Collagen cross-linked with human stem cell factors, a preparation method of collagen and applications of collagen in fields of beauty treatment and skin repair

[0037] Obtaining Human Stem Cell Factors with Collagen Affinity

[0038] Coat the bottom of the cell culture flask with collagen, coating method: 10mg collagen I, 99.167mL H 2 0.0.833mL 6M HCl is used for reagent preparation, and after standing at room temperature for 3 hours, use a 0.45um filter to filter, and the plating concentration in the cell bottle is 0.5ml / cm 2 , the bottom is naturally dry, and the coated culture bottle or culture plate can be stored at a low temperature of 4 degrees for one year.

[0039] Obtain P3 umbilical cord mesenchymal stem cells from the cell bank, inoculate them in culture flasks coated with collagen type I, and press 1-3×10 4 / cm 2 For inoculation, place at 37 °C, 5% CO 2 Culture in the incubator for 2-3 days, and when the cell confluency reaches 85-90%, the cell supernatant is collected.

[0041] Human Stem Cell Factor Concentrate

[0042] The collected stem cell culture supernatant was filtered through a 0.22 μm filter membrane, and then passed through a Vivaflow 50 swirl / tangential flow ultrafilter with a molecular weight of 50KD to concentrate the stem cell supernatant, and then the concentrated solution was passed through a Vivaflow with a molecular weight of 3KD 50 swirl flow / tangential flow ultrafilter to collect the retentate, which is the cytokine concentrate.

[0044] ELISA detection of concentrated human stem cell factor concentration

[0045]ELISA quantitative kits were used to detect the contents of SCF, VEGF, IL-10, and TGF-β in stem cell factors respectively. Taking TGF-β as an example, the detailed steps of ELISA kit determination were described.

[0046] (1) Mix all reagents thoroughly before use. Do not make the liquid produce a lot of foam, so as not to add a lot of bubbles when adding the sample, resulting in errors in the sample addition.

[0047] (2) Determine the number of strips required based on the number of samples to be tested plus the number of standards. Each standard, blank well, and sample were repeated three times. The sample was diluted 1:1 with the sample diluent and added 50uL to the reaction well.

[0048] (3) Add 50uL of the diluted standard substance to the reaction well, and add 50uL of the sample to be tested into the reaction well. Immediately add 50uL of biotinylated antibody. Cover the membrane pla