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tebu-bio - GlobalStem Feeder cells FAQ

Mouse Embryonic Fibroblast (MEF) Cells

1. What are feeder cells?
Mouse embryonic fibroblasts (MEF) are often referred to as feeders or feeder cells and have routinely been used to support the culturing and maintenance of embryonic stem cells (ESCs) in an undifferentiated state without losing their pluripotency . Human Newborn Fibroblast are also used as feeder cells for supporting healthy undifferentiated human ES and iPS cells.

2. Why are mouse embryonic fibroblast (MEF) used when culturing ES cells?
MEF serve as a substrate for the ES cells to grow on. They secret a number of essential growth factors that are important for maintaining pluripotency. MEFs also represent a very reliable, readily available, and reproducible source of feeder cells.

3. What is the difference between the two mouse strains of MEF feeders, CF‐1 and C57B/6 (Black 6)?
The only difference between the CF‐1 and the C57BL/6 (also known as Black6) MEF is the mice that they are made from. CF‐1 mice are outbred, while C57BL/6 are inbred. All of our MEF are tested for their ability to support human ESC in the undifferentiated state.

4. At what density should I plate my feeder cells (MEF)?
There is a wide range of MEF plating densities used by researchers for mouse and human pluripotent stem cell culturing. The density depends on the ESC or iPSC lines you are culturing. We recommend that you use the feeder density previously used by the source lab from which you received the cell line; or the density determined through personal experience to be appropriate for your specific stem cell line. Our MEF have been successfully used to support undifferentiated pluripotent stem cells at densities ranging from 20,000 to 53,000 cells/cm2.

5. Can I use your MEF for human ES cells?
Yes, every lot of our MEFs is comprehensively tested for its ability to support both mouse and human ES cells to ensure robust and consistent performance.

6. How many passages can MEF be expanded?
It is recommended that our untreated MEFs be expanded only up to two passages. MEFs should be mitotically arrested so that they will not proliferate when used as feeders for ES/iPS cell culture. We suggest purchasing already Mitomycin C‐treated or irradiated MEF that have been thoroughly tested to support ES/iPS culture.

7. Can I use gelatin when plating MEF?
We do not recommend the use of gelatin. Our MEFs adhere well to tissue culture plastic without coating the plastic with matrix proteins. Gelatin introduces potential variability and toxicity into the cell culture system. It must be carefully screened before use. Once solubilized, shelf life must be carefully monitored.

8. How soon after plating my MEF can I plate cells?
MEF should be plated 24 hours before being used as a feeder layer

9. What type of tests do you perform to ensure consistence between different lots of cells?
For each lot of MEF and NuFF we perform all the following quality tests:
1.) Viability – typically >95%
2.) Pathogen Screening – MEF (mouse), NuFF (Human)
3.) Sterility‐ Bacterial and Fungal
4.) Mycoplasma testing – PCR analysis
5.) Identity Testing for NuFF‐ Cell identity is tested by Short Tandem Repeat (STR) analysis using 16 probes – 15 STR loci plus Amelogenin.
6.) Supports mouse and/or human pluripotent stem cell culture through multiple passages based on the morphology, growth, and immunocytochemisty of multiple undifferentiation markers.

10. What are the mouse pathogens that you screen for?
We screens for 22 different pathogens: Sendai virus, Mouse hepatitis virus, Pneumonia virus of mice, Minute virus of mice, Mouse parvovirus (MPV1, MPV2, & MPV3), Theiler's murine encephalomyelitis virus, Murine norovirus, Reovirus 3, Mouse rotavirus, Ectromelia virus, Lymphocytic choriomeningitis virus, Polyoma virus, Lactate dehydrogenase‐elevating virus, Mouse adenovirus (MAD1, MAD2), Mouse cytomegalovirus, K virus, Mouse thymic virus, Hantaan virus, and mycoplasma.

11. Are the cells prepared under GMP conditions?
No. Our cells are not prepared under GMP conditionals, but they are produced under a very well controlled laboratory process that includes defined SOPs and controls to ensure quality and consistency lot‐to‐lot.

12. What method do you use for screening mouse pathogens?
All of the mouse pathogens listed above (#9) are assayed by PCR.

Human Newborn Fibroblast (NuFF) Cells

13. What are NuFF cells?
Human Newborn Fibroblasts (NuFF) are feeder cells for supporting healthy undifferentiated human ES, and iPS cells. They can also be successfully reprogrammed.

14. What medium should I use to grow my mitomycin‐C treated NuFF ?
Our NuFFs have been expanded in DMEM containing 2 mM glutamine with the addition of 15% FBS. We recommend that the treated NuFFs be thawed and plated into the same medium. If using the NuFFs as a feeder layer, the medium can be switched to stem cell medium when the stem cells are plated on the feeders.

15. How many times can NuFF cells be expanded?
We guarantee that our NUFFs can be passaged to P11 and used to culture pluripotent stem cells. While cells can be expanded much further, and most likely be fine, we do not test past passage 11.