A method for inducing differentiation of adipose stem cells into chondrocytes
A technique for inducing differentiation of adipose stem cells, applied in the field of inducing differentiation of adipose stem cells into chondrocytes, can solve the problems of slow cell proliferation, low osteoblast differentiation efficiency, and decreased expression of marker genes in chondrocytes, so as to promote cell proliferation, The effect of improving the efficiency of induction of differentiation
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Embodiment 1
[0045] Example 1: Culture and passage of adipose stem cells
[0046] 1) Adipose tissue was extracted from the abdomen of the volunteer, washed three times with PBS solution under aseptic conditions, washed away red blood cells, and cut into small particles with a scalpel;
[0047] 2) Add 0.1% collagenase solution twice the volume of adipose tissue, shake at 37 °C for 2-3 hours, and digest;
[0048] 3) Collect the liquid after shaking digestion, 1500rpm / 10min, discard the supernatant, and resuspend the pellet with low-glucose DMEM medium containing 10% FBS;
[0049] 4) Filter with a 200-mesh fine sieve to filter out the residual tissue impurities in the suspended cell fluid, and the filtrate is placed in a centrifuge tube;
[0050] 5) 1500rpm / 10min, discard the supernatant, resuspend the pellet in low-glucose DMEM medium containing 10% FBS, and place it in a 37°C, 5% CO2 incubator for culture, this is the primary cell, after 5 days, change the medium for the first time;
[00...
Embodiment 2
[0053] Example 2: Induction of differentiation of adipose stem cells into chondrocytes
[0054] (1) Preheating
[0055] Preheat 1.2% sodium alginate solution (alginate suspension prepared with 0.9% NaCl) and calcium chloride solution to 37°C.
[0056] (2) Preparation of adipose stem cell-sodium alginate suspension
[0057] Adipose-derived stem cells (ADSCs) cultured to P3 passage in T175 flasks, about 90% of the bottom of the flask, rinse the cells 3 times with PBS solution, add 2-3ml 0.25% trypsin digestion solution to prepare ADSCs suspension, the cell concentration is 4.5 ×10 6 pcs / ml;
[0058] The ADSCs suspension was thoroughly mixed with 1.2% sodium alginate solution at a volume ratio of 3:1 to make a cell concentration of 6.0 × 10. 6 adipose stem cells-sodium alginate suspension per ml, gently pipet with a pipette to prevent the generation of air bubbles.
[0059] (3) Preparation of adipose stem cells-calcium alginate microbeads
[0060] Pipette the adipose stem c...
Embodiment 3
[0073] Example 3: Detection of cell proliferation activity by MTT method
[0074] The adipose stem cells-calcium alginate microbeads from the above four groups on the 3rd, 7th, and 14th days were respectively inoculated into 96-well plates, and 100 μl of cell suspension was added to each well, and the cell density was 5×10 4 pcs / m. There were 3 wells in each group, and a blank control group was set at the same time.
[0075] Add 10 μl MTT solution (5 mg / ml) to each well, continue to incubate for 4 h in a 37°C, 5% CO2 incubator, carefully discard the supernatant in the wells, add 150 μl dimethyl sulfoxide (DMSO), shake for 10 min, and add dimethyl sulfoxide (DMSO) for 10 min. The absorbance value of each well was measured at OD490nm of the immunodetector.
[0076] According to the MTT test, on the 3rd, 7th, and 14th days after induction, the OD values of experimental groups B, C, and D were significantly higher than those of control group A. On the 14th day, the OD value o...
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