1. What are the formulations of the PeproGrow™ hESC Media Products?
The serum- and phenol red-free, complete, chemically-defined formulations of PeproTech’s PeproGrow™ hESC Media Products are proprietary. Designed and developed by PeproTech in collaboration with the Stem Cell Training Course at Rutgers University, each PeproGrow™ hESC Media Kit includes a bottle of basal medium and a separate, lyophilized component of PeproTech’s recombinant growth factors.
2. What is the difference between PeproTech’s PeproGrow™ hESC Media Kits?
The PeproGrow™ hESC Medium Kit (catalog number HESC) is an insulin-free formulation, in which insulin is replaced by a unique activator of similar pathways to eliminate possible interference during the measurement of insulin-production in subsequently differentiated cells. The PeproGrow™ hESC Plus Medium Kit (catalog number HESCP) is an insulin-inclusive formulation ideal for use when switching from other insulin-inclusive media products.
3. How should each PeproGrow™ hESC Media Kit component be stored?
The basal media can be stored in the dark at 2°C to 8°C for up to 6 months. The lyophilized growth factor component can either be stored at 2°C to 8°C for up to 6 months, or -20°C to -80°C for up to five years.
4. How should the PeproGrow™ hESC Medium be prepared for use?
Centrifuge the vial of lyophilized growth factor component prior to opening, and reconstitute with sterile, cell culture grade water (using either 500μL of water for 500mL-size Media Kits, or 100μL of water for 100mL-size Media Kits). For use within two weeks, aseptically add the entire reconstituted growth factor component to the basal medium and mix well by swirling or pipetting. Otherwise, aseptically transfer the necessary volume of basal medium into a sterile polycarbonate bottle or conical-bottom polypropylene tube, aseptically add the necessary proportion of reconstituted growth factor component and mix well by swirling. Filtration is not necessary when prepared aseptically. Label with the date of mixture and newly calculated expiration date (2 weeks from date of mixture). Store in the dark at 2°C to 8°C until needed, and only warm the amount of media needed for each given feeding.
5. Are accommodation protocols necessary when switching from my current medium to a PeproGrow™ hESC Medium?
Accommodation protocols are not necessary when switching from one insulin-inclusive formulation to another, but may be necessary to avoid issue when switching from an insulin-inclusive formulation to an insulin-free formulation. Accommodation requirement can also depend on cell type. When an acute method (no accommodation) does not function as expected, then additional accommodation protocols (e.g. short or long) may be used. Where accommodation protocols do not function as expected, then a long accommodation can be used in combination with a gradually increasing percentage of new medium (e.g. 100:0, 80:20, 60:40, 20:80, 0:100). Please refer to the PeproGrow™ hESC Medium Instruction Manual for additional information.
6. Is differentiation common during adaptation protocols?
Some amount of differentiation is expected with both acute and accommodation methods. Differentiated colonies can either be scraped from the culture, or non-differentiated colonies can be transfered onto newly-coated cell culture dishes. Please refer to the PeproGrow™ hESC Medium Instruction Manual for additional information.
7. Will cultures change in PeproGrow™ hESC medium?
While exact changes cannot be predicted for all applications, some changes in colony morphology (e.g. cell flattening with PeproGrow™ hESC Medium) are possible. Changes in pluripotency and cell phenotype have not been observed. Colonies in adapted cultures may tend to become more elastic and resilient to passaging. Please refer to the PeproGrow™ hESC Medium Instruction Manual for additional information. Routine laboratory protocols can be followed where no changes are observed.
8. Should cells be cultured on coated or uncoated cultureware?
Culturing on uncoated plastic is not recommended. Corning Matrigel® is recommended as an extracellular matrix (ECM) during initial adaptations; however, other suitable ECM products can be used thereafter. Specifically formulated for use as a cell culture surface-coating reagent for our stem cell media products, PeproTech’s Animal-Free Recombinant Human Vitronectin Matrix and Buffer Kit (catalog number AF-VMB-220) can be used following either a surface-coating or premix method.
9. Why are cells attaching poorly after enzymatic passaging?
This is more than likely the results of over-digestion; however, if some colonies have attached, cultures can continue to be fed, as some cells will grow robustly after 4-6 days and cultures can be maintained from 2-4 colonies. Over-digestion can be avoided by using non-ezymatic passaging methods. Note: Enzymatic methods should not be used during cell culture steps with Animal-Free Human Vitronectin Matrix-coated cultureware. PeproTech’s osmotically-compatible Cell Passaging/Non-Enzymatic Detachment Buffer (catalog number CPD-125), which only contains PBS, HEPES, and EDTA, should be used instead.
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 Medium 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 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 using 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 those recommended above to coat cell culture dishes?
Yes, we have tried several other coating reagents including: Laminin/Entactin (BD™), Recombinant Human Vitronectin (PeproTech; catalog number 140-09 and AF-140-09), 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.