I am trying to culture the HMEpC cell line but the cells are slowly dying. Upon arrival of the cell line from the supplier, I put them in a T75 flask using the Human Mammary Epithelial Cell Growth Medium from Sigma (815-500). The cells don't seem to grow but are slowly dying. You can see clumps of cells but they are all floating. It seems that once the cells are proliferating they lift of the surface of the culture flask.
Does anyone have any idea on how I can rescue the cell line before all cells have died?
Hi Julie
I have never worked with this cell line but detachment is a problem... Have you checked the viability of the floating cells? I heard it once from a colleague, that his cells starts to float due to failure in Talin production. I think he used some endothelial cells. Interestingly, the cells were still alive and also proliferative.
You may also try to coat your culture-plates with Fibronectin or Laminin? Check the recommended amount of cells per cm2..
Best,
Martin
Hello Julie:
Most tissue/cell culture vessels come already treated for that purpose with agents such as listed above or, specifically, with poly-L-Lysine. If your Supplier cannot assure you that their culture flasks have been so treated, look elsewhere for a different supplier. Beckton Dickinson (blue cap), Corning (yellow/orange or red cap) or Sarstedt (red cap) may be appropriate for your needs.
How your cells were shipped to you will determine how you seed or reseed them. If you received the cells frozen in a vial, thaw at 37 degrees Celsius with shaking, by hand, and disperse the sample completely into single cells, check % viability, e.g., by Trypan Blue exclusion, before seeding. Completely dispersing the cells will help minimize the clumping that has occurred in your culture. If the cells were received at room temperature (RT), and already seeded into a flask, immediately rinse the cells with your own RT culture media, then exchange this rinse with a 37 degree Celsius culture media and incubate overnight or till confluent before reseeding.
Initially seeding cells in a TC 75 cm2 flasks may not be optimal for the viability of the cells given that they have just been traumatized by storage and by shipment. First, seed the cells into a TC 25 cm2 flask, grow to confluency, then reseed into larger culture vessels. When seeding into TC 25 cm2, seed the cells at a density that is appropriate for the flask. Each Culture flask has an effective seeding area which you can obtain either from the product insert of from your Vendor. Initial culture media volume should be such that the media meniscus is barely above the seeding surface of the flask (between 5 and 10 mL should suffice) but sufficient to cover the cells completely once they have settled to the seeding surface . Too dense an initial seed would cause the cells to clump due to over proliferation given that your cells being a cell line suggests that they possess a very high proliferative potential as opposed to their cognate primary ancestor. Ultimately, you'd have to try several different combinations of these suggestions to find the most suitable ones for your cells.
Finally, you may want to consider checking your cells for possible contamination with an agent such as Mycoplasma.
Best of luck.
Enrico.
Try adding glutamine 3% solution of 1/100. Cow bMEC cells do not grow without it.
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