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.

Safety information

It is recommended to wear personal protective devices.

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.

Safety information

This step shoul be performed under chemical hood wearing personal protective devices.

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 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 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 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 .

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.

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