TITLE: Human myocardium decellularization
AUTHORS: Immacolata Belviso, Anna Maria Sacco, Domenico Cozzolino, Daria Nurzynska, Franca Di Meglio, clotilde.castaldo, Veronica Romano

[DESCRIPTION]
The protocol represents a step-by-step method to obtain a decellularized cardiac matrix through the combination of sodium dodecyl sulphate (SDS) and Triton X-100. Briefly, cardiac samples obtained from left ventricles of explanted, pathological human hearts were dissected and washed to remove residual body fluids. Samples were then snap-frozen and sliced by a cryostat into 350 µm thick sections. The sections obtained were decellularized using a solution containing 1% Triton X-100 and 1% SDS in combination, for 24 hours, until observing the color change from brownish-red to translucent-white. As a result, the protocol shows efficiency in preserving extracellular matrix architecture and protein composition during the whole process, suggesting that it is worthwhile, highly reproducible and produces a well- preserved decellularized extracellular matrix from cardiac samples.

[STEPS]
SECTION: Preparation of decellularizing solution
1. Preparation of 600 mL of decellularizing solution




SECTION: Preparation of decellularizing solution
1.1. Prepare 300 mL of 2% Triton X-100 solution by measuring 294 mL of double-distilled water in a graduated cylinder and transferring it to a 500 mL beaker. 

SECTION: Preparation of decellularizing solution
1.4. Prepare 300 mL of 2% SDS solution by measuring 275 mL of double-distilled water in a graduated cylinder and transferring it to a 500 mL beaker. 

SECTION: Preparation of decellularizing solution
1.8. Pour 2% Triton X-100 and 2% SDS solutions, previously prepared, in a 1 L cylinder to obtain a total volume of 600 ml of 1% decellularizing solution. Cover with parafilm and gently mix by inversion to obtain a homogeneous solution.

 


SECTION: Preparation of decellularizing solution
1.9. Transfer 1% decellularizing solution in a 1 L graduated bottle using a funnel to reduce foaming. 
Store at +4 °C  until use.
 


SECTION: Preparation of 1x phosphate buffered saline (PBS) solution
2. Preparation of 500 mL of 1x PBS 

SECTION: Preparation of antibiotic solution
3. Preparation of 10 mL antibiotic solution
SECTION: Preparation of antibiotic solution
3.1. Accurately weigh 625 µg using an electronic balance and add it to a 8 mL. Mix vigorously until it is completely dissolved.

  
  
SECTION: Preparation of antibiotic solution
3.2. Pour the solution in a graduated cylinder and adjust the volume to 10 mL adding pen/strep mixture. 
Store at +4 °C  until use.

SECTION: Preparation of samples and decellularization procedure
4. Preparation and decellularization of samples
SECTION: Preparation of samples and decellularization procedure
4.7. Prepare and label with all the information identifying the samples a 50 mL tube for each section. Add 40 mL of decellularizing solution previously prepared  , place one section in each tube. 
 


SECTION: Preparation of samples and decellularization procedure
4.8. 
 
Place the tubes on an orbital shaker and start the procedure setting moderate speed of agitation for 24 hours, at Room temperature.

SECTION: Preparation of samples and decellularization procedure
4.10. Stop the agitation and check the color of the sections. 
 

SECTION: Preparation of samples and decellularization procedure
4.9. Replace the decellularizing solution in each 50 mL tube with 40 mL of 1x PBS   and 0.2 mL of antibiotic solution  . 

SECTION: Preparation of samples and decellularization procedure
4.11. Start the agitation on the orbital shaker at a moderate speed overnight, at Room temperature.

SECTION: Preparation of samples and decellularization procedure
4.12. Stop the agitation. Replace the solution in each 50 mL tube with 40 mL of double-distilled water. 

SECTION: Preparation of samples and decellularization procedure
4.13. Start the agitation on the orbital shaker at a moderate speed for 30 minutes at Room temperature.

SECTION: Preparation of 1x phosphate buffered saline (PBS) solution
2.1. Weigh all the salt powders in recommended amounts using an electronic balance, a spatula and a spoon.

0.1 g 
0.1 g 
4.0 g 
0.575 g 
 
Transfer the salts into a 500 mL beaker.

   
 
   
   



SECTION: Preparation of 1x phosphate buffered saline (PBS) solution
2.2. Take a graduated cylinder to measure 400 mL of double-distilled water and pour it into the beaker.

SECTION: Preparation of 1x phosphate buffered saline (PBS) solution
2.3. Add a stir bar and place the beaker on a magnetic stirrer to completely dissolve the salts.
SECTION: Preparation of 1x phosphate buffered saline (PBS) solution
2.4. Pour the solution in a graduated cylinder and adjust the volume to 500 mL by adding double-distilled water.
SECTION: Preparation of samples and decellularization procedure
4.2. 
 
Prepare a set of large surgical scissors, long forceps, fine forceps and scalpel needed to dissect the heart. Use a dissecting board with graduations to measure sample size.
SECTION: Preparation of samples and decellularization procedure
4.6. 
 
Mount samples  on cryostat chuck and slice them one by one to obtain   thick sections.
 




SECTION: Preparation of samples and decellularization procedure
4.1. Identify and wash the cardiac tissue samples obtained from explanted hearts into a plastic tray using a 0.9 Mass / % volume  to remove any residual fluid.

  


SECTION: Preparation of 1x phosphate buffered saline (PBS) solution
2.5. Check the pH value and adjust to pH 7.4 if needed. 
Store at +4 °C untile use.

SECTION: Preparation of samples and decellularization procedure
4.3. Cut unrefined samples from full-thickness left ventricle wall avoiding injured areas and wash with 0.9 Mass / % volume . 

SECTION: Preparation of samples and decellularization procedure
4.4. Place them on the dissecting board and cut, by a dissecting scalpel, 2 cm x 2 cm (lenght by width) fragments using the graduation on the dissection board as a reference.
 

SECTION: Preparation of decellularizing solution
1.3. 
 
Add a stir bar into the beaker and place it on a magnetic stirrer to mix the solution until completely dissolved.
SECTION: Preparation of decellularizing solution
1.7. Pour the solution in a graduated cylinder and adjust the volume to 300 mL by adding double-distilled water.
SECTION: Preparation of decellularizing solution
1.2. Add 6 mL of Triton X-100 to the beaker containing the double-distilled water  using a serological pipette. 

  
 



SECTION: Preparation of decellularizing solution
1.5. 
 
Weigh 6 g  of SDS powder in a weighing boat using a spoon and an electronic balance. Transfer the powder to the beaker containing the double-distilled water.  

  
 

SECTION: Materials List
6. Addictional materials


 

SECTION: Preparation of decellularizing solution
1.6. Add a stir bar into the beaker and place it on a magnetic stirrer to mix the solution until completely dissolved.
SECTION: Preparation of samples and decellularization procedure
4.5. Snap freeze at -80 °C.
SECTION: Preparation of samples and decellularization procedure
4.14. Stop the agitation. Open each tube and gently dry sections to remove the excess of double-distilled water.
SECTION: Sample storage
5. Fix decellularized sections for histological analyses.
Store at +4 °C in a   for further cell seeding or snap-freeze at -80 °C for other applications.