Dear all,
Our lab works a lot with cells and since now we didn't have any problems with cell culturing. But since few months ago we can not do it right because cells are growing very slow and when the dish is confluent they are differentiated. We don't have any contamination and also we haven't got micoplasma. Any ideas to look for or what to do?
Dear Judit Bigas-Corominas
Since you have excluded any bacterial contamination you can look at several things. Check this list one by one.
1. Serum: if you are using FBS make sure you are using an aliquot just thawed from -20 deg C.
2. Media: make sure you are using optimum amount of glucose that keratinocytes require.
3. Incubator: make sure that the Co2 level displayed on the incubator is actually the same. Use a CO2 sensor to verify.
4. Cells: if it is an primary culture then cells will senescence after some passages, if you are using a transformed cell line then revive another cryovial.
5. seeding density: make sure you are seeding the cells at the recommended cell density.
6. Culture vessel: if using a t flask without air vent make sure that lid is slightly lose.
Hope this helps.
Regards,
Prasad.
Since you’ve ruled out common bacterial and mycoplasma contamination, perhaps there is cross-contamination going on between some of the cells that your lab is handling? Other than that, have you recently purchased or started using different materials and/or medium? Sometimes, different culture medium may incorporate different amount of growth factors which might be essential for your keratinocytes to thrive. Also, is this slow growth contained to just 1 of your keratinocyte cell line or all of the keratinocyte cell lines in your lab?
Hi, I dont' have experience with keratinocyte lines, when I read " the dish is confluent they are differentiated " I think you could work with muscle cell lines.
I have to exclude contamination as you said, but for mycoplasma you have to checked only with PCR, to be sure. You have experience with cells so excluding also old cells passage, it is possible that you thawn an aliquote that is not good, so checked all the aliquotes frozen in a certain period.
From my experience when something start to go wrong I also think about what I changed as reagent batch, incubator, if I have to buy some reagent from another vendor, if the vendor change something in buffer recipe.
Dear Judit Bigas-Corominas
Since you have excluded any bacterial contamination you can look at several things. Check this list one by one.
1. Serum: if you are using FBS make sure you are using an aliquot just thawed from -20 deg C.
2. Media: make sure you are using optimum amount of glucose that keratinocytes require.
3. Incubator: make sure that the Co2 level displayed on the incubator is actually the same. Use a CO2 sensor to verify.
4. Cells: if it is an primary culture then cells will senescence after some passages, if you are using a transformed cell line then revive another cryovial.
5. seeding density: make sure you are seeding the cells at the recommended cell density.
6. Culture vessel: if using a t flask without air vent make sure that lid is slightly lose.
Hope this helps.
Regards,
Prasad.
It seems to be a general phenomenon in cell culture. Please, consider my paper ("HepG2 Fucoidan") that normal Hc cells have 4.7% viruses together with bacteria and plant (Viral, bacterial and plant proteins are present at 18.6%), and hepatoma HepG2 has 6.6% viruses (bacteria is not present due to antibacterial antibiotics).
Other growth factors may be related.
Activations of purified human biotinidase at 128 and 150% have been observed at 50 nM mercuric ions and at 5 μM cupric ions, respectively (please see file; Thiol-type BIN). Therefore, the ancient Chinese-Emperor of Shi Huangdi (Qin dynasty; BC 3 C), who believes the “Taoism” and finds “the elixir of immortality by mercury”, may have possessed a good intuition. Although gold ion has not yet been tested on the thiol-BIN/LIP activity, an English philosopher of Roger Bacon (1214-1292) has already recommended the oral intake of gold as “the elixir of immortality” in his book of “Liber Sex Scientiarum”. The addition of Fe+++ at 250 nM in the cell-culture medium is interestingly also effective to fast growth (our unpublished observation), and the additions of Hg and Au ions to the culture medium should then also be re-studied (please see file again; Feed by Measure).
Effects of biotin (vitamin H; 20 μM, please see file, JCB Fucoidan transport), lipoic acid (240 μM, thioctic acid; toxic to rat cells), and oligo-saccharides such as nigero-oligosaccharide (0.1 mg/mL) on hastening the cell growth have been observed (my unpublished observation),
Thank you all. It seems there was a problem with cell freezing.
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