A kind of anti-aging medicine or cosmetic and preparation method thereof
A cosmetic and anti-aging technology, which is applied in the field of biomedicine, can solve the problems of insufficient research and unsatisfactory anti-aging effects, and achieve the effects of improving anti-aging performance, good market application value, and promoting anti-aging effects
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Embodiment 1
[0029] Embodiment 1 The cultivation of highly active stem cells
[0030] Take 1ml of bone marrow and add an equal amount of PBS to mix well, slowly inject it into 1.073g / ml Percoll lymphocyte separation medium along the tube wall with a needle to make it a single cell suspension, and centrifuge at 2000r / min for 30min. Gently suck out the white turbid bone marrow mesenchymal stem cell layer in the middle, put it into another centrifuge tube, add PBS, centrifuge at 1500r / min for 15min, and wash 3 times in total. Discard the PBS, use the cell suspension made of L-DMEM culture medium containing 10% FCS for counting, and count at 1X10 9 The number of cells per L was inoculated in L-DMEM culture medium containing 10000 U / 100 ml of penicillin and streptomycin and 10% FCS. After 48 hours of culture, the first change of medium shows that the adherent cells become spindle-shaped, and when the cell fusion reaches 85%, subculture and inoculate in 25ml 60 Place the cell culture flask tre...
Embodiment 2
[0033] Example 2 Preparation of stem cell exosomes
[0034] The bone marrow stem cells identified in Example 1 were resuspended in serum-free MSC medium and inoculated on a bottom area of 25 cm 2 In a Petri dish, the number of seeded cells was 1×10 6 Each, culture medium 20ml. When the cell proliferation reached 90%, the cell surface was washed with sterile PBS solution, repeated twice, and 20 ml of α-MEM basal medium supplemented with 5 mg / L FGF was added in 1% O 2 , 3%CO 2 The culture supernatant was collected for exosome extraction after 3 days of culture under the above-mentioned culture conditions.
[0035] After cell counting, the collected cell supernatants were placed in 50ml centrifuge tubes, and centrifuged at 3500rpm at room temperature for 25min to remove cells and cell debris. The supernatant after centrifugation was collected, passed through a 0.22 μm filter membrane to remove large particles and vesicles, added 5×PEG 6000 reagent at a final concentration o...
Embodiment 3
[0036] Example 3 Preparation of control stem cell exosomes
[0037] The bone marrow stem cells identified in Example 1 were resuspended in serum-free MSC medium and inoculated on a bottom area of 25 cm 2 In a Petri dish, the number of seeded cells was 1×10 6 Each, culture medium 20ml. When the cells proliferate to 90%, wash the cell surface with sterile PBS solution, repeat twice, add 20ml α-MEM basal medium in 3% CO 2 The culture supernatant was collected for exosome extraction after 3 days of culture under the above-mentioned culture conditions.
[0038] After cell counting, the collected cell supernatants were placed in 50ml centrifuge tubes, and centrifuged at 3500rpm at room temperature for 25min to remove cells and cell debris. The supernatant after centrifugation was collected, passed through a 0.22 μm filter membrane to remove large particles and vesicles, added 5×PEG 6000 reagent at a final concentration of 5%, and stood overnight at 4°C. Centrifuge the overnigh...
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