3 Methods
3.1 Protocol 1
Suitable for small heart valve tissue samples ( 3 mL total volume,
sample plus solution) with maximum retention of cell viability and
matrix integrity.
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).
2. The top of the vitrification solution is then covered with
2-methylbutane (isopentane; see Note 3).
3. The samples should be cooled rapidly to À100
C followed by
slow cooling to ! À135
C (see Note 4).
4. Finally the samples should be stored at À135
C in a mechanical storage freezer or at ! À135
C near the top in a vaporphase nitrogen freezer.
5. Rewarming of vitrified tissues is a two-stage process including
slow warming to À100
C and then rapid warming to ~4
C
(see Note 5). Wipe the container with 70% ethanol to minimize
the risk of microbial contamination and use sterile pickups to
transfer the tissue to the first VS removal solution.
6. 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.
3.2 Protocol 2
Suitable for large heart valve tissue samples (up to 100 mL total
volume) with preservation of matrix integrity and minimal residual
cell viability.
1. Tissues are gradually infiltrated with VS83 in six 15 min steps of
increasing concentration at 4
C.
2. After the final step, the tissues are placed individually in sterile
polyester bags containing 70–80 mL of the vitrification solution. Each bag is then evacuated of air by squeezing (see Note
3) and heat-sealed.
3. The bags containing tissues will then be cooled rapidly to
À100
C in a precooled 2-methylbutane bath and then transferred to storage (see Note 4).
4. Store at À80
C in a mechanical storage freezer (see Note 6).
5. Rewarming is performed by placing each bag in a 37
C water
bath until the solution moves freely.
Vitrification of Heart Valve Tissues
599
3.1 Protocol 1
Suitable for small heart valve tissue samples ( 3 mL total volume,
sample plus solution) with maximum retention of cell viability and
matrix integrity.
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).
2. The top of the vitrification solution is then covered with
2-methylbutane (isopentane; see Note 3).
3. The samples should be cooled rapidly to À100
C followed by
slow cooling to ! À135
C (see Note 4).
4. Finally the samples should be stored at À135
C in a mechanical storage freezer or at ! À135
C near the top in a vaporphase nitrogen freezer.
5. Rewarming of vitrified tissues is a two-stage process including
slow warming to À100
C and then rapid warming to ~4
C
(see Note 5). Wipe the container with 70% ethanol to minimize
the risk of microbial contamination and use sterile pickups to
transfer the tissue to the first VS removal solution.
6. 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.
3.2 Protocol 2
Suitable for large heart valve tissue samples (up to 100 mL total
volume) with preservation of matrix integrity and minimal residual
cell viability.
1. Tissues are gradually infiltrated with VS83 in six 15 min steps of
increasing concentration at 4
C.
2. After the final step, the tissues are placed individually in sterile
polyester bags containing 70–80 mL of the vitrification solution. Each bag is then evacuated of air by squeezing (see Note
3) and heat-sealed.
3. The bags containing tissues will then be cooled rapidly to
À100
C in a precooled 2-methylbutane bath and then transferred to storage (see Note 4).
4. Store at À80
C in a mechanical storage freezer (see Note 6).
5. Rewarming is performed by placing each bag in a 37
C water
bath until the solution moves freely.
Vitrification of Heart Valve Tissues
599