FAQ keywords:
(See FAQs below)

Antibiotics FAQ 3
Cell count FAQ 6
Clonal assay FAQ 13
Dissociation enzyme FAQ 18
Markers FAQ 12
Media color FAQ 7
MEF FAQ 5, 11
Protocols FAQ 1, 2, 9, 10
Quality control FAQ 12, 14
Serum FAQ 16, 17
Splitting FAQ 22
Thaw viability FAQ 4, 8
Transfection FAQ 19

8. The day after I thawed my HES cells, cell attachment appears to be sparse. Is this normal? It is common to experience a low level of cell attachment the day after thawing your hES cells. Occasionally, because of the low recovery from cryostorage and a moderate doubling time, it may take your hES cells several days to become visible, and it may take a few weeks to have wells dense enough to expand surface area. However, you should passage to fresh MEF before the MEF are two weeks old, as MEF  deteriorate over time. 9. I have cultured mouse ES cells in my lab. Should I expect human ES cells to behave and look similar to mouse ES cells? No, human ES cells will not grow as robustly or in the same manner as mouse ES cells. Your human ES cells will be slower to recover and will generally need more attention than mouse ES cells. 10. I have cultured primate ES cells in my lab, and the components/feeder/media I used were different from what you recommend for human ES cells. Why aren't the conditions identical, given the similarty of non-human to human primates? Although non-human primate and human ES cells are quite similar, we have observed significant differences in their responses to different media. Use the provided protocols for your hES cells for best results. 11. My irradiated MEF look strange after having been exposed to human ES cell media (i.e., when I plated my ES cells on them). Is this normal? Are my irradiated MEF dead? MEFs, like many other cell types, do not proliferate well in serum replacer (SR) because it lacks many of the components of FBS. This may alter the appearance of irradiated MEFs in media containing SR. However, irradiated MEF in SR-containing media will support hES cells. 12. How often should I karyotype my HES cells? How often should I test my cells for stem cell-specific antigens and teratoma formation? There are no hard and fast rules. We karyotype cells before each freeze, and following thaw as a lot release test for all cells distributed. In our research, we karyotype before and after each experiment to ensure our cells are normal. We also karyotype after the cells have been heavily selected (pick to keep), as that may contribute to abnormalities.  It is very important that you expand, freeze, and karyotype to create a back stock as soon as possible so that you can routinely return to lower-passage cells as necessary. Ideally you should use the cells for several months and then expand from a new thaw you banked back from the initial vial we sent. 13. How do you clone HES cells? Do HES cells clone efficiently as single cells? To clone HES cells, remove colonies with trypsin, and pipet to form single cells. Pipet cells to new wells at low density. Efficiency will be low. In order to determine cloning efficiency, do a titration. It is possible that, as a result of recovery from a cloning efficiency, colonies may arise from a >1-cell origin. It is important that this possibility is considered when setting up controls. 14. How many passages or doublings are HES cells good for? We routinely use hES cells for experimental purposes for 20-40 passages before retiring cultures and obtaining new cultures from fresh thaws. We have however maintained cultures for as long as 200+ passages. We recommend routinely karyotyping cells to determine cell normality and ensure the optimal experimental results. 15. When I see differentiated cells that appear spontaneously in my culture, what cell type are they? It is generally a mixture of cell types, and the exact cell types depend on the culture conditions. Staining for specific markers would be required to determine specific cell types present. 16. Why do you use "defined" serum for freezing HES cells? What is defined serum? Defined serum is a high-quality serum. You can read about defined serum here: 17. Why heat-inactivate the serum used for MEF culture media? While we heat-inactivate the serum in our MEF Medium, we do not heat-inactivate the serum in our hES Cryopreservation Medium. The decision to heat-inactivate may vary by lab philosophy. You can read about heat-inactivation of serum from Hyclone here: Heat Inactivation Note: Do not inactivate defined serum. 18. What is the difference between dispase and collagenase? Why choose one over the other? In our hands, the best results are obtained by using Collagenase Type IV for cells grown on MEF feeder layers. Dispase is used when cells are cultured in a feeder independent or feeder-free system. 19. What reagent should I use to transfect HES cells? Can I use lentivirus? See: Eiges, R., M. Schuldiner, et al. (2001). "Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells." Curr Biol 11(7): 514-8. Ma, Y., A. Ramezani, R. Lewis., R.G. Hawley, and J.A. Thomson. "High level sustained transgene expression in human embryonic stem cells using lentiviral vectors." Stem Cells 21 (2003): 111-117. 20. Why do you recommend the use of glass serological pipets as opposed to plastic? We find fewer cell particles attach to the glass as opposed to plastic, and feel there may be a reduced risk of plastic toxicity exposure to the hES cells. For this reason, we recommend glass pipets. 21. Why does so much published HES cell research reflect the use of the H9 line as compared to our other four lines? There is no apparent reason it was historically used more. 22. How often should I split my cells? What ratio should I split my cells? This varies by the application. When you are just maintaining cells or expanding for stocks, use the following for a guide. When to split: a. Split cells when colonies are getting larger or denser. b. Split cells when MEF are getting old (more than 14 days) c. Some spontaneous differentiation is expected, so pick off differentiation regularly, but split cells if differentiation becomes increasingly excessive. This may indicate a bad MEF plate. d. Soon after thaw, be conservative about splits. Once culture is established, you can be more selective and aggressive. e. Generally passage cells every 4-6 days. What ratio to split: a. In general, after a thaw, cells would be split about 4-8 days after thaw.  Usually first split is 1:1 or 1:2. b. When cells have expanded and are more dense they can be split about 1:2 to 1:5, about every 4 to 7 days. Regarding splitting, please note: These are general guidelines, not absolute, as there can be many factors which affect proliferation and differentiation. Assess whether to split your cells on a daily basis based on their appearance because your cells may not follow a predictable schedule.