Preparation method and product of biological cosmetic raw material derived from human stem cells
A stem cell and quality stem cell technology, which is applied to the preparation of bio-cosmetic raw materials derived from human stem cells and its product field, can solve the problems of skin allergy, low biological activity, lack of modification, etc.
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Embodiment 1
[0065] Example 1 Culture of human mesenchymal stem cells
[0066] Collect discarded human umbilical cords and cut them into 1mm pieces 3 Size in 10ml Mesen Gro serum-free medium (containing 50μg / ml hydrocortisone, 10μg / ml ascorbic acid and 50μg / ml human insulin), 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 10ml 75cm 2 Culture bottle proliferation culture starts, pass to the second generation 20ml 225cm 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 microcarrier rotat...
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 the human mesenchymal stem cells, mix well, add 1ug of protease inhibitor (Dalian Bao Biology Co.), place on ice, and crush in an ultrasonic breaker (Thermo Electric Corporation) for 8 minutes at high speed. Centrifuge at 15,000 rpm for 20 min, 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 (Spectrum, In...
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 the Mesen Gro 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 ...
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