Hi! Over the past weeks, I have observed an anomaly in one of the cell lines I routinely culture (CHSE-214 cells, derived from Chinook salmon). My routine culture cells are grown in flasks and appear to be perfectly healthy. Lately however, I have noticed that when using these cells to seed 24-well cell culture plates, the majority of the cells will die within just a week or two. After a couple of days, the cells start rounding up, detach from the monolayer (they are adhesive cells) and float to the surface of the medium, often forming large clumps of dead cells.
Since then, I have collected cell lysates and performed mycoplasma tests and also QPCR tests for every disease that we routinely work with in our lab. Every one of these tests was negative. What is strange is that I do not observe this in any of my culture flasks. It only seems to happen as soon as I plate them, and they only recently started to do this. I have read that this can happen when too many cells are seeded in the wells and are too confluent, but I only seed my cells at about 70% confluency and count them every time before plating them, so I do not think that is the issue.
Last week I seeded some cells in 3 different types of cell culture plates (tried 2 different brands of TC treated plates and also Corning cell-bind plates). I have not observed any difference between these 3 brands of plates; the cells keep dying. I have also screened the growth media I am using for mycoplasma and some viruses, all of which were negative. There is nothing visible under the microscope either, so I am not suspecting bacterial contamination, especially since the cells appear healthy in their culture flasks and I use the same media bottle for all my cultures.
We work with a few other cell lines in the lab as well, and all of them perform well when seeded to culture plates. It is just this one cell line, and I was not observing this until a few weeks ago. Anybody have any ideas? I am stumped!
Bacterial contamination is usually easily detected by eye, the cells will be covered in a fine dusty layer and often the media will quickly turn yellow. Using a medium/high magnification any bacteria will be clearly apparent under the microscope,
Are the cells starting to lift once they form an intact monolayer sheet? This is quite common behaviour with many cell type.
I would suspect the seeding density may be the issue, have you tried seeding the cells onto the plates at a range of different densities?
Hi Sion,
My media is not yellow when this happens. It remains a red-ish color and I do not see any bacteria moving under the microscope. We had bacterial contamination maybe 3 or 4 years ago but I have not had it happen since then.
The cells start to lift after maybe 3 or so days. There is still plenty of empty space in the wells, as it usually takes at least a whole week before the wells reach 100% confluency. I have performed assays in the past in order to determine the optimal number of cells to plate (to determine the best level of density) and have remained consistent ever since then. It had worked for the longest time and my issue has only started very recently. What is very strange to me is that I have had a number of wells reach complete destruction of the monolayer with a week, although most of the time, the cells will manage to recover and the monolayer will begin to grow again.
Thinking horses, not zebras -
Are all conditions, medium, incubator, seeding density, etc, identical between flasks and wells?
When you split a flask, and put half into another flask and half into wells, do the cells still lift?
Do you have a master or working cell bank you can use to get lower passage cells?
Closer to zebras -
Can you co- culture your suspect salmon cells with another cell type which is easily distinguishable and see if both lift in the wells, or if the problem is cell-autonomous to the salmon cells?
Hi Bruce,
Thinking horses : the only condition that is not identical is that my routine cultures are grown at 20 degrees Celsius, while the culture plates eventually get transferred into an incubator kept at 15 degrees. This is because the viruses we cultivate in the lab and perform infection experiments with will only replicate at lower temperatures. Usually, after I passage and seed the cells into culture plates, they are returned at 20 degrees for a 24 hour period so that they can recover and re-attach to the surface. Afterwards, they are inoculated (or not) with a virus and then incubated in the 15 degree incubator, where they remain until the end of the experiment. It did occur to me that perhaps the temperature shock is to blame, but I have been following this protocol for a year and this is the first occurrence of this. It is possible though that the incubator is no longer holding its temperature properly, so I am going to look into this possible issue.
Otherwise, when I split a flask, the cells only lift in the culture plates, not in the flasks. Also, the cells I am currently working with are relatively fresh - I thawed out a master stock a few months ago and the cells have only went through roughly 10 or 12 passages since then.
I do have another salmon cell line currently in culture and do not see this happening. It is really just this one cell line. They share the same medium.
Thanks for the thoughts! I am going to try and check if the problem is related to the temperature in the incubator.
Two technical suggestions/questions: it occurs sometimes that plastic used to produce various multi-well plates is not exactly the same. Some surfaces are smoother than the others and do not support adherence. Are all plates you were using produced by the same company from same material? If not try several others... If for some reason you want to stick to exactly these 24-weel plates, try to coat them e.g. with collagen (or polylysine, that my primary trout hepatocytes used to like so much). This helps at least with endos ...
I agree with Anna. With cell lines that have difficulty attaching to the plastic surfaces, I normally coat the plates with collagen prior to seeding the cells. Do you know how old your plates are? We have experiences in the lab where culture plates older than a year "lose" their coating. We think this is happening because the cells have difficulty attaching in these old plates but not in the same brand of plates that were recently purchase. So we also monitor the Lot no. of the plates we are using...
Hi Jean René
As Bruce suggested, I guess you should try lower passage cells. 10-12 passages may be to much for some cell types.
Since, temperature conditions may also be critical.
Good luck
Hi everyone,
I was finally able to get some good results this week. I simply calibrated the temperature of my incubators and the last batch of cells I plated adhered just fine and there is almost no cell mortality. I think temperature is very critical for cell adhesion of this specific cell line.
Thanks for all your suggestions!
Hi Jean-Rene,
I am having similar problem with you. The cells do not die but do not grow in outer wells containing cells of 96-well plate. I have used this protocol and I know they work and trouble started when we moved to other building.
I think the problem is caused by evaporation in the CO2 incubator. There is no problem in the T-25 and T-75 flasks during cell growth but the issue started in 96-well plate stage. Do you have any suggestion about that?
Thanks in advance.
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