A Novel Cell Injection Method with Minimum Invasion

The animal experiments were conducted according to the Kansai-Medical University ethical guidelines for animal experiments and were approved by the ethical committees (approval number: 23-104). All the animals were raised under a constant light-dark cycle in a specific pathogen-free environment. All the sterilized surgical tools, such as scissors, forceps, and retractors, were autoclaved and dried thoroughly.

1. Preparation of the neonatal rat cardiomyocyte balls

1. Neonatal rat heart collection
   1. For the heart collection, follow a procedure similar to that described in a previous report⁷.
   2. Immerse neonatal (0-2 days after birth) Sprague-Dawley (SD) rats in povidone-iodine and 70% ethanol solutions sequentially, and then transfer them into an air-tight case filled with vaporized isoflurane (the concentration should be over 10% v/v) for deep anesthesia.
   3. After the confirmation of unconsciousness by a loss of locomotor activity, decapitate the ratwhile holding the body from the back with the hand, and then cut 2-4 mm of tissue from the frontal center of the ribcage to the caudal and then in the rostral direction using sharp scissors.
   4. Grip the back skin to pull the cut open and push out the heart from the ribcage. Cut the ventricles using scissors and immerse them in phosphate-buffered saline (PBS) without calcium or magnesium (PBS(−)).
2. Dispersion of the neonatal rat cardiomyocytes
   1. Mince the collected ventricles dispersed in a minimal amount of Ads buffer (Ads buffer: 116 mM NaCl, 20 mM HEPES, 12.5 mM NaH₂PO₄, 5.6 mM glucose, 5.4 mM KCl, 0.8 mM MgSO₄, pH 7.35) in an autoclaved concave glassware into small pieces (1 mm x 1 mm) using curved scissors.
   2. Transfer the minced tissues and a micro-magnetic stirrer into a 50 mL centrifugation tube, and disperse the tissues into single cells with 0.1% collagenase, 0.1% trypsin, 20 µg/mL DNase I, and 50 nM tetramethylrhodamine methyl ester in Ads buffer at 37 °C by stirring for 30 min.
   3. Separate the aggregates and dispersed cells through natural sedimentation, collect only the dispersed cells into a tube, and digest the residual cell aggregates again with the same digestion medium.
   4. Continue this procedure until all the cells are completely dissociated. To confirm the complete dispersion of the cells, observe the tubes under a microscope (4x objective lens).
   5. Collect the dispersed cells using centrifugation at 150 x g for 5 min and dissociate them in 1-2 mL of Ads buffer.
3. Fluorescence-activated cell sorting of cardiomyocytes
   1. Analyze the cells using fluorescence-activated cell sorting (FACS) using 556-601 nm bandpass filters to detect red fluorescence signals.
   2. Carefully perform pre-gating to eliminate doublet fractions¹⁴. The gate settings for doublet elimination should be as per the manufacturer's instructions.
   3. Set the forward scatter on the x-axis and the red fluorescence signal on the y-axis. Three populations were observed: a lowermost population containing erythrocytes and dead cells, a middle population containing non-cardiomyocytes, including fibroblasts and endothelial cells, and a top population containing pure ventricular cardiomyocytes⁷.
4. Preparation of red fluorescence-labeled cardiomyocyte balls
   1. Selectively sort the cardiomyocytes, and centrifuge for 5 min at 150 x g. Completely dissolve the cell pellets in 1 mL of alpha-modified minimal essential medium (alpha-MEM) containing 10% heat-inactivated fetal bovine serum (FBS).
   2. Measure the cell concentration using a hemocytometer and dilute the cell suspension solution to 3,000 cells/mL with alpha-MEM 10% FBS.
   3. Distribute them into cell non-adhesive 96-well plates (100 µL per well), centrifuge for 5 min at 100 x g, and culture for 2-3 d in a cell culture incubator with 5% CO₂ at 37 °C.
   4. Before the injection experiments, harvest a cardiomyocyte ball from each well via aspiration with the culture medium using a 1,000 µL pipette, and collect them in a 15 mL tube.
   5. Stain with PKH26 following the manufacturer's instructions for tracking after engraftment.

