Frequently Asked Questions (FAQs)
Q: Where can I find written Standard Operating Protocols for the Massachusetts Human Stem Cell Bank?
A: The MHSCB Standard Operating Procedures can be found online here: http://umassmed.edu/MHSCB/SOPs.aspx
Q: I followed your protocols to prepare hESC/iPSC culture media and my media turned orange. After incubation the culture media is now yellow-orange. Should I be concerned?
A: No. Fresh hESC/iPSC culture media is normally varying shades of red-orange. DMEM-F12 base media mixed with knock-out serum replacer normally turns the phenol-red color of DMEM to red-orange. Once the media is incubated in 5% CO2 it turns into an orange-yellow and when the growing cells are becoming quite confluent the media can become a yellowish-straw color.
Q: Can I add antibiotics to my hESC/iPSC media?
A: It is best to follow the recommendations received from the source of the line that you are growing. Generally, the MHSCB does not recommend adding antibiotics to the cell lines that it distributes. Prior to thawing the vial it is suggested to thoroughly read the protocols and ensure that the proper materials are available.
Q: What are my mouse embryonic fibroblast (MEF) feeder cells supposed to look like?
A: The photo below shows MEFs at the correct confluence. We recommend keeping your MEF cultures in MEF media until they are about to be used for hESC/iPSC culture. MEF media, which contains fetal bovine serum, supports the health of irradiated MEFs in culture much better than hESC/iPSC media, which contains knock-out serum replacer.
Image: MEF cells at correct confluence
Q: I have some immortal mouse embryonic fibroblast cell lines in my lab already; can I culture my hESC/iPSCs with them instead?
A: It is strongly suggested to use primary embryonic fibroblasts that have been derived from CF-1 mice. It may be possible to use other sources, but they must be thoroughly tested.
Q: What is heat-inactivated serum?
A: Heat Inactivated serum is a serum that has been heated to 56°C for 30 minutes. Heat inactivation has been reported to inactivate serum complement. We use heat inactivated FBS in MEF culture medium, but defined media, such as Knockout serum replacer, used in our hESC/iPSC culture protocols do not require heat inactivation.
Q: Do human ES cells and iPS cells behave and look similar to mouse ES cells?
A: In general, human ES cells grow slower and have a much lower viability from thaw. Human ES cells and iPS cells are maintained in different media conditions and also require more attention and care in order to maintain quality cell culture.
Image: hESC colonies on MEF feeders
Q: I have previously grown hESC/iPSCs from another source, can I follow those protocols?
A: It is recommended to follow the protocols from the source of the vial being cultured. Prior to thawing the vial it is suggested to thoroughly read the protocols and ensure that the proper materials are available.
Q: I thawed my cells yesterday and I do not see a lot of colonies. What is the timeframe to view many colonies?
A: The viability of thawed hESC/iPSCs can be as low as 0.1% to 1%. It is not uncommon to only see a few colonies several days after thawing. Continue to feed the well daily, even if you do not see any colonies emerging. It can take up to a week for them to become visible. It is advised to manually passage the first passage after thaw using the pick to keep method. However, if there is a good yield and there are more than 50 colonies visible, mild or gentle enzymatic passaging in accompaniment with the pick to discard method is recommended.
Q: Can I count my hES or iPS cells with a hemacytometer?
A: Yes. In order to achieve the most accurate count, you must create a single cell suspension. For more information on making an hESC or iPSC single cell suspension using Trypsin, see our Flow Cytometry Protocol. Keep in mind that your population will also include MEF feeders if the hESC/iPSCs are being cultured in a feeder system.
Q: I keep seeing some different cell types in my hESC/iPSC culture. What are they?
A: It can depend on the cell line in culture, as well as the cell culture conditions. Some cell lines tend to differentiate mostly into specific lineages, while others appear to spontaneously develop into many different lineages. The differentiating cells at the colony periphery (which are often the first signs of differentiation) are often described as epithelioid. These cells can be flattened or have a cobblestone appearance. These cells may be ectodermal, but endoderm cells may also be found in a differentiating culture. Further testing, through immunofluorescence or RT-PCR can be used to help determine the characteristics of differentiating cells.
Image: Differentiation in hESC/iPSC culture.
Undifferentiated Colony | Colony with differentiation (arrows) |
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Q: Why do you passage your hESC/iPSCs on MEF feeders with collagenase?
A: When passaging hESC/iPSCs on feeders it is recommended to enzymatically disassociate the culture using collagenase type IV. Collagenase has been shown to aid in the disruption of larger colonies of hESC/iPSCs into smaller aggregates without disassociating them into a single cell suspension. Collagenase type IV has been designed to have low tryptic activity and therefore limits the damage to membrate proteins and receptors during its incubation with the culture. When passaging cells in feeder-free or feeder independent conditions, dispase is commonly used. When a single cell suspension is needed for downstream applications or assays, we recommend trypsin.
Q: I have thawed my cells successfully, but now I don’t know when to passage, or what ratio to split them. Any recommendations? What are the next steps?
A: There are many parts that contribute to successful hESC/iPSC culture. For successful culture of hESC/iPSCs a fair amount of planning and preparation and daily monitoring are necessary. In addition to planning when to passage hESC/iPSCs, MEFs must be prepared accordingly and in advance. Also, it is recommended to use a log sheet in order to track cell growth over time. In general, we recommend passaging hESC/iPSCs every 4 to 6 days. Cultures may be split in low ratios such as 1:1 or 1:2 after thawing. Once the cultures have been passaged several times they will settle into a more predictable growth pattern. The split ratio can vary from line to line and will depend on the number of passages post thaw, as well as the kinetics of the line being cultured. Because there are many conditions that effect cell culture conditions, listed below are several guidelines that we use to help determine when to passage hESC/iPSCs.
hESC/iPSCs should be passaged if:
1. MEF feeder layer is two weeks old.
2. Most of the colonies are large (greater than 700 microns)
3. Colonies are too dense (at approximately 70% confluence)
4. Colonies exhibit increased differentiation. See protocol on Passaging Stem Cells for more information on which method to use when passaging your cells.
Q: I understand that karyotyping is important, but how often should I have my cells tested?
A: We recommend karyotyping cells in culture every 15 to 20 passages, as well as karyotyping samples just prior to cryopreservation and after thawing a lab stock (it is important to note that karyotyping is often an expensive test). It is advisable to create a lab stock as soon as possible in the case of any future abnormalities.
Q: How many passages are acceptable for hESC/iPSCs ?
A: As with most cell culture, it is recommended to obtain the lowest passage possible. Many lines of hESC/iPSCs have been successfully passaged while maintaining their pluripotency for >100 passages. We advise to karyotype your hESC/iPSC cultures regularly in order to ensure their quality.