Download Frequently Asked Questions Stem Cells - Coriell

Coriell Institute For Medical Research
Frequently Asked Questions – Stem Cells
Q: Do you add antibiotics to your media?
A: Coriell does not use antibiotics when culturing stem cells. Customers should be aware
that inclusion of antibiotics in media may change growth characteristics of the cells. Also,
continued culturing of cells in antibiotics can result in growth of antibiotic-resistant strains
of bacteria
Q: I am replenishing the culture medium daily; however, the medium is changing to a light
orange/yellow color. Am I doing something wrong?
A: The basal culture medium contains the pH indicator phenol red. When the media
color changes from pink to yellow, it is indicative of the pH becoming more acidic, which
commonly occurs when cells are dividing rapidly and producing acidic waste products.
The medium should never be a bright yellow as this is an indication that the cells have
overgrown.
Q: Why do you recommend that I cryopreserve a small number of vials of stem cells for my
laboratory?
A: Establishing a master stock is critical so that a vial can be recovered and expanded if
a problem arises with the growing stem cells such as: contamination, spontaneous
differentiation and changes in chromosomal integrity. If any of these occur, you can
recover a vial of your master stock and expand it to make a working stock for the
laboratory.
Q: Why do you recommend that I perform karyotype analysis on my stem cells?
A: Due to the possible genomic instability that can be introduced with the reprogramming
process, iPSC lines need to have routine genomic analysis performed. Additionally,
chromosomal abnormalities may arise if iPSC lines are not cultured appropriately. We
recommend that you analyze the stem cells using G-band karyotype analysis when you
create a master stock, a working stock and every 10 to 15 passages. We also
recommend that karyotyping be performed if there is a noted change in morphology or
growth kinetics.
Human induced pluripotent stem cells (iPSC)
Q: I plan to obtain human induced pluripotent stem cells (iPSC) from Coriell. What reagents
should I have on hand to successfully culture iPSCs?
A: The table below provides a list of reagents necessary for culturing human iPSCs.
There are no preferred vendors. Reagents can be obtained from a variety of vendors;
the list below is given for convenience only. Refer to the appropriate Stem Cell Protocol
for additional information.
Form 1300-02 Rev B-061516
Coriell Institute For Medical Research
Frequently Asked Questions – Stem Cells
Reagent
Potential Sources
6-well plates
Nunc, BD biosciences,
Corning
Millipore, Stem Cell
Technologies
Global Stem, Applied
Stemcell, ATCC
Thermo Fisher Scientific ,
Stem Cell Technologies
Thermo Fisher Scientific
0.1% gelatin
Mouse embryonic fibroblasts
(MEFs)
DMEM/F12
Knock-out Serum
Replacement
L-glutamine
Basic fibroblast growth factor
(bFGF)
Non-essential amino acids
2-mercaptoethanol
Rho Kinase (ROCK) Inhibitor
Collagenase
TrypLE Express
Dispase
Versene
Thermo Fisher Scientific ,
Mediatech
Thermo Fisher Scientific ,
Global Stem, Stemgent
Thermo Fisher Scientific ,
Mediatech
Thermo Fisher Scientific
Stemgent, Millipore
Sigma, Fisher
Thermo Fisher Scientific
Thermo Fisher Scientific
Thermo Fisher Scientific
Q: Why do you suggest using a Rho Kinase (ROCK) Inhibitor in the recovery medium?
A: iPSCs have a very low viability following cryopreservation. Inclusion of the Rho
Kinase (ROCK) Inhibitor in the recovery medium increases the viability of the cells
increasing the number of colonies observed within the newly thawed colony and
decreasing the number of days it takes for the colonies to develop.
Q: I have thawed my iPSCs according to the stem cell protocol. It is 24 hours following recovery
and I do not see colonies. What should I do?
A: Continue replenishing the culture medium daily. The viability of iPSCs following
recovery from cryopreservation is very low (between 0.1% and 1%). Colonies may take
up to one week to begin growing. If after one week, colonies have not developed
contact [email protected].
Q: How do I know when it is time to passage my iPSCs?
A: iPSCs should be passaged for any of the following reasons:
Form 1300-02 Rev B-061516
Coriell Institute For Medical Research
Frequently Asked Questions – Stem Cells
1.
2.
3.
4.
