6. The bag is then transferred onto ice and externally cleaned with
70% ethanol to reduce the risk of microbial contamination. Cut
the bag open with sterile scissors and use sterile pickups to
transfer the tissue to the first VS removal solution.
7. A multistep VS removal protocol was employed in our first
unpublished sheep study, similar to that used in Protocol
1, above. Simpler, washing methods have been employed in a
subsequent sheep study [19, 20] and a study with arterial
patches [12] (see Note 7).
3.3 Protocol 3
Simplified versions of Protocol 2 in development for heart valves.
1. Place the tissues in sterile polyethylene bags containing 80 mL
of VS83 at room temperature (see Note 1).
2. Evacuate the air and heat seal the bags (see Note 3).
3. Incubate the tissues with continuous shaking for at least 1 h at
room temperature prior to cooling.
4. The cooling process can then be achieved by placing the bags
for 10 min in a precooled bath of 2-methylbutane (< À100
C;
see Note 4).
5. The tissues can then be placed at À80
C for storage.
6. Rewarming of the tissue is then performed by submersion of
the tissue in its bag in a 37
C water bath.
7. The bag is then dried and externally cleaned with 70% ethanol
to reduce the risk of microbial contamination. Cut the bag
open with sterile scissors and use sterile pickups to transfer
the tissue to the first wash step.
8. Washing to remove CPAs has been performed using five wash
steps of 5 min duration each with ice EuroCollins solution or
with several washes with Lactated Ringer’s solution containing
5% dextrose (see Note 8).
3.4 Protocol 4
Modified version of Protocol 1 in development for heart valves for
retention of tissue cell viability in relatively large samples
(>30 mL) (see Note 9).
1. Tissues should be gradually infiltrated with VS55 at 4
C (see
Note 1). Precooled diluted VS solutions are employed in six
15 min steps at 4
C of increasing CPA concentration as
previously described [17] (see Note 2). Sugars, sucrose, or
trehalose or a mixture is added in the final addition step at
0.1–0.6 M concentrations.
2. Evacuate the air and heat seal the bags (see Note 3).
3. The cooling process can then be achieved by placing the bags
for 10 min in a precooled bath of 2-methylbutane (< À100
C)
(see Note 4).
600
Kelvin G. M. Brockbank et al.
70% ethanol to reduce the risk of microbial contamination. Cut
the bag open with sterile scissors and use sterile pickups to
transfer the tissue to the first VS removal solution.
7. A multistep VS removal protocol was employed in our first
unpublished sheep study, similar to that used in Protocol
1, above. Simpler, washing methods have been employed in a
subsequent sheep study [19, 20] and a study with arterial
patches [12] (see Note 7).
3.3 Protocol 3
Simplified versions of Protocol 2 in development for heart valves.
1. Place the tissues in sterile polyethylene bags containing 80 mL
of VS83 at room temperature (see Note 1).
2. Evacuate the air and heat seal the bags (see Note 3).
3. Incubate the tissues with continuous shaking for at least 1 h at
room temperature prior to cooling.
4. The cooling process can then be achieved by placing the bags
for 10 min in a precooled bath of 2-methylbutane (< À100
C;
see Note 4).
5. The tissues can then be placed at À80
C for storage.
6. Rewarming of the tissue is then performed by submersion of
the tissue in its bag in a 37
C water bath.
7. The bag is then dried and externally cleaned with 70% ethanol
to reduce the risk of microbial contamination. Cut the bag
open with sterile scissors and use sterile pickups to transfer
the tissue to the first wash step.
8. Washing to remove CPAs has been performed using five wash
steps of 5 min duration each with ice EuroCollins solution or
with several washes with Lactated Ringer’s solution containing
5% dextrose (see Note 8).
3.4 Protocol 4
Modified version of Protocol 1 in development for heart valves for
retention of tissue cell viability in relatively large samples
(>30 mL) (see Note 9).
1. Tissues should be gradually infiltrated with VS55 at 4
C (see
Note 1). Precooled diluted VS solutions are employed in six
15 min steps at 4
C of increasing CPA concentration as
previously described [17] (see Note 2). Sugars, sucrose, or
trehalose or a mixture is added in the final addition step at
0.1–0.6 M concentrations.
2. Evacuate the air and heat seal the bags (see Note 3).
3. The cooling process can then be achieved by placing the bags
for 10 min in a precooled bath of 2-methylbutane (< À100
C)
(see Note 4).
600
Kelvin G. M. Brockbank et al.