16. When the sample has reached the end temperature of À80
C,
transfer to liquid nitrogen storage or to mechanical freezer
with À150
C or below temperature for the further storage.
17. Application of CPAs alternative to DMSO requires individual
optimization of cooling rates, concentration of these CPAs in
cryopreservation suspension, equilibration time, as well as
thawing procedures.
3.3 Thawing
of iPSC-Derived MKs
1. Work quickly during the thawing steps to minimize the stress
on cells as well as cytotoxic effects of DMSO.
2. Heat up water bath to 37
C.
3. Take out cryopreserved MK suspension in 1.8 mL cryogenic
vial from liquid nitrogen storage (or from À150
C mechanical
freezer), and quickly place on the floating shaking holder in the
water bath at 37
C.
4. Gently shake the sample in the water bath until the ice is melted
(approximately 1 min).
5. Work in a laminar flow hood under sterile conditions.
6. Quickly move thawed suspension (1 mL) to a 15 mL centrifuge
tube filled with 5 mL MK culture medium.
7. Centrifuge the mixture at 120 Â g for 10 min at room temperature to remove DMSO.
8. Aspirate the supernatant and resuspend the MK pellet in 5 mL
MK culture medium.
9. Centrifuge again the suspension at 120 Â g for 10 min at room
temperature to remove remaining DMSO.
10. Resuspend the MK pellet in a necessary amount of MK culture
medium, plate the cells onto non-adherent suspension culture
plate, and place into humidified CO 2 incubator.
3.4 Evaluation
of Survival
of iPSC-Derived MKs
After Cryopreservation
with Trypan Blue
Exclusion Test
Trypan blue exclusion assay is a simple conventional method for
primary evaluation of cell viability, which is widely applied in cryobiology for the evaluation of survival rates after cryopreservation.
As an azo dye, trypan blue cannot penetrate an intact membrane,
but easily enters inside cells with damaged membranes, binds to
intracellular proteins, and changes the color of dead cells to blue.
Thus, trypan blue exclusion assay is an easy test for direct identification and quantification of live (non-stained) and dead (stained
blue) cells in a population.
1. Work in a laminar flow hood under sterile conditions.
2. Concentrate thawed MKs from a suspension by centrifugation
(120 Â g, 10 min) and resuspend in 1 mL of MK culture
medium.
3. Prepare a tube with 10 μL trypan blue.
544
Denys Pogozhykh et al.
C,
transfer to liquid nitrogen storage or to mechanical freezer
with À150
C or below temperature for the further storage.
17. Application of CPAs alternative to DMSO requires individual
optimization of cooling rates, concentration of these CPAs in
cryopreservation suspension, equilibration time, as well as
thawing procedures.
3.3 Thawing
of iPSC-Derived MKs
1. Work quickly during the thawing steps to minimize the stress
on cells as well as cytotoxic effects of DMSO.
2. Heat up water bath to 37
C.
3. Take out cryopreserved MK suspension in 1.8 mL cryogenic
vial from liquid nitrogen storage (or from À150
C mechanical
freezer), and quickly place on the floating shaking holder in the
water bath at 37
C.
4. Gently shake the sample in the water bath until the ice is melted
(approximately 1 min).
5. Work in a laminar flow hood under sterile conditions.
6. Quickly move thawed suspension (1 mL) to a 15 mL centrifuge
tube filled with 5 mL MK culture medium.
7. Centrifuge the mixture at 120 Â g for 10 min at room temperature to remove DMSO.
8. Aspirate the supernatant and resuspend the MK pellet in 5 mL
MK culture medium.
9. Centrifuge again the suspension at 120 Â g for 10 min at room
temperature to remove remaining DMSO.
10. Resuspend the MK pellet in a necessary amount of MK culture
medium, plate the cells onto non-adherent suspension culture
plate, and place into humidified CO 2 incubator.
3.4 Evaluation
of Survival
of iPSC-Derived MKs
After Cryopreservation
with Trypan Blue
Exclusion Test
Trypan blue exclusion assay is a simple conventional method for
primary evaluation of cell viability, which is widely applied in cryobiology for the evaluation of survival rates after cryopreservation.
As an azo dye, trypan blue cannot penetrate an intact membrane,
but easily enters inside cells with damaged membranes, binds to
intracellular proteins, and changes the color of dead cells to blue.
Thus, trypan blue exclusion assay is an easy test for direct identification and quantification of live (non-stained) and dead (stained
blue) cells in a population.
1. Work in a laminar flow hood under sterile conditions.
2. Concentrate thawed MKs from a suspension by centrifugation
(120 Â g, 10 min) and resuspend in 1 mL of MK culture
medium.
3. Prepare a tube with 10 μL trypan blue.
544
Denys Pogozhykh et al.