5. Corbit-Balsam mounting medium (Hecht, Hamburg,
Germany).
6. Hematoxylin-eosin staining solution. Filter solution prior
to use.
3 Methods
3.1 Preparation
of Heart Valves
and Decellularization
1. Obtain porcine hearts from a local slaughterhouse and dissect
heart valves from the hearts.
2. Disinfect by incubation in Braunol solution for 5 min, and then
remove this solution by incubation in PBS+ for 20 min.
3. Place tissues into Triton-X100 decellularization solution for
24 h at room temperature, while continuously shaking the
solution and replacing the Triton-X100 solution with fresh
solution after 12 h.
4. Place tissues into SDS decellularization solution for 24 h at
room temperature, while continuously shaking the solution
and replacing the SDS solution with fresh solution after 12 h.
5. Remove the detergents by washing two times 12 h in distilled
water.
6. Conduct another 12 washing cycles in PBS+, while continuously shaking and changing the solution every 12 h (see
Note 3).
7. Store decellularized heart valves in PBS+ at 4
C, until further
processing (see Fig. 1).
3.2 Loading
Decellularized Heart
Valve Tissue
with Lyoprotectants
and Freeze-Drying
1. Incubate decellularized heart valves in 100 mL 5% sucrose
solution, at 37
C for 4 h (see Note 4).
2. Transfer the heart valves in 100 mL 80% sucrose solution, at
37
C for 30 min, changing the sucrose solution with fresh
sucrose solution every 10 min (see Notes 5 and 6).
3. Dip the valves on tissue paper to remove excess solution.
Transfer the heart valves to an open or semi-open container
(i.e., a Petri dish with knuckles allowing vapor transport), and
put on the freeze-dryer shelves with the shelve temperature set
at 20
C.
4. Start the freeze-drying protocol.
(a) Freeze the samples by cooling the shelves at a cooling rate
of 1
C/min until the shelve temperature reaches À40
C,
and keep at À40
C for 3 h.
(b) Increase shelf temperature to À30
C at a rate of 1
C/
min.
734
Willem F. Wolkers and Andres Hilfiker
Germany).
6. Hematoxylin-eosin staining solution. Filter solution prior
to use.
3 Methods
3.1 Preparation
of Heart Valves
and Decellularization
1. Obtain porcine hearts from a local slaughterhouse and dissect
heart valves from the hearts.
2. Disinfect by incubation in Braunol solution for 5 min, and then
remove this solution by incubation in PBS+ for 20 min.
3. Place tissues into Triton-X100 decellularization solution for
24 h at room temperature, while continuously shaking the
solution and replacing the Triton-X100 solution with fresh
solution after 12 h.
4. Place tissues into SDS decellularization solution for 24 h at
room temperature, while continuously shaking the solution
and replacing the SDS solution with fresh solution after 12 h.
5. Remove the detergents by washing two times 12 h in distilled
water.
6. Conduct another 12 washing cycles in PBS+, while continuously shaking and changing the solution every 12 h (see
Note 3).
7. Store decellularized heart valves in PBS+ at 4
C, until further
processing (see Fig. 1).
3.2 Loading
Decellularized Heart
Valve Tissue
with Lyoprotectants
and Freeze-Drying
1. Incubate decellularized heart valves in 100 mL 5% sucrose
solution, at 37
C for 4 h (see Note 4).
2. Transfer the heart valves in 100 mL 80% sucrose solution, at
37
C for 30 min, changing the sucrose solution with fresh
sucrose solution every 10 min (see Notes 5 and 6).
3. Dip the valves on tissue paper to remove excess solution.
Transfer the heart valves to an open or semi-open container
(i.e., a Petri dish with knuckles allowing vapor transport), and
put on the freeze-dryer shelves with the shelve temperature set
at 20
C.
4. Start the freeze-drying protocol.
(a) Freeze the samples by cooling the shelves at a cooling rate
of 1
C/min until the shelve temperature reaches À40
C,
and keep at À40
C for 3 h.
(b) Increase shelf temperature to À30
C at a rate of 1
C/
min.
734
Willem F. Wolkers and Andres Hilfiker