1. What is the PeproGrow™ hESC medium formulation?
PeproGrow™ hESC is 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.
2. How do I store the PeproGrow™ hESC medium?
When 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.
3. How do I prepare the PeproGrow™ hESC medium?
Reconstitute the lyophilized growth factor component with 100µl or 500µl (depending on size) 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.
4. How do I switch from my current medium to PeproGrow™ hESC medium?
You 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/accomodation).
5. Should I use any precautions during an adaptation protocol?
This 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.
6. What do I do if I see differentiation during adaptation?
Some 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.
7. Will my cell cultures change in PeproGrow™ hESC medium?
The 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.
8. How will my cell cultures change in PeproGrow™ hESC medium?
Although 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.
9. How come the cells did not attach well after enzymatic passaging?
The 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.
10. Can a reagent be added to my culture if the cells have not attached well after enzymatic passaging?
The 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.
11. Are there any additional recommendations for passaging the cells?
Additional recommendations have been included below; however, please also refer to the PeproGrow™ hESC Instructions Manual.
- Appropriate 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 upon attaching.
- After 5-7 days the colony will most likely be large enough to passage.
- Some 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.
12. Do you have any additional recommendations for the timing of passaging the cells?
The 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.
13. How does cell density affect the colony growth?
If 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 PBS/EDTA method.
14. How does spacing affect the cell density?
Spacing 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.
15. Are there any specific recommendations for feeding the cells?
Feed the cells daily, with the exception of one “double volume” feed over the weekend.
16. Can I use matrices other than the recommended BD Matrigel™ to coat cell culture dishes?
Yes, 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 additional time.