I isolated endothelial progenitor cells (EPCs) from mice bone marrow and cultured them in a human fibronectin coated plate for 4 weeks. The cells grew well in the plate albeit there were some other cells except EPCs. But the cells didn't respond to the trypsin (0.25%) when I tried to typsinize them. Some of the cells could be detached while most of the cells were still on the plate although their morphology became round. Is there any method other than trypsinization I could use to passage my cells, such as degrading the fibronectin?
Do you need to coat the plates with fibronectin? Maybe polylysine would suffice and you maybe need to pipette up and down with a narrowed Pasteur etc...
Check the activity of your trypsin. Use a fresh thawed vial. In my experince cells which are growing for a long time without subcuring are more difficult to trypsinize.
I used to work with human EPC (and usually I coated them on collagen IV) but maybe the concentration of your fibronectin is too high and then the cells are very well attached to it !! An other thing, as Francisco said your trypsin must be fresh but most importantly the time of tryspin-treatment must be longer since your cells were growing for 4 weeks !!
Best of luck.
Try washing before trypsinising better/more and trypsinise longer with tapping the dish from time to time
Yes, the longer is the time of growth without trypsination, the longer is the time necessary to detach the cells! Try to use EDTA+Trypsin (MEC fibers are protected against proteases by Ca) and obviously, fresh trypsin.
Hello,
You can also try cell scraping. There are special plastic scrapers that you can use. In such case, you don't need enzymatic detachment.
If it's only possible to do it with trypsin you should check your trypsin activity, prolongate the incubation time at 37°C and it's necessary to wash the cells prior to trypsinisation.
Good luck!
If you do not want mechanical stress by scraping and trypsin for too long is also not a way, try dispase - it cleaves fibronectin
Thanks Samia. I tried to scrape off the cells after 2mins incubation with 0.025% typsin + EDTA, the cells were dead and became debris. Before this, I tried to scrape off another cell line, it did work well.
Also, according to Damir's advice, I tried to incubate cells with typsin + EDTA for around 8 minutes, half of the cells were still unable to detached and the attachment of those typsinized cells were not good neither.
I wonder what if I typsinize the cells after 2 weeks instead of 2 weeks? I'm also considering to use dispase.
Généralement les cellules adhèrent de manière plus forte sur le plastique que sur le verre. je vous propose d'utilser des lames LAB TEK , vous détacherez les cellules avec de la trypsine ou trypsine EDTA ou vous pourriez utiliser le scrape ( proposée par ROUROU) , la difficulté est de savoir comment mener ce travail sans contaminer les cellules. Aussi, les cellules se détachent plus facilement quand elles sont denses et quand elles ont plusieurs jours de culture.
bonne chance
M. Brahimi
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