Isolation and characterization of intact cardiomyocytes from frozen and fresh human myocardium and mouse hearts

Abstract

Procurement and characterization of intact human cells is essential for studies in regenerative medicine and translational medical research. The selection of the currently available approaches to isolate intact cells depends on the age of the hearts. To isolate cardiomyocytes from the fetal or neonatal myocardium, the myocardium can be minced into small tissue blocks followed by enzyme incubation. However, the fetal and neonatal cardiomyocytes are very soft and the morphology changes from long-rod or spindle shape to spheres after isolation. Because the dense packing of the cardiomyocytes and the strong cell-cell connection in adult myocardium, it is difficult to isolate the cardiomyocytes from adult myocardium only by enzyme incubation. Perfusion method is necessary to deliver the enzyme solution to the deep layers of the myocardium. However, intact hearts, which is very rare, are required for perfusion method. Therefore, lacking methods to efficiently isolate cardiomyocytes from myocardium of various ages builds a barrier between basic research and clinical studies. Here, we describe a method for the isolation of intact cardiomyocytes from fresh or frozen human myocardium or fresh mouse hearts and the quantification of multinucleation, cardiomyocyte size, cell cycle activity, and total cardiomyocyte count per heart. We generalize this fixation-digestion method by isolating cells from a variety of mouse organs, including the liver, lung, and thymus.

1. Introduction

Regenerative medicine strategies have the potential to revolutionize therapies for important diseases [1]. These approaches require rigorous characterization of single cells and make the collection of intact cells essential for studies in regenerative medicine. The central experiments in the study of regenerative medicine require the determination of cell cycle activity, proliferation, cell size, and total number of the cells. At present, the methods to characterize intact cells are based on immunofluorescence microscopy, which is currently the major approach to characterize intact cardiomyocytes [2–4]. However, the nature of cell-cell connection and limited thickness of the tissue sections make rigorously measuring single cell characteristics (e.g. nucleation, cell volume) and total number of cardiomyocytes in tissue sections challenging. Some researchers have isolated cardiomyocytes from heart samples using enzyme digestion for quantification of cardiomyocyte binucleation [3, 5, 6] and evaluating the total number of cardiomyocytes [5]. However, the cell death and insufficient digestion decrease the quality and reproducibility of the results. Moreover, this kind of enzyme digestion requires re-optimization for context-specific myocardium, for instance, isolating cardiomyocytes from infant, aged, or diseased human myocardium. In addition, enzymatic digestions techniques cannot isolate cardiomyocytes from frozen human myocardium samples.

We have developed the fixation-digestion method to isolate intact cardiomyocytes from fresh and frozen human myocardium through enzyme dissociation of formaldehyde-fixed samples [7]. The fixation-digestion method eliminates the viability issue of the cardiomyocytes, as it preserves the cardiomyocyte morphology and minimizes the tissue loss during cell dissociation. Here, we describe the details of the fixation-digestion method and demonstrate its use for characterization of intact cardiomyocytes from fresh and frozen human and mouse myocardium and on isolated cells from the mouse liver, lung, and thymus.

2. Materials

2.1. Tools and Instruments

1. Curved fine forceps.

2. Fine scissors.

3. Disposable scalpels.

4. Eppendorf tubes (2 mL).

5. Lab shaker.

6. Hybridization oven (37 °C).

7. Multi-key differential counter.

8. Epifluorescence microscope connected to a CCD/CMOS camera.

9. Confocal microscope that acquires z-stack 3D images.

10. Fiji software (https://fiji.sc/).

2.2. Reagents

1. Fixation reagent: 3.7% formaldehyde solution in distilled water.

2. Digestion buffer: collagenase B (3.6 mg/mL), collagenase D (4.8 mg/mL). Dissolve the enzyme in PBS.

3. Blocking and permeabilization solution: donkey or goat serum (2.5%), Triton X-100 (0.05%). Dilute the chemicals in PBS.

4. Cardioplegia solution: KCl (25 µM). Dissolve the chemical in PBS.

3. Methods

3.1. Sample Fixation and Enzyme Digestion

1. For freshly isolated mouse hearts, remove the atria and other non-ventricle tissue (i.e. only ventricular cardiomyocytes will be isolated). Wash off the blood with ice-cold cardioplegia. For fresh-collected human myocardium, cut sufficient tissue but not larger than 5 mm × 5 mm × 2 mm for the experiment and immerge it in cardioplegia solution. For frozen myocardium, thaw the tissue on ice (~5 min) and immerge the tissue in ice-cold cardioplegia.

2. Cut a small piece of the myocardium (e.g. 5 mm × 5 mm × 2 mm), put it on dry surface of a Petri dish, and drop 100 µL of cardioplegia to cover the myocardium piece. Alternative: sample preparations will be conducted using a series of mechanical manipulation: cut a small piece of myocardium and place it onto a dry surface of a petri dish and drop 100 µL of cardioplegia to cover the myocardium piece. Return the remaining tissue to ice.

3. Use a scalpel to mince the small piece of myocardium into ~0.5–1 mm tissue blocks, then transfer all the minced tissue to a 2 mL Eppendorf tube containing 3.7% formaldehyde.

