A kind of proliferator and its application
An accelerator, fibroblast technology, applied in the field of biology, to achieve the effect of anti-aging skin, reducing scar formation, and remarkable efficacy
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Embodiment 1
[0090] Example 1: Primary isolation, culture, subculture and identification of skin fibroblasts
[0091] (1) Primary isolation, culture and passage of skin fibroblasts
[0092] After wiping the skin with iodine and subcutaneously injecting a small amount of anesthetic, the skin tissue is taken by professional medical personnel with a medical skin sampler to obtain a skin block with a size of about 3mm in diameter. Put the skin in a PBS solution containing 1000U / mL penicillin and 1000μg / mL streptomycin, fully wash off the blood on the tissue block, cut off the peduncle tissue appropriately and cut the remaining skin tissue into 4-8 pieces, and then Transfer the tissue pieces to a culture dish containing PBS solution containing 100 U / mL penicillin and 100 μg / mL streptomycin and wash thoroughly; cut up the skin tissue pieces and transfer them to a 15mL centrifuge tube, add 1-2mL with a mass volume ratio of 0.25 % neutral protease at 37°C for 1-3 hours, separate the epidermis and...
Embodiment 2
[0095] Example 2: Preparation process of fibroblast extract freeze-dried powder
[0096] (1) Preparation of Human Skin Fibroblast Extract Solution
[0097] Human skin fibroblasts in good growth state were subcultured and expanded in T-175 culture flasks, and the culture medium was phenol red-free DMEM high-glucose medium containing 10% (v / v) serum substitute (penicillin 100 U / mL, strepto Mycin 100 μg / mL, pH7.2). After the cell confluence reaches 80%-90%, use trypsin digestion solution (0.25% (w / w) trypsin + 0.02% (w / w) EDTA) to digest the cells, press 5000-6000cell / cm 2 Subculture. When the cells were subcultured to the P6-P8 generation, the subculture was stopped, and the skin fibroblast supernatants of each passage were collected and stored at -80°C until processing. Trypsinization was used to collect the skin fibroblasts by centrifugation for ultrasonication. The ultrasonication conditions were: 4°C, 400W, ultrasonication for 3s, interval of 4s, 99 cycles. After the son...
Embodiment 3
[0100] Example 3: Effects of luffa saponin and bitter melon saponin composition on skin fibroblast collagen synthesis and cytokine secretion
[0101] After identification, the P3-P5 skin fibroblasts in good growth state were taken for culture, and the cells were collected to adjust the cell density to 5×10 4 cell / mL, inoculate cells into a six-well plate, 2 mL per well. After the cells were cultured for 24 hours, the original culture medium was removed, and the skin fibroblast culture medium containing the combined stimulator of loofah saponin and bitter melon saponin was added as shown in Table 1 for culture. After the cells were cultured for 3 days, the cell culture supernatants of each group were collected. Using human type I collagen (Collagen I), human transforming growth factor (TGF-β), human platelet-derived growth factor (PDGF-AB), human basic fibroblast growth factor (bFGF) ELISA kits, The contents of collagen and major cytokines secreted by human skin fibroblasts w...
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