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

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Scope of the method

The Method relates to

Other

Characterization techniques

The Method is situated in

Translational - Applied Research

Type of method

In vitro - Ex vivo

This method makes use of

Human derived cells / tissues / organs

Description

Method keywords

Model characterization
Assay optimization
spheroids
Longitudinal analysis
endpoint analysis
phenotyping
automation

Scientific area keywords

3D in vitro models
In vitro analysis
Translational Research
cancer research

Method description

Here we present the self-assembly of single-cell suspensions into spheroids by the liquid overlay method, followed by a modular framework for a multifaceted phenotyping of spheroids. Cell seeding, supernatant handling and compound administration are elaborated by both manual and automated procedures. The phenotyping modules contain a suite of orthogonal assays to analyze spheroids longitudinally and/or at an endpoint. Longitudinal analyses include morphometry with or without spheroid or cell state specific information and supernatant evaluation (nutrient consumption and metabolite/cytokine production). Spheroids can also be used as a starting point to monitor single and collective cell migration and invasion. At an endpoint, spheroids are lysed, fixed or dissociated into single cells. Endpoint analyses allow the investigation of molecular content, single-cell composition and state and architecture with spatial cell and subcellular specific information. Each module addresses time requirements and quality control indicators to support reproducibility. The presented complementary techniques can be readily adopted by researchers experienced in cell culture and basic molecular biology. We anticipate that this modular protocol will advance the application of three-dimensional biology by providing scalable and complementary methods.

Method status

Published in peer reviewed journal

Pros, cons & Future potential

Advantages

This protocol describes the self-assembly of single-cell suspensions into spheroids by the liquid overlay method, alongside a modular framework of orthogonal assays to phenotype spheroids both longitudinally and at an endpoint. This method ensures production and long-term culture of spheroids with defined size, morphology and composition. In addition, the workflow addresses a comprehensive number of spheroid metrics including supernatant analysis.

Future & Other applications

The presented pipeline can be readily adopted by researchers working with other 3D models (e.g., patient-derived tissue fragments and organoids) by making small adaptations to fit the model.