Isolation and culture experiments of neonatal rat heart cells
Isolation and culture experiments of neonatal rat heart cells
This experiment is based on the "Guide to Cellular Experiments", translated by Huang Peitang et al.
Operation method
Isolation and Culture Procedures
Materials and Instruments
Sprague Dawley pups Move Tissue digestion and isolation of cells For more product details, please visit Aladdin Scientific website.
Pancreatic enzyme solution Ethanol
Stirring table Beaker
1. In the cell manipulation room, place a heated stirring table with a 600 ml beaker containing 100 ml of sterilized water and heat it to 37°C.
2. Prepare the trypsin solution. Maintain the temperature of the trypsin solution at 37°C in a water bath or incubator.
3. Make an ice table: fill a dish with ice and invert it. Wipe the dish with 70% ethanol and place it on the table. Take three 60 mm disposable tissue culture dishes, add 5 ml of saline A and mark them with 1, 2, 3 and place them on the ice bench.
4. Place a clean, lidded beaker with a piece of Metofane-saturated gauze and anesthetize 0-1 day old Sprague Dawley pups in the beaker. 20 pups will take approximately 5 minutes to anesthetize. The beaker is wiped clean with 70% ethanol and placed in the operating room.
5. Remove the pups one at a time, making sure to close the lid again, then place the pups lying down on a piece of sterile gauze and scrub their chests and abdomens with ethanol. Clamp the rat's shoulder blades back so that they protrude into the chest cavity, pull a bite under the ribs in the direction of the sternum, and remove the heart and place it in the petri dish marked 1.
6. Continue with the remaining pups, making sure to keep gloves and instruments clean, and allow approximately 1 minute for each dissection.
7. After the above operations are completed, use two No. 5 forceps to remove connective tissue and blood clots from the heart before transferring it to the petri dish labeled 2. Rinse again and transfer to a petri dish labeled 3.
8. Take a sterile Pasteur pipette and gently aspirate saline solution A from dish 3 and add 2 ml of incubated trypsin solution. Chop the heart to the size of a grain of sand with the second sterile scalpel prepared.
9. Transfer the chopped heart and trypsin solution to a conical flask with a stirrer. Rinse the dish and scissors with an additional 3 ml of fresh trypsin and transfer to the conical flask, top up with trypsin to a final volume of 10 ml, stopper and place in a 37°C water bath. Turn on the stirrer, adjust the speed to about 60 r/min and digest for 15 minutes.
10. Remove the conical flask from the water bath and carefully aspirate the supernatant, which is mainly composed of hemoglobin, and add 10 ml of fresh trypsin to the flask and incubate at 37°C for 15 minutes.
11. Discard the supernatant. Add 10 ml of fresh trypsin, blow the solution twice with a disposable graduated pipette to mechanically disperse the cells, and incubate for 10 minutes.
12. Simultaneously with the above digestion, add 10 ml of cold inoculation medium to a 50 ml sterile disposable conical tube.
Cultivation of isolated cells
13. After digestion, carefully transfer the supernatant to the above conical tube with inoculation medium, which is the first collection of isolated cells. Add 10 ml of fresh trypsin to the remaining tissue mass and continue digestion.
14. While the digestion is going on, the conical tube containing the medium and the cell supernatant is centrifuged at 1200 r/min for 4 minutes. Gently aspirate the supernatant and resuspend the cell mass with 5 ml of inoculation medium.
15. Repeat steps 11 to 14 nine times or until only a small amount of tissue remains. Concentrate the cell suspension in a conical tube.
16. Precipitate the cells one last time by adding 10 ml of inoculation medium and blowing several times to disperse all the cell mass.
17. Transfer the 10 ml of suspension to a 100 mm sterile tissue culture dish and place in a 37°C incubator with 5% CO2 and high humidity for 1 to 1.5 hours to minimize contamination by fibroblasts.
18. After incubation, the culture solution containing non-adherent cells was collected from all dishes by flushing each dish with 10 ml of pre-warmed inoculum culture solution. The volume of the final collection solution was measured and the cells were counted using the Tepan blue reject method. Generally, the total cell number was between 5X107~1X108, and the viability rate was about 85%.
19. Adjust the cell density to 5X105~6X105 cells/ml with inoculation medium and make inoculation solution.
20. Add 2 ml of the above inoculum solution to each 35 mm-size dish and 5 ml of inoculum solution to a 60 mm-size dish and incubate for 4-6 hours.
21. After 4-6 hours, gently wash the dish twice with medium or saline solution A, pour it off, and add exchange medium to continue incubation overnight.
22. Rinse the dish twice with medium or saline solution A, add freshly prepared exchange medium and continue incubation, changing the solution once every two days.