My mouse embryonic stem cell line are not working. They are not able to attach to the surface of coated dishes ( I have tried both Geltrex and 0.5% gelatin) remain in suspension. I am using ESGRO LIF. Please help
Media composition = KO-DMEM+15% FBS+1xGluatmax+1xNEAA+BME+PS
Hi Saras,
I'm not sure if this helps, but I can tell you how our group cultures ES cells:
DMEM containing 15% FBS, 0.1 mM MEM non-essential amino-acids, 1 mM sodium pyruvate, 2 mM GlutaMAXTM, 0.1 mM 2-mercaptoethanol, and 1000 U/ml LIF. Further medium supplement with two inhibitors (2i), 3 μM CHIR99021 and 1 μM PD0325901.
We generally use 0.1% gelatin.
Hello
I agree with Virlana M Shchuka , Kindly look at my research and PDF here
Good Luck
Article Ultrastructural Characteristics of Three Undifferentiated Mo...
Hello Saras,
Firstly are your cells not attaching after trypsinization or post revival. One reason could be too much trypsinization can affect the attachment. In both scenarios check with trypan blue if your cells are alive. I think the problem is with cell viability, your viability staining will give you some answer.
Wish you the best.
Thank you.
Regards,
Prasad.
Thanks, Prasad Sir for replying
Yes, I have done viability with trypan blue. It's after revival they are not attaching. Viability was less, they attach as single cells, not as colonies.
Thank you Virlana M Shchuka Yes I have used this composition.
Thank You, Saleh Alkarim I will go through the article
Dear Saras, we had similar problem but with different cell type and as Prasad said, it could be a viability problem. I also advise you to get in touch with Dr Gauthaman Kalanegam at [email protected] and he could give you more advises, best.
Hi Saras,
Here is the media I used when working with mES:
Ko-DMEM;
Ko-Serum 15%
Nonessencial amino acid 1%
L-glutamine 2mM
Β-Mercaptoethanol 50mM 0.1mM
LIF 1000 UI
We used 0.1% Gelatin (Sigma) and Esgro Complete Accutase to dissociate cells.
Hi Saras!
In my experience, mouse embryonic stem cells begin to differentiate when attaching. It could be a big benefit not having attaching cells! 3D culture is better anyway. If you would like to get more information, please write me a mail to [email protected].
Warm regards,
Patrick
You should start by checking your FBS. Always use ES-qualified FBS. We have had batches of regular FBS that do not promote adhesion of ES cells. Different lots of ES-qualified FBS still give variability, but at least they are screened to confirm adhesion.
You should also check how the ESCs were cultured before they were frozen. If they were on MEF feeder cells, you will want to thaw them on feeders. If they were adapted to feeder-free, confirm the matrix. There will be less stress to maintain the same conditions they were adapted to before frozen.
Confirm your final concentration of b-mercapto. I have had techs use the wrong bottle at the wrong dilution and wonder why the cells are not attaching.
ES cell culture is less forgiving than other cell cultures. If you are still having problems, find a PI or mentor for more hands-on training and practice.
They are several ways to cultivate mouse ESC. Therefore you should check how the cells were cultivated before and follow the same protocol (ie with serum or KSR, with or without feeders, with or Without 2 inhibitors, what kind of basal medium,...)
FCS lot is crucial, as well as the feeder density, number of cells par cm2, bME concentration, sanitary status, confluency at passaging,....and yes if you are new in the fiels of ESC, please find a mentor around you as mentioned by Robert Cabrera.
In my experience, this generally happens when you are moving from feeder-dependent culture to feeder-free culture. The cells do not attach properly on gelatin. This is specially true if you are just reviving the cells after receiving from ATCC, etc where feeder-dependent culture was done.
In this case, split 1:2 or 1:3 and do it regularly. Initially the colonies will form and start floating. Do not worry and try to change the media. After 5-6 passages, the cells will start adhering better on gelatin.
I hope this helps.
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