Despite their advantages, spheroids also have some limitations. One of the main challenges is the difficulty in maintaining uniform size and shape, which can lead to variability in experimental results. Additionally, spheroids are less accessible for certain types of analyses, such as imaging and biochemical assays, compared to 2D cultures. Ensuring adequate nutrient and oxygen supply to the innermost cells is another challenge, as larger spheroids can develop necrotic cores due to insufficient diffusion.
