4. The tissues can then be placed at À135
C for storage.
5. Rewarming of the tissue is then performed by submersion of
the tissue in its bag as described in Protocol 1, step 5.
6. 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.
7. The vitrification solution is then removed in a stepwise manner
at 4
C in six or seven sequential, 15 min steps of increasingly
dilute VS solutions containing mannitol as previously described
[17] (see Note 1). Finally the tissues are placed in DMEM
culture medium.
4 Notes
1. Stepwise VS addition and removal procedures are employed in
Protocol 1 in order to minimize damage to cells as they
respond to osmotic changes in their environment as a consequence of being placed in VS solutions with high concentrations of CPAs [reviewed in 17]. This is not an issue in Protocols
2 and 3 because we are not trying to maximize cell viability.
Loss of cell viability is considered advantageous because it
appears to reduce immunogenicity in vivo [19, 20].
2. In the last step, the tissues are placed in vials; we usually use
glass scintillation vials (25 Â 60 mm), containing 1–2 mL of
precooled full-strength VS solution. In circumstances where
unexpected cytotoxicity is observed, the last one or two steps
can be performed at subzero temperatures in a cold bath to
minimize cytotoxicity.
3. We use 0.7–1.0 mL of 2-methylbutane for the glass vials
described in Note 1. The 2-methylbutane has a freezing
point of À160
C and density of 0.62. The 2-methylbutane
layer prevents direct air contact, minimizing the risk of ice
nucleation. Alternatively, the vial can be purged with a dry
gas, such as nitrogen, or a container without excess air space
can be used.
4. Rapid cooling to < À100
C is done by placing the vials in a
precooled (À135
C) 2-methylbutane bath; slow cooling is
performed by placing the vials in air in the top of storage
freezer, either a À135
C mechanical freezer or near the top
of a vapor-phase nitrogen-cooled freezer. More rapid cooling
can be obtained, if needed, using liquid ethane at approx.
À183
C. However, this strategy may result in the development of thermal stresses within the sample because the edges
will cool rapidly and the inside more slowly. Such stresses may
lead to cracking during storage or upon rewarming.
Vitrification of Heart Valve Tissues
601
C for storage.
5. Rewarming of the tissue is then performed by submersion of
the tissue in its bag as described in Protocol 1, step 5.
6. 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.
7. The vitrification solution is then removed in a stepwise manner
at 4
C in six or seven sequential, 15 min steps of increasingly
dilute VS solutions containing mannitol as previously described
[17] (see Note 1). Finally the tissues are placed in DMEM
culture medium.
4 Notes
1. Stepwise VS addition and removal procedures are employed in
Protocol 1 in order to minimize damage to cells as they
respond to osmotic changes in their environment as a consequence of being placed in VS solutions with high concentrations of CPAs [reviewed in 17]. This is not an issue in Protocols
2 and 3 because we are not trying to maximize cell viability.
Loss of cell viability is considered advantageous because it
appears to reduce immunogenicity in vivo [19, 20].
2. In the last step, the tissues are placed in vials; we usually use
glass scintillation vials (25 Â 60 mm), containing 1–2 mL of
precooled full-strength VS solution. In circumstances where
unexpected cytotoxicity is observed, the last one or two steps
can be performed at subzero temperatures in a cold bath to
minimize cytotoxicity.
3. We use 0.7–1.0 mL of 2-methylbutane for the glass vials
described in Note 1. The 2-methylbutane has a freezing
point of À160
C and density of 0.62. The 2-methylbutane
layer prevents direct air contact, minimizing the risk of ice
nucleation. Alternatively, the vial can be purged with a dry
gas, such as nitrogen, or a container without excess air space
can be used.
4. Rapid cooling to < À100
C is done by placing the vials in a
precooled (À135
C) 2-methylbutane bath; slow cooling is
performed by placing the vials in air in the top of storage
freezer, either a À135
C mechanical freezer or near the top
of a vapor-phase nitrogen-cooled freezer. More rapid cooling
can be obtained, if needed, using liquid ethane at approx.
À183
C. However, this strategy may result in the development of thermal stresses within the sample because the edges
will cool rapidly and the inside more slowly. Such stresses may
lead to cracking during storage or upon rewarming.
Vitrification of Heart Valve Tissues
601