I feel your cells may have PPLO infection or in other words they maybe contaminate! You can check using RT-PCR kit and meanwhile revive a new stock for your work.

Hope this helps!

Pratibha

Check the medium that you are using - if you're having the cells supplied by a commercial supplier, make sure to confirm that you're following their protocol. Although HEK 293 are easy to transfect, they can be picky about their required media.

Pascal, there are a number of things that can affect the growth rate of a cell line. Normally, I suggest that one 'keeps it simple' and look at the basics. This is going to be a long answer...

But, before I get into the basic things, Alex Kashkin (above) is right. Are you talking about HEK 293 cells BEFORE they've been transfected or AFTER? If they have been transfected, then it's entirely possible for them to grow at a different rate depending on what they've been transfected with and how they've been transfected.

However, assuming it is not the transfection that is causing the problem...

• Are you using the same media that you have been using?

• Are you plating the cells with the same protocol? When you split your cells, are you

- detaching them the same way every time?

- Washing them with the same buffer each time?

- Doing a cell count and plating them at the same density every time?

I've seen students get lazy about washing the cells after detaching them. If you do not wash the cells properly, you risk leaving small amounts of enzyme (trypsin) or EDTA in with the cells when you re-plate them.

I've seen students take a short cut and not do a cell count before re-plating the cells. If you are in a hurry, you might simply do a 1 to 3 or 1 to 5 split, but you don't REALLY know the density at which you are plating the cells. While this is fine for keeping a cell line alive and growing, it's not ideal for using those cells during an experiment. I'll give you an example of one thing that could go wrong (and something you said made me think of this)

If you plate the cells at too low a density, you risk getting what I call, "Islands of confluence." Imagine you have very few cells and plate them into the flask. The cells that attach and grow are very far apart. So, when each cell divides, the new cell will be right next to the parent cell. Then they will both divide and so on and so fourth. after a few days, you will likely see 'groups' of cells scattered about the plate/flask with a fair amount of space between them. When this happens, you might be tempted to simply let them continue growing as there is plenty of space and the media is still the right color and pH. But here is the problem - the cells that are in the middle of these 'groups of cells' are trapped in an area of dense cell population, whereas, the cells at the perimeter of the cluster sill have room to divide. So, within these groups of cells (Islands of cells) you have cells that are confluent, but at the perimeter the cells have space to grow. SOOOO, the cells within the clusters will stop dividing and start dying while the cells at the perimeter continue to grow quite well... You mentioned that you are seeing a lot of dead cells... Could this be a possibility?

This "islands of confluence" scenario also happens when one lets the cells sit in the tube, in new media, too long before plating the cells. If left too long in the new media (in a tube) the cells can start sticking to each other, so that when you plate them, they plate in clusters rather than individuals. You'll get the same problem that you get when you plate with too low a density...

• Are you using the same type of flasks/plates

Now, I can't explain this, but I have definitely seen this. It happened to me not long ago in fact. I was culturing cells in Corning T-75 tissue culture treated vented flasks with the angled necks. I ran out of T-75 flasks and switched to medium sized tissue-culture treated round plates (nalgene brand I think.) and suddenly, the cells grew more slowly and just didn't look quite right. After a couple of weeks my new T-75 flasks came in and i switched back. The cells went back to being happy.

• Infection

Other than your technique or reagents, you can look closely for any signs of infection. Things like yeast and Mold usually grow so fast that they are obvious. You'll see them... but some microbes can be small enough and grow so slowly, that you might not detect them unless you specifically look for them.

The first and simplest thing to do, is to put the cells under the microscope with at least a 40X or 60X objective and see if you can see anything moving or swimming around. There shouldn't be ANYTHING in there swimming around...

Next, take some of the media directly from the cell culture flask and put it on a slide by itself and examine the culture media. Again, there shouldn't be anything swimming around.

next, put some of the media (from the culture flask) on an agar pate, spread it around, and incubate it overnight at 37 C... See if anything grows. If so, you have an infection in your cell culture.

I hope this helps...

Good luck!

Tim, thank you so much for the detailed answer. I had to go back to the details as you suggested and i realized the problem might have been multifactorial for sure. I had plated the cells at too low a density, and, of note, i observed the Islands of confluence. Secondly, my advisor's lab has not been using Trypsin to detach cells, they prefer using mechanical detachment (pipetting the cells several times) and i felt that perhaps i had been too harsh when pipetting, leading to massive cell death in my culture. To enhance the growth of the already adherent cells, i had to change the medium and that worked very well. I was very careful with the subsequent passaging, applied all the tips you suggested and others that i led elsewhwere. Suffice to say that i have seen tremendous improvement since then. Thank you so much once again everyone for the tips...

Elie, sure thanks.