Experiments on the isolation and culture of ventricular muscle cells from adult rats
Experiments on the isolation and culture of ventricular muscle cells from adult rats
This experiment is based on the "Guide to Cellular Experiments", translated by Huang Peitang et al.
Operation method
Isolation of ARVM cells
Materials and Instruments
Mouse Move I. Preparation for ARVM cell isolation For more product details, please visit Aladdin Scientific website.
KH buffers Gas mixtures
Convection bench or laminar flow ultraclean table Ether hood Condenser Circulating water bath and water bath shaker Circle rack Peristaltic pump Tabletop medical centrifuge Sterile beaker ColorpHast pH test paper
1. Prepare the following equipment and devices:
Convection bench or laminar flow ultra-clean bench
Ether hood
Condenser
Circulating water baths and water bath shakers
Circular racks with clips
Peristaltic pump free
Benchtop medical centrifuge
95% O2 5% CO2 gas mixture
400 ml sterile beaker
ColorpHast pH test paper (precision)
2. In convection or laminar flow benches, the Langedorff system is assembled by clamping the condenser and tee tubes to a ring stand and connecting the tubes to a peristaltic pump. The condenser is connected to a 37°C circulating water bath so that warm water flows from the bottom of the condenser into the bath and back from the top.
3. In the sterile area of the bench, arrange the following sterile instruments, which may be autoclaved or disposable sterile devices:
Tissue forceps
Large scissors
Bent Small Clamps 2 pcs
Small Scissors
Mosquito type hemostatic forceps
Swinnex 25 mm size filter holder
250 ml beaker
Glass and Silicone Tubing
Screw cap, 50 ml conical flask with small stirring bar
250 ml conical flask with screw cap
Disposable Petri dishes, pipettes, 60 ml syringes, 15 ml and 50 ml tubes, sutures
4. Prepare Krehs-Henseleit (KH) medium with and without CaCl2, filtered and sterilized and held at 37 °C.
5. Oxygenate and saturate KH buffer and calcium-free KH medium to a pH of approximately 7.4. Transfer 150 ml of oxygen-saturated calcium-free KH buffer to sterile 250 ml conical flasks with screw caps and place in a 37°C water bath. Make sure that the screw cap is closed.
6. Prepare Enzyme Solution I. Add 75 mg of collagenase and 45 mg of hyaluronidase to a conical flask containing oxygen-saturated calcium-free KH buffer.
7. Prepare Enzyme Solution II. Transfer 21 ml of solution I to a new tube, add 2 ml of trypsin and 20 μl of sterile 1 mol/L CaCl2 and filter to remove bacteria. The collagenase/hyaluronidase solution was transferred to a sterile 250 ml conical flask with screw cap and the collagenase/hyaluronidase/trypsin solution was transferred to a sterile 50 ml conical flask with screw cap.
8. Add 150 ml of KH buffer to the first 250 ml beaker and add to the perfusion system to avoid air bubbles. Check the pH of the flow-through solution at the cannula and return the pH to 7.4 by gassing with 95% O2/5%CO2. The flow-through solution is recovered into a sterile 400 ml beaker.
9. Cover the entire bottom of the dish with pre-warmed KH buffer.
10. Place the first rat in an ether mask for anesthesia.
11. Place the oxygenated calcium-free KH buffer into a second sterile 250 ml beaker, increase the flow rate to 5.5 ml/min, and perfuse for 6 minutes.
12. Enzyme Solution I is placed in a third 250-ml beaker, pumped to 8 ml/min, and perfused for 5 minutes. Place this 250 ml beaker under the heart and continue to reperfuse the heart for 15 minutes or more.
Animal Dissection and Perfusion of the Heart
1. After the animal is fully anesthetized, it is placed near the Langendorff device. The animal is placed flat on its back and the chest is moistened with ethanol and the chest skin is clipped.
2. a transverse incision is made under the ribs with large scissors to open the thoracic cavity, followed by incision of the thoracic diaphragm.
3. Lift the raphe and make a radial incision along the ribs about 2 cm on either side of the midline of the chest, never touching the heart.
4. the heart is palpated with clips, the lower connections are cut, and the heart and thymus gland are removed. the removed heart is placed in a Petrie dish containing KH solution and transferred to a table supporting the perfusion equipment.
5. Move the heart to the edge of the Petri dish, lift the thymus to expose the aorta, and cut off the thymus with small scissors.
6. Turn on the pump and adjust the flow rate to approximately one drop per second. Holding the two small forceps firmly in both hands, slide the aorta along the cannula until the bottom of the cannula sits on top of the heart.
7. Secure the heart with mosquito hemostats, with the pliers on the bottom of the aorta and making sure the hemostats are clamped on the top of the aorta.
8. the heart is securely tied with sutures in the lower portion of the hemostat and perfused for 3 to 5 minutes to allow complete drainage of blood from the heart.
9. Remove residual tissue from the heart, leaving the pulmonary artery and arterial appendages intact.
Digestion of Cardiac Tissue
1. During the last 2 minutes of perfusion with enzyme solution I, check the pH of the fluid with colorp Hast paper and adjust the pH to 7.4 with ventilation if necessary.
2. Cut the ventricle from the cannula and carefully slice it into 1 mm2 pieces. Transfer to a 50 ml conical flask containing Enzyme Solution II and incubate for 15 minutes at 37°C in a water bath with shaking at 100 r/min.
3. Blow the samples slightly with a 5 ml pipette and transfer to a 50 ml conical centrifuge tube with 20 ml of wash medium.
4. A 60 ml syringe, with the pusher removed, is fitted with a Swinnex filter. Add the cell suspension to the syringe and filter the cells into a new tube.
5. Centrifuge at 500 r/min (50 g) for 30 seconds, remove the supernatant and re-dissolve the cells in 10 ml of fresh wash medium.
6. Allow the spindle cells to settle for 20 minutes, carefully remove the supernatant and resuspend the cells in 10 ml of new wash medium.
7. Allow the cells to settle for 15 minutes, remove the supernatant and resuspend the cells in 10 ml of wash medium.
8. Repeat the above washing procedure 4 times, and after the last wash, pour off the supernatant. Add 5 ml of wash medium.
9. After resuspending the cells, carefully lay them on the top layer of 6% BSA solution prepared in DMEM and let the shuttle cells settle for 10-15 minutes.
10. The final cell mass should contain a high percentage of live clostridial heart cells. It would be good to receive 3X106 cells per heart.
ARVM Cell Culture
1. While the cells are being washed as described above, prepare coated plates/coverslips/slides at room temperature and submerge them in 10 μg/ml laminin solution for 30 minutes.
2. Resuspend the final cell mass by adding an appropriate amount of Petri dish and inoculate the coated plate with 1X106 cells per 100 mm dish.
3. Allow the cells to adhere to the wall for 1 hour and then incubate continuously in new medium. Continue incubation for 1 or 2 weeks in serumed or non-serumed medium.
4. Replace cells every other day.