Apr 24, 2023
Version 3
Human myocardium decellularization V.3
- Immacolata Belviso1,
- Anna Maria Sacco1,
- Domenico Cozzolino1,
- Daria Nurzynska2,
- Franca Di Meglio1,
- clotilde.castaldo1,
- Veronica Romano1
- 1Department of Public Health, University of Naples Federico II, Naples, Italy;
- 2Department of Medicine, Surgery and Dentistry, Scuola Medica Salernitana, University of Salerno, Baronissi, Italy
Protocol Citation: Immacolata Belviso, Anna Maria Sacco, Domenico Cozzolino, Daria Nurzynska, Franca Di Meglio, clotilde.castaldo, Veronica Romano 2023. Human myocardium decellularization. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2o22xv1y/v3Version created by clotilde.castaldo
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 14, 2023
Last Modified: April 24, 2023
Protocol Integer ID: 80556
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.
(The last step contains a supplemental video with extra context and tips, as part of the protocols.io Spotlight series, featuring conversations with protocol authors.)
Protocol materials
Sodium Phosphate Dibasic Merck MilliporeSigma (Sigma-Aldrich)Catalog #S9763-1Kg
Step 2.1
Potassium Phosphate MonobasicCatalog #P5655-1Kg
Step 2.1
Sodium ChlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #S7653
Step 2.1
Penicillin-StreptomycinMerck MilliporeSigma (Sigma-Aldrich)Catalog #P4333
Step 3.1
Amphotericin BMerck MilliporeSigma (Sigma-Aldrich)Catalog #Y0000005
Step 3.1
Sodium Chloride SolutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #S8776
Step 4.1
Triton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #X100-1L
Step 1.2
Potassium ChlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #P9333
Step 2.1
Sodium dodecyl sulfate Merck MilliporeSigma (Sigma-Aldrich)Catalog #62862
Step 1.5
Preparation of decellularizing solution
Preparation of decellularizing solution
50m
50m
Preparation of 600 mL of decellularizing solution
50m
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.
2m
Add 6 mL of Triton X-100 to the beaker containing the double-distilled water using a serological pipette.
Triton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #X100-1L
Safety information
It is recommended to wear personal protective devices.
2m
Equipment
Heating Magnetic Stirrer
NAME
VELP SCIENTIFICA
BRAND
VP-F20520162
SKU
LINK
Add a stir bar into the beaker and place it on a magnetic stirrer to mix the solution until completely dissolved.
15m
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.
2m
Equipment
Explorer Pro Precision EP413
NAME
Precision balance
TYPE
Ohaus
BRAND
80108921
SKU
LINK
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.
Sodium dodecyl sulfate Merck MilliporeSigma (Sigma-Aldrich)Catalog #62862
Safety information
This step shoul be performed under chemical hood wearing personal protective devices.
5m
Add a stir bar into the beaker and place it on a magnetic stirrer to mix the solution until completely dissolved.
10m
Pour the solution in a graduated cylinder and adjust the volume to 300 mL by adding double-distilled water.
4m
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
Parafilm M
NAME
Thermoplastic film
TYPE
Sigma-Aldrich
BRAND
P7793-1EA
SKU
LINK
5m
Transfer 1% decellularizing solution in a 1 L graduated bottle using a funnel to reduce foaming.
Store at +4 °C until use.
Note
The final volume of the decellularizing solution can vary according to the number of samples to decellularize. The volume reported in the protocol is intended for 15 samples.
5m
Preparation of 1x phosphate buffered saline (PBS) solution
Preparation of 1x phosphate buffered saline (PBS) solution
29m
29m
Preparation of 500 mL of 1x PBS
29m
Weigh all the salt powders in recommended amounts using an electronic balance, a spatula and a spoon.
0.1 g Potassium Phosphate Monobasic
0.1 g Potassium Chloride
4.0 g Sodium Chloride
0.575 g Sodium Phosphate Dibasic
Transfer the salts into a 500 mL beaker.
