Composition and application thereof to preparation of product for improving chloasma

[0050] Example 1 Preparation of umbilical cord mesenchymal stem cells

[0051] After rinsing the umbilical cord with PBS solution containing 200U / m double antibody for 3 times, cut it into 2cm long pieces, peel off the arterial and venous tubes and adventitia, and cut them into pieces of 1-2mm 2Small pieces, evenly attached to a 10cm culture dish, placed upside down in a 5% CO2 incubator at 37°C for 1-2 hours; the medium is X-VIVO15 serum-free medium;

[0052] Add X-VIVO15 serum-free medium containing 10 ng / mL EGF, culture at 37°C with 5% CO2 for 2 to 3 days, then change the medium in half, continue to culture for 6 to 8 days, and discard the tissue after the cells crawl out of the tissue continue to train;

[0053] After growing to 80% confluence, discard the culture medium, wash once with PBS, add 0.25% trypsin to digest for 1-2 minutes, add FBS to stop digestion, and collect umbilical cord mesenchymal cells by centrifugation at 400g for 5 minutes.

[0054] Resuspend umbi

[0056] The preparation of embodiment 2 stem cell extract A

[0057] The P2 and P3 generation stem cell culture supernatants prepared in Example 1 were collected, concentrated using a Slice200 tangential flow filter, and the cycle filtration time was 3 hours. During this process, large particles were removed by filtration through a 0.45 μm filter membrane, and then Filter through a 0.22 μm filter membrane to remove small particles below 3 kDa; the concentrated solution is made into a freeze-dried powder by a freeze dryer, which is the stem cell extract A.

[0058] The preparation of embodiment 3 stem cell extract B

[0059] Collect the P2 or P3 generation cells prepared in Example 1, add pure water and resuspend until the cell density is 1×106cell / mL~5×10 6 cell / mL, quick-frozen in liquid nitrogen for 5 minutes, and thawed at 37°C after taking out;

[0060] After thawing, put it into liquid nitrogen for quick freezing, repeat 3 times;

[0061] Ultrasonic oscillation for 3 minutes in an ice-water bath at 0-4°C (power 300W, frequency 20Hz-40Hz), ultrasonic oscillation 3 times;

[0062] The resulting suspensions were combined and filtered through a 0.22 μm filter membrane under sterile conditions to obtain stem cell extract B.