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Keratinocyte Culture: Cells detach - (Jan/20/2014 )



I am currently culturing primary human keratinocytes. Until last week it always worked rather well. But since last monday, the cells in the culture flasks started to spantaneously detach. There are still lots of cells attached and prloiferating, but this slows everything down enormeously.

After splitting and passaging only about half of the cells actually seem to attach at all. This is especially annoying for my assays, as it takes much much longer for cells to get confluent and to get valuable results. Another phenomenon is, that in some flasks all of the keratinocytes seem to differentiate.

All my cells are in first to 4th sub-culture, so not too old.
The problem is that it all started so abruptly. Before cell attachment was just fine.

I am using Serum Free Keratinocyte Medium 2 from PromoCell (with Supplement, Streptomycine+Penicilline and CaCl2), and Falcon T75 Flasks (we never coated them). Medium is changed twice a week. No bacterial contamination is visible. I also tried a new bottle of medium, but I cant really say if things are getting better now. I know that calcium induces differentiation, but I am using this CaCl2 supplement since november (the producer implies that the addition to the serum is required for effective cell growth).


Has anyone else ever experienced this? Any ideas whats happening?


Thanks a lot!


Any ideas or tips? :(




I observed some other phenomenons as well:


Fibroblasts start to emerge and start to grow rampant. They were absent before. I would say before the keratinocytes used all the nutrients of the specific medium, and now as keratinocytes seem to stop doing anything there is enough for the once suppressed fibros.


Besides many differentiated keratinocytes, I also observed vacuoles inside many of the keratinocytes, which could be a sign for elevated apoptosis?


On ResearchGate I found a similar thread:
So after reading it I asked the manufacturer of my medium if they changed any ingredients, but they negated it.


Thanks a lot!


Hmmm...have you switched flask manufacturers recently?  Or anything else?  Trypsin, pipets, batches of media, batches of calcium chloride?  If you have some of old previous batches/lab supplies that worked fine, try going back to using them and see if the problem clears up.


I have had issues with muscle cells (permanent lines) that would occasionally just start dying.  We were never able to really definitely nail down the cause.  It may have been due to seasonal fluctuations in the water quality, since it always seemed to happen at the same time of year.  It was old school lab:  we washed the glass bottles and glass pipets in the water and used distilled water from the tap to make our own media.  So it could have been a pyrogen or something in the water.  Finally resorted to using sterile water for irrigation to make media and switched to plastic disposable bottles and pipets.


I looked at the website you referenced above.  They have lots of good ideas, especially about checking your cultures for mycoplasma.  Also evaluate the CO2 levels and temperature.


Good luck.



no everything remained the same. We only order 2-3 bottles of the medium at a time, because I am the only one working with these cells. The only time we use water on the cells is with PBS, but I dont know if the water for it is bought or from the tap. Well the PBS Flasks are washed under tap water too before being sterilized. The rest is "one use only"-material.


Co2 is normal at 5%, and the temperature remains at 37°C.


I dont know if mycoplasma testing would help, because the manufacturer guarantees that its free of microbial contaminants.


Thanks a lot.


Oh and I remember something else now as well perhaps it contributed to the development as well:

From the 28.12.13 until the 6.1.14 I didnt change the medium (I went home for new years eve), but I put 17 ml of medium into each flask instead. The assistant in the lab told me that should work. But the 6.1. was the day I observed all these problems first.

I just dont know how that might affect freshly isolated keratinocytes.


Thanks a lot.




Are you using explant technique or enzymatic ? Which protease did you use? Anyway this problem is common for keratinocytes culture. Trust me I have been culturing oral keratinocytes over a year, still could not obtain sustainable cell culture. There are many factors involved, especially when you dealing with biological sample, you might need to choose your sample carefully, I mean in term of age of patient etc


I'd rather perform your experiments using immortalized keratinocytes, such as HaCaT cells.  They are easier to work with.