Our labs mammalian cells - A375, and HEK293- seem to be unhappy and are rounding up during culturing.
I have checked mycoplasma contamination using a PCR kit, that turned back negative. I have also changed all plastics, media components, and CO2 tanks. I clean our incubators every two months.
I recently killed all cells, bleached and sterilized the entire room and thawed new stocks purchased from ATCC and the problem consisted. I have attached photos of our cells showing the rounded morphology.
Any suggestions would be useful as I am not sure what else to try at this point.
Thanks!
Mammalian cell rounding occurs during mitosis to facilitate tissue organization and homeostasis.
Firstly, mitotic cell rounding in combination with maintenance of apical cell-cell junctions appears to be necessary for correct mitotic spindle alignment, so that daughter cells divide parallel to the tissue plane, thus sharing apical surface to maintain tissue homeostasis. If not, then we would have mis-localization of one daughter cell to the basal region on the tissue layer which will have to be cleared by apoptotic cell death.
Secondly, mitotic rounding has been proposed to be a driver for morphological events during tissue development.
Thirdly, mitotic rounding has been shown to be important to generate sufficient space and appropriate geometry for proper mitotic spindle function, which is necessary for timely and accurate progression through mitosis.
Hello, If no any contamination the main reason that mammalian cells rounding up during culture is absence of adhesion of cells on surface of plastics used. Try different clean plastics.
Usually process by which otherwise hydrophobic polystyrene surfaces are made to become hydrophilic, usually by increasing negative charge through a chemical means. This increased negative charge is important for cell attachment, and may explain why cells cultured in your non-treated plasticware rounded (namely that they are not attaching adequately to the surface).
Sound like it's genetics and environment , as usual.
Very interesting.
Best,
Jerry
Please check the quality parameters of water you are using in preparation of your laboratory solutions, I think you have a problem in water quality
I have been facing the same issue with A375. It is showing the rounded morhology during the late passages, say after 5 or 6 passages.
Rachael Day Deepti Mittal Interesting! But If its just the mitotic cell rounding, cell numbers should be maintained as you passage them. How is the comparison between the cell numbers post revival? Also, may be do not change media very frequently.
Perhaps you could try plating them in small culture dishes first so they form networks and grow as a sheet. If optimal seeding density is used its quite unusual as they look otherwise healthy (in picture).
If happens consistently, I would have looked into this genetically as well.
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