3.2 Cryopreservation
of iPSC-Derived MKs
1. Work in laminar flow hood under sterile conditions.
2. Prepare a 50% (v/v) DMSO solution, by mixing equal volumes
of DMSO and MK culture medium.
3. Pre-cool the freezing container capable of providing 1
C/min
cooling rate (e.g., Nalgene
® Mr. Frosty) in the fridge at 4
C.
In case a controlled rate freezer is used, switch it on, adjust the
program to cooling down to À80
C with 1
C/min cooling
rate, and pre-cool to 10
C (see Note 7).
4. Concentrate MKs from a suspension by centrifugation
(120 Â g, 10 min), and resuspend in 1 mL of MK culture
medium.
5. Prepare a tube with 10 μL trypan blue.
6. Prepare a cell counting chamber with cover glass.
7. Resuspend MK cell suspension, take 10 μL, and mix with 10 μL
of trypan blue in a tube (see Note 11).
8. Fill a counting chamber with 10 μL mixture of cell suspension
and trypan blue.
9. Count non-stained with trypan blue cells at 10Â magnification
of a phase-contrast microscope.
10. Calculate the amount of MKs per mL using the following
formula:
Total cells counted in 4 squares
4 number of squares
ð
Þ
 2 dilution factor
ð
Þ Â 10, 000
¼ cells=mL
11. Label cryogenic vial accordingly, taking into account the stability of labeling material in subzero temperatures (e.g., type
of sample, total number of cells cryopreserved, type of CPA,
cooling rate, date of cryopreservation, etc.).
12. Resuspend 1 Â 10
6 cells in 850 μL of MK culture medium
(calculated per total 1 mL cryopreservation suspension in one
1.8 mL cryogenic vial), and transfer to 1.8 mL cryogenic vial.
13. Add 150 μL of 50% DMSO solution dropwise on top of cell
suspension in cryogenic vial from the previous step to achieve
7.5% (v/v) of final DMSO concentration in cryopreservation
suspension.
14. Incubate for 10 min at room temperature to equilibrate the
cells with CPA.
15. Insert cryogenic vial into pre-cooled freezing container, and
place in the À80
C freezer to cool down the sample to
À80
C with a cooling rate of 1
C/min. In the case a
controlled rate freezer is used, cool down the sample to
À80
C with 1
C/min cooling rate.
Cryopreservation of iPSC-Derived Megakaryocytes
543
of iPSC-Derived MKs
1. Work in laminar flow hood under sterile conditions.
2. Prepare a 50% (v/v) DMSO solution, by mixing equal volumes
of DMSO and MK culture medium.
3. Pre-cool the freezing container capable of providing 1
C/min
cooling rate (e.g., Nalgene
® Mr. Frosty) in the fridge at 4
C.
In case a controlled rate freezer is used, switch it on, adjust the
program to cooling down to À80
C with 1
C/min cooling
rate, and pre-cool to 10
C (see Note 7).
4. Concentrate MKs from a suspension by centrifugation
(120 Â g, 10 min), and resuspend in 1 mL of MK culture
medium.
5. Prepare a tube with 10 μL trypan blue.
6. Prepare a cell counting chamber with cover glass.
7. Resuspend MK cell suspension, take 10 μL, and mix with 10 μL
of trypan blue in a tube (see Note 11).
8. Fill a counting chamber with 10 μL mixture of cell suspension
and trypan blue.
9. Count non-stained with trypan blue cells at 10Â magnification
of a phase-contrast microscope.
10. Calculate the amount of MKs per mL using the following
formula:
Total cells counted in 4 squares
4 number of squares
ð
Þ
 2 dilution factor
ð
Þ Â 10, 000
¼ cells=mL
11. Label cryogenic vial accordingly, taking into account the stability of labeling material in subzero temperatures (e.g., type
of sample, total number of cells cryopreserved, type of CPA,
cooling rate, date of cryopreservation, etc.).
12. Resuspend 1 Â 10
6 cells in 850 μL of MK culture medium
(calculated per total 1 mL cryopreservation suspension in one
1.8 mL cryogenic vial), and transfer to 1.8 mL cryogenic vial.
13. Add 150 μL of 50% DMSO solution dropwise on top of cell
suspension in cryogenic vial from the previous step to achieve
7.5% (v/v) of final DMSO concentration in cryopreservation
suspension.
14. Incubate for 10 min at room temperature to equilibrate the
cells with CPA.
15. Insert cryogenic vial into pre-cooled freezing container, and
place in the À80
C freezer to cool down the sample to
À80
C with a cooling rate of 1
C/min. In the case a
controlled rate freezer is used, cool down the sample to
À80
C with 1
C/min cooling rate.
Cryopreservation of iPSC-Derived Megakaryocytes
543