Unexplained Cell Culture Death - Expertise needed (Apr/17/2008 )

I had posted in this forum some 2 months ago and tried the suggestions made but my cells just seem to die without any conceivable reason.

The cells that I am using now are COS7 and various strains of VERO. These cells had been adapted to MEM/10% FCS and had been growing well with all the other people in my laboratory with no signs of contamination.

I am currently sharing incubators with other people and they are not facing any cell growth problems. This is a brief protocol on what I am doing now:

1. Obtain healthy confluent cells and spilt (T25, 1x10*4, 2x10*4 ~ 2x10*5 sequentially). Removed old media, added 0.1% trypsin-EDTA (0.5ml), leave it until cells round up (3~4 min, 37oC), remove excess trypsin with pippette, add new medium, repeated pipetting and incubation at 5%CO2, 37oC.

2. The flask caps were left loose to allow gas exchange. However, the caps appear tight on the day after and loosening it causes a gas leakage sound. The cells then had a look of dark appearance with highly refractile nucleolus (bright, almost empty look under light microscope) and do not divide henceforth. The cells appear flat, smaller than usual and dark linings surround the nuclues. The sheets are resistant towards trypsinization.

3. The problems also appear in dishes and plates which I had made.

4. I then threw away all the FBS, medium, antibiotics and made fresh ones nearly every week with no avail. The cells still die upon passaging.

I was wondering are there any errors in my techniques? I had been working on cell culture for 5 years and had never faced such a problem. Three of my lab seniors (and my Professor) had watched me do cell culture and they said my technique was alright. I had even used media provided by other lab members (they all had heathly cultures growing) and the problem does not seem to go away.

Problems in cell culture are mainly:
A) Incubator/CO2 : mine is shared and other people had theirs growing well
B ) Medium: Used theirs too. Added glutamine/ added/omitted antiniotics, antimycotics but cells are still dying on the next day upon passaging.
C) Cell seeding: Did cell counts, various number of cells were seeded, they still die the next day
D) Trypsinization: Removed extra trypsin with/without centrifugation (did various speed/time) but to no avail

I am in real need of suggestions and advice (same problem for the past 6 months, no experiments performed). Please help if you can. Thanks in advance.

I had checked the pH as well

1. Unused media MEM (Earle's Salt, bicarbonate 2.2g/l) 2 days old, (with 10% FCS, 2mM Glutamine, 1%NEAA) pH7.2
2. Unused media MEM 1 week old, Invitrogen M4655 (with 10% FCS, 2mM Glutamine, 1%NEAA) pH7.6
3. Used media (from dead cells, 1 day after passaging) pH 7.4

All media were ready made, from Invitrogen and the FCS were from GIBCO. Labels mentioned sterile filtered medium and endotoxin tested.

Oxygen tensions are atmospheric.

Temperature in the CO2 incubator gave a constant 37oC reading with thermometer placed at various corners.

Disposable pippetes are used in all experiments and plastic flasks from TPP, Corning (with or without collagen, CellBind) were used.

Water baths were washed periodically (once a week). Water tray in incubator is replaced with fresh, autoclaved MilliQ every week. Incubators were clean periodically as well and scanned for fungal growth daily.

Efforts seems futile. My boss said that I am wasting my time.

I would add the trypsin and just leave it on and add fresh media once the cells have rounded. By sucking it off after the cells have rounded up you are likely to have removed a good proportion of the actively dividing cells, leaving the senescent ones behind, which are then the ones you are trying to re-grow your cells from. The medium containing FCS will inactivate the trypsin.

How frequently do you split your cells? If they are too confluent or the seeding density is too low, some cell lines will just die. I can't comment on your cell lines as I haven't used them but they seem to be pretty standard cell lines. The ATCC recommends a 1:3 -1:8 split ratio for both cell types and also recommends just lifting them by straight dilution of the trypsin with medium.

I would be very surprised if it was a problem with the CO2 or the presence of antibiotics (antimycotics are a different story, they are pretty toxic to most cell types as fungi are eukaryotes).

Thank you Bob1. I had tried both methods. Removing the trypsin and not removing it. Both came up with the same results. I had also counted the cells and seeded the T25 flask with
1x10*4 5x10*4 1x10*5
2x10*4 6x10*4 2x10*5
3x10*4 7x10*4
4x10*4 8x10*4

and splitting them at 1:3, 1:4, 1:5

in different flasks but they still end up dead on the next day.

Very absurd, something is very wrong with only my cells.

Please help me.

Hi again.

I managed to get my boss to show me how to passage the cells again (VERO). So he did all the trypsinization, dilution and added the media to the right concentration. I watch all the steps as usual and asked him to let me have the leftovers after he was done with it. So he got up from the clean bench and let me pipetted the extra cells into 3 new flasks (T75).

Then he checked the flask caps for all the flasks and put them all in the same incubator.

The next day, the 2 flasks which I pipetted cells into ended up DEAD!! Only 1 flask left but all the flasks and plates done by my boss was all right (30x 12 well plates and 1x T75 and 2x T150).

Is this just plain luck?

[attachment=4575:contamin...on071218.jpg][attachment=4573:contamin...n071218b.jp
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Hi, I loaded some pictures of the dead COS7 cells. Wonder it would be of any reference. Please advice.

[attachment=4581:contamin...0423pile.JPG]
I have also included the cells which I pipetted yesterday. The healthy cells were the cells from the same batch, pipetted into new flasks by my boss. I really do not get it, he watched me pipetted yesterday and the trypsinization and media was all his.



Any comments will be appreciated.

Hi,

Thats amazingly strange! Last month, I had the same problem with my COS-7 cells. I started with a new media,trypsin etc but it would'nt help. Everything was alright. Ultimately I ended up throwing away all the COS-7 I had and wake up some frozen cells. They started to grow well. Do you sort of 'hit' the flask after trpsinisation to detach the remaining cells? That might create shearic stress on the cells. Often the COS give a pain in the neck during trpsinisation. I usually use 4 mls and leave for 5-7 mins. and they come off well (though it sounds a lot!). Have you tried spinning down the cells at about 1000rpm after trypsinisation and resuspending them in new media and then pippeting into new flasks??