4. Repeat steps 2 and 3 until all of the myocardium for the experiment is minced and transferred to the same Eppendorf tube (see Note 1).

5. Place the Eppendorf tube on a lab shaker at room temperature, and fix the tissue blocks for 1 h and 40 min (see Note 2).

6. Wash the fixed tissue blocks with PBS 3 times.

7. The procedure is presented in Fig. 1.

8. Add 1 mL of the digestion buffer to the 2 mL Eppendorf tube containing the fixed heart tissue.

9. Put the Eppendorf tube in the hybridization oven (37 °C) and shake the tube 10 times per minute for 24 h.

10. Transfer the digested cells from the supernatant to a clean 2 mL Eppendorf tube and store upright at 4 °C.

11. Re-suspend the undigested heart tissue with 1 mL fresh digestion buffer and shake the tube in the hybridization oven (37 °C) at 10 rpm for 24 h.

12. The dissociated cardiomyocytes in the first digestion will have formed a pellet at the bottom of the Eppendorf tube (step 10). Carefully remove the supernatant in the tube immediately prior to step 13.

13. Transfer the newly digested cells to the same Eppendorf tube, and store them at 4 °C for 24 h to allow the cells in suspension to pellet at the bottom of the tube (see Note 3).

14. Repeat the above digestion procedure until all of the tissue is digested (see Note 4). Store the tube containing all the dissociated cardiomyocytes at 4 °C for 24 h and remove the supernatant.

15. Resuspend the cell pellet at the bottom containing all the cells dissociated from the tissue with PBS. Store the tube at 4 °C for 24 h (see Note 5).

16. Remove the PBS from the supernatant and re-suspend the cell pellet with a clean PBS solution. Store the tube at 4 °C for 24 hours.

17. Remove the PBS from the tube as much as possible. Do not disturb the cell pellet at the bottom.

Figure 1.

Isolation and collection of cardiomyocytes from tissue blocks.

3.4. Count the Total Number of Cardiomyocytes in the Mouse Heart

1. Resuspend the pellet with PBS to make the volume of the cell solution 2 mL.

2. Transfer 10µL of the cell solution to the hemocytometer to measure the concentration of the rod-shape cardiomyocytes, C_{cardiomyocytes} (cells/mL) under an invert bright field microscope (Fig. 2) (see Note 6).

3. Calculate the total number of the cardiomyocytes in the tube (2 mL, i.e. the total number of the cardiomyocytes of the heart), N_total = C_{cardiomyocytes}*2 mL.

4. Store the Eppendorf tube containing cell solution at 4 °C for 24 h to allow the cells to settle at the bottom.

Figure 2. Count the total number of cardiomyocytes in a mouse heart.

The cardiomyocytes were isolated from adult mice (P30) using fixation-digestion method. No further treatment for immunofluorescence. The cells with blunt ends are considered as half cells (white arrows). Scale bar: 100 µm.

3.5. Immunofluorescence Microscopy

1. Remove the supernatant carefully. Do not disturb the cell pellet at the bottom.

2. Use 100 μL of blocking and permeabilization solution to resuspend the cells.

3. Incubate at room temperature for 30 min (see Note 7).

4. Add 100 µL of 2X primary antibody working solution to the tube (e.g. for α-actinin and H3P antibodies, the working solution is 1:200 of stock solution; dilute the stock solution with PBS by 1:100 to make the 2X concentration of the working solution). Invert the solution several times to mix the solution.

5. Lay down the tube at room temperature for 1 hr (Fig. 3) (see Note 8).

6. Add 1.5 mL of PBS to the tube.

7. Store the tube upright at 4 °C for 24 h. Let the cells settle at the bottom.

8. 8. Remove the supernatant carefully. Do not disturb the cell pellet at the bottom of the tube (see Note 9).

9. Prepare the 2X concentration of the secondary antibody working solution in PBS (e.g. we usually dilute the secondary antibody by 1:200 for immunostaining, the dilution for 1:100 is 2X concentration.) (see Note 10).

10. Add 100 µL of the secondary antibody working solution (2X) to the tube. Resuspend by gently flicking the tube.

11. Lay down the tube at room temperature for 1 hr.

12. Add 100 µL of Hoechst solution (PBS, 1:500) to the tube.

13. Lay down the tube at room temperature for 5 min.

14. Add 1 mL of PBS to the tube.

15. Store the tube upright at 4 °C for 24 h.

16. Remove the supernatant carefully. Do not disturb the cell pellet at the bottom.

17. Resuspend the pellet with 2 mL of PBS.

18. Store the tube upright at 4 °C for 24 h.

19. Remove the supernatant carefully. Do not disturb the cell pellet at the bottom.

20. Resuspend the pellet with 1 mL of PBS.

21. Measure the concentration of the cardiomyocytes (C_CM, unit: cardiomyocytes/mL) using a hemocytometer (see Note 11).

22. Transfer 100 µL of the cell solution to the wells of an 8-well chambered cover glass.

23. Check the wells containing cells under an invert bright field microscope. Transfer more solution to the wells if 100 µL of the solution contains very few cells. Dilute the cell solution and redo the transfer if too many cells are transferred to the wells.

