6. Basal medium for differentiation of iPSCs into MKs (e.g.,
STEMdiff™ APEL™ Medium, STEMCELL Technologies,
Vancouver, Canada) (see Note 4).
7. Cytokines and growth factors for differentiation of iPSCs into
MKs (PeproTech, NJ, USA) (see Note 5).
8. IPSCs line capable of differentiation into MKs (see Note 6).
2.3 Cryopreservation
Equipment,
Consumables,
and Reagents
1. Freezing container capable of providing 1
C/min cooling rate
(e.g., Nalgene
® Mr. Frosty, Thermo Fisher Scientific, MA,
USA). Alternatively, a controlled rate freezer capable of
providing 1
C/min cooling rate can be used (e.g., Planer
PLC, Sunbury-on-Thames, UK) (see Note 7).
2. Freezer capable of providing À80
C storage temperature (e.g.,
Sanyo VIP Ultra Low Temp Freezer À86
C, Sanyo, Osaka,
Japan).
3. Liquid nitrogen storage systems (e.g., Thermo Fisher Scientific, MA, USA) or mechanical freezers capable of providing
À150
C or below storage temperature.
4. Water bath (e.g., Precision™, Thermo Fisher Scientific,
MA, USA).
5. Dimethyl sulfoxide (DMSO) (e.g. Sigma-Aldrich, MO, USA)
(see Note 8).
6. Cryogenic vials, 1.8 mL (e.g., Nunc
® CryoTubes
® , SigmaAldrich, MO, USA).
7. DMEM (Dulbecco’s Modified Eagle Medium) (e.g., Thermo
Fisher Scientific, MA, USA). Alternatively, basal medium for
differentiation of iPSCs into MKs (e.g., STEMdiff™ APEL™
Medium, STEMCELL Technologies, Vancouver, Canada).
2.4 Equipment,
Consumables,
and Reagents
for Microscopic
Analysis
1. Fluorescence microscope equipped with a digital camera (e.g.,
Olympus IX81 microscope, Olympus, Shinjuku, Japan).
2. Personal computer with software for acquiring and analysis of
fluorescence microscopy images (e.g., Xcellence Pro image
software, Olympus, Shinjuku, Japan).
3. 12-well suspension culture plates (e.g., Greiner CELLSTAR
® ,
Greiner Bio-One, Kremsmu ¨nster, Austria).
4. Cell counting chamber (e.g., Neubauer-improved hemocytometer, Marienfeld-Superior, Lauda-Ko ¨nigshofen, Germany).
5. Trypan blue solution, 0.4% (e.g., Thermo Fisher Scientific,
MA, USA).
6. DPBS (Dulbecco’s Phosphate Buffered Saline) (e.g., Lonza,
Basel, Switzerland).
7. Basal medium for differentiation of iPSCs into MKs (e.g.,
STEMdiff™ APEL™ Medium, STEMCELL Technologies,
Cryopreservation of iPSC-Derived Megakaryocytes
541
STEMdiff™ APEL™ Medium, STEMCELL Technologies,
Vancouver, Canada) (see Note 4).
7. Cytokines and growth factors for differentiation of iPSCs into
MKs (PeproTech, NJ, USA) (see Note 5).
8. IPSCs line capable of differentiation into MKs (see Note 6).
2.3 Cryopreservation
Equipment,
Consumables,
and Reagents
1. Freezing container capable of providing 1
C/min cooling rate
(e.g., Nalgene
® Mr. Frosty, Thermo Fisher Scientific, MA,
USA). Alternatively, a controlled rate freezer capable of
providing 1
C/min cooling rate can be used (e.g., Planer
PLC, Sunbury-on-Thames, UK) (see Note 7).
2. Freezer capable of providing À80
C storage temperature (e.g.,
Sanyo VIP Ultra Low Temp Freezer À86
C, Sanyo, Osaka,
Japan).
3. Liquid nitrogen storage systems (e.g., Thermo Fisher Scientific, MA, USA) or mechanical freezers capable of providing
À150
C or below storage temperature.
4. Water bath (e.g., Precision™, Thermo Fisher Scientific,
MA, USA).
5. Dimethyl sulfoxide (DMSO) (e.g. Sigma-Aldrich, MO, USA)
(see Note 8).
6. Cryogenic vials, 1.8 mL (e.g., Nunc
® CryoTubes
® , SigmaAldrich, MO, USA).
7. DMEM (Dulbecco’s Modified Eagle Medium) (e.g., Thermo
Fisher Scientific, MA, USA). Alternatively, basal medium for
differentiation of iPSCs into MKs (e.g., STEMdiff™ APEL™
Medium, STEMCELL Technologies, Vancouver, Canada).
2.4 Equipment,
Consumables,
and Reagents
for Microscopic
Analysis
1. Fluorescence microscope equipped with a digital camera (e.g.,
Olympus IX81 microscope, Olympus, Shinjuku, Japan).
2. Personal computer with software for acquiring and analysis of
fluorescence microscopy images (e.g., Xcellence Pro image
software, Olympus, Shinjuku, Japan).
3. 12-well suspension culture plates (e.g., Greiner CELLSTAR
® ,
Greiner Bio-One, Kremsmu ¨nster, Austria).
4. Cell counting chamber (e.g., Neubauer-improved hemocytometer, Marienfeld-Superior, Lauda-Ko ¨nigshofen, Germany).
5. Trypan blue solution, 0.4% (e.g., Thermo Fisher Scientific,
MA, USA).
6. DPBS (Dulbecco’s Phosphate Buffered Saline) (e.g., Lonza,
Basel, Switzerland).
7. Basal medium for differentiation of iPSCs into MKs (e.g.,
STEMdiff™ APEL™ Medium, STEMCELL Technologies,
Cryopreservation of iPSC-Derived Megakaryocytes
541