Sequential orthogonal assays for longitudinal and endpoint characterization of three-dimensional spheroids

Area of Science:

• Biotechnology
• Cell Biology
• 3D Cell Culture

Background:

• Spheroids, reaggregated multicellular 3D structures, are crucial in research for both healthy and pathological tissues.
• Current spheroid analysis methods often lack modularity for comprehensive phenotyping.
• There is a need for adaptable protocols to advance 3D biology applications.

Purpose of the Study:

• To present a modular framework for multifaceted phenotyping of spheroids.
• To detail manual and automated procedures for spheroid generation and analysis.
• To provide a scalable and reproducible protocol for 3D cell culture research.

Main Methods:

• Self-assembly of single-cell suspensions into spheroids via the liquid overlay method.
• Development of a modular framework with orthogonal assays for longitudinal and endpoint analyses.
• Incorporation of manual and automated procedures for cell seeding, supernatant handling, and compound administration.

Main Results:

• A suite of phenotyping modules enabling longitudinal analyses (morphometry, supernatant evaluation, migration/invasion monitoring) and endpoint analyses (molecular content, single-cell composition, spatial architecture).
• Inclusion of time requirements and quality control indicators to ensure reproducibility.
• Demonstration of complementary techniques for advanced spheroid analysis.

Conclusions:

• The presented modular protocol facilitates multifaceted spheroid phenotyping.
• The framework is adaptable for researchers experienced in cell culture and molecular biology.
• This approach advances 3D biology by offering scalable and complementary methods for spheroid analysis.