24. Calculate the total number of the cardiomyocytes (N_CM= C_CM×100µl) transferred to the wells.

25. Seal the wells with clear tape and cover the wells with a chamber lid (see Note 12).

26. The cells are ready for image acquisition.

Figure 3.

Lay down the tube at room temperature and incubate the cardiomyocytes with antibody solution.

3.6. Image Acquisition and Cardiomyocytes Characterization

1. Quantify the proportion of bi-/multinucleated cardiomyocytes using an epifluorescence microscope

2. Some rod-shaped cardiomyocytes can be broken during tissue block preparation, digestion, and the following procedures. Check the pan-cadherin to identify the intact cardiomyocytes (Fig. 4) (see Note 13).

3. Identify the cardiomyocytes and number of their nuclei through the eye port of the microscope to determine their nucleation.

4. Swipe through the well to count the number of mononucleated, binucleated, and multinucleated cardiomyocytes using a multikey cell counter until the pre-designed total number of cardiomyocytes (e.g. 500 cardiomyocytes) has been reached.

5. Calculate the percentage of the bi-/multinucleated cardiomyocytes.

6. Quantify the proportion of H3P⁺ cardiomyocytes using an epifluorescence microscope (Fig. 5).

7. Count the total number of the H3P⁺ cardiomyocytes (N_H3P-CM) in the well.

8. Calculate the percentage of the H3P⁺ cardiomyocytes in the myocardium or heart (P_H3P-CM= N_H3P-CM/N_CM×100%).

9. Measure the size of the intact cardiomyocytes by measuring the projection area (2D, unit: µm²) of a cardiomyocyte on the CCD/CMOS camera attaching to an epifluorescence microscope.

10. Acquire multichannel images of the cardiomyocytes (bright field, α-actinin, pan-Cadherin, DAPI/Hoechst) (Fig. 6).

11. Open the image file with Fiji software in color 16-bit (8-bit) type, not RGB type. Use the (Image->Color->Channels Tool->Composite) tool to make all the channels visible.

12. Use the (Analyze->Set scale) function to set the scale of the image if the scale is not set automatically.

13. Set the bright field channel as the working channel.

14. Identify the cardiomyocytes (α-actinin⁺) and the nucleation (the number of nuclei, DAPI⁺/Hoechst⁺).

15. Use the “Freehand selections” tool to outline the bright field boundary of the target cardiomyocytes.

16. Use the (Analyze->Measure) function to measure the “Area” value of the selected area. The “Area” value will appear in the popup “Results” window.

17. Use the (Edit->Cut) function to permanently mark the selected area. It is important that the bright field channel is the current working channel; it is only in this channel the selected area is cut off but the immunostained pattern is preserved.

18. Save the modified image in tiff format to revisit in the future if needed.

19. Measure the volume (3D, unit: µm³) of a cardiomyocyte.

20. Using a confocal microscope, acquire a multichannel z-stack of the target cardiomyocytes (α-actinin, pan-Cadherin, DAPI/Hoechst). The smaller the step distance(d_slice) of the z-stack, the more accurate the measurement.

21. Open the z-stack with Fiji software and use the (Analyze->Set scale) function to set the scale if the scale is not set automatically.

22. Measure the “Area” value (S) of the α-actinin⁺ area on each slice of the z-stack using the method described above (Fig. 7).

23. Calculate the volume of the target cardiomyocyte V_CM=∑S·d_slice.

Figure 4. Determine the nucleation of the intact cardiomyocytes of different nucleation dissociated from mice and humans of different ages.

(A) Cardiomyocytes were isolated from mouse hearts. (B) Cardiomyocytes were isolated from human hearts with diseases. Scale bar: 20 µm.

Figure 5. The intact mouse cardiomyocytes at M-phase.

Cardiomyocytes were isolated from mouse hearts at different ages (E19.5: 19.5-day embryo; P4: 4 days after birth). Scale bar: 20 µm.

Figure 6. Measure the projection area of cardiomyocytes.

The target cell is indicated by white arrows. (A) The snapshot of the isolated cardiomyocytes by the CCD/CMOS camera attaching to an epifluorescence microscope. (B) The target cardiomyocyte is outlined for area measurement. (C) The measured area is reserved by cutting off in the bright field channel. Scale bar: 40 µm.

Figure 7. Measure the volume of cardiomyocytes from z-stack.

(A) The z-stack of the isolated cardiomyocytes are demonstrated as volume view. (B) Individual slices of the cardiomyocyte indicated by white arrow in (A) for the measurement of cardiomyocyte volume.

3.7. Other species and organs

We have applied the fixation-digestion method to isolate intact cells from different organs of different species at different ages (Fig. 8).

Figure 8. The cells isolated from a variety of species and organs.

(A) Cardiomyocytes from different species. The zoomed region of the human cardiomyocyte (lower panel) shows the characteristic striated structures. (B) The cells isolated from different mouse organs. Scale bar: 50 µm.