3 Methods
3.1 Coating Culture
Plates with
Recombinant
Vitronectin
This step is important for promoting cell attachment and growing
undifferentiated hiPSC colonies.
1. Dilute recombinant human vitronectin to a final concentration
of 10 μg/mL by adding 1Â PBS+ (see Note 5).
2. Add sufficient amounts from the diluted vitronectin solutions
to each culture well. Typically, 1.5 mL and 0.5 mL are added to
each well of 6-well and 12-well plates, respectively.
3. Gently shake the plates to spread the vitronectin solution across
the entire surface of each well.
4. Incubate the plates at ambient temperature for at least 1 h or at
4
C overnight.
5. Aspirate the coating solution, and plate iPSCs without allowing
any well to dry out.
3.2 Culturing hiPSCs
Different chemically defined media have been developed for culturing hPSCs and are commercially available [17–19]. To date, we
have used E8 medium (also called TeSR-E8) and, thus, are suggesting its use; however, other chemically defined media might be as
good as E8 medium.
1. Before plating hiPSCs, aspirate the coating solution, and
quickly add E8 medium containing 10-μM Y-27632 to each
well to prevent drying out of the coated surfaces.
2. Plate detached and partially dissociated hiPSC colonies on the
coated wells upon suspension in E8 medium supplemented
with 10-μM ROCK inhibitor Y-27632 (see Note 6).
3. Replace 2/3 of the medium volume with fresh E8 medium
every day to maintain effective concentrations of the growth
factors and, thus, to grow undifferentiated colonies (see
Note 7).
3.3 Passaging of
hiPSCs
hiPSC colonies should be passaged before the colonies start merging—usually when they are around 70–80% confluent. Overgrown
colonies start to spontaneously differentiate. hiPSCs can be passaged both enzymatically (e.g., using accutase and TrypLE) and
nonenzymatically (e.g., by manual cutting or EDTA treatment)
although they are sensitive to dissociation to single cells. For better
cell survival, partial dissociation to small cell clumps is a preferred
choice, which can be achieved by a simple EDTA treatment.
1. Before dissociation, replace E8 flex medium with fresh medium
containing 10-μM ROCK inhibitor (Y-27632), and incubate at
37
C for 2 h.
574
Juliette Seremak and Ali Eroglu
3.1 Coating Culture
Plates with
Recombinant
Vitronectin
This step is important for promoting cell attachment and growing
undifferentiated hiPSC colonies.
1. Dilute recombinant human vitronectin to a final concentration
of 10 μg/mL by adding 1Â PBS+ (see Note 5).
2. Add sufficient amounts from the diluted vitronectin solutions
to each culture well. Typically, 1.5 mL and 0.5 mL are added to
each well of 6-well and 12-well plates, respectively.
3. Gently shake the plates to spread the vitronectin solution across
the entire surface of each well.
4. Incubate the plates at ambient temperature for at least 1 h or at
4
C overnight.
5. Aspirate the coating solution, and plate iPSCs without allowing
any well to dry out.
3.2 Culturing hiPSCs
Different chemically defined media have been developed for culturing hPSCs and are commercially available [17–19]. To date, we
have used E8 medium (also called TeSR-E8) and, thus, are suggesting its use; however, other chemically defined media might be as
good as E8 medium.
1. Before plating hiPSCs, aspirate the coating solution, and
quickly add E8 medium containing 10-μM Y-27632 to each
well to prevent drying out of the coated surfaces.
2. Plate detached and partially dissociated hiPSC colonies on the
coated wells upon suspension in E8 medium supplemented
with 10-μM ROCK inhibitor Y-27632 (see Note 6).
3. Replace 2/3 of the medium volume with fresh E8 medium
every day to maintain effective concentrations of the growth
factors and, thus, to grow undifferentiated colonies (see
Note 7).
3.3 Passaging of
hiPSCs
hiPSC colonies should be passaged before the colonies start merging—usually when they are around 70–80% confluent. Overgrown
colonies start to spontaneously differentiate. hiPSCs can be passaged both enzymatically (e.g., using accutase and TrypLE) and
nonenzymatically (e.g., by manual cutting or EDTA treatment)
although they are sensitive to dissociation to single cells. For better
cell survival, partial dissociation to small cell clumps is a preferred
choice, which can be achieved by a simple EDTA treatment.
1. Before dissociation, replace E8 flex medium with fresh medium
containing 10-μM ROCK inhibitor (Y-27632), and incubate at
37
C for 2 h.
574
Juliette Seremak and Ali Eroglu