Potassium Phosphate MonobasicCatalog #P5655-1Kg
Potassium ChlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #P9333
Sodium ChlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #S7653
Sodium Phosphate Dibasic Merck MilliporeSigma (Sigma-Aldrich)Catalog #S9763-1Kg
5m
Take a graduated cylinder to measure 400 mL of double-distilled water and pour it into the beaker.
2m
Add a stir bar and place the beaker on a magnetic stirrer to completely dissolve the salts.
15m
Pour the solution in a graduated cylinder and adjust the volume to 500 mL by adding double-distilled water.
2m
Check the pH value and adjust to 7.4 if needed.
Store at +4 °C untile use.
5m
Preparation of antibiotic solution
Preparation of antibiotic solution
5m
5m
Preparation of 10 mL antibiotic solution
5m
Accurately weigh 625 µg Amphotericin B using an electronic balance and add it to a 8 mL pen/strep mixture . Mix vigorously until it is completely dissolved.
Amphotericin BMerck MilliporeSigma (Sigma-Aldrich)Catalog #Y0000005
Penicillin-StreptomycinMerck MilliporeSigma (Sigma-Aldrich)Catalog #P4333
3m
Pour the solution in a graduated cylinder and adjust the volume to 10 mL adding pen/strep mixture.
Store at +4 °C until use.
2m
Preparation of samples and decellularization procedure
Preparation of samples and decellularization procedure
1d 13h 16m
1d 13h 16m
Preparation and decellularization of samples
1d 13h 16m
Identify and wash the cardiac tissue samples obtained from explanted hearts into a plastic tray using a 0.9 Mass / % volume Sodium Chloride isotonic solution to remove any residual fluid.
Sodium Chloride SolutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #S8776
10m
Equipment
Dissecting Board
NAME
Board for Anatomical Dissection
TYPE
VWR
BRAND
100498-398
SKU
LINK
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.
5m
Cut unrefined samples from full-thickness left ventricle wall avoiding injured areas and wash with 0.9 Mass / % volume Sodium Chloride isotonic solution .
10m
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.
Note
Fragments should not be larger than 2 cm wide by 2 cm long.
15m
Snap freeze at -80 °C .
1h
Equipment
new equipment
NAME
Cryostat
TYPE
Leica
BRAND
CM1950
SKU
LINK
Mount samples on cryostat chuck and slice them one by one to obtain 350 µm thick sections.
Note
It is recommended to cut at least three 350‐μm‐thick sections of each sample, using as a reference the same number of sections of native tissue.
1d 13h 16m
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.
Note
Make sure the tubes are appropriately locked to avoid solution leakage.
20m
Equipment
Platform Rocker STR6
NAME
Orbital Shaker
TYPE
Stuart Scientific
BRAND
L065
SKU
LINK
Place the tubes on an orbital shaker and start the procedure setting moderate speed of agitation for 24 hours, at Room temperature .
1d
Replace the decellularizing solution in each 50 mL tube with 40 mL of 1x PBS and 0.2 mL of antibiotic solution .
30m
Stop the agitation and check the color of the sections.
Note
Samples should shift from the native red to translucent white.
10m
Start the agitation on the orbital shaker at a moderate speed overnight, at Room temperature .
8h
Stop the agitation. Replace the solution in each 50 mL tube with 40 mL of double-distilled water.
30m
Start the agitation on the orbital shaker at a moderate speed for 30 minutes at Room temperature .
30m
Stop the agitation. Open each tube and gently dry sections to remove the excess of double-distilled water.
30m
Sample storage
Sample storage
30m
30m
Fix decellularized sections for histological analyses.
Store at +4 °C in a 0 Mass / % volume Sodium Chloride isotonic solution for further cell seeding or snap-freeze at -80 °C for other applications.
Note
A cycle of sterilization under UV is highly recommended before cell seeding, and d-ECM must be rehydrated with an appropriate culture medium prior to use.
30m
Materials List
Materials List
Addictional materials
| EQUIPMENT | BRAND | CATALOG NUMBER | SPECIFICATION | |
| 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 |
Spotlight video
Spotlight video
(The following video contains extra context and tips, as part of the protocols.io Spotlight series, featuring conversations with protocol authors.)