CANINE CARDIAC MYOCYTE ISOLATION PROTOCOL November 2000 I. Preparation for cell isolation: A. Make sure the following items are autoclaved: 1. 1-8X8 baking dish 6. 3-1 liter bottles 2. 2-500 ml beaker 7. cellector/triturators 3. 3-250 ml beaker 4. 3-100 ml beaker 5. 4-50 ml beaker ***Surgical instruments: rib spreader, metzenbaum scissors, 10 blade scalpel, castro-veijo needle driver, vascular forceps, debakey forceps, small hemostat, measuring spatula B. Prepare solutions as shown on protocol sheet: 1. Ca 2+ KHB 2 liters 2. Ca 2+ free-modified Tyrode s 1 liter 3. Incubation solution with 2% BSA 100 ml 4. DMEM + P/S 5. DMEM + P/S + 10% FBS C. Wash out the perfusion apparatus: 1. ~100 ml of EtOH + filtered DI (MQ) water for each syringe and tubing 2. ~200 ml of MQ water in each syringe and tubing 3. ~100 ml of MQ water + P/S in each syringe and tubing 4. clean out the water bath with EtOH and Lysol, then fill it with DI water and turn on D. Inject dog with 1 ml IM BAG in housing kennel, bring up to the lab, inject with 5000 units Heparin IV (500 units/kg), wait 5 minutes E. Place Ca 2+ KHB in the left syringe and Ca 2+ free-modified Tyrode s in the right syringe. Flush system to make sure no bubbles remain and oxygenate left syringe for 10-15 min prior to experiment F. Set up dissecting area: 1. Need 4 blue pads down, roll of tape, opaque trash bag 2. Full bucket of ice X 3 3. 1-500 ml beaker filled with Ca 2+ KHB. Place in ice bucket 4. 1-8X8 baking dish half filled with Ca 2+ KHB. This will be used to dissect the left ventricular free wall away from the remainder of the heart. Place in ice bucket 5. One sterile 100 mm petri dish. Half of petri dish should be filled with Ca 2+ KHB. Place in ice bucket
a. For the cannulation dish, mount a 10 cc syringe containing Ca 2+ KHB 6. One 250 ml beaker with 95% EtOH (~50-100 ml) for sterilizing instruments. G. Prepare collagenase solution in sterile 50 ml beaker: Add 12 mg hyaluronidase, 32 mg collagenase and 40 mg of BSA to 20 ml of Ca 2+ -free modified Tyrode s. Ultimately this will be diluted up to 80 ml. 1. Prepare extra solution in sterile 250 ml beaker: Add 30 mg hyaluronidase, 80 mg collagenase, and 100 mg of BSA to 200 ml of Ca 2+ -free modified Tyrode s. II. Heart perfusion A. Euthanize dog with Pentobarbital (390 mg/ml) 1 ml/5 kg IV, place in lateral decubitus position, prep left chest with 95% EtOH, perform left thoracotomy, remove heart with several centimeter cuff of aortic root. B. Rinse heart in ice cold Ca 2+ KHB, making sure as much blood as possible is gently squeezed from chamber. Then transfer to 8X8 baking dish. C. Excise left ventricular free wall perfused by LAD, remove with left aortic cusp and wedge of aortic wall. D. Loosely approximate the epicardium and endocardium with a running 6-0 prolene suture. Carefully ligate major perforating vessels and the left circumflex artery. Trim aortic wall around left coronary ostia. Place a pursestring suture around the left coronary ostia with a 4-0 sik suture. E. Cannulate the left coronary ostia with a 14 gauge angiocath. Advanced into the proximal LAD. Secure in place with the silk pursestring. Wrap extra suture around the catheter and retain long ends to further secure to the perfusion apparatus. Mount catheter on the syringe containing Ca 2+ KHB in the mounting petri dish. Gently perfuse the heart to assess flow and identify any major leaks. Adjust catheter as needed. Ligate any persistent leaks. F. Start slow drip on perfusion apparatus, check for bubbles. Transfer cannula with heart to perfusion apparatus and turn on flow completely. G. Perfuse with Ca 2+ KHB for 10 minutes. Wipe off oxygenator, transfer to Ca 2+ -free modified Tyrode s after ~8 minutes. Check to make sure effluent is clear. H. Perfuse with Ca 2+ -free modified Tyrode s 1. After 1 min, collect fluid for 1 minute to determine flow rate (~15 ml/min is desired).
