Pretreatment method of human adipose-derived mesenchymal stem cells
A technology of adipose-derived stem cells, applied in animal cells, extracellular fluid diseases, vertebrate cells, etc., can solve the problems of difficult control of parameters, stem cell death, etc., and achieve the effect of increasing survival rate and migration ability
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Embodiment 1
[0026] Example 1: Isolation and primary culture of hAD-MSC
[0027] hAD-MSCs were obtained by digesting and separating aseptic adipose tissue collected from adults after liposuction. Adult fat samples were obtained from a plastic surgery hospital. All samples were signed with informed consent.
[0028]Specifically, the fat collected by liposuction was stored and transported in D-Hanks' solution (purchased from Beijing Suolaibao Technology Co., Ltd.) containing double antibiotics (penicillin and streptomycin (purchased from North China Pharmaceutical Factory)). The adipose tissue collected by liposuction was washed twice with the above-mentioned D-Hanks' solution containing double antibiotics to remove blood cells and anesthetics, and centrifuged at 800rpm for 3min. Use a pipette to suck out the washed lower layer liquid, transfer the adipose tissue to a new 50ml centrifuge tube, add 0.2% collagenase P (purchased from Sigma) (fat:collagenase P volume ratio 3:1) to digest , inc...
Embodiment 2
[0029] Example 2: Subculture of hAD-MSCs
[0030] The primary cultured hAD-MSCs obtained in Example 1 were subcultured, specifically, 1) when the cells reached 70%-80% confluence, the medium was discarded, the cells were washed twice with D-Hanks' solution, and 0.25 % trypsin (containing 0.01% EDTA) digestion solution (Gibco), digested at room temperature, observed under a microscope, when the cells became round, FBS terminated the action of trypsin;
[0031] 2) Gently blow the cells in the culture flask with a pipette to separate the cells from the wall of the culture flask, and collect the cell suspension in a 15ml centrifuge tube, take out a small amount of culture solution and dilute to an appropriate multiple for counting with a cell counter, and the remaining cells are at 1200rpm Centrifuge for 5 minutes, discard the supernatant;
[0032] 3) Passage: The cells were resuspended in new medium, passaged at a ratio of 1:3, inoculated into a new culture bottle, and placed in...
Embodiment 3
[0033] Embodiment 3: low energy laser (LLL) irradiation treatment
[0034] The hAD-MSC subcultured in Example 2 was irradiated with a low-energy laser, and the cells between the first generation cells and the third generation were irradiated, and the adherent cells were directly irradiated. The irradiation time was about 1h ± 10min (irradiation dose 11- 16J / cm 2 ). Specifically, in this embodiment, hAD-MSCs were irradiated by using LXW660-II Quantum External Irradiation Apparatus from Shenyang Jixing Medical Instruments Co., Ltd. The working conditions of the quantum vascular external irradiation instrument are: power supply 220V±22V, 50hz±1HZ; light output wavelength 660nm±20nm, red to near infrared range (630-1000nm), output power of single point light source 3mw-4.5mw. All irradiations were carried out in a clean bench at room temperature, and the control group was treated under the same conditions without laser irradiation.
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