of iPSC-derived MKs is therefore of a high priority. However,
sensitivity of cells to cryopreservation procedures, including the
rates of cooling and thawing, the choice of type and concentration
of cryoprotective agent (CPA), steps of preincubation and conditioning of the cells prior freezing, as well as recovery steps may
result in highly variable survival rates and alteration of structural
and functional properties after thawing [15–19]. In this chapter a
method to cryopreserve iPSC-derived MKs is described. Furthermore, several methods to assess retention of phenotypic and functional parameters of MKs are shown. The proposed method allows
recovering over 80% of cells after cryopreservation with dimethyl
sulfoxide. The protocols can be used as a basis for the further
optimization of cryopreservation methods for various populations
of iPSC-derived MKs, including the search of alternative CPAs, and
adjustment of cooling and thawing rates.
2 Materials
2.1 Cell Culture
Maintenance
Equipment
and Consumables
1. Humidified CO 2 incubator (e.g., Thermo Fisher Scientific,
MA, USA).
2. Laminar flow hood (e.g., Thermo Fisher Scientific, MA, USA).
3. Inverted microscope (e.g., Olympus IX81 microscope, Olympus, Shinjuku, Japan).
4. Cell counting chamber (e.g., Neubauer-improved hemocytometer, Marienfeld-Superior, Lauda-Ko ¨nigshofen, Germany).
5. Motorized pipet controller for use with serological pipets from
0.5 to 100 mL volume ranges (e.g., Falcon
® Pipet Controller,
Corning, NY, USA).
6. Set of pipets of volume ranges: 0.5–10 μL, 10–100 μL, and
100–1000 μL (e.g., Eppendorf, Hamburg, Germany).
7. Cell culture maintenance consumables: appropriate cell culture
dishes and plates, serological pipets, pipet tips, and centrifuge
tubes (e.g., Thermo Fisher Scientific, MA, USA) (see Note 1).
2.2 Cell Culture
Maintenance Medium,
Reagents,
and Cell Lines
1. DMEM (Dulbecco’s Modified Eagle Medium) (e.g., Thermo
Fisher Scientific, MA, USA).
2. DPBS (Dulbecco’s Phosphate Buffered Saline) (e.g., Lonza,
Basel, Switzerland).
3. Optional: water for cell culture applications (e.g., Lonza, Basel,
Switzerland) (see Note 2).
4. Optional: TrypLE™ Express Enzyme (Thermo Fisher Scientific, MA, USA) (see Note 3).
5. Trypan blue solution, 0.4% (e.g., Thermo Fisher Scientific,
MA, USA).
540
Denys Pogozhykh et al.
sensitivity of cells to cryopreservation procedures, including the
rates of cooling and thawing, the choice of type and concentration
of cryoprotective agent (CPA), steps of preincubation and conditioning of the cells prior freezing, as well as recovery steps may
result in highly variable survival rates and alteration of structural
and functional properties after thawing [15–19]. In this chapter a
method to cryopreserve iPSC-derived MKs is described. Furthermore, several methods to assess retention of phenotypic and functional parameters of MKs are shown. The proposed method allows
recovering over 80% of cells after cryopreservation with dimethyl
sulfoxide. The protocols can be used as a basis for the further
optimization of cryopreservation methods for various populations
of iPSC-derived MKs, including the search of alternative CPAs, and
adjustment of cooling and thawing rates.
2 Materials
2.1 Cell Culture
Maintenance
Equipment
and Consumables
1. Humidified CO 2 incubator (e.g., Thermo Fisher Scientific,
MA, USA).
2. Laminar flow hood (e.g., Thermo Fisher Scientific, MA, USA).
3. Inverted microscope (e.g., Olympus IX81 microscope, Olympus, Shinjuku, Japan).
4. Cell counting chamber (e.g., Neubauer-improved hemocytometer, Marienfeld-Superior, Lauda-Ko ¨nigshofen, Germany).
5. Motorized pipet controller for use with serological pipets from
0.5 to 100 mL volume ranges (e.g., Falcon
® Pipet Controller,
Corning, NY, USA).
6. Set of pipets of volume ranges: 0.5–10 μL, 10–100 μL, and
100–1000 μL (e.g., Eppendorf, Hamburg, Germany).
7. Cell culture maintenance consumables: appropriate cell culture
dishes and plates, serological pipets, pipet tips, and centrifuge
tubes (e.g., Thermo Fisher Scientific, MA, USA) (see Note 1).
2.2 Cell Culture
Maintenance Medium,
Reagents,
and Cell Lines
1. DMEM (Dulbecco’s Modified Eagle Medium) (e.g., Thermo
Fisher Scientific, MA, USA).
2. DPBS (Dulbecco’s Phosphate Buffered Saline) (e.g., Lonza,
Basel, Switzerland).
3. Optional: water for cell culture applications (e.g., Lonza, Basel,
Switzerland) (see Note 2).
4. Optional: TrypLE™ Express Enzyme (Thermo Fisher Scientific, MA, USA) (see Note 3).
5. Trypan blue solution, 0.4% (e.g., Thermo Fisher Scientific,
MA, USA).
540
Denys Pogozhykh et al.