I have been working with 3d spheroids for a few months. I started with HCT-116 colon cancer cell line and have not had any problems, this cell forms tightly aggregated spheroids. However, I have not been able to form spheroids from HT-29 cell line. I've tried different agarose concentrations (1-3%) and different cell concentrations (800-10.000 per well) and this cell line won't form the spheroids, just cell clumps. Does anyone have any idea what could be happening? I've seen in many papers spheroids from HT-29 and theorically they are formed easily. I use the following protocol for the HCT-116:
96 well plate flat bottom coated with 50uL of 1.5% agarose. I plate 2000 cells and centrifuge 1000rpm for 5 minutes. Then, I incubate for 4 days untill the spheroids are formed.
Thanks in advance.
Hello Gabriel,
This is indeed worrying, because, in my experience, HT-29 spheroids are very easy to form (and are usually even more uniform than HCT116 spheroids - still, both cell lines form fantastic spheroids).
Two possible explanations for your problem come to my mind:
1. There is something wrong with the cell line. In other words, you're not really using HT-29. I recommend checking when and where from the cells were obtained. Have they been authenticated by the vendor? Have you performed any authentication at your facility? Do you use suitable medium for your cell culture?
2. Coating is uneven. Check your coating protocol and try out some pre-coated plates (e.g. Corning Spheroid plates, available in both 96 and 384-well versions). Still, you say that you have no problems with HCT116, so I would suspect the cell line.
I hope this helps!
Cheers,
Wojtek
Dear Wojciech,
Thanks for your answer. We haven't done cell authentication yet, but we look forward to doing the STR profilling. I am indeed very worried about the cell line. I'll definetely try changing the coating protocol!
Thanks,
Gabriel.
Hi,
Infection might be a possible cause, and the agarose might not cover the whole bottom of the plate which makes the cells adhere in different sites in the individual plate.
Good luck,
Hi Gabriel,
There maybe a problem with your cell line. You need to check; what do the cells form instead of spheroid generation? do they form spreading layer through the agarose, then the agarose quality or preparation could be the reason. If you see multiple small spheroids here and there so the cell line and contamination could have a role in this. Using V or U shape bottom plate is more proper for this procedure as the cells would be efficiently triggered to be aggregated. If HT-29 is will known to form spheroids easily, then I will not advise you to use matrigel. Hope this can help and please feed us back in what would happen with you.
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