2. Preparations for the slow injection method

1. Preparation of the gelatin stock solution
   1. Weigh the gelatin, dissolve it in Ads buffer to produce a 10% w/v solution, and autoclave it.
2. Production of a cell injection device
   1. Overall device design: Prepare the apparatus shown in Figure 1. This system is combined with a syringe cooling apparatus and main injection apparatus.
   2. Prepare the main injection apparatus described below:
      1. For a neonatal cardiomyocyte ball injection, use a 29 G, 50 mm long needle equipped with a syringe.
      2. Connect a power transfer syringe (18G, 1 mL) back-to-back using a cell injection syringe (Figure 1). Connect the needle of the power transfer syringe to a thin polypropylene tube with low lumen expandability.
      3. Then, connect the other side of the power transfer tube to the needle of the same syringe (18G, 1 mL), and set it in a syringe pump. Fill the two power transfer syringes and tubes with water without any air bubbles.
         NOTE: When one plunger of the power transfer syringe is pushed in, the pressure is directly transferred to the other syringe, and the plunger protrudes.
   3. Set up the injection syringe cooling system as described below.
      1. Wind a copper tube (external diameter = 1 mm; internal diameter = 0.3 mm; thickness = 0.35 mm) tightly around the cell-containing part of the cell injection syringe, leaving 10 mm of excess pipe at both ends.
      2. Connect the copper tube to flexible plastic tubing. Further, connect the other ends of the plastic tubing to an external pump, fill the line with cooling water, and cool the water by immersing the excess copper tube into crushed ice (Figure 1).
         NOTE: This cooling system maintains the cell suspension solution in the cylinder at approximately 2 °C.

3. Development of a rat myocardial infarction model by obtuse coronary artery occlusion

1. Anesthetize male immune-deficient nude rats (F344/Njcl-rnu/rnu) with air containing 3% isoflurane. Insert a cannula into the trachea and connect it to the respirator.
2. Connect a cannula to an isoflurane vaporizer with a controller to maintain an 3% isoflurane concentration and, thus, sustain sufficient anesthesia. Confirm no response to pain stimuli. Apply veterinarian ointment on the eyes to prevent dryness.
3. Fix the limbs with cut surgical tapes on a 40 °C warmed surgical plate. Twist the body of the rat to the right of the body axis and use the left armpit as the surgical field.
4. Remove the hair in the surgical field using a depilatory cream and wipe the skin with povidone-iodine. Using sharp scissors, cut a 1.5 cm incision in the skin and pectoralis major muscle.
5. Confirm the third intercostal space and rip the intercostal muscles and costal pleura using micro forceps with blunt tips. Keep the chest open by using a retractor. Gently remove the thin pericardium with forceps.
6. To construct an infarction model of the lateral wall of the heart, find the position 1 mm caudal to the tip of the left atrium to identify the obtuse coronary artery, pass a 7-0 silk suture, scoop up tissue that is 2.5 mm wide and 2.5 mm deep from dorsal to ventral, and ligate the tissue tightly.
7. Confirm successful ligation by the weak contraction distal from the ligature. After gently removing the retractor, place a 5-0 silk suture between the second and fourth intercostal spaces, and close the thoracotomy.
8. Decrease the isoflurane concentration to 1%. Gently suture the muscle and skin with 5-0 silk. Decrease the isoflurane concentration to 0% and wait for approximately 5 min until spontaneous breathing starts.
9. Topically apply 2 mg/mL lidocaine in saline to the incision. Administrate 1 mL of saline via a subcutaneous injection. Apply veterinarian ointment topically to prevent infections.
10. Remove the rat from the intubation tube, and return to the animal cage; then, raise the rats in individual cages for 1 week.
11. Analyze the changes in the cardiac systolic pump function using echocardiography.
    ​NOTE: The cardiac function will be reduced owing to lateral myocardial infarction.

