I am doing 3D tissue culture. These black dots show up in my spheroids from time to time, please take a view of the picture below. Does anybody know about these black dots?
Hi Yimeng Guan
If you are culturing your spheroid for long time, it is the dead cells present in the core which show up in form of black dots.
I agree with Swarali SUHAS Joshi they are likely necrotic regions, caused by poor nutrient diffusion. Cancer cells rely primarily on glycolysis for energy so can essentially survive indefinitely without oxygen. However, this is not the case with other nutrients like glucose, amino acids etc.which have a diffusion distance limit. Cells closer to the periphery grab these nutrients first, leaving little for cells deeper in the spheroids.
You should not make the sphere too big. If it is, nutrients from the medium cannot reach there and cells in the middle become apoptosis and death. You need to keep your sphere to be around 200 um the most. Even though 200 um, you cannot keep too long too. You need to dissociate them and seed them again to form your spheres again and again until the qty you want.
Thanks for your answers. The architecture of spheroids is indeed our model: invading zone - proliferating zone - G1-arrested zone - necrotic core. Yimeng was not asking about the central necrotic core. It was about a random distribution of 'circular black structures'. Yimeng, you should upload some example pictures.
We now know that these black dots are solid structures, potentially of crystalline nature. We still don't understand their origin, but believe that the medium and/or the agarose, on which the spheroids are formed, play a role.
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