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Spheres adhere in non-adherent conditions


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#1 Rute

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Posted 23 January 2012 - 03:25 AM

Dear all,

I am facing a problem which a cannot explain and I would very much appreciate your help.

I am culturing putative stem cells in non-adherent condiiton to grow as spheres.
I successfully obtained spheres which I was expanding. However, all of the sudden I started to have these circular strucutures at the bottom of the low attachemnt culture flask.
It resembles an adheren culture.

What I believe happen is that a shpere started to adhere and formed a circular monolayer attached culture.

Has anyone came across this problem and knows how to explain the phenomenon?

Thank you for your help.

Cheers.

#2 Tabaluga

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Posted 30 January 2012 - 12:29 PM

Which tissue are the cells from ? Is it just a few spheres that become adherent or is it a great percentage ?
I'm working with glioblastoma stem cell spheres, and the phenomenon you describe can happen, usually to a few spheres, when they have been in the same flask for several passages. Do you regularly move them to a new flask ?
By the way, spheres becoming adherent is an early step of differentiation, but I suppose there is nothing which could have induced differentiation in your cells ?

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#3 Rute

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Posted 31 January 2012 - 06:07 AM

Thanks for the reply.
I am working in pancreas so i am using the adult tissue and wait for sphere formation.
They have formed spheres within 3 to 4 days (whch i find amazing) and by the 7th or 8th day some of them have started to adhere. I haven't split the spheres, the only thing i did was to supplement the culture with a litle bit of more media.
I thought this could be media saturation so i slightly centrifuged the spheres and gently ressuspended them in fresh stem cell media.
This was they are know in culture for 15 days and all od them are adhering.
Do you have any advise??
I have previously worked with lung stem cells and this did not happen. I don't know if it's smothing characteristic of pancreatic tissue or if i;m doing something wrong.

Tks




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