Human myocardium decellularization
Immacolata Belviso, Anna Maria Sacco, Domenico Cozzolino, Daria Nurzynska, Franca Di Meglio, clotilde.castaldo, Veronica Romano
Abstract
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
Preparation of decellularizing solution
Preparation of 600 mL of decellularizing solution
Prepare 300 mL of 2% Triton X-100 solution by measuring 294mL of double-distilled water in a graduated cylinder and transferring it to a 500 mL beaker.
Add 6mL of Triton X-100 to the beaker containing the double-distilled water using a serological pipette.
Equipment
| Value | Label |
|---|---|
| Heating Magnetic Stirrer | NAME |
| VELP SCIENTIFICA | BRAND |
| VP-F20520162 | SKU |
Add a stir bar into the beaker and place it on a magnetic stirrer to mix the solution until completely dissolved.
Prepare 300 mL of 2% SDS solution by measuring 275mL of double-distilled water in a graduated cylinder and transferring it to a 500 mL beaker.
Equipment
| Value | Label |
|---|---|
| Explorer Pro Precision EP413 | NAME |
| Precision balance | TYPE |
| Ohaus | BRAND |
| 80108921 | SKU |
Weigh 6g 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.
Add a stir bar into the beaker and place it on a magnetic stirrer to mix the solution until completely dissolved.
Pour the solution in a graduated cylinder and adjust the volume to 300 mL by adding double-distilled water.
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.
Equipment
| Value | Label |
|---|---|
| Parafilm M | NAME |
| Thermoplastic film | TYPE |
| Sigma-Aldrich | BRAND |
| P7793-1EA | SKU |
Transfer 1% decellularizing solution in a 1 L graduated bottle using a funnel to reduce foaming.
Store at +4°C until use.
Preparation of 1x phosphate buffered saline (PBS) solution
Preparation of 500 mL of 1x PBS
Weigh all the salt powders in recommended amounts using an electronic balance, a spatula and a spoon.
0.1g
0.1g
4.0g
0.575g
Transfer the salts into a 500 mL beaker.
Take a graduated cylinder to measure 400mL of double-distilled water and pour it into the beaker.
Add a stir bar and place the beaker on a magnetic stirrer to completely dissolve the salts.
Pour the solution in a graduated cylinder and adjust the volume to 500 mL by adding double-distilled water.
Check the pH value and adjust to 7.4 if needed.
Store at +4°C untile use.
Preparation of antibiotic solution
Preparation of 10 mL antibiotic solution
Accurately weigh 625µg using an electronic balance and add it to a 8mL. Mix vigorously until it is completely dissolved.
Pour the solution in a graduated cylinder and adjust the volume to 10 mL adding pen/strep mixture.
Store at +4°C until use.
Preparation of samples and decellularization procedure
Preparation and decellularization of samples
Identify and wash the cardiac tissue samples obtained from explanted hearts into a plastic tray using a 0.9Mass / % volume to remove any residual fluid.
Stop the agitation and check the color of the sections.
Start the agitation on the orbital shaker at a moderate speed overnight, at Room temperature.
Stop the agitation. Replace the solution in each 50 mL tube with 40mL of double-distilled water.
Start the agitation on the orbital shaker at a moderate speed for 30 minutes at Room temperature.
Stop the agitation. Open each tube and gently dry sections to remove the excess of double-distilled water.
Equipment
| Value | Label |
|---|---|
| Dissecting Board | NAME |
| Board for Anatomical Dissection | TYPE |
| VWR | BRAND |
| 100498-398 | SKU |
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.
Cut unrefined samples from full-thickness left ventricle wall avoiding injured areas and wash with 0.9Mass / % volume .
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.
Snap freeze at -80°C.
Equipment
| Value | Label |
|---|---|
| Cryostat | TYPE |
| Leica | BRAND |
| CM1950 | SKU |
Mount samples on cryostat chuck and slice them one by one to obtain 350 µm thick sections.
Prepare and label with all the information identifying the samples a 50 mL tube for each section. Add 40mL of decellularizing solution previously prepared , place one section in each tube.
Equipment
| Value | Label |
|---|---|
| Platform Rocker STR6 | NAME |
| Orbital Shaker | TYPE |
| Stuart Scientific | BRAND |
| L065 | SKU |
Place the tubes on an orbital shaker and start the procedure setting moderate speed of agitation for 24 hours, at Room temperature.
Replace the decellularizing solution in each 50 mL tube with 40mL of 1x PBS and 0.2mL of antibiotic solution .
Sample storage
Fix decellularized sections for histological analyses.
Store at +4°C in a Mass / % volume for further cell seeding or snap-freeze at -80°C for other applications.
Materials List
Addictional materials
| A | B | C | D |
|---|---|---|---|
| 1 L beaker | VWR | 511-0318 | Clean and autoclave before use |
| 10 mL serological pipette | Falcon | 357551 | Sterile, polystyrene |
| 50 mL sterile tubes | Falcon | FC-1 352070 | Sterile tubes, polypropylene |
| 10 mL graduated cylinder | VWR | 612-1518 | Clean and autoclave before use |
| 1L graduated cylinder | VWR | 612-1524 | Clean and autoclave before use |
| 1 L bottle | VWR | 215-1596 | Clean and autoclave before use |
| 25 mL serological pipette | Falcon | 357525 | Sterile, polystyrene |
| 500 mL beaker | VWR | 511-0317 | Clean and autoclave before use |
| Dissecting scalpel | VWR | 233-5526 | Sterile and disposable |
| Fine forceps | VWR | 232-1317 | Clean and autoclave before use |
| Funnel | VWR | 221-1861 | Clean and autoclave before use |
| Hexagonal weighing boats size M | Sigma-Aldrich | Z708585 | Hexagonal, polystyrene, 51 mm |
| Hexagonal weighing boats size S | Sigma-Aldrich | Z708577 | Hexagonal, polystyrene, 25 mm |
| Large surgical scissors | VWR | 233-1211 | Clean and autoclave before use |
| Long forceps | VWR | 232-0096 | Clean and autoclave before use |
| Pipette gun | Eppendorf | 613-2795 | Eppendorf Easypet® 3 |
| Plastic tray | VWR | BELAH162620000 | Corrosion-proof polypropylene |
| Spatula | VWR | RSGA038.210 | Clean and autoclave before use |
| Spoon | VWR | 231-1314 | Clean and autoclave before use |
| Stir bar | VWR | 442-0362 | Clean and autoclave before use |
