This experiment was mainly used to culture porcine thyroid cells.
Operation method
Cell Culture Technology
Principle
Because the thyroid gland is rich in fibrous tissue, the digestive isolation of the thyroid gland is generally done by a combination of collagenase and trypsin, and digestion with a single trypsin is generally effective; thyroid-stimulating hormone (TSH) is necessary for the culture of thyroid cells, and it can play a role in promoting the growth of thyroid cells.
Materials and Instruments
Porcine Thyroid Move I. Experimental steps Figure 1 Porcine thyroid cells after 24 hours of incubation. Caveat 1. In primary culture, the activity of the inoculated cells is directly related to the success or failure of the culture. 2. The cell activity of the tissue with short thermal ischemia time is good. It is advisable to process and culture the tissue as soon as possible without delay. 3. The digestion time can be shortened for soft tissues that are purely independent thyroid adenomas, because they are relatively easy to digest. 4. When the primary cells inoculated into the culture flask are observed under the microscope, they are most likely to grow if most of them are in small clusters of 5 to 10 connected cells. Common Problems Trypsin is suitable for digesting soft tissues with little intercellular matrix such as epithelial tissues, liver, kidney, etc., and is also very good for passaged cells. However, it is less effective in digesting fibrous tissues or harder cancerous tissues. Collagenase has a strong digestive effect on collagen and is suitable for digesting fibrous tissues, epithelial tissues and cancerous tissues. Because the thyroid gland is rich in fibrous tissues, the digestive separation of the thyroid gland is usually carried out by the combined digestion method of collagenase and trypsin, and the effect of digestion with a single trypsin is generally effective; thyroid-stimulating hormone (TSH) is necessary for the cultivation of thyroid cells, and it can play a role in promoting the growth of thyroid cells. For more product details, please visit Aladdin Scientific website.
F-12 medium Fetal bovine serum Trypsin Collagenase IV Bovine TSH
Ophthalmic Scissors Drip Tube Centrifuge Petri Dish CO2 Incubator
1. Remove 1 or 2 thyroid glands quickly after killing the pig, place them in a beaker with 75% alcohol and send them to the laboratory in an ice box (within 1 h).2. Immediately place in PBS buffer (containing penicillin) at pH 7.4 and rinse repeatedly until the rinse solution is clear.3. Remove the envelope and connective tissue, and use ophthalmic scissors and forceps to cut the thyroid tissue into 1 mm3 pieces.4. Place in a container containing 0.25% trypsin and 300 U/ml collagenase, and place in a 37°C incubator for 60 min, shaking every 15 min.5. Two thirds of the supernatant is pipetted into a centrifuge tube and digestion is terminated by the addition of medium containing fetal bovine serum.6. Centrifuge at 1000 rpm for 10 minutes and discard the supernatant.7. The cell precipitate is made into a cell suspension with F-12 medium, blown well and filtered through a stainless steel mesh (200 mesh), then centrifuged at 1000 rpm for 10 minutes and the supernatant discarded.8. The precipitate was suspended in F-12 medium containing 15% fetal bovine serum and 1 U/L bovine TSH, and the number of viable cells was demonstrated to be greater than 95% by Taipan blue staining, and the cell concentration was adjusted and inoculated into culture plates (1 ml/well).9. Incubate at 37℃ in 5% CO2 incubator and change the solution every 2-3 days until the cells are fused into pieces and then used for experiments.
Results
Porcine thyroid cells were cultured for 24 hours, and under the microscope, the cells were adherent to the wall and showed aggregated growth, and the cells were round or oval in shape, as shown below. 