autoclave with an appropriate liquid program. Once sterilized,
the remaining sterile ingredients of the defined cryopreservation medium can be added under the biosafety hood without
the need to filter.
3. KSR contains undefined lipids that can unintentionally be
removed during filtration. Therefore, sucrose can first be dissolved in DMEM/F-12 and filtered before adding the remaining sterile components under the biosafety hood.
4. The sterilization of the defined thawing medium is also challenging. We recommend first dissolving methylcellulose and
sucrose in HBSSÀ and then autoclaving the resulting solution
before aseptically adding the remaining sterile components
under the biosafety hood.
5. Recombinant human vitronectin is available as a concentrated
solution usually at 0.5 mg/mL or lyophilized powder. When
received as lyophilized powder, an appropriate volume of PBS+
can be added directly to the vial to dissolve it. Next, the dissolved
vitronectin can be transferred to a larger sterile tube to make a
working solution at 10 μg/mL by adding more PBS+. Then,
the working solution should be aliquoted and stored at À20
C
or À80
C to avoid repeated freeze/thaw cycles.
6. When dissociated to single cells, hiPSCs require the presence of
a ROCK inhibitor to survive passaging. It is not necessary to
supplement E8 medium with a ROCK inhibitor if hiPSC colonies partially dissociated by EDTA treatment. Nevertheless, we
prefer to supplement E8 medium with 10 μM ROCK inhibitor
Y-27632 during the first 48 h of passaging independent of to
what extent hiPSC colonies are dissociated.
7. Particularly, native bFGF is highly unstable and quickly
degraded under cell culture conditions, requiring changing
the culture medium every day. Recently, a heat-stable recombinant human bFGF has been engineered and added to E8
medium (called E8 flex) to skip changing the culture medium
at weekends. In our experience, skipping 1 day was okay, but
hiPSCs seemed to be stressed when the E8 flex medium was
not changed during entire weekends. Therefore, we cannot
recommend skipping 2 days.
References
1. Thomson JA, Itskovitz-Eldor J, Shapiro SS,
Waknitz MA, Swiergiel JJ, Marshall VS, Jones
JM (1998) Embryonic stem cell lines derived
from
human
blastocysts.
Science
282:1145–1147
2. Takahashi K, Yamanaka S (2016) A decade of
transcription factor-mediated reprogramming
to pluripotency. Nat Rev Mol Cell Biol
17:183–193
3. Evans MJ, Kaufman MH (1981) Establishment in culture of pluripotential cells from
mouse embryos. Nature 292:154–156
4. Martin GR (1981) Isolation of a pluripotent
cell line from early mouse embryos cultured in
578
Juliette Seremak and Ali Eroglu
the remaining sterile ingredients of the defined cryopreservation medium can be added under the biosafety hood without
the need to filter.
3. KSR contains undefined lipids that can unintentionally be
removed during filtration. Therefore, sucrose can first be dissolved in DMEM/F-12 and filtered before adding the remaining sterile components under the biosafety hood.
4. The sterilization of the defined thawing medium is also challenging. We recommend first dissolving methylcellulose and
sucrose in HBSSÀ and then autoclaving the resulting solution
before aseptically adding the remaining sterile components
under the biosafety hood.
5. Recombinant human vitronectin is available as a concentrated
solution usually at 0.5 mg/mL or lyophilized powder. When
received as lyophilized powder, an appropriate volume of PBS+
can be added directly to the vial to dissolve it. Next, the dissolved
vitronectin can be transferred to a larger sterile tube to make a
working solution at 10 μg/mL by adding more PBS+. Then,
the working solution should be aliquoted and stored at À20
C
or À80
C to avoid repeated freeze/thaw cycles.
6. When dissociated to single cells, hiPSCs require the presence of
a ROCK inhibitor to survive passaging. It is not necessary to
supplement E8 medium with a ROCK inhibitor if hiPSC colonies partially dissociated by EDTA treatment. Nevertheless, we
prefer to supplement E8 medium with 10 μM ROCK inhibitor
Y-27632 during the first 48 h of passaging independent of to
what extent hiPSC colonies are dissociated.
7. Particularly, native bFGF is highly unstable and quickly
degraded under cell culture conditions, requiring changing
the culture medium every day. Recently, a heat-stable recombinant human bFGF has been engineered and added to E8
medium (called E8 flex) to skip changing the culture medium
at weekends. In our experience, skipping 1 day was okay, but
hiPSCs seemed to be stressed when the E8 flex medium was
not changed during entire weekends. Therefore, we cannot
recommend skipping 2 days.
References
1. Thomson JA, Itskovitz-Eldor J, Shapiro SS,
Waknitz MA, Swiergiel JJ, Marshall VS, Jones
JM (1998) Embryonic stem cell lines derived
from
human
blastocysts.
Science
282:1145–1147
2. Takahashi K, Yamanaka S (2016) A decade of
transcription factor-mediated reprogramming
to pluripotency. Nat Rev Mol Cell Biol
17:183–193
3. Evans MJ, Kaufman MH (1981) Establishment in culture of pluripotential cells from
mouse embryos. Nature 292:154–156
4. Martin GR (1981) Isolation of a pluripotent
cell line from early mouse embryos cultured in
578
Juliette Seremak and Ali Eroglu