Preparation of Human Myocardial Tissue for Long-Term Cultivation

Using this protocol, human left ventricular tissue slices can be cultivated ex vivo in a biomimetic chamber. The application of pre and afterloads improves the resemblance of the physiological environment. This technique allows for continuous registration of tissue contraction.

User-defined stimulation protocols allow for the assessment of vital contraction parameters like post-pause potentiation, stimulation threshold, force-frequency relation, and refractory period. The long-term cultivation of myocardial slices, using this setup, will pave the way for future ex vivo research, facilitating this screening for therapeutic and cardiotoxic drug effects in cardiovascular medicine. To begin, submerge the cultivation chambers and graphite electrodes in one liter of a 10%isopropanol solution and agitate overnight.

The following day, transfer the chambers to a 100%isopropanol solution for 3 minutes, then allow the chambers and graphite electrodes to air dry under a laminar flow hood. Attach a circuit board to each chamber according to the available positions on the rocker. Place two graphite electrodes on the circuit board as per the manufacturer's instructions, then place a 35-millimeter Petri dish lid on the chamber to prevent infection.

Next, fill the cutting tray up to 90 to 95%with slicing buffer. Transfer the tissue sample to a 100-millimeter Petri dish filled with cold slicing buffer, and place the dish on a cooling plate at 4 degrees Celsius. Remove endocardial trabeculae by holding the endocardium with tweezers, then use scissors to cut away approximately 3 millimeters of endocardial tissue.

Fixate the cut tissue sample, endocardial side up to a 2-by-2-centimeter rubber patch using four 0.9-by-70-millimeter 20-gauge needles that are fixed in a square position. Ensure that the diagonal edge of each needle tip is pointing inward, enhancing fixation and preventing damage to the myocardium. Using a scalpel, cut away all the excess tissue outside the four needle squares.

Using tweezers, place the trimmed sample on a sterile piece of tissue for 10 seconds to remove excess slicing buffer. Next, remove the agarose syringe from the water bath and submerge the sample in agarose. Let the agarose solidify for five minutes on a cooling plate.

The endocardial side of the sample must be visible in the agarose. Using tweezers, place the epicardial side of the sample contained in agarose on top of the glued area, then gently press the agarose-containing sample from the top with a blunt tool without cutting or damaging the agarose. Let the glue solidify for 1 minute.

Set the vibration amplitude to 1 millimeter, initial cutting speed to 0.07 millimeter per second, and thickness of the slice to 300 microns. After connecting the cultivation system to a computer, start the corresponding software program. Set the rocker speed to 60 rpm and preset the stimulation parameters.

For human cardiac slices, set the standard stimulation to biphasic impulses with 50 milliamp or current consisting of 3 milliseconds positive current, 1 millisecond pause, and a 3-millisecond pulse of inverted current at a pacing rate of 30 beats per minute or bpm, then check the electrode indicators of the software and verify that the electrodes of the cultivation chambers are working correctly. Using tweezers, separate the agarose from the tissue. Avoid touching the tissue and handle it carefully as any damage to the tissue will reduce the success rate of the cultivation.

To attach two plastic triangles to a sample, place 1 microliter of glue on a sterile Petri dish lid. Use a hooked tweezer to pick up one autoclaved plastic triangle. Quickly dip the front edge of the triangle into the glue and paste the triangle onto the sample perpendicular to the cardiomyocyte alignment.

Repeat for the other triangle. Using a scalpel, trim off tissue exceeding the triangle width and place the slice with the two mounted triangles back into the cutting tray containing slicing buffer. Remove the medium-filled cultivation chamber from the incubator.

Select one prepared slice and insert it into the chamber by connecting one triangle to each pin. Adjust the distance between the mounting pins to the sample size. Ensure that the sample is submerged in the medium.

After placing the dish onto the rocker, decrease the preload by turning the adjusting screw counterclockwise until the baseline of the corresponding graph on the computer screen does not change anymore, then carefully increase the preload or tension by turning the adjusting screw clockwise. For chambers with high stiffness, continue until the corresponding baseline in the graph has increased by 1, 000 to 1, 200 units corresponding to one millinewton preload. After preparing the fresh cultivation medium, pre-warm the medium in a water bath or hot air incubator at 37 degrees Celsius for 30 to 45 minutes.

Remove medium from the chamber, leaving approximately 0.8 milliliters in the chamber, then add 1.6 milliliters of fresh medium to the same chamber, making a total volume of medium to 2.4 milliliters per chamber. Finally, place the cover of the chamber back and place the cultivation chamber into its respective position. The readout of the contraction of five myocardial slices during a typical stimulation consisted of four distinct sections of post-pause potentiation, stimulation threshold, force-frequency relation, and refractory period.

Compared to control, the contraction force of the slices treated with calcium antagonists, nifedipine and calciseptine, was decreased within 10 minutes. In contrast, the voltage-gated calcium channel agonist, Bay-K8644, increased the contraction force. The post-pause potentiation of the control and treated slices was assessed for the intracellular calcium release from the sarcoplasmic reticulum.

The control slice did not show any changes. In the presence of calciseptine and nifedipine, the inhibition of the L-type calcium channels led to potentiation of the first contraction after a pause of 50 seconds, reflecting a higher relative contribution of intracellular calcium release to total contractility. The opposite effect was observed with Bay-K8644, which stimulates the entry of extracellular calcium via the L-type calcium channels.

In force-frequency relation analysis, no change was observed in the control slice. The calciseptine treatment did not change the ability of the slice to follow the stimuli upon an increase of the stimulation frequency when comparing the pre and post-treatment data. Compared to calciseptine, nifedipine prevented an increase in contractility at higher pacing rates and reduced the maximum capture rate at 80 bpm.

With Bay-K8644, increased contraction force at very low stimulation frequencies was observed. However, at frequencies higher than 50 bpm, the contraction force was lower than during the pre-treatment condition. Excised tissue should be quickly transferred to 4 degrees cardioplegia.

After slicing, plastic triangles must be attached perpendicular to the fiber direction. The preload should not exceed 1500 millinewton. Biomimetic slice culture can help researchers to utilize genetic manipulations as well as cell-based therapies for studying regeneration and repair in adult human myocardium.