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1. Quickly remove 1 or 2 thyroids after killing pigs, place them in a beaker containing 75% alcohol, and send them to the laboratory (within 1 h) using an ice box.
2. Immediately place in PBS buffer (with streptomycin) at pH 7.4 and rinse repeatedly until the rinse is clear and transparent.
3. Remove the capsule and connective tissue, and cut the thyroid tissue into pieces of 1mm3 size with ophthalmic scissors and tweezers.
4. Place the container containing 0.25% trypsin and 300 U/ml collagenase in a 37 ° C incubator for 60 min and shake once every 15 min.
5. Pipette two-thirds of the supernatant into the centrifuge tube with a pipette and add the medium containing fetal bovine serum to stop digestion.
6. After centrifugation at 1000 rpm for 10 minutes, discard the supernatant.
7. The cell pellet was made into a cell suspension using F-12 medium, thoroughly blown and filtered through a stainless steel mesh (200 mesh), centrifuged at 1000 rpm for 10 minutes, and the supernatant was discarded.
8. The pellet was suspended in F-12 medium containing 15% fetal bovine serum and 1 U/L bovine TSH. Trypan blue staining confirmed that the number of viable cells was greater than 95%, and the adjusted cell concentration was inoculated into the culture plate (1 ml/ hole).
9. Place in a 5% CO2 incubator at 37 °C and change the solution every 2-3 days until the cells are fused into pieces and used for the experiment.
2. Results Pig thyroid cells were cultured for 24 hours. The cells were attached to the microscope and aggregated. The cells were round or oval.
Precautions
1. In primary culture, the activity of the inoculated cells is directly related to the success or failure of the culture.
2. Tissues with short tissue warm ischemia time have good cell viability. Try not to delay the time after taking the material, it should be handled and cultivated as soon as possible.
3. For tissues that are softer than simple thyroid adenomas, the digestion time can be appropriately shortened because it is relatively easy to digest.
4. Under the microscope, observe the primary cells inoculated into the culture flask. If most of them are 5 to 10 connected cell small clusters, they will grow easily.
Experimental considerations for porcine thyroid cell culture
Pig thyroid cell culture experiment step I, experimental steps