Method for preparing human stem cell sourcing biobeauty raw material and product thereof
A technology of stem cells and mesenchymal stem cells, which is applied in the preparation of bio-cosmetic raw materials derived from human stem cells and its products, can solve the problems of lack of modification, low biological activity, functional and biological activity limitations, etc.
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Embodiment 1
[0065] Embodiment 1 The cultivation of human mesenchymal stem cells
[0066] Collect discarded human umbilical cords and cut them into 1mm pieces 3 Size in 10 ml MesenGro serum-free medium (containing 50 μg / ml hydrocortisone, 10 μg / ml ascorbic acid, and 50 μg / ml human insulin) at 37°C, 5% CO2 The mesenchymal stem cells were obtained by primary culture in the incubator, and then subcultured.
[0067] Digested with 0.25% trypsin (0.02% EDTA), from passage 1 10ml75cm 2 Culture bottle proliferation culture starts, pass to the second generation 20ml225cm 2 In the culture bottle, culture in 100ml system in the third generation 2g / L microcarrier rotary bioreactor, digest and scale up culture, then culture in 500ml system in the fourth generation 2g / L microcarrier rotary bioreactor, continue to scale up and then to The 2000ml system culture in the 5th generation 2g / L microcarrier rotating bioreactor, then continue to expand to the 5000ml system culture in the 6th generation 2g / L mic...
Embodiment 2
[0069] Example 2 Preparation of cosmetic raw materials derived from human mesenchymal stem cells
[0070] Take the 2×10 obtained in Example 1 9 Add 10ml of 1×PBS (pH=7.4) to human mesenchymal stem cells, mix well, add 1ug of protease inhibitor (Dalian Bao Biological Company), place on ice, and crush in an ultrasonic breaker (Thermo Electric Company, USA) for 8 minutes at a high speed of 15,000 Centrifuge at rpm for 20 minutes, take the supernatant and add it to the culture medium in which human mesenchymal stem cells have been cultured.
[0071] The culture solution containing human mesenchymal stem cell lysate was added to a dialysis bag with a molecular weight of 500 (Spectrum, USA), and placed in a refrigerator for dialysis concentration, and the volume was concentrated by 5 times.
[0072] In addition, the culture solution containing the human mesenchymal stem cell lysate can also be concentrated by ultrafiltration using a 500 molecular weight cellulose acetate membrane (...
Embodiment 3
[0074] Example 3 Detection of cytokine expression and secretion levels in human mesenchymal stem cells
[0075] Take the human mesenchymal stem cell culture fluid prepared in Example 1, the culture fluid containing human mesenchymal stem cell lysate and its concentrate, and detect the expression and secretion levels of cytokines therein by ELISA kit (R&D Systems, Inc., USA). For the steps, refer to Instructions for use are all used as a blank control with MesenGro medium that has not been cultured with human mesenchymal stem cells. Table 1 shows the expression and secretion levels of various cytokines in the culture medium of human mesenchymal stem cells, the culture medium containing lysate of human mesenchymal stem cells and the concentrate, and the unit is ng / ml.
[0076] Table 1
[0077]
[0078] Table 1 shows that the culture medium of human mesenchymal stem cells, the culture medium containing lysate and the concentrated solution all secrete high levels of cell growt...
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