50 milliliter conical tubes to extract the collagen. Next, wash the samples two times in two milliliters per gram, 0.075 molar sodium citrate buffer for one minute at six meters per second in the benchtop homogenizer, discarding the supernatants and compressing the samples after each wash as just demonstrated after the second wash at a fresh aliquot of sodium citrate buffer to each sample, and then perform six sequential one minute six meter per second cycles of agitation without removing the buffer between each cycle. Now transfer the thick clear supernatants to individual collection tubes and add an additional one milliliter per gram 0.075 molar sodium citrate buffer to the supernatants and perform one final agitation cycle.
Add these final supernatants to collection tubes and then centrifuge the collection tubes for 10 minutes at 3000, 200 times gravity and four degrees Celsius. Transfer the supernatants to the top compartment of a 100, 000 molecular weight cutoff centrifugal filter device, and then spin them down for 30 minutes at 3000, 200 times gravity and four degrees celsius. Finally, transfer the purified collagen one from the upper compartment to individual clean conical tubes and store the samples at four degrees Celsius.
The final product achieved from this procedure is a concentrated, highly pure preparation of soluble collagen, one that can be further polymerized by adding sodium bicarbonate and incubating the sample at 37 degrees Celsius. Phase contrast microscopy, images, and transmission electron micrographs of this solidified product reveal collagen, one fis that could serve as a substrate for cell attachment to tissue culture plates, or be used as a scaffold for 3D tissue engineering applications Once mastered, this technique can be done in two hours and 15 minutes if it is performed properly.
