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

Area of Science:

• Cell Biology
• Biotechnology
• Microscopy

Background:

• Three-dimensional (3D) cell cultures offer a more physiologically relevant model than traditional 2D cultures.
• Standard analytical methods like light microscopy face challenges in effectively analyzing 3D cell structures.
• Understanding factors influencing 3D spheroid formation and stability is crucial for reliable experimental outcomes.

Purpose of the Study:

• To investigate the impact of cell seeding density, culture duration, and fixation methods on the morphology and stability of 3D cell cultures.
• To quantify changes in 3D cell culture size and shape over time.
• To compare the effects of different fixation agents (paraformaldehyde, glutaraldehyde/dehydration) on 3D cell aggregate structure.

Main Methods:

• Generation of 3D Normal Human Dermal Fibroblast cultures using the liquid overlay technique with varying cell densities (10,000–200,000 cells).
• Analysis of cultures at 1, 2, and 3 days post-seeding using light microscopy, scanning electron microscopy, and digital light microscopy (height profile).
• Quantification of spheroid roundness versus indented aggregates under different fixation conditions and cell densities.

Main Results:

• 3D cell cultures exhibited size reduction over time, mathematically described by exponential decay, attributed to cell shrinkage and reorganization.
• The proportion of round spheroids versus indented aggregates was dependent on cell number, culture age, and fixation method.
• Higher cell densities and aldehyde fixation (paraformaldehyde, glutaraldehyde) reduced the formation of round spheroids, particularly on day 1, suggesting steric hindrance and protein cross-linking effects.

Conclusions:

• Cell seeding density, incubation time, and fixation protocols significantly influence the stability and morphology of 3D spheroids generated by the liquid overlay technique.
• Aldehyde fixation can induce structural changes leading to more indented aggregates, an effect that diminishes over time.
• Careful consideration of these parameters is essential for reproducible results in downstream applications utilizing 3D cell culture models.