Isolation and Characterization of Cardiac Mesenchymal Stromal Cells from Endomyocardial Bioptic Samples of Arrhythmogenic Cardiomyopathy Patients

This approach complies with the Declaration of Helsinki and the collection of right ventricular samples was approved (07/06/2012) by "Centro Cardiologico Monzino IRCCS" Ethics Committee.

1. Solutions

1. Make TMES medium for C-MSC culture with Iscove's Modified Dulbecco's Medium (IMDM), supplemented with 20% Fetal Bovine Serum (FBS), 10 ng/mL basic fibroblast growth factor, 10,000 U/mL Penicillin, 10,000 µg/mL Streptomycin, and 0.02 M L-Glutamine. Filter the TMES medium using a 0.22 µm sterile filter unit, and store at 4 °C.
2. To prepare collagenase solution, resuspend the collagenase NB4 mix in IMDM medium to obtain a final solution with a concentration of 3 mg/mL. Vortex gently to dissolve the powder, and filter the collagenase solution using a 0.2 µm syringe filter. Aliquot 1 mL volume into 2 mL sterile tubes and store at -20 °C until needed for use.
3. To prepare T. ADIPO medium, make adipogenic medium with IMDM medium supplemented with 10% FBS, 0.5 mM 3-isobutyl-1-methylxanthine (pre-diluted in dimethyl sulfoxide (DMSO) at a concentration of 0.5 M and kept at -20 °C until use), 1 µM hydrocortisone (pre-diluted in DMSO at 100 mM and further diluted to 10 mM in sterile distilled water and kept at -20 °C until use) and 0.1 mM indomethacin (pre-diluted in DMSO at a concentration of 0.5 M and kept at -20 °C until use). Filter the T. ADIPO medium using a 0.22 µm sterile filter unit and store at 4 °C.
4. Prepare washing buffer composed of phosphate-buffered saline 0.0067 M PO₄ (PBS), ethylenediamine tetra acetic acid (EDTA) 5 mM, and 5% bovine serum albumin, and store at 4 °C.
5. Prepare oil red O (ORO) working solution by dissolving 1% ORO powder in 60% isopropanol, mixing for 2 h, and filtering using a 0.22 µm filter unit to remove precipitates. Store the working solution at room temperature (RT).

2. Isolation of Cardiac Mesenchymal Stromal Cells

1. Cardiac biopsy collection and processing
   Note: Before starting, make sure to have the collagenase solution and the TMES medium ready.
   1. Put the ACM endomyocardial biopsy (usually about 5 mg of tissue) in a sterile tube filled with TMES and transport it to the laboratory.
      Note: ACM biopsies are collected in the electrophysiology operating room, according to previous reports¹⁰. Briefly, the integration of electro-anatomical mapping and intracardiac echocardiography in the right ventricle (see Video 1) is used to guide the endomyocardial biopsy (see Video 2, fluoroscopy) in correspondence to the border zone of the diseased myocardium. Healthy Control (HC) samples are supplied by a tissue bio-bank, obtained from the right ventricular free wall of cadaveric donors within 24 h of death (accidental death, healthy subjects). During transport and prior to dissection, store the biopsy in TMES at 4 °C. Limit the time between biopsy collection and tissue processing (max 24 h).
   2. Set up the biological safety cabinet with required solutions to carry out the procedure up to the enzymatic digestion.
   3. Put the sterilizer under the hood and turn on the instrument 15 min before use until the temperature rises 200 – 250 °C.
   4. Sterilize scissors and tweezers for 10 s. Make sure the sterilized instruments are cold before use. Prepare disposable sterile scalpels.
   5. Place the tubes with the biopsy in the sterile hood. Open the tube and transfer the biopsy into a sterile plate.
   6. Wash the right ventricle biopsy twice with 3 mL of sterile PBS. If you want take a picture of the biopsy at the microscope.
   7. Transfer the right ventricle biopsy, using sterile tweezers, into a sterile 2 mL tube containing 1 mL of the collagenase solution.
   8. Cut the sample into 0.5 – 1 mm³ pieces with sterile scissors.
   9. Incubate for 1.5 h at 37 °C under continuous rotating agitation.
   10. Centrifuge the digested solution at 400 x g for 10 min at RT.
   11. Remove the supernatant and add 1 mL of sterile PBS to wash the pellet.
   12. Centrifuge at 400 x g for 10 min at RT.
   13. Remove the supernatant and resuspend the pellet in 1 mL of TMES medium.
   14. Plate the obtained suspension onto sterile 60-mm tissue culture treated plate and add TMES medium to the final volume of 3 mL. Incubate the plate in a cell culture incubator at 37 °C with 5% CO₂.
       Note: A higher volume of medium may prevent the correct adhesion of a digested biopsy fragments.
   15. After 24 h, discard the medium to remove non-adherent cells and debris.
       Note: Single dissociated mesenchymal cells can attach to the plastic surface and give rise to clones; also, undigested small biopsy fragments can attach to the plastic dish and allow cell sprouting.
   16. Wash the plate twice with 5 mL of sterile PBS, and add 3 mL of fresh TMES medium.
   17. Replace the TMES medium three times a week until the cells are 80% confluent.
       Note: The number of attached cells depends on the quality, amount of tissue, and on the efficiency of digestion.