MEFs are over 2 weeks old
Individual colonies are large (over 700 microns)
Colonies are close together and reaching confluence (~70-80%)
Significant differentiation is observed
There are many factors that contribute to the successful culturing of iPSCs. Cells should
be observed microscopically daily and MEFs should be plated the day before an
anticipated split. In general, iPSC colonies should be passaged every 4-7 days at a 1:3
to 1:6 split ratio. However, the number of days between passaging and the split ratio are
dependent on the individual cell line and its growth. We recommend that you record the
passage information for each cell line to determine the exact growth kinetics of a
particular line. As soon as the iPSCs have been passaged, we highly recommended
that you cryopreserve, at miniumum, a small number of vials as a master stock. Refer to
“Cryopreservation of Human iPSC” in the stem cell protocol.
Q: Why do you suggest I centrifuge my cells at such a low speed?
A: iPSCs should be passaged as clusters and not as single cells. By using centrifuge
speeds that are lower than typically used to pellet other cell types, the clusters will pellet
to the bottom of the centrifuge tube while the single cells remain floating in the media.
Q: Why is the concentration of basic fibroblast growth factor (bFGF) variable?
A: Each iPSC line is grown in bFGF at the concentration specified by the submitter. The
concentration of bFGF for a specific line is indicated on the Certificate of Analysis.
Murine Embryonic Stem Cells (mES)
Q: I plan to obtain murine embryonic stem cells from Coriell. What reagents should I have on
hand to successfully culture mES cells?
A. Refer to the Murine Embryonic Stem Cell Culturing Protocol for detailed information.
The table below provides a list of reagents necessary for culturing mES cells. There are
no preferred vendors. Reagents can be obtained from a variety of vendors; the list below
is given for convenience only.
Reagent
Potential Sources
6-well plates
Nunc, BD biosciences,
Corning
Millipore, Stem Cell
Technologies
Global Stem, Applied
Stemcell, Open Biosystems
Thermo Fisher Scientific ,
Sigma, Hyclone
0.1% gelatin
DR4 Mouse embryonic
fibroblasts (MEFs)
DMEM (high glucose)
Form 1300-02 Rev B-061516
Coriell Institute For Medical Research
Frequently Asked Questions – Stem Cells
ES cell Fetal Bovine Serum
Glutamax
B-mercaptoethanol
Non-essential amino acids
Sodium pyruvate
Leukemia Inhibitory Factor
Doxycycline
Puromycin
Applied Stem Cell, ATCC,
Millipore, Invitrogen
Thermo Fisher Scientific
Thermo Fisher Scientific ,
Sigma
Thermo Fisher Scientific ,
Mediatech
Thermo Fisher Scientific ,
Mediatech
Millipore, Stem Cell
Technologies, Stemgent
Clontech
Thermo Fisher Scientific
Q: I have thawed my mES cells according to the Murine Embryonic Stem Cell Protocol. What
should my cells look like 24 hours after plating?
A: The picture above is of a cell line 24 hours after thaw. Note the round colonies and
even distribution of the cells throughout the dish. If colonies have not developed after
two days, contact [email protected].
Q: How do I know when it is time to passage my mES cells?
A: Typically, mES cells passage every 2-3 days and plate at a density between 1.0 X 106
and 1.8 x 106 cells per well of a 6 well plate. The cells should be passaged when they
are between 70-80% confluence using split ratios ranging from 1:4 to 1:10. However,
the number of days between passaging and the split ratio are dependent on the
Form 1300-02 Rev B-061516
Coriell Institute For Medical Research
Frequently Asked Questions – Stem Cells
individual cell line and its growth. We recommend that you record the passage
information for each cell line to determine the exact growth kinetics of a particular line.
As soon as the mES cells have been passaged, we highly recommended that you
cryopreserve, at miniumum, a small number of vials as a master stock. Refer to
Cryopreserving mES cells.
Q: Initially my cells were growing and passaging as expected. Now my cells are not growing
and seem to be lifting off the plate. What is happening?
A: The sudden change in growth kinetics of a cell line can be indicative of problems with
media components. Specifically, puromycin concentration as well as FBS and MEF
quality can impact the growth of your cells. Testing new lots of reagents prior to using
them in growth medium for your cells can decrease the probability of observing negative
effects on the growth of your cells. For information, we recommend testing new lots of
MEFs to determine the optimal plating density, see the protocol “Determining Optimal
Plating Density for Mouse Embryonic Fibroblasts (MEFs)”. We further suggest
performing a puromycin kill curve as the concentration can vary slightly with each lot of
antibiotic.
Form 1300-02 Rev B-061516