Recently, i found that 293T cells did not grow at all in the 6-wells plate, most of cells were attached to the wells though. However, those cells grow very well in the T75 flask. (flask and plate come from the same company). Before culturing and plating cells, I usually coat flask and plate with poly-l-lysine for 1 hour in the room temperature, and then wash with PBS. Moreover, i also tried to coat plate with 0.1%gelatin under 37 degree for 2 hours or more. Unfortunately, i still got the same problem.
Dose anyone ever had the same problem with 293T cells? How did you solve it? Thanks for your suggestions in advance!
I used HEK293 cells before, so not the 293T, but we didn't coat the flasks or plates at all. The grew fine without coating. On coverslips we used collagen (rail tail type 1), because during washing and staining they attached better to the coverslips when we used coating. As I understand the cells do attach, but do not grow in the 6-well plates, but in the T75 flask it is no problem? Sounds like the 6-well plates are then not suited to grow the cells, or the cells are not viable. We cultured them in MEM-alpha, supplemented with 2 mM L-glutamine and 10% FCS.
Kristin, Thanks for your answer and link!
I tried twice to plate cells on poly-l-lysine coated 6-wells plate,but they both did not work. I will check the lot number, probably it will affect the cell growth.
I plated 100,000 cells on each well. The cell number are less so that they don't grow, which might be the reason? It is my first time to do experiment with this cells line, i'm not quite familiar with it's growth condition.
I just lay it flat in the incubator without shaking or rotation.
Dafna, Thanks for your answer!
What kind of flask and plate you are using for culturing HEK 293? I never tried to culture this cells line without coating, I was told this cells line has weak adherence to culture plate and flask, so i just follow the same protocol as previous people used. yeah, cells don't like glass so much, eg. coverslip.
I tried few times to plate cells in flask and plate as the same time, but they only do not grow on the plate.
I think it is not necessary to coat 6-well plate with poly-lysine, since you grow 293T well in T75 flask. Please check the 6-well plate whether it is cell culture treated. Try other culture dishes like 6-cm dish or 10-cm dish to see if the problem still occur.
For the flasks we used Greiner Bio one, for the plates I'm not sure. I think they were from Corning Costar. All were tissue culture treated. We use 2ml medium/well for the 6-well plates. What happens when you culture them in other plates, or on coverslips...do they grow then? Are the T75 flasks you use from a different company than the 6-well plates?
I grow HEK293T in flasks/multi-well without coating, I use pll only for glass coverslips. How do you split your cell? Is it possible that you are using trypsin and leaving it on cells too long? If they suffer during the passages, you might not see it in the flask because you have more cells, while if you plate them at low density in the wells and they suffered from trypsinazion, you might end up with unhealthy cells that won't grow and a few healthy ones that will take longer to recover.
Valeria, usually I put 300ul trypsin into T25 flask, trypsinize cells and slap the flask by hand for about 1min in room temperature, i don't think they suffer from it too much. Probably because of this batch of plate is not fine and the cell density i plated is not high enough.Did you use already treated cell culture plate? i never tried to culture this cell line without coating.
This is how I trypsinize cells. Rinse the flask with 2 ml of warm Sterile PBS to remove any residual presence of medium . Add 300µl of warm 0.25% trypsin (containing EDTA), gently tilt the flask so the adherent layer of cells get a coating of trypsin. Incubate flask at 37 °C incubator for 2-5 min. Gently shake the flask ( you will see the opaque layer (trypsinized cells ) detaching , add 5 ml medium to your flask. Gently mix the cell suspension using the 5ml pipet 2 times. ( warm the PBS and Trypsin in 37°C water bath)
this should work for the HEK293 cells . I do not tap the petri dish or the flask to dislodge the cells.
Tissue culture dishes/multi-wells work fine with me without coating. If you are concerned about the lot of plates, can you ask a few dishes to a close by lab just to test?
Thanks a lot for answers and suggestions from all of you!
I solved this problem already, cells grow good in coated 12-wells plate. I also tested it on 6-wells plate from other lab, it was better as well, so it is the problem of plate of this batch.I just still need to increase the cell density, they will be much better.
We split the cells 1:10 in T75 flasks. For a whole 6-well plate, I take a confluent T75 flask, add trypsin and afterwards I add medium to get 10ml suspension. I take 1ml of this and add 11ml medium (1:10). I distribute this over the 6 wells (2ml/well). When I do this, the cells grow fine. Also 1:20 dilution works fine for the HEK293 cells. Good luck with culturing them! :)
Some of the tissue culture devices I own do not support the adhesion of 293T. I am not sure what the variable is, but currently my cells will not attach to the 24 well plates or 12 well plates that I have. They do attach on the same plates after pre-coating with rat tail collagen. I am not sure whether PLL would also favor transfection efficiency on top of attachment. One of my previous lab member noticed an improvement after using PLL. I believe that collagen pre-coating is dispensable, for the purpose of attachment. Takara advises to grow their LX cells onto collagen coated plates, at least after thawing.
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