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Cardiomyocyte culture process

【Cultivation process】
1. Insert two 5ml syringes into DMEM and pancreatin respectively, and add 5ml of DMEM culture solution to the two dishes. Use a pair of large tweezers to take out an SD rat 2~3 days after birth, put it in 0.1% neocerin and soak it out, put it on the foam board, and fix the rat with a needle (right position). Use another large tweezers to take the iodine cotton ball and wipe the skin, and then use the alcohol cotton ball to deiodize, take a small scissors and small tweezers to open the skin, fully tear it apart, and then use the alcohol cotton ball to disinfect, then change to another one The small scissors and tweezers are placed slightly to the left of the midline of the xiphoid process to open the chest and take the core.
2. Place the removed heart in a round dish containing DMEM prepared just now and then remove one. Repeat the above process. (After all the rats are killed, take the rat tweezers, foam board, and Xinjieer extinguishing tank out of the table. Disinfect, clean, spread gauze, and change gloves.
3. Use the third pair of scissors and tweezers to remove the blood coagulation and fibrous tissue around the heart in the round dish, and place it in another round dish pre-loaded with DMEM. Take 5ml of pancreatin, then put a little of it into a small beaker of 50ml, and pour most of it into a conical flask. Use the fourth pair of scissors to cut the heart in the beaker into 1mm3 pieces, pour them into an Erlenmeyer flask (can be scraped in with scissors), and then clean the beaker with the remaining pancreatin.
4. Put the thermometer and the triangular beaker into a 250ml flask, (adjust the amount of water in advance, the Glass Conical Flask will not be more than that, and the temperature will be uneven if it is less). Turn on the power of the magnetic stirrer, adjust the temperature (slowly add to 34~35℃) and speed (the speed should not be too fast, otherwise the cells will be broken, basically marked as parallel). Must pay attention to monitoring.
5. The timing starts when the temperature reaches 34°C, the first time is 10 minutes, and the rest can be 8 minutes, depending on the situation. During this period, 5 or 6 centrifuge tubes were placed on the test tube rack, labeled 1, 1, 2, 2. Among them, add 2 ml of DMEM solution to the two tubes of No. 1 in advance
6. After 10 minutes, remove the Erlenmeyer flask, turn off the magnetic stirrer, and gently pipette with a pipette (to make the digested cells fall off the tissue mass quickly). The pipette (1) is fixed and placed in one after use. In the centrifuge tube, after each digestion, use it for a few pipetting, the first time the supernatant is discarded, and then pour 2ml of the supernatant into the two No. 1 tubes (try not to pour out the tissue or leave it. Too much liquid, so as to avoid repeated digestion of the digested cells and cause damage), use another pipette (2) to mix and balance the two tubes (that is, the two tubes have the same level), and gently pipette to avoid damaging the cells.
7. Then put the two centrifuge tubes in the centrifuge, adjust for 10 minutes, 800 or 1000 revolutions, put a small lock, and turn on the switch.
8. At the same time, add 5ml of pancreatin to the Erlenmeyer flask, continue to digest for 8 minutes, pay attention to control the temperature. At this time, add 2ml DMEM solution to the two No. 2 tubes respectively.
9. Remove the Erlenmeyer flask and blow it with the No. 1 pipette, then let it stand for a while and pour 2 ml of supernatant into the two No. 2 tubes respectively, take out the two No. 1 tubes after centrifugation, and stick the supernatant along the cells. Pour out slowly, then add 4ml of DMEM solution to one of the tubes, use a No. 2 pipette to take a small amount of liquid into the other centrifuge tube, wash off the sediment cells at the bottom of the tube, transfer all the liquid into one of the three tubes (1 No. 1 tube, two No. 2 tubes) balance, put in a centrifuge tube, and centrifuge for ten minutes. At the same time, add 5ml of pancreatin, put it in the Erlenmeyer flask, and continue to digest (pay attention to control the temperature and count when it reaches 34℃).
10. After the digestion is about 4 times, pipette evenly, suck a drop on the microscope glass slide, get the microscope to observe the digestion, and determine the number of digestions.
11. Place the pipette after centrifugation twice on the test tube rack. After the centrifugation process is complete, pour out the supernatant of each tube, add 3ml (do not be precise, roughly this) DMEM solution to each tube, remember how much DMEM you add Change the pipette (No. 3) to suck down the cell block at the bottom of each pipe and pipette evenly, then transfer it into a 50ml beaker, then use a syringe to draw the remaining amount of DMEM into the beaker, add calf serum and antibiotics, and change the pipette (4 No.) Blow evenly.
12. Take out the 24-well culture plate, one person pipettes, and the other person uses a pipette to add medicine. When the lid is overheated, close the lid and place it in the CO2 incubator. After 24 hours, observe whether it adheres to the wall.
(For 24-well plate, add up to 2ml per well, generally add 1~1.5ml per well when adding liquid)