3. Cell Expansion

Note: Before starting make sure to have TMES medium ready.

1. When the cells are 80% confluent, transfer the digested small bioptic samples into a new sterile 60-mm tissue culture treated plate, and add 3 mL of fresh TMES medium.
   Note: The bioptic samples can be re-used for further C-MSC isolation. It is possible to repeat this procedure until there is a significant number of isolated cells.
2. Wash the attached cells twice with 3 mL of sterile PBS. Add trypsin-EDTA solution (0.5 mL for a 60-mm dish), and incubate at 37 °C for 3 – 5 min to allow cell detachment.
3. When the cells are detached from the surface of the plate, add 0.5 mL of sterile FBS to inactivate the trypsin and collect cells into a new 50 mL sterile tube.
4. Wash the plate twice with 5 mL of sterile PBS to add the remaining cells to the same tube.
5. Centrifuge the cells at 400 x g for 10 min at RT.
6. Remove the supernatant and resuspend the pellet in 1 mL of TMES medium.
7. Load 10 µL of the cell suspension in the cell counting chamber.
   Note: If the cells are too concentrated, dilute the initial suspension 1:10 in PBS and load 10 µL of the diluted cells in the cell counting chamber.
8. Under the microscope, count cells in 3 large squares and on the lines of two of their sides and calculate the average number of cells.
   Note: To obtain the number of cells/mL, the average of counted cells is multiplied by 10⁴, the dilution factor of the cell counting chamber. If the cells have been further diluted, multiply for the dilution factor. The ratio between the cell number to seed and the cell concentration (number of cells/mL) corresponds to the volume (in mL) of initial suspension to be plated for the next step (point 3.9).
9. Plate the resuspension onto sterile 100-mm tissue culture treated plates at a final concentration of 5,000 – 10,000 cells/cm² and add TMES medium to the final volume of 8 mL. Incubate the plate in a cell culture incubator.
   Note: Repeat the cell expansion procedure when the cells are 70 – 80% confluent. At every passage (P), it is recommended to cryopreserve part of the cells and plate the remaining amount. Expand the cells until P3 or P4 and use for fluorescent-activated cell sorter (FACS) analysis (section 4) and adipogenic differentiation (section 5).
10. For the cryopreservation after detachment, centrifuge the cells at 400 x g for 10 min at RT.
11. Count cells as described (steps 3.7 – 3.8) and resuspend 1 x 10⁶ cells in 900 µL of sterile FBS. Transfer to a sterile cryo-vial and add 100 µL of sterile DMSO and mix.
12. Quickly store the cryo-vials into a freezing container at -80 °C for at least 2 days and then transfer the vials into liquid nitrogen.

