Hi, I encountered an issue while detaching some cells. I had both HEK 293 and RAW 264.7 cells. When I added 0.05% trypsin, the cells did not round up and instead detached in the form of a sheet. Upon centrifugation, mucus formed, and when I tried to seed them, they no longer adhered. I have always used the same brand of trypsin (Gibco), the bottle was not new, and I diluted it with PBS. Has this ever happened to you? What could be the cause?
Dear Dr. Lizeth Guadalupe Campos-Múzquiz
When cells reach confluence, they adhere to each other and develop a confluent monolayer on a culture surface, with tight adhesions between them. Generally, an enzyme such as trypsin is employed to harvest cells, which results in dispersed single cells, since adhesion proteins are non-specifically destroyed. As in your case, at this stage, cells detach in a state of cell sheet if adhesions between the cells and culture surface are selectively lost.
The use of 0.05% trypsin might be too low a concentration to trypsinize the cells. You may use 0.25% trypsin for trypsinization. But prior to adding trypsin, you should rinse the cell monolayer with 2-3 ml of warm and sterile PBS (without Ca2+ and Mg2+) for a T-25 cm^2 flask after having removed the complete media.
Much better than trypsin, is the use of trypsin-EDTA (0.25% trypsin + 0.02% EDTA). The presence of EDTA will help since EDTA is a chelator that binds calcium and magnesium ions that may otherwise inhibit the trypsin activity, which then hydrolyzes and gains access to the intercellular bonds (cell-cell and/or cell-substrate bonds).
Regards,
Malcolm Nobre
Hello Lizeth Guadalupe Campos-Múzquiz ,
Did you encounter the problem with both cell lines, or only with one of them ?
I don't know exactly what your protocol for detaching cells is, it might help to know in order to find where the problem comes from. Note that HEK 293 cells are generally loosely adherent (we use CellBind flasks to cultivate them), on the contrary to RAW 264.7 cells which are strongly adherent, not easily detached and also re-adhere to the culture flask very quickly (sometimes in the matter of 5 minutes).
For HEK 293 cells, I would not suggest a stronger trypsin than 0.05% : at the facility where I work, we use even "lighter" trypsin (made in-house) to detach these cells. Also we reserve trypsin-EDTA for cells that are strongly bound together, for example cells that grow in tight clusters.
First we discard the medium, then we rinse the flask with PBS (not necessarily warm, room temperature is fine), we add max. 0.5mL of trypsin/T25 flask and let sit at room temperature for 3-5 minutes, checking regularly if the cells detach. When they do, we inactivate with some fresh serum-containing medium (~5mL/T25) and dissociate the cells by doing a few up-and-downs, then we dilute. If you inactivate with enough medium (at least 10 volumes of medium for 1 volume of trypsin) and don't use trypsin-EDTA, centrifugation is not absolutely required.
For me, HEK 293 cells detaching in layers were maybe a bit too confluent (more bound together), and letting the trypsin act 1(-2) minutes longer, then dissociating with the pipette, would have solved the problem.
Regarding the fact that they were not adhering after the dilution : how long did it last, did you observe anything peculiar in the flasks, or in the medium you used ? It might help also, to find a possible cause. Generally when cells don't adhere it is because they are in bad shape, sometimes dead, and is usually due to the way they were detached and diluted, or because of contaminations.
For RAW 264.7 cells, as they are strongly adherent to the culture surface, we don't even use trypsin as it destroys adhesion proteins (and it would be really constraining and time-consuming): rather, we use a cell scraper and the protocol is mechanical (not chemical). I can ask why exactly we use this method, maybe if you encountered the problem of cells not readhering with this cell line it is because of the trypsin.
Also, you say you diluted the trypsin with PBS : at which stage, and maybe what ratio did you use ? When you say the bottle was not new, does it mean that you had already used it in the past and it worked, or that you don't know since when it was open ?
Sorry for the long answer, maybe with all these questions and answers we can find where your problem comes from ! ;-)
Hello, have you found a solution to your problem? and if yes could you share it please. I am facing the same problem.
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