4. Echo-guided percutaneous cell transplantation using the slow injection method

1. Pre-warm 10% gelatin stock at 37 °C until it becomes liquid.
2. Dilute 10% gelatin stock with pre-warmed Ads buffer to obtain the final injectable 5% w/v gelatin solution (100 µL is required per animal).
3. Suspend 96 cardiomyocyte balls (total: 28,800 cardiomyocytes/animal) in 100 µL of pre-warmed injection solution.
4. Load the suspension prepared in step 4.3 into the cell injection syringe, avoiding the aspiration of excess air.
5. To eliminate bubbles in the syringe, hold it vertically with the needle upward, tap the syringe, and collect any bubbles at the upper ridge of the syringe.
   NOTE: During this step, observe the cardiomyocyte balls gradually settle onto the rubber seal of the plunger.
6. Maintain the vertical position of the syringe, push up the plunger slowly, and discard the bubbles and excess cell suspension solution until 20 µL of the cell suspension remains in the syringe. Push the plunger carefully at a constant speed so that the cardiomyocyte ball remains resting on the rubber seal of the plunger.
7. Immerse the capped syringe directly in an ice bath for 5 min.
8. Place a cooled syringe in the injection apparatus. Tightly fix the injection syringe apparatus settled on a fine movement device on the animal stage using an X-Y-Z position-adjustable handmade clamp. The fine movement device can move the needle position using the x-direction 20 mm slide, y-direction swing, and z-direction bowing movements.
9. Anesthetize the rats in a sealed box filled with air containing 3% isoflurane. Confirm anesthesia by no response to pain stimuli. Apply veterinarian ointment on the eyes to prevent dryness.
10. Fix the limbs with cut surgical tapes on a 40 °C warmed echo plate. To sustain sufficient anesthesia, ensure the inhaled air contains an approximately 3% concentration of isoflurane.
11. Remove the hair at the injection field (2 cm in diameter) and chest with depilatory cream and wipe the skin with povidone-iodine.
12. Apply echo-gel on the chest and echo probe. Place the echo probe close to the chest along the cranial and caudal axes and start to obtain B-mode echocardiography following the manufacturer's manual.
13. Advance the tip of the injection needle into the myocardium at the frontal view (Figure 2 and Supplementary Video 1).
14. To activate the main syringe pump, push the Start button, and rotate the dial to adjust to a predetermined number for an injection speed of approximately 0.02 µL/s. Apply veterinarian ointment topically around the needling position to prevent infections.
    NOTE: It is necessary to determine the appropriate dial number for the intended injection speed using an injection solution. After the injection, remove the injection needle using an exemplary movement system.
15. Decrease the isoflurane concentration to 0% and wait for approximately 5 min until the animal regains sufficient consciousness to maintain sternal recumbency.

5. Evaluation of cardiac function

1. Anesthetize the rats in a sealed box filled with air containing 3% isoflurane. Confirm anesthesia by no response to pain stimuli. Apply veterinarian ointment on the eyes to prevent dryness.
2. Apply electrifying cream for ECG acquisition on the limb tips. Fix the limbs with cut surgical tapes on a 40 °C warmed echo plate. Use a physiological monitoring system to detect real-time ECG and heart rate.
3. According to the manufacturer's instructions, first determine the long-axis angle using the B-mode echo image showing the left ventricular apex to the outflow tract, and then rotate the echo-probe 90° to change to the short-axis view.
4. Using only the caudal to rostral axis fine movement system of the animal stage, adjust the short-axis view to the papillary muscle level. Then, change the image mode to M-mode by pressing the M-mode button, and record a video for 5 s by pressing the Cine-loop button.
5. To analyze and calculate the fraction shortening using the software, press the Measure button and a vertical line tool to define the end-systolic and end-diastolic left ventricular internal dimensions. The software automatically calculates the percent fraction shortening (FS).
6. Decrease the isoflurane concentration to 0% and wait for approximately 5 min until the animal regains sufficient consciousness to maintain sternal recumbency.

6. Immunohistochemistry

1. Fix the euthanized body (perform euthanasia as in step 1.1.3) on the table using the cut surgical tapes, excise the hearts, wash with PBS, and immerse the heart in 4% paraformaldehyde/PBS.
2. Dissect the hearts into three sections, immerse them in 40% sucrose for cryoprotection, embed in an optimal cutting temperature (OCT) compound, and freeze them at −80 °C.
3. Attach the cryosectioned tissues (8 µm thickness) to aminosilane-coated glass slides. After sufficient drying using non-warmed wind generated by a general hair dryer, immerse the glass slides in tris-buffered saline containing 0.2% Tween-20 (TBS-T).
4. Immerse them in a blocking solution for 30 min at 25 °C. Pour 100 µL of the primary antibody-containing blocking solution onto the slide, and incubate overnight at 4 °C with paraffin sealing to keep the antibody solution spread on the tissue.
   NOTE: The antibody concentration is shown in the Table of Materials.
5. Lay the slides horizontally in a handmade high-humidity box utilizing the natural vapor from wet paper to prevent evaporation. Wash the slides three times with TBS-T.
6. Treat with the secondary antibody-containing blocking agent for 1 h at 25 °C in the same manner as the primary antibody. After three washes, observe the fluorescence signals using a fluorescence microscope and confocal laser microscope.
   NOTE: The antibody concentration is shown in the Table of Materials.
7. Statistical analysis: For the cell volume comparison, as shown in Figure 3A, perform a non-paired t-test; to assess the cardiac functional recovery following cardiomyocyte administration using the slow injection method, as shown in Figure 4A, use a paired t-test. In this work, differences were considered statistically significant at P < .01. Error bars represent the standard deviation.