This protocol is for slicing and culturing heart tissue sections for six days under physiological conditions which can be used for acute cardiac toxicity testing as well as testing efficacy of heart failure therapies. This culture system has the potential to become a powerful predictive human in situ model for acute cardiac toxicity testing that closes the gap between pre-clinical and clinical testing results. Demonstrating the procedures will be Qinghui Qu, a lab manager from our laboratory.
To obtain pig heart tissue slices, first add ice to the tissue bath cooling jacket of a vibratome and add Tyrode solution to the bath. Use a one liter plastic jar to collect the melted ice runoff from the tissue bath cooling jacket and transfer the pig heart to a tray containing one liter of fresh cold cardioplegic solution in a biosafety cabinet. Use retractible sterile scalpels to dissect the heart to isolate the left ventricle and use rectangular razor blades to cut the left ventricle into one to two cube centimeter blocks.
Set aside one tissue block for slicing and place the remaining tissue block pieces into a 50 milliliter tube of cold Tyrode solution on ice. Gently massage the reserved tissue block and add one to two drops of tissue glue to the metal sample holder of the vibratome. Stick a piece of 4%agar block with a surface area of approximately one square centimeter to the glue and add one to two drops of tissue glue to the agar.
Attach the heart block to the agar cardiac epicardium side down with the tissue as flat against the holder as possible and place the tissue holder with the heart block into the slicing bath of the vibratome. It is crucial to stick the heart block to the agar with the cardiac epicardium side facing down on the tissue glue and to make sure the tissue is as flat as possible. Then attach the oxygen tube to the slicing bath and the metal tray filled with Tyrode solution and place 40 micrometer cell strainers onto the metal tray to collect the heart tissue sections as they're sliced.
Use the vibratome operating software to adjust the height of the blade and sample so that the blade is close to the top of the tissue but below the papillary muscles and liquid is covering the tissue and the blade. Select advance to adjust where to begin slicing the tissue. Press slice to increase the speed and use the knob to move the blade toward the edge of the tissue.
Press slice again to stop and advance to inactivate the process. Then adjust the cutting parameters as indicated and press slice to begin slicing the tissue. When the blade reaches the end of the tissue but before it hits the end of the specimen holder, press slice again to stop and press return to go back to the start position.
When the slices reach full length and appear to be of good quality, use a plastic Pasteur pipette filled with cold Tyrode solution to gently collect the tissue from the bath using forceps and spring scissors to dislocate the slice from the heart as necessary. Place each collected slice in a single cell strainer in the oxygenated Tyrode bath and use the solution in the pipette to press the tissue on the cell strainer surface. Then place a metal washer onto the top of the tissue to hold it down.
After at least one hour in oxygenated Tyrode solution, trim the outside of each slice to remove any uneven edges and glue the end of each slice onto a six millimeter wide piece of sterilized polyurethane printer timing belt with embedded metal wires. Place the supported heart slices into six-well plates containing six milliliters of culture medium per well and use sterile forceps to make sure that the tissue is in the center of the well. Take care that the plate cover will not touch the tissue and place the plate onto the culture plate so that the curved side of the plate cover matches up with the angled side of the plate and that the square corners line up.
Place the slices into a 37 degree Celsius and 5%carbon dioxide incubator. Replace the culture supernatants with six milliliters of fresh oxygenated culture medium three times per day. Connect the cover to the cell culture electrical stimulator and adjust the stimulator to continuously deliver 10 volts of electricity and a 1.2 hertz frequency to the heart slices.
After plugging in the stimulation, the heart slices will begin to beat. Everyday during the midday media change, replace the stimulation plate cover from the culture dish to prevent the release of toxic graphite particles into the culture medium. Insert the white foam plug where the used cover connects to the cable for the cell culture electrical stimulator to prevent water damage to the electric circuit and place the plate cover into a bath of autoclaved water supplemented with 2X antibiotic/antimycotic.
The next day, decontaminate the plate cover in a 70%ethanol bath for five to 15 minutes before transferring the cover into a fresh autoclaved water bath supplemented with 2X antibiotic/antimycotic for three minutes. After rinsing the plate cover, use an ethanol-sprayed clean lint-free wipe to remove any residual water from the plastic parts and remove the white foam plug. The cover is then ready to be placed back into the culture plate.
Using this new biomimetic culture setup as demonstrated, pig heart slices viability was maintained for six days as assessed by MTT assay. In the first six days, similar to the responses observed for fresh heart slices, there are no spontaneous calcium transients within the pig heart slice cardiomyocytes. The cardiomyocytes did respond to external electrical and beta-adrenergic stimulation.
In addition, the contractile force and responses to isoproterenol were maintained in heart slice culture for up to six days similar to that observed in fresh heart slices. Several structural and functional assessments can be performed on the heart slices including MTT viability assays, immunostaining, electron microscopy, calcium transient measurements, contractile function assessments, metabolic assessments, and analysis. We are currently using this culture system to test the acute cardiac toxicity and efficacy of drugs and gene therapies of interest in pig and human heart slices.
