I got embryonic stem cells 1 month ago and cultured them according to the protocol from its company. After 3 X making them passage, they start growing very slowly and not confluent well as we always expect. Everything is optimized and it shouldn't be existed any problem but this time I face to the problem. Does anyone know how I can overcome to the problem or have any suggestion to improve cells growing. I should mention that I use cell medium from company for seeding and growing the cells as well which needs to keep pluripotency of cells . I would be appreciated if anyone give me a clue to overcome the problem.
If you use feeder free condition for your cells maybe you can add bFGF in orfer to feed, support pluripotency and proliferation.
Dear Nadia I use the medium without FBS. I feed the cells by supplement that contain GF.
i used to culture hESC in 20% KO-serum replacement, 5% l-glut, 5% NEAA and 4ng/ml bFGF and 910ul b-mercapto in KO-DMEM which had been previously cultured on MEF after 24hrs on MEF we then added a further 4ng/ml bFGF and filtered prior to use on hESC.
Hello Mandana
Can you provide a little bit more info:
-Which cell line is it?
-How many cells are you plating and what is the code of the plate?
-What is the media formulation?
-Are they grown in Mefs, gelatin or matrigel?
-Can you provide pictures?
Thank you
Hi,
I suppose you are working with human ESCs under feeder- and serum-free conditions.
If this is the case: take care with handling medium and supplements as KO-DMEM and KO-serum replacement: they are extremely unstable and thermo-labile: never defreeze KO-SR at 37° (leave it at RT or o/n in the 4°), once complemented, never over-warm or warm the KO medium several times: don’t use the entire bottle but use instead small aliquots. If you need few ml, put them into a falcon tube and warm it up few min at 37°C.
Moreover: which type of detaching reagent do you use? If you don’t use FBS (that inhibits trypsin and co), you must centrifuge the detached cells in order to physically remove the detaching reagent. Otherwise, even if diluted it is still active and it will cause irreversible cell membrane damages in your cell cultures....
All the best,
Laura
Hello,
Could you maybe provide more Information about the cell line you are using? Are they human or mouse and which strain? Do you have to grow them on a Feeder layer or gelatin?
If you can provide more details about your cells, we can all help you in a better way!
You should also make sure that you transfer sufficient numbers of cells, otherwise they will grow slower or might even die.
In case you dissociate them to a single cell suspension, count them and optimize the density. Density is also a bit cell line dependent. For black6 mES i used ~40.000 cells per cm2 in 6 cm dishes with 3-4 ml medium, and passaged every 2-3 days. For another mESC line i used 26.000 cells per cm2.
For hES that may vary!
I am agree with Martin , ...But may be you should check mycoplasma infection .
Best
Hello
I am agree with researchers .... add for that , lots of debris in the medium can affect the growth of the ES cells. also embryonic stem cells should always be passag to a plate of feeder cells less than seven days old. Check the feeders before use to be sure they are healthy The Feeder Medium should be aspirated from the plate and replaced with ESC medium prior to use.
Thank you for all useful tips that you gave me. Now, It's working out and the cells start growing well. It supposed that it was caused by supplement from company because we used first seeding cells on supplement from company and then changing in differentiated medium for transplantation. Supplemets from company doesn't include many feeders for keeping pluripotency that's why they were growing slowly and I was worried to decline cell number on plate and also growing time but solved it now.
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