I noticed that spheroids vary depending on the cell type. When I seed the same number of cells for different types, the resulting spheroids are of different sizes. I need to understand whether the size of the spheroids is a critical factor in assays or if there are any established guidelines regarding optimal spheroid size.
Hello Gisela Leticia Isasi Rivas
You have observed rightly. When you seed the same number of cells for different cell types, the resulting spheroids are of different sizes. This is because different cell types will have different proliferative rates, which means a higher proliferative rate can lead to big sized spheroids. So, spheroid size, shape, and density/opacity will vary for each cell line. The spheroid size can range from 200µm to 850µm.
The size of the spheroids is a critical factor. Large spheroids have more significant molecular (nutrient, metabolite, drug, probe, etc.) gradients that can affect the apparent phenotypic outcome of the drug treatment. Thus, in such case, the lesser is the chance of the cells placed in the core zone to gain access to nutrients and drug as well as there is a high probability of them being under hypoxia.
The optimal spheroid size will depend on the assay requirements. For instance, the presence or not of a hypoxic core, So, when performing long-term spheroid monitoring, special attention must be paid to cell seeding density and growth rate. These two parameters determine spheroid size. The size is also limited by the instrument imaging capacity. Considering that spheroids are not always perfectly centered within the wells and that spheroids disrupted by drugs may have an irregular shape, this size limit must be smaller (
Hello Gisela Leticia Isasi Rivas
The optimal size of a tumor spheroid for drug screening assays typically ranges from 200 to 500 micrometers in diameter. This size range allows for a balance between mimicking in vivo-like tumor architecture and ensuring sufficient diffusion of nutrients and drugs to the core of the spheroid.
Smaller spheroids (around 100-150 micrometers) may be more prone to rapid nutrient depletion in the center, affecting the relevance of drug responses, while larger spheroids (greater than 800 micrometers) may present challenges due to poor drug penetration, leading to gradients of drug concentration that complicate results. Therefore, the 200-500 micrometer range is often considered ideal for assessing drug efficacy, penetration, and potential toxicity in a more physiologically relevant manner.
Thank you very much Sara Osama. In this context, the number of cells will need to vary based on the proliferation capacity of each individual cell. Could you provide a relevant reference on this topic?
Yes of course here it is
Paine, T. M., et al. (2023). “Optimizing three-dimensional cancer cell culture systems for drug discovery.” Cell Systems, 14(1), 9-20.
• This review highlights advances in spheroid culture techniques, including the preferred size ranges for efficient nutrient diffusion and drug penetration, with particular attention to the 200-500 µm range as optimal for assessing drug responses.
Hello Gisela Leticia Isasi Rivas ,
You might want to check this fluorescent dye for spheroid labelling:
https://luminicell.com/products/cellaris-540-cell-labelling-kit-green
Cellaris is an AIE dye specifically designed for live-cell imaging. It is suitable for labeling both 2D cells (before spheroid formation) and 3D cells (after spheroid formation) due to its high biocompatibility and stability. The dye provides long-term fluorescence for up to 10 cell generations in vitro!
Hope this helps with your research 😊
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