Something is wrong in our cell culture system. It has been like this for months. Cells cannot survive after several passages. They are gradually dying. At the beginning, only some stable cells with virus vectors weren't easy to culture. They would die after a few passages without apparent reason. Later, even some easy culture cancer cells like A549 or A2780 couldn’t be kept long in incubator. It has never happened before. We have changed the serum, medium, trypsin, dishes and monitored the pH value in medium but can not solve the problem. Now it is getting worse. Also, it’s not cell specific.
Hello Jun, when you say, that this problem is not cell type specific and you have already tried different media, serum, etc., then it could be the incubator. You should probably check the CO2 level. CO2 should be at 5 percent!
hi Jun, please check the antibiotics and the composition of the media as the media might be lacking some of the content essential for the growth of the cells. Please prepare your media properly and also take care of proper trypsinization. Check the concentration of the the antibiotics.
Hello Jun, when you say, that this problem is not cell type specific and you have already tried different media, serum, etc., then it could be the incubator. You should probably check the CO2 level. CO2 should be at 5 percent!
Since the effect is on all your cells in culture and A549 is a very hardy line I would guess you have a mycoplasma infection. Consider checking for myco contamination and treating with plasmocin if necessary. Myco cannot be seen under normal light microscopy and does not accumulate visibly in the media like a bacterial contamination and it can severly impair metabolism and auto/paracrine signalling over time.
In contrary, mycoplasma do affect cell growth and vitality depending on contamination grade. But before you try something like plasmocin, test your cultures on mycoplasma by PCR - it is the most sensitive method. If tests are negative, I would agree with Thomas Lettner and advice you to check incubator for other possible contamination and CO2 concentrations.
Hi, I agree with evryone here that mycoplasma may be the problem. The PCR kit for mycoplasma detection works very well. If you r cells are clear then its probably the incubator, you should check the calibration it can be telling you that it is 5% when it is actually 10% or more. You need a CO2 meter. Same for the temparature is it actually 37oC or less or more? Do you have a line feed into the system or run off gas tanks? Has the pressure been turned up. Is the incubator humidifed with a water pan (use only autoclaved tap water not deionized or distilled ). I would call a service engineer before you order more cells. Hope things work out
Hello Jun, its me again. I agree with the colleagues in this forum. Mycoplasma is a big suspect in this matter. A PCR test for mycoplasma is quick and easily done. If it is really mycos then your liquid nitrogen tank is probably also contaminated, and all your cell lines that you keep in there are in danger. Mycoplasma perfectly survives in liquid nitrogen, so dont forget about the tank as the source of contamination.
Hello, two things you should check.
1. Mycoplasma contamination QC PCR.
2. Level of CO2 in the incubator.
Regards,
Sanjay H
Hello Jun, If the liquid nitrogen tank is indeed contaminated with mycoplasma then there is not much that you can do. In the worst case scenario you will have to replace the entire thing, sorry! Therefore, I hope that this is not the case.
I honestly dont know of any method to decontaminate a nitrogen tank. May be there are some companies that can do that. May be the company that manufactured the tank has some ideas about getting it clean. If not, then get a new one, fill it with fresh N2 and make sure, that it stays clean.
Mycoplasma and other contaminations usually come from primary cells and from biopsy material. Also cells that you get from a friend (another lab) are often contaminated. Cell lines that you have purchased from a company are usually perfectly clean. Bovine and fetal calf serum can also be a source of contamination. And last but not least, the person who works with the cell cultures and is in charge of the cryoconservation is also a major source of contaminations.
A little hope for you: If the N2 tank is contaminated that does not necessarily mean that you have to buy a new one. If you have very important and valuable cell lines, store them at -80°C (at least below -70°C) for the time being. I have stored human cell lines at -80°C for many month and they were still perfectly OK. If it is really a contamination problem then you have to get your cell cultures clean first (and dont forget to clean the incubator). Plasmocin was recommended here in the forum. We use Primocin. Primocin destroys all prokaryotes but has no effect on mammalian cells. Change the medium very often, wash thoroughly with sterile PBS, add Plasmocin/Primocin and hope for the best. If the mycoplasma contamination is really bad, then you can see it under the microscope (at least I can at 200x magnification, very little tiny tiny black dots), and sometimes the culture medium gets very cloudy.
When you have treated your cell lines with Primocin and the PCR confirms that there is no mycoplasma contamination any longer, then store the cells at -80°C. The cell culture textbook recommends to use cryovials with a "male" cap and a coarse thread. It is not recommended to use cryovials with a "female" cap and a fine thread. Make sure the cap sits tight. After having them stored at least over night at -80°C you can put them into the liquid N2 tank EVEN IF THE TANK IS CONTAMINATED. If the cryovial is sealed tight, then you dont have to worry too much about mycoplasma contamination in the N2 tank. When you take a vial from the N2 tank in order to thaw cells and bring them into culture, make sure you dont carry contamination from the N2 tank to the cell culture hood. Clean the vial with EtOH, clean the hood with EtOH, change the gloves as often as you think its necessary and also sterilize your gloves between single working steps. This should help to keep your cell cultures clean.
Thats the best advice that I can give you so far. I really hope that it is not a mycoplasma contamination. I hope for you that it is only the CO2 level in the incubator, because this problem is much easier to solve.
Greetings!
Tom
There are some companies that provide anti-mycoplasma spray and solutions for decontamination of incubator and flow. I never tested them myself, but seen some at the exhibition during conferences. I think, that can be a solution for you if you clean the tank.
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