Mitomycin C Treatment Protocol to Create Feeding Layer

Mitomycin C, a broad-spectrum antitumor antibiotic isolated from the culture of Streptomyces capitis, has anticancer effects on a variety of cancers. It works by depolymerizing the DNA of cells while hindering DNA replication, thereby inhibiting tumor cell division.
A certain dose of mitomycin can stop the cells from dividing, but it does not cause immediate death and can sustain life for a period of time in vitro. Therefore, mitomycin can be used to treat feeder cells so that the feeder cells do not divide, but can survive and secrete factors that inhibit the differentiation of embryonic stem cells (ESCs) and promote the value-added of embryonic stem cells to ensure the growth of ES cells.
Mitomycin C is able to inhibit cell proliferation of human or murine embryonic fibroblasts in the concentration range of 0.2-20 μg/mL. These treated cells are subsequently used as an adherent feeder layer in long-term culture assays, primarily for the growth of pluripotent stem cells.

When the mouse embryonic fibroblasts (MEFs) fuse into a monolayer, the culture medium is discarded and the solution containing 10 µg/mL of mitomycin C (covering the cell monolayer) is added. The culture flasks are placed in an incubator containing 5% CO₂ for 2-3 hours.
The mitomycin C treatment solution is discarded, and the flask is washed 3-5 times with PBS to remove the residual mitomycin C, which can affect the growth of ES cells.
The cells are digested with 0.05% trypsin solution for 2-3 minutes, and then the digestion is terminated by adding an equal amount of cell base culture solution.
Finally, the cell culture solution is transferred to a 15 mL centrifuge tube and centrifuged at 1000 rpm/min for 3 minutes.
The cells are resuspended with the cell base culture medium to make a cell suspension with a concentration of 3×10⁵ cells/mL and planted into the culture flasks or dishes pre-treated with 0.1% gelatin.
The cells are incubated in an incubator with 5% CO₂, and the cells quickly adhered to the wall and spread as a monolayer. If the cells are too dilute, mitomycin-treated cells can be added and the feeder monolayer can be used for 6-10 days.
MEFs monolayers can also be used without digestion after treatment with mitomycin and sufficient washing with PBS.

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Cover the surface of the culture flask or dish with 0.1% gelatin and leave it at room temperature for more than 2 hours. Aspirate the excess gelatin before use.
Mouse embryonic fibroblasts are functionally active, with significant protein synthesis and secretion activities. And under certain conditions, they can interconvert with fibroblasts.

For research use only. Not for any other purpose.