2. After 3 minutes, make sure syringe has 30 ml of Ca 2+ -free KHB and add the 20 ml collagenase solution to make a final volume of 80 ml. (15 ml in tubing). 3. After 15 minutes, change the enzyme solution if original is cloudy. 4. Total perfusion time with collagenase solution should be 30 minutes. III. Cell Isolation A. Remove heart and cannula from perfusion apparatus. B. Remove cannula and suture. Transfer heart to petri dish. Lift the endocardial flap. Remove the digested myocardium from between the epicardium and endocardium. Mince any partially digested pieces. Transfer entire digested portion of the myocardium to 2-100 ml beakers. Add 25 ml of fresh collagenase solution to each beaker. C. Swirl gently for 5 minutes in water bath. Remove supernatant with pipette and save. Resuspend ventricle in 25 ml of collagenase + Ca 2+ -free modified tyrodes. 1. Repeat for the second and third digest. 2. Dispose of first and second digest. D. For the fourth and fifth digest, gently triturate the minced tissue with wide bore (the 2 largest bores only) silanized Pasteur pipette, then swirl in the water bath. Repeat trituration at the end of the digests. Each digest should be 5-7 minutes. 1. Pour off supernatant into two 15 ml conical tubes; resuspend ventricle 2. Centrifuge conical tubes on setting 4 until max speed is reached; then turn off. Pour off supernatant and combine the two pellets in 5 ml of Incubation solution. Place a drop of cell suspension on a coverslip and view on light microscope to check viability. Save resuspended cells and place in 37ºC incubator. E. For the sixth and subsequent digests repeat step D. Collect the cells by passing through the cellector, which is sitting in a 100 mm petri dish. Transfer suspension to 2 X 15 ml tubes and then continue as described in D1. Approximately 7-9 digests will be needed. F. Combine digest 3-5 in one conical tube, and all digest run through the cellector in another conical tube. Suspend in 10 ml of incubation solution. Add back 100 mm CaCl 2 at 14.6 µl every 5 minutes for one hour. After adding back calcium, centrifuge each tube on setting 4 for 6 seconds. Resuspend the pellet in DMEM + P/S (volume will depend on projected myocyte yield (5-15 ml) IV. Plating Cells A. In preparation for plating cells, you will need to do the following in the hood prior to getting the cells out of the incubator. 1. Laminin coat coverslips: start ~40 min before ready to plate, place 1x18 mm coverslip per well, UV (uncovered for 5 min. Coat each coverslip
with 100 µl laminin (40µg/ml), UV (uncovered) for 10 min to sterilize, cover dishes and leave in hood until plating. 2. Warm up DMEM + P/S and DMEM + P/S + 10% FBS at 37ºC. 3. Clean and dry off hemocytometer. B. Count cells: Transfer a small aliquot of resuspended cells (make sure cells are resuspended completely and are not settling out) with sterile Pasteur pipette to hemocytometer. Count 9 fields: live (rod-shaped) cells vs. dead (non rod-shaped) cells. Calculate the percentage of rod shape cells and the concentration of cells Concentration= # of cells in 1 field x 10 4 Expect 50-70% viability C. Remove excess laminin from coverslips with sterile Pasteur pipette connected to vacuum. D. Transfer desired number of cells to coverslips. 1. Cells are plated in DMEM + P/S + 5% FBS 2. To determine the # of cells needed: a. For square coverslips need 2 x 10 4 cells/coverslip; 200 µl/coverslip b. Dilute cells so that concentration is 1 X 10 5 rod-shaped cells /ml in 50% DMEM + P/S and 50% DMEM + P/S +10% FBS; therefore final FBS concentration is 5% c. Plate 200 µl/coverslip, which equals 2 x 10 4 cells/coverslip. E. Place plate in incubator carefully so as to not disturb cells. Incubate for 2 h ours, remove serum containing media with Pasteur pipette, making sure to avoid the cells which are concentrated at the middle of the coverslip. Then add back 2 ml of DMEM + P/S. Incubate in 37ºC incubator until ready to use.
PROTOCOL SOLUTIONS KHB MW Stock g in 1 L stock 2 L KHB 118 mm NaCl 58.44 1 M 58.44 g 236 ml 4.8 mm KCl 74.53 0.5 M 37.27 g 19.2 ml 25 mm HEPES 260.3 add fresh 13 g 1.2 mm KH 2 PO 4 136.1 0.5 M 68.05 g 4.8 ml 1.2 mm MgSO 4 x 7 246.47 100 mm 24.65 g 24 ml H 2 O 1.0 mm CaCl 2 x 2 H 2 O 147 100 mm 14.70 g 20 ml 11 mm Glucose 180.2 add fresh 3.96 g ***ph to 7.4 Ca 2+ Free Modified Tyrode s MW Stock g in 1L stock 1 L FMT 118 mm NaCl 58.44 1 M 58.44 g 118 ml 4.8 mm KCl 74.53 0.5 M 37.27 g 9.6 ml 1.2 mm MgSO 4 x 7 H 2 O 246.47 100 mm 24.65 g 12 ml 1.2 mm KH 2 PO 4 136.1 0.5 M 68.05 g 2.4 ml 0.68 mm Glutamine 146.1 add fresh.1 g 11 mm Glucose 180.2 add fresh 1.98 g 25 mm HEPES 260.3 add fresh 6.5 g 5 mm Sodium Pyruvate 110.0 add fresh 0.55 g 2 mm Mannitol 182.2 add fresh 0.364 g 10 mm Taurine 125.1 add fresh 1.25 g ***ph to 7.4 Incubation Solution MW Stock G in 1 L stock 100 ml IS 118 mm NaCl 58.44 1 M 58.44 g 11.8 ml 4.8 mm KCl 74.53 0.5 M 37.27 g 0.96 ml 1.2 mm MgSO 4 x 7 246.47 100 mm 24.65 g 1.2 ml H 2 O 1.2 mm KH 2 PO 4 136.1 0.5 M 68.05 g 0.24 ml 0.68 mm Glutamine 146.1 add fresh 0.01 g 11 mm Glucose 180.2 add fresh 0.198 g 25 mm HEPES 260.3 add fresh 0.65 g 5 mm Sodium Pyruvate 110.0 add fresh 0.055 g 10 mm Taurine 125.1 add fresh 0.125 g 2% BSA add fresh 2 g DMEM + P/S 1 L of DMEM add 10 ml P/S and then filter sterilize with 0.2 µm filter in hood DMEM + P/S + 10% fetal bovine serum (FBS) For 100 ml of DMEM + P/S, add 10 ml FBS stock in hood 100 mm CaCl 2 = 1mM/1µl