is the PeproGrow-hESC medium formulation?
a proprietary formulation that was designed and developed by PeproTech in
collaboration with the Stem Cell Training Course at Rutgers University.
The medium is serum- and phenol red-free, and a complete, chemically defined
formulation designed for the expansion of both human embryonic stem cells
(hESCs) and induced pluripotent stem cells (iPSCs) using BD Matrigel™ as a
surface coating matrix. This formulation does contain a unique activator
of the TGF-Beta pathway, a substitute for insulin that activates similar
pathways and also FGF-Basic. There are no inhibitors of TGF-Beta.
do I store the PeproGrow-hESC medium?
using within 1 week of receipt, the medium and growth factor component should
both be stored in the dark at 4°C. For
extended storage past one week, the medium should be stored in the dark at 4°C
for up to 6 months, and the lyophilized growth factor component should be
stored at -20°C for up to five years.
do I prepare the PeproGrow-hESC medium?
the lyophilized growth factor component with 500µl of sterile cell-culture
grade water. Allow the vial to sit for
several minutes, gently mix several times by inversion, and then centrifuge. Aseptically add the entire contents of the
vial to the medium bottle. Label the
bottle with both the date of mixture and the newly calculated expiration date
(2 weeks from the date of mixture). Store
the media in the dark at 4°C until needed, and only warm the amount of media
needed for each given feeding.
do I switch from my current medium to PeproGrow-hESC medium?
can switch the medium immediately (acute protocol) without an adaptation step,
or slowly increase the percentage of PeproGrow-hESC media daily over the course
of a week (adaptation/accommodation).
I use any precautions during an adaptation protocol?
can be done at your discretion as an adaptation is not required and the cells
can adapt immediately to the new medium.
If using an adaptation protocol, then you can maintain a culture in its
current media as a precautionary step.
When adapting cultures, you may choose to slowly change the ratio of the
current media to PeproGrow-hESC; again, this is not necessary. A change in morphology, such as flattening, may
be observed; this change should not be a reason for concern. The cultures may look slightly different than
those observed in other vendor’s media, but will maintain their pluripotency.
do I do if I see differentiation during adaptation?
differentiation is expected, and you will need to either scrape off the
differentiated colonies, or “pick-to-save” the non-differentiated colonies onto
newly coated cell culture dishes.
my cell cultures change in PeproGrow-hESC medium?
morphology of some cell colonies may change.
You may also observe some amount of differentiation during media
switching via both type of protocols (acute or adaptation). This is not unexpected, and you may need to
clean and passage cells up to 3 times to sort out poorly responding
colonies. Please refer to the
PeproGrow-hESC Instructions Manual for suggested protocols.
will my cell cultures change in PeproGrow-hESC medium?
we cannot exactly predict the changes, we found the colonies became more
elastic and resilient to passaging in the adapted cultures. There is detailed passaging technique information
for more elastic colonies in the PeproGrow-hESC Instructions Manual. If you find that there is no change in the
texture of the colonies, then continue using your routine laboratory protocols.
come the cells did not attach well after enzymatic passaging?
colonies have most likely been over-digested, but the remaining cells should
not be discarded. If some colonies have attached, then continue to feed the
cultures as some cells will grow robustly after 4-6 days, and your cultures can
be maintained from 2-4 colonies. Also,
you may consider using a non-enzymatic passing method to avoid over-digestion.
a reagent be added to my culture if the cells have not attached well after
addition of 2-10 µM (preferably 2 µM) Y-27632, or a ROCK inhibitor, has been
shown to enhance cellular survival and attachment during enzymatic passaging (when
using either dispase or Accutase™). Increased
amounts of Y-27632 have been shown to cause neural differentiation, in addition
to morphological changes that have been shown to be reversible.
there any additional recommendations for passaging the cells?
Additional recommendations have been
included below; however, please also refer to the PeproGrow-hESC Instructions
timing is the most difficult part of the passaging method as it is extremely
important to avoid the over-digestion of the colonies with dispase, since this
can prevent colonies from attaching, or to cause them to fold in on themselves
5-7 days the colony will most likely be large enough to passage.
cell death is normal. When passaging using
the dispase method you can expect some percentage of cells not to attach, but
if you get less than 50% viability, then the cells have been passaged too
roughly. The PBS/EDTA method is gentler
and leads to better recovery if cells are passaged correctly.
you have any additional recommendations for the timing of passaging the cells?
timing of the passaging is a critical step.
Cells that are passaged too early will plate
poorly since the colonies will be too small and will not survive the passaging
technique. Also, it is important to
avoid overexposure to collagenase. We
recommend dispase or PBS/EDTA. Both are
optimal at 5 minutes, but you should observe the cells at 3 minutes for both
methods to determine if the full 5 minutes is needed.
does cell density affect the colony growth?
the cells are too sparsely, or too densely, populated, then this can lead
to differentiation. If the split ratio is too high, then the
cells will not have enough autocrine factors to enhance viability and many
cells will die and/or differentiate. We
recommend starting with a near confluent dish and trying a 1:6, 1:12, or 1:18
split using dispase, or a 1:6, 1:12, 1:18, or 1:24 split using a PBS/EDTA
method. Further dilutions can be made if
needed. We recommend adding Y27632 when
using dispase at a low cell plating density, but this is not needed for a
does spacing affect the cell density?
is arbitrary. When plating the cells we
recommend that you transfer cells around the rim of the well (only for 6-well
dish) and then gently move the dish back and forth to mix. For other sized dishes, disperse cells by
moving the pipette as you add the cells.
there any specific recommendations for feeding the cells?
the cells daily, with the exception of one “double volume” feed over the
I use matrices other than the recommended BD Matrigel™ to coat cell culture
we have tried several other coating reagents including: Laminin/Entactin (BD™),
recombinant human Vitronectin (PeproTech), and Synthemax II® (Corning). For the Laminin/Entactin, we observed nearly
identical growth as compared to BD Matrigel™ on the standard cell culture
plastics. For both the human Vitronectin
and Synthemax II®, we recommend CellBIND® or an equivalent type of cell culture
plastic for optimal binding of factors.
We observed less spreading of colonies and slightly less robust
attachment, and this method appears to require the addition of Y-27632 for