Hello everyone!
I have a question regarding the attached picture. You can see fibroblasts from an animal under 10x magnification that form very dense spots although in the rest of the cell culture flask they distribute very evenly and there is still space.
We cultivate with P/S and the culture has already been tested negatively for the presence of mycoplasma.
Sometimes we manage to solve this problem by adding trypsin and then fresh medium but I really would like to know why they form like that and if there's any way how we could avoid that in the future!
Thanks a lot for every proposal!!
I've found 2 things helpful: seed at lower cell density and allow attachment with the dishes isolated from vibrating surfaces (like swirling the dish or direct contact with the floor of the laminar flow hood). Vibration can cause eddies that bring the cells together.. i use a flexible rubbery sheet under the dish. A nice squishy mouse pad works.
Hello,
Just your cells in trypsin do not separation one from one and after this clot of cells adgesion on flask. You must thoroughly break clot of cells in trypsin at next passage.
Following up on Ellen and Elena‘s answers, if you visualize clumps after trypsinization, you can always pass the cell suspension through 40 um nylon mesh to reduce them. With a low seeding density, any remaining clusters should spread and migrate.
Also, your pic looks like cells are over confluent. It’s possible for some lots of FBS to encourage piling up when cells are crowded. Best of luck solving the problem!
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