When I culture the HEK293T cells in cell culture plates (60 or 100mm ), they grow well but when I culture the cells in 24-well cell culture plate then after one day of incubation at 37'C, the cells start to detach from the culture plate surface. What could be the possible reason? Thank you.
Hi Behzad,
Did you take account the seeding density for the 24-well culture plate? This table from ThermoFisher could be useful to adjust the conditions of your culture:
https://www.thermofisher.com/es/es/home/references/gibco-cell-culture-basics/cell-culture-protocols/cell-culture-useful-numbers.html
Hi Behzad Hussain,
HEK293T cells are very delicate cells and they easily detach from the culture plates. I have experienced the same. Firstly, make sure that while sub-culturing/passaging, you do not leave your cells out for longer periods and give them a thorough washing. Secondly, don't seed too many cells as HEK293T have the tendency to behave in this manner at higher confluency. Do not cross more than 60-70% confluency with 293T. In case you visualize the cells under a microscope or take the culture dishes out of the incubator, remember to keep them back inside without much delay since regular temperature shift and conditions results in some cellular shock. Also, check your incubator that does it maintain the right temperature without much fluctuations. HEK293T cells grow best without antibiotics. Moreover, if they do tend to be loose, you can make use of lower serum concentrations (2-2.5%) in your media. Good luck!
In my experience HEK cells can often suffer from low adherence, and are typically enhanced in retention and growth by pre-coating of cultureware with a substrate such as PDL or Collagen. As noted in other comments the lower media volume in 24w plates will offer less thermal and chemical buffering, so minimizing time out of the incubator and checking the equipment for stability might also help.
Seeding density like María Luisa Caballero mentioned should be the fix here. I remember encountering a similar problem but with HEK293 cells. We use to precoat the culture vessels with polylysine and observed far better attachment. A brief protocol by SIGMA can be found here https://www.sigmaaldrich.com/IN/en/technical-documents/protocol/cell-culture-and-cell-culture-analysis/3d-cell-culture/poly-lysine-product
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