Bone marrow stromal stem cells (BMSC) are the most common adult stem cells, and their operation process is relatively stable and mature. Taking BMSC as an example, the specific application steps and procedures of adult stem cells are preliminarily described. (1) Preparation of bone marrow stromal stem cells for isolation and culture 1. Prepare DMEM culture medium to take DMEM medium 10g, NaHC03 2.2g, L-glutamine 300mg, vitamin C 37.5mg, penicillin 100,000 units, streptomycin 100,000 units After adding appropriate amount of three distilled water to dissolve completely, adjust the pH to 7.4, and make up the steamed water to 900m1. After filtration through a sterile 0.22 btm filter, FBS 100 ml was added and stored at 4 °C. 2. Prepare trypsin-EDTA digestive juice, weigh 0.125g of trypsin, EDTA 0.01g, dissolve the appropriate amount of PBS, adjust the pH to 7.4, make up the distilled water to 100ml, 0.22um filter filter sterilization, packaging , stored at 4 ° C. 3. Prepare Percoll Separate Percoll stock solution and 1.5 mol/L NaCl in a 9:1 ratio. 4. Formulated into osteoinductive culture medium DMEMlog/L, sodium p-glycerate 10nmo/L, dexamethasone 10nmol/l L-a-ascorbate 50btmol/L, L-glutamine 300mg/L, l, 25(OH 2 - Vitamin D3 10nmol / L, 10% fetal bovine serum. 5. Prepared chondrocyte-inducing medium DMEM 10g/L, dexamethasone 10 nmol/L, L-α-phosphate vitamin C 50 umol/L, L-glutamine 300 mg/L, TGF-βl 10ng/ml, IGFl00ug /ml, 10% fetal bovine serum. 6. Formulated as adipocyte induction solution DMEM 10g/L, 10% FBS, 1-methyl-3-isobutylxanthine 0.5mmol/m1, dexamethasone 1mmol/m1, insulin 10ug/m1, indomethacin 100 mmol/m1. (II) Isolation and culture of bone marrow stromal stem cells 1. Adult bone marrow extraction Adult volunteers should have no systemic disease, and the age should be between 20 and 40 years old. The instrument used was previously wetted with heparin and a 10 ml syringe was used to inhale 1 ml of heparinized PBS (1 000 U/ml). Bone marrow provider supine position, anterior superior iliac spine routine disinfection, toweling, local anesthesia with 2% lidocaine, bone marrow puncture needle vertically into the marrow cavity, slowly extract 2m1 bone marrow, immediately into a 15ml centrifuge tube (if bone marrow A large amount, the puncture point must be replaced to prevent the peripheral blood from causing bone marrow dilution). The bone marrow aspiration process of animals is basically similar. 2. The separation of BMSCs was carried out by density gradient centrifugation. The bone marrow was repeatedly blown into a single cell suspension by a 24 gauge needle, centrifuged at 600 g for 5 min, and the upper layer of suspended fat was carefully aspirated. The Percoll separation solution was preliminarily placed in a centrifuge tube, and the specific gravity of the Percoll stock solution was 1.13 g/m1, and adjusted to a density of 1.073 g/m1 with 0.9% physiological saline. Note that the bone marrow was slowly dropped onto the Percoll surface along the wall of the tube. The volume of both (Bone marrow: Percoll) was 1:2; 900 g was centrifuged for 30 min. It can be seen that the liquid in the centrifuge tube is divided into 5 layers, and the capillary pipette absorbs the cloud-like nucleated cell layer on the percoll layer, adds PBSloml, centrifuges at 600 g for 15 min, discards the supernatant to obtain nucleated cells, and counts the cells. The cells were inoculated at a density of lXl0'/cm2, and 10 ml of a low-sugar DMEM medium was added thereto, and the mixture was cultured at 37 ° C, at a temperature of 5% C 2 , and 95% O 2 . 3. After inoculation of primary cultured cells of BMSC, the cells were adhered to the microscope by inverted microscope every day. According to the adherence of the cells, the cells were changed 2 to 5 days after inoculation. When changing the solution, gently shake the culture dish to float the unattached red blood cells, aspirate the culture medium containing a large amount of red blood cells, and wash the PBS 2 to 3 times. At this time, a plurality of clones were clearly observed under a low magnification microscope, fresh culture medium was added, and cultivation under the same conditions was continued. Primary cells generally reach 80% confluence in 7 to 14 days and are subcultured. 4. BMSC digestion and passage to remove the culture solution, add PBS 10 m1, wash 2 times; after draining PBS, add 0.25% trypsin-EDTA4m1, place 37~C for 2rain; wait until the cells become round and float, add human serum The culture solution was 4 ml and the digestion was terminated. The cells were collected into a centrifuge tube, centrifuged at 500 r/min for 5 min, and the supernatant was aspirated; 10 ml of PBS was added, mixed in a vortex shaker, and the viability of the chondrocytes was observed by trypan blue staining, and the blood cell count plate was counted. Incubate in a 10 cm culture dish at 4×10'/cm2, add 10 ml of the culture solution, and incubate at 37 ° C with constant temperature. 5. BMSC multi-directional induction experiment was carried out by using the above prepared osteogenic, chondrocyte, and adipocyte-inducing liquid cultures. The morphology of the cells was observed under an inverted microscope, and the preset slides at the bottom of the culture dish were used for detection. The control group was still cultured and passaged with DMEM containing 10% FBS, and passaged with the induction group at the same time.
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