4. Characterization of Cardiac Mesenchymal Stromal Cells by Flow Cytometry

Note: Before starting, make sure to have the cell dissociation reagent, washing buffer, and the specific antibodies prepared.

1. When the cell number reaches at least 3 x 10⁶ (count the cells as described in 3.7 – 3.8), wash the 100-mm plate twice with 10 mL of sterile PBS.
2. Add 5 mL of a cell dissociation reagent and wait 7 – 10 min at RT to allow the cell detachment.
3. When the cells are detached from the surface of the plate, add 15 mL of sterile washing buffer and collect the suspension into a new 50 mL sterile tube.
4. Wash the plate twice with 10 mL of washing buffer and add the remaining cells to the same tube.
5. Centrifuge the cells at 400 x g for 10 min at RT and resuspend the pellet in 1 mL of washing buffer.
6. Count the cells as described (steps 3.7 – 3.8), and transfer 3 x 10⁶ cells into a new sterile tube and add washing buffer to the final volume of 1.5 mL.
   Note: The total cell number and the total volume of washing buffer depends on the number of selected markers used for the analysis (3 x 10⁵ cells in 100 µL for each FACS polystyrene tube).
7. Distribute 100 µL of cellular suspension in 12 different FACS polystyrene tubes, and add the specific antibody at the concentration indicated in the product datasheet.
   Note: Antibodies used for C-MSC characterization are CD34, CD105, CD45, CD29, CD90, CD44, CD31, CD14, and HLA-DR (Table 1). It is important to prepare a control sample composed of 100 µL of resuspended cells stained with the isotype control to check the specificity of the signal.
8. Incubate the samples for 15 min in the dark.
9. Add 1 mL of sterile washing buffer to each tube to stop the reaction.
10. Centrifuge the cells at 400 x g for 10 min at RT.
11. Remove the supernatant and resuspend the pellet in 250 µL of washing buffer.
12. Proceed to C-MSC characterization with flow cytometry.

5. Adipogenic Differentiation of Cardiac Mesenchymal Stromal Cells

Note: Before starting, make sure to have prepared T. ADIPO medium and ORO working solution.

1. Culture in T. ADIPO medium
   1. Detach and count the cells as described in section 3.
   2. Plate 3 x 10⁵ cells onto each well of a sterile 6-well tissue culture treated plate in 2 mL of T. ADIPO medium.
   3. Let C-MSC differentiate in T. ADIPO medium for 72 h or 1 week, changing medium every 2 – 3 days.
2. Oil Red O staining
   Note: Use Oil Red O (ORO) staining to test the lipid accumulation in C-MSC.
   1. Place the 6-well tissue culture plateunder the fume hood.
   2. Remove the adipogenic medium and wash the cells twice with 2 mL of PBS.
   3. Add the enough volume of 4% paraformaldehyde (PFA) to cover each well. Fix the cells for 5 min at RT.
   4. Discard PFA and wash the cells twice with 2 mL of PBS.
   5. Incubate the fixed cells with ORO working solution for 1 h at RT, using enough volume to cover each well.
      Note: Do not keep the 6-well tissue culture plate under the fume hood during ORO incubation to avoid the evaporation of ORO working solution.
   6. Remove all ORO working solution and wash with 2 mL of PBS 3 – 5 times until the plate is completely cleaned; discard washes. Stop the washes when the PBS used is clear and when you don’t see, under the microscope, unspecific staining out of the cells.
   7. Capture 20 images at 20X magnification for each well using the inverted tissue culture phase-contrast microscope.
   8. Open the pictures with the image processing program to quantify the cell ORO accumulation.
   9. Separate the different color channels through the "split channels" function in order to quantify only the luminance in the 255-red channel.
   10. Count the number of cells for each picture by counting the nuclei. Normalize the ORO signal intensity on the cell number.
   11. Calculate the average of the results obtained for each picture of the same sample.