Dear all,
I am currently working with Caco-2 cells and need to culture them to achieve high cell numbers. However, due to my protocol, I cannot use trypsin for detachment. My main challenge is that when I collect the cells using a cell scraper, they tend to form visible, gel-like clusters. Despite extensive pipetting and gentle vortexing, these clusters persist, making it necessary to reseed them in this state.
While the cells still grow, it would be preferable if they were more evenly dissociated. Is there a way to overcome this issue and achieve better cell dispersion?
P.S. I have found a solution for counting the cells: I take 0.5–1 mL of the collected cell suspension and incubate it with trypsin in a tube. Once the gel-like clusters disappear, I proceed with counting. Since cell viability is not a concern for me, I incubate them with 1X trypsin for a longer duration than standard protocols suggest.
Hello Elif Öztemiz
The use of cell scrapers is actually a harsh method to detach cells from the substratum. It does cause plasma membrane breakage and cell death. Due to cell death, free DNA from dying cells is released in culture media. The sticky nature of DNA causes cells and other debris to aggregate into large clumps.
You will have to remove the DNA released by dead cells by including 25-50 µg/ml DNase I in the presence of at least 1mM Mgcl2 (DNase I requires a concentration of at least 1mM Magnesium to work effectively) in your cell suspension as well as prevent cation dependent cell-cell adhesion by adding EDTA at a concentration of up to 5mM.
Best,
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