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Short answer: Mouse embryonic fibroblasts (MEFs, also written MEF cells) are primary fibroblasts taken from mouse embryos, most often at 12.5 to 13.5 days of gestation. They are best known as feeder layers that support embryonic stem cell (ESC) culture. Because they are primary cells, they last only a few passages, and once mitotically inactivated they cannot be expanded.
For research use. This page summarises published methods and manufacturer technical notes; it is not a protocol and not a substitute for the instructions that come with a specific product.
What are mouse embryonic fibroblasts (MEFs)?
A mouse embryonic fibroblast is a connective-tissue cell obtained from a mouse embryo and grown in culture. Cytiva’s HyClone technical note states that MEFs have been used as feeder layers for embryonic stem cell culture since the first mouse ESCs were derived in 1981, and that they remain the most commonly used feeder cell type for mouse and human ESC lines. The same note describes the MEF layer as providing a complex but unknown mixture of nutrients and substrata for long-term growth of undifferentiated pluripotent cells.
MEFs are primary cells, not an immortalised cell line. That one fact drives most of the practical decisions below: how old the embryos were, how many passages are usable, and why feeders are prepared shortly before they are needed.
MEF cells as feeder layers
In feeder-based culture, a layer of MEFs that can no longer divide is plated first, and the stem cells are grown on top. The feeders are rendered non-dividing so they support the stem cells without overgrowing them. The Cytiva note gives two timing points: inactivated MEFs fed every other day make good feeders for mouse ESCs for 5 to 7 days, and for human ESC culture the MEFs should be inactivated the day before the human ESCs are passaged. A published Cytotechnology method (2015, 68(4)) reports that human ESCs grown on feeders it prepared showed no visible difference in proliferation, morphology or differentiation compared with commercial gamma-irradiated feeders.
For the wider context of keeping cultures healthy, see the cell culture basics guide.
Passage limits
The two sources describe the usable window slightly differently, which is useful to see side by side.
| Question | Cytiva HyClone technical note | Cytotechnology 2015 (68:4) |
|---|---|---|
| Embryo age | Cells isolated from 12.5 to 13.5 day embryos | 12.5 to 13.5 dpc gave the best MEFs; 13.5 to 15.5 dpc acceptable; older embryos gave poorer MEFs |
| Usable passages | Limited lifespan of 1 to 5 passages | P2 to P5 usable as feeders; P3 recommended for hESC culture; P2 generally excluded because of unwanted mixed cell types |
| Sign a culture is past its useful state | Cells elongate and doubling time rises significantly; overgrowth can cause early senescence | Growth rate declines and deterioration increases at higher passages |
Both sources describe the passage window for the mouse material they worked with, not as a universal rule for every strain or preparation. The practical reading is to record the passage number of every feeder batch and stop using a culture once it shows elongation or slowed doubling.
Mitotic inactivation: mitomycin C or irradiation
Feeders are inactivated either chemically with mitomycin C or physically with radiation. The Cytotechnology method compared parameters directly:
| Method | Parameters reported | Reported outcome |
|---|---|---|
| Mitomycin C, routine | 10 μg/ml for 3 h | Routine condition |
| Mitomycin C, alternatives | 10 μg/ml for 2.5 h, or 15 μg/ml for 2.0 h | Acceptable alternatives; higher concentration or longer exposure gave many dead cells |
| X-ray, preferred | 1.5 Gy/min, total 25 Gy, on P3 to P5 cells | Preferred condition |
| X-ray, alternative | 2.0 Gy/min, total 20 Gy, on P3 to P4 cells | Alternative condition |
The Cytiva note independently states that exposure to mitomycin C at 10 μg/mL for 2 to 3 hours is sufficient to mitotically inactivate MEFs. It also warns that mitomycin C inhibits DNA synthesis and nuclear division, is toxic and possibly carcinogenic, and should be handled with skin and eye protection, good ventilation and the supplier’s safety data sheet for disposal. Both documents are labelled for research use.
What to check when sourcing MEFs
The Cytiva note lists three recommended criteria when sourcing MEF cells:
- Isolated from 12.5 to 13.5 day mouse embryos.
- Mycoplasma free (see the comparison of mycoplasma testing methods and the guide to cell culture contamination).
- Tested for mouse antibody production (MAP).
Two further points follow from the same sources. First, the note advises not to inactivate MEFs until they are needed, since inactivated cells cannot be expanded; inactivated MEFs can, however, be cryopreserved (see cryopreservation of cells). Second, the Cytotechnology authors report that commercially purchased MEFs often lost attachment ability after dry-ice transport, which they attribute to transport damage; this was their observation, not a measured comparison, so it is a reason to check attachment and condition on arrival rather than a verdict on any supplier.
Common misunderstandings
“MEF cells are a cell line”
They are not. They are primary cells with a limited culture lifespan (1 to 5 passages per Cytiva), unlike immortalised lines that can be passaged indefinitely.
“I can expand a large stock of inactivated feeders”
Mitotically inactivated MEFs cannot be expanded. Expand the live cells first, inactivate what is needed, and cryopreserve inactivated cells if they must be stored.
“Any embryo age works”
Both sources point to 12.5 to 13.5 days. The Cytotechnology study found 13.5 to 15.5 days acceptable but older embryos gave poorer MEFs.
“The inactivation dose is the same everywhere”
The reported conditions vary by agent and by passage, as the table above shows, and higher or longer exposure produced more dead cells. Follow the documented conditions for the specific material in use.
FAQ
What does MEF stand for?
MEF stands for mouse embryonic fibroblast; “MEF cells” and “mouse embryonic fibroblasts” refer to the same cells.
What are mouse embryonic fibroblasts used for?
According to Cytiva’s technical note, they are most commonly used as feeder layers for mouse and human embryonic stem cell culture.
How many passages can MEFs be used for?
Cytiva gives a limited lifespan of 1 to 5 passages; the Cytotechnology study used P2 to P5 as feeders and recommended P3 for hESC culture.
Why are MEFs inactivated?
So the feeder layer supports the stem cells without continuing to divide. Mitomycin C and irradiation are the two methods compared above.
Sources
- Cytiva (HyClone), Mouse embryonic feeder cell protocol: mitotic inactivation of MEF cells by mitomycin C, document 29154592 AC, read 2026-11-07.
- A reliable and economical method for gaining mouse embryonic fibroblasts capable of preparing feeder layers, Cytotechnology 2015;68(4), PMC4960116, read 2026-11-07.
