Morphological characterization of 3D cell cultures generated by liquid overlay technique

Cultivating cells in 3D is considered a significant advancement in cell culture models, as it better reflects natural cellular environments compared to 2D cultures. However, analytical methods like standard light microscopy are less effective for 3D cultures. In this study, 3D cell cultures were generated using the liquid overlay technique with 10,000, 50,000, 100,000 and 200,000 Normal Human Dermal Fibroblasts, analyzed on days 1, 2, and 3 post-seeding. We quantified the influence of fixation with paraformaldehyde or glutardialdehyde/dehydration on their morphology compared to living 3D cell cultures. They were analyzed by light microscopy, scanning electron microscopy as well as by digital light microscopy (height profile measurement). Over time, the cultures decreased in size, likely due to cell shrinkage and structural reorganization. The size reduction could be mathematically described by an exponential decay function. The proportion of round spheroids versus indented aggregates depended on cell number, culture age, and fixation method. On day 1, cultures seeded with 10,000 cells formed nearly 100% round spheroids, regardless of fixation. Higher cell numbers led to fewer round spheroids, and fixation further reduced their number. This suggests that large cell quantities sediment in layers due to steric hindrance, forming indentations. Since aldehydes are responsible for cross-linking proteins, we hypothesize that this chemical reaction, combined with low stability of the 3D cell cultures, leads to the increased formation of the indented 3D cell aggregates. This is consistent with an overall increase in the number of round spheroids and a decrease of the negative influence of fixation over time. In summary, it is important to consider the number of seeded cells, the incubation time, as well as the possible fixation effects when generating stable spheroids using the liquid overlay technique for down-stream experiments.