Composition comprising sapogenin and exosome as effective ingredient
A composition and technology of sapogenin, applied in the field of skin external preparations, can solve the problems of not paying much attention, and achieve the effects of promoting fat cell proliferation and reducing cytotoxicity
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Embodiment 1
[0072] Example 1: Cell Culture
[0073] At 37°C, 5% CO 2 Next, the human dermal fibroblast (human dermal fibroblast, HDF) HS68 cells purchased from ATCC were added to 10% fetal bovine serum (fetal bovine serum, FBS; purchased from Thermo Fisher Scientific) and 1% antibiotic-antimycotic agent (purchased from ThermoFisher Scientific) DMEM (purchased from ThermoFisher Scientific) medium for subculture. In addition, at 37°C, 5% CO 2 Next, 3T3-L1 preadipocytes purchased from ATCC were subcultured in DMEM (purchased from ThermoFisher Scientific) containing 10% NBCS (New Born Calf Serum) and 1% penicillin / streptomycin.
[0074] According to the cell culture method known in the technical field of the present invention, at 37 ℃, 5% CO 2 cultured adipose-derived stem cells. Next, the cells were washed with phosphate-buffered saline (purchased from ThermoFisher Scientific), and then the medium was replaced with a serum-free, phenol red-free medium, and the cells were cultured for 1 t...
Embodiment 2
[0076] Example 2: Exosome Isolation and Purification by the TFF Method
[0077] To isolate, concentrate and diafilter exosomes from the conditioned medium filtered through a 0.22 μm filter in Example 1, the TFF method was used. As a filter for the TFF method, a cartridge filter (also called a hollow fiber filter; available from GE Healthcare) or a cassette filter (available from Pall, Sartorius, or Merck Millipore) was used. TFF filters with different molecular weight cut-offs (MWCO) are available. Using a filter with a selected MWCO, exosomes are separated and concentrated, and particles, proteins, lipids, nucleic acids, low molecular weight compounds, etc. smaller than that MWCO are removed.
[0078] For separation and concentration of exosomes, TFF filters with MWCO of 100,000 Da (Daltons), 300,000 Da or 500,000 Da were used. Exosomes are isolated from conditioned medium by the TFF method by removing material smaller than the MWCO and concentrating the conditioned medium ...
Embodiment 3
[0080] Example 3: Characterization of Isolated Exosomes
[0081] The amount of protein in isolated exosomes, conditioned media and fractions during TFF isolation was measured using the BCA colorimetric assay (from ThermoFisher Scientific) or the FluoroProfile fluorescence assay (from Sigma). For the exosomes separated and concentrated by the TFF method according to one embodiment, the degree to which proteins, lipids, nucleic acids, low molecular weight compounds, etc. are removed is monitored by protein assay, and the monitoring results are shown in figure 2 middle. As a result, it can be seen that proteins present in the conditioned medium are very efficiently removed by the TFF method according to one embodiment.
[0082] image 3Shown are the results of comparing the productivity and purity of exosomes in each of five independent batches when exosomes are isolated by the TFF method according to one embodiment. The results obtained from five independent batches were ana...
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