Hello everyone.
I'm trying to form spheroids fom hct116 cells. The first time I did the experiment I could see a defined edge around the spheres and they looked tight. Now all I get are those agregates in the picture. Are those true spheroids? I'm sorry for the picture quality, it was the best I could take.
Thank you!
P.S: Here is the protocol I use:
- I coat the wells of a 96 well flat bottom plate with 50 uL of agarose 1.5%;
- I seed 800 cells per well in 200 uL of medium;
- Centrifuge 1000xg for 10 min;
- Incubate for 4 days.
These are aggregates of spheroids. Although, aggregation is a common hurdle within spheroids culture, it might be at a non significant level since it considerably results(hypoxia effect...).
You might try to generate new spheroids and reduce aggregation by avoiding plates motions at least for 4 days.
Good luck.
Best,
Hamadi
Hi, according to me, these are not spheroid. It looks like an unadhered cell monolayer. Did you mesure the thickness? By the way you can have a look at some of my posters, I'm working on 3D cell culture with hyaluronic acid scaffold and you'll see what are, according to me, multicellular shperoids.
I agree with Guillaume - this appears to be a rather loose aggregation of cells.
hct116 normally forms rather tight spheroids, so your observation in your first round was a typical one, while this time your cells seem to have a problem, they do not look too viable to me.
Your liquid overlay protocol seems quite ok to me. Centrifugation might be skipped, but should not be able to kill cells at such low g rates. Imaging in 96 well plates is usually not optimal because of the curved surface of the sulture medium - for better resolution under the microskope, you might use 48 well plates and double agarose, medium and cell quantities.
So I would just encourage you to try again with new cells.
Good luck,
andreas
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