Primary culture method of normal human thyroid cells
Experimental Materials:
1. Â Â Â Â Â Thyroid cell material source: normal tissue next to human benign thyroid adenoma, hyperthyroidism thyroid tissue or thyroid tumor tissue can be used as research materials;
2.      1×PBS containing no Ca 2+ and Mg 2+ , adding 200,000 IU/L penicillin, 200 mg/L streptomycin, pH 7.2;
3.      Culture medium: DMEM medium containing 15%-20% calf serum, 0.6 mmol/L HEPES, 12.5 mmol/L glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin. Adjust the pH to about 7.0 with 5.6% NaHCO 3 solution;
4. Â Â Â Â Â Digestive juice: 0.25% trypsin solution;
5.      Culture apparatus: culture flask or dish, stainless steel mesh screen with a pore size of 100 μm, ophthalmic scissors, tweezers, etc.;
Training method:
1. The material taken is placed in a sterile container filled with PBS and immediately sent to the culture chamber for no more than 30 minutes. If it can not be cultured immediately, it can be placed in the culture solution station and stored at 4 °C, but it is not suitable for 4 hours.
2. Under sterile conditions, the tissue was transferred to a dish and washed 2-3 times with PBS. Removing the film and connective tissue with an ophthalmic sputum and squeezing out the blood components therein;
3. The tissue was cut into a homogenate of 1 mm 3 size with an ophthalmic scissors and transferred into a bottle. Add 5 to 10 times more digestive solution than the cells, and digest for 30-60 minutes in a water bath at 37 °C. Shake every 5-10 minutes;
4. After digestion, the digestion was terminated by the addition of cold PBS. It was filtered through a stainless steel mesh sieve having a pore size of 100 μm. The filtrate was collected in a centrifuge tube and centrifuged at 1500 r/min for 10 min. The supernatant was aspirated and the cell pellet was rinsed twice with PBS. Discard the supernatant by centrifugation. The tissues that have not passed through the sieve are collected, and the digested solution is further digested for 15-30 minutes under a water bath condition of 37 ° C, and sieved and combined. Adding a certain amount of the culture solution to the cell pellet after rinsing, and pipetting several times with a pipette to prepare a cell suspension;
5. A small amount of the cell suspension was aspirated, and the trypan blue dye solution was added and counted on a blood cell counting plate. The proportion of viable cells should be greater than 90%. Dilute the cells with the culture medium to a density of 5 × 10 5 - 10 × 10 5 / ml;
6. Inoculate the cell suspension into a culture flask or petri dish. Incubate at 37 ° C in a 5% CO 2 incubator;
7. On the next day, the culture solution was aspirated to remove the unattached cells, fresh culture medium was added, and the solution was changed once every 3-5 days;
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