Begin by measuring the neonatal Rat Ventricular Cardiomyocytes concentration using a hemocytometer. Dilute the cell suspension solution to 3, 000 cells per milliliter, with alpha MEM containing 10%heat inactivated FBS. Distribute 100 microliters of the diluted ventricular cardiomyocyte in each well of the cell's non-adhesive 96 well plates.
Centrifuge the cells for five minutes at 100 G.And culture them for two to three days in the cell culture incubator, with 5%carbon dioxide at 37 degrees Celsius. After incubation, aspirate the culture medium from the Rat Cardiomyocyte balls seeded in the non-adhesive 96 well plate. Then, transfer the cardiomyocyte balls from each well to a 15 milliliter tube.
Centrifuge the cardiomyocyte balls and aspirate the supernatant. Then, wash them using an ADS buffer. To facilitate the tracking of the cardiomyocyte cells in the Rat after engraftment, stain the cells with PKH26.
Before centrifuging the cells, track the development of bidim cardiomyocyte balls by observing them under the microscope regularly after seeding. Begin preparing the main injection apparatus for a neonatal cardiomyocyte ball injection using a 29 gauge, 50 millimeter long needle equipped with a syringe. Connect an 18 gauge one milliliter power transfer syringe needle to a thin polypropylene tube with low lumen expandability.
Aspirate the water using a syringe, and remove all the bubbles from the syringe and the connected tubes. Repeat it for another 18 gauge needle. Then, connect the other side of the power transfer tube to the 18 gauge needle of the same one milliliter syringe.
Confirm the successful power transition by pushing one syringe plunger down, and another syringe plunger up. Once done, set the syringe in a syringe pump. Next, set up the cooling system by attaching the tightly coiled copper tube to the cell containing part of the cell injection syringe.
Leaving 10 millimeter of excess pipe at both ends. Connect the copper tube to the flexible plastic tubing before connecting the other ends of the plastic tubing to an external pump. Fill the line with cooling water, and immerse the excess copper tube into crushed ice to cool the water.
This cooling system maintains the cell suspension solution in the cylinder at approximately 2 degrees Celsius.
