4. Add 95 μL of the CPA cocktail to each vial containing the
coverslip in 95 μL of EGM-2 (final concentrations: 5%
DMSO, 6% HES, 2% CS). Mix gently by swirling the vial,
and then cover with the lid.
5. Keep vials in ice slush for 15 min to allow DMSO to permeate
the cell monolayer.
6. Put each vial in a Styrofoam floater and place in the methanol
bath, making sure the bottom of the vial containing medium is
immersed. Set the timer for 2 min to allow the cell monolayer
to attain equilibrium with the methanol bath temperature
(À5
C).
7. Induce ice nucleation in the solution by touching the vial with
cold metal forceps (previously immersed in liquid nitrogen) (see
Note 6).
8. Return the glass vial to the methanol bath and let stand for
3 min to release the latent heat of fusion.
9. Set the cooling rate in the methanol at 0.2
C/min or 1.0
C/
min and the final temperature at À45
C.
10. When the temperature reaches À45
C, remove the vial from
the methanol bath and transfer to liquid nitrogen.
11. Store in liquid nitrogen.
3.3 Thawing Cell
Monolayers
1. After removing a vial from liquid nitrogen and before placing in
the water bath, loosen the lid of the vial (see Note 7).
2. Place the vial in a 37
C water bath, and swirl the vial in the bath
to induce rapid thawing.
3. Remove the CPA from the monolayer as described below for
viability and functional assessments or downstream
experiments.
3.4 CPA Removal by
Single Wash or Serial
Dilution
1. Single wash: The CPA may be removed immediately after thaw
by adding 500 μL of 1Â PBS. Let stand for 5 min at room
temperature. Remove solution carefully without disturbing the
cell monolayer. Replace with 190 μL 1Â PBS and proceed with
downstream assessments or experiments.
2. Serial dilution: Add 200 μL of 20% fetal bovine serum (FBS) in
1Â PBS to the glass vial and incubate for 2 min at room
temperature. Then add 200 μL of 10% FBS in 1Â PBS, and
let stand for 2 min at room temperature. Repeat the last step
two more times. Remove the last solution carefully, replace
with 190 μL 1Â PBS, and proceed with downstream assessments or experiments.
Cryopreservation of Endothelial Monolayers
587
coverslip in 95 μL of EGM-2 (final concentrations: 5%
DMSO, 6% HES, 2% CS). Mix gently by swirling the vial,
and then cover with the lid.
5. Keep vials in ice slush for 15 min to allow DMSO to permeate
the cell monolayer.
6. Put each vial in a Styrofoam floater and place in the methanol
bath, making sure the bottom of the vial containing medium is
immersed. Set the timer for 2 min to allow the cell monolayer
to attain equilibrium with the methanol bath temperature
(À5
C).
7. Induce ice nucleation in the solution by touching the vial with
cold metal forceps (previously immersed in liquid nitrogen) (see
Note 6).
8. Return the glass vial to the methanol bath and let stand for
3 min to release the latent heat of fusion.
9. Set the cooling rate in the methanol at 0.2
C/min or 1.0
C/
min and the final temperature at À45
C.
10. When the temperature reaches À45
C, remove the vial from
the methanol bath and transfer to liquid nitrogen.
11. Store in liquid nitrogen.
3.3 Thawing Cell
Monolayers
1. After removing a vial from liquid nitrogen and before placing in
the water bath, loosen the lid of the vial (see Note 7).
2. Place the vial in a 37
C water bath, and swirl the vial in the bath
to induce rapid thawing.
3. Remove the CPA from the monolayer as described below for
viability and functional assessments or downstream
experiments.
3.4 CPA Removal by
Single Wash or Serial
Dilution
1. Single wash: The CPA may be removed immediately after thaw
by adding 500 μL of 1Â PBS. Let stand for 5 min at room
temperature. Remove solution carefully without disturbing the
cell monolayer. Replace with 190 μL 1Â PBS and proceed with
downstream assessments or experiments.
2. Serial dilution: Add 200 μL of 20% fetal bovine serum (FBS) in
1Â PBS to the glass vial and incubate for 2 min at room
temperature. Then add 200 μL of 10% FBS in 1Â PBS, and
let stand for 2 min at room temperature. Repeat the last step
two more times. Remove the last solution carefully, replace
with 190 μL 1Â PBS, and proceed with downstream assessments or experiments.
Cryopreservation of Endothelial Monolayers
587