[0057] Example 2. Primary isolation and culture of human adipose-derived mesenchymal stem cells and detection of proliferation ability
[0058] 1. Primary isolation of human adipose-derived mesenchymal stem cells
[0059] 1. Aseptically collect the liposuction fluid for liposuction, wash with PBS (GIBCO, catalog number C10010500BT) for several times, remove the drugs and blood cells used in liposuction, and obtain adipose tissue.
[0060] 2. Cut the adipose tissue obtained in step 1 into pieces, collect them in a 50ml centrifuge tube, add an equal volume of PBS, and shake vigorously. After shaking, let stand at room temperature until layers appear in the centrifuge tube, and collect the upper part.
[0061] 3. Wash the upper part collected in step 2 with PBS, add an equal volume of 0.1% collagenase type I (Sigma company, catalog number 9001-12-1) to digest at 37°C for 1 hour, centrifuge at 400g/min for 5min, suck up The upper layer of fat was mixed, and then filtered through a 200-mesh sieve, and the filtrate was collected to obtain a cell suspension.
[0062] 4. Centrifuge the cell suspension obtained in step 3 at 1200 r/min for 8 min to collect the cell pellet. Add 2 times the volume of red blood cell lysate (BD Company, USA, catalog number: 349202) to the cell pellet to resuspend for 3 minutes, centrifuge at 1200 r/min for 8 minutes, discard the supernatant, and collect the pellet; Filter and collect the filtrate to obtain human adipose-derived mesenchymal stem cells, which are recorded as P0 generation cells.
[0063] 2. Culture and passage of human adipose-derived mesenchymal stem cells
[0064] 1. Culture of human adipose-derived mesenchymal stem cells
[0065] The P0 generation human adipose mesenchymal stem cells (referred to as P0 generation cells) isolated in step 1 were inoculated into the special medium for human adipose mesenchymal stem cells in Example 1, so that the cell concentration was 1 × 10 9 pcs/L, then place the culture system in a 10cm petri dish under 5% CO 2 , Suspension culture in a 37°C incubator. After culturing for 48 hours, the suspended cell liquid was aspirated and replaced with a new special medium; the medium was changed every 3 days, and when the cells reached more than 80% confluence for about 10 days after culture, the cells were collected and recorded as P1 generation human adipose mesenchyme. Stem cells (referred to as P1 generation cells), and subcultured.
[0066] 2. Subculture of human adipose-derived mesenchymal stem cells
[0067] The P1 generation cells were centrifuged at 1200 r/min for 8 min, the precipitate was collected, and the cells were counted with a Count Star cell counter. 6 Optimal seeding density per mL and inoculated into 10cm petri dishes in 5% CO 2 , 37 ℃ incubator to continue the cultivation. When the cell confluence reaches 80% to 90%, the cells are collected and recorded as P2 generation human adipose mesenchymal stem cells (referred to as P2 generation cells), and so on, repeated passages for large-scale expansion and culture, and passed to P15 generation When using human adipose-derived mesenchymal stem cells, a large amount of human adipose-derived mesenchymal stem cells can be obtained that meet the clinical therapeutic dose.
[0068] The special medium for human adipose-derived mesenchymal stem cells of the present invention is referred to as Medium-I. At the same time, serum medium and commercial serum-free medium (StemPro MSC SFM serum-free medium for human mesenchymal stem cells, Invitrogen, product number A10332-01) were used as control medium. Serum medium is a medium obtained by mixing a-MEM basal medium and 10% (volume percent) fetal bovine serum (FBS), which is denoted as medium-II; commercially available serum-free medium (StemProMSC SFM) Human mesenchymal stem cell serum-free medium) was designated as Medium-III.
[0069] Human adipose-derived mesenchymal stem cells were cultured in medium-II and medium-III according to the methods in steps 1 and 2 above, respectively, to obtain P0-P15 generation control cells.
[0070] 3. Comparison of the proliferation ability of human adipose-derived mesenchymal stem cells cultured in different media
[0071] The P1 generation human adipose-derived mesenchymal stem cells prepared in step 2 were seeded into medium-I, medium-II and medium-III at a density of 5000 cells/ml, and then placed in 5% CO. 2 , 37 ℃ cell culture incubator, add 0.25% (v/v) Trypsin (ProSpec company, product catalog number: PRO-770) and 0.04% (v/v) when the cells grow to 80%-90% confluence ) EDTA (Solarbio, product catalog number is E1170) is digested, the number of cells is calculated, and then subculture is carried out at a density of 5000 cells/ml according to the method in step 2, until the P2, P3, P4, P5, P6 generations are obtained. Adipose-derived mesenchymal stem cells. According to the number of cells in each passage, the average number of cell population doublings (Cumulative Population Doublings, CPDs) in each passage was calculated, and the cell population doubling curve was drawn with the cell passage as the abscissa and the CPDs as the ordinate. CPDs=lg (post-culture cell count/pre-culture cell count)/lg2.
[0072] The result is as figure 1 shown. It can be seen from the figure that the cell population doubling number of human adipose-derived mesenchymal stem cells cultured in medium-I is significantly greater than that in medium II and medium-III, and the proliferation rate is significantly increased. It shows that the special medium for human adipose-derived mesenchymal stem cells of the present invention can be used instead of serum medium, and the proliferation effect is significantly better than that of serum medium.
