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Incucyte® 3D Tumor Spheroid Assays

Spheroids Showcase

Effectively Modeling Tumors In Vitro

In vitro culture models of cancer cells are integral to pre-clinical drug development and advancing our understanding of cancer cell biology. While still relatively new compared to traditional 2D monolayer cultures, 3D cell culture models have emerged as advanced tools to accelerate drug discovery efforts.

It is well established that conventional monolayer cultures of tumor cells on plastic do not adequately reflect the in vivo situation:  Cancer cells within a solid tumor are part of a complex 3-dimensional (3D) structure with a specific organization and cytoarchitecture. Tumor cells interact with each other and with the surrounding microenvironment. These complex interactions in combination with the unique properties of a 3D structure affect tumor cell properties and behavior, gene expression, distribution of proteins, and ultimately, response to anticancer drugs. Thus, 2D culture models have limited value for investigating tumor pathophysiologic pathways or predicting the potential efficacy of drug candidates.

To develop tumor models that better mimic the complexity and heterogeneity of clinical tumors, 3D culture methods have been developed in which tumor cells are grown as multicell aggregates, or spheroids. There are a multitude of approaches to generating these models, and can generally be described as liquid-based or scaffold-based and detailed elsewhere1. Liquid-based models are easily achieved using round-bottom ultra-low attachment (ULA) microplates to promote spheroid self-assembly. These models, generally yielding a single tumor spheroid per well, exhibit key features of solid tumors, with larger spheroids consisting of proliferating, quiescent, and necrotic zones resulting from a radial gradient of nutrients, metabolites and oxygen2. Scaffold-based models used today primarily rely on an extracellular matrix (ECM) such as Matrigel or collagen to recapitulate both physical and biochemical characteristic of the tumor microenvironment. Generally, these models are generated using flat bottom plates and result in multiple tumor spheroids per well.

  1. Thoma et al. 3D cell culture systems modeling tumor growth determinants in cancer target discovery. Adv. Drug. Deliv. Rev. 69-70, 29-41 (2014).
  2. Vinci et al. Advances in establishment and analysis of three-dimension tumor spheroid-based functional assays for target validation and drug evaluation. BMC Biol. 10, 29-7007-10-29 (2012).

However, many available three-dimensional techniques for generating and quantitatively analyzing spheroids are time consuming, laborious, costly, and/or lack reproducibility, prohibiting the effective utilization of these more stringent and representative in vitro tumor models.

Introducing Incucyte® 3D Tumor Spheroid Assays

Effective analysis of 3D tumor spheroids can be challenging. These heterogeneous models are multi-faceted and dynamic. Traditional plate reader assays are endpoint and lack multiple aspects of image-based analysis, including morphological information and ability to confirm data within images. Conventional imaging systems are inherently difficult to adapt to kinetic analyses of in vitro culture models, suffering from:

  • Incomplete data: Missed information between imaging intervals
  • Multiple uncontrolled environmental fluctuations: Repeated transportation from the incubator to the imaging system and lengthy 3D image acquisition protocols outside the incubator leading to temperature differentials and loss of control of oxygen and carbon dioxide conditions
  • Time-consuming development of optimal image acquisition parameters
  • Complex image processing requiring expert operators to generate quantitative information

Incucyte® 3D Tumor Spheroid Assays offer an integrated turnkey solution to automatically monitor and quantify tumor spheroid formation, growth and health in real time inside your tissue culture incubator.

Choose from two assays depending on your scientific questions:

Single Spheroids

Incucyte Assays for Scaffold-Free Tumor Models (Single-Spheroid)

An integrated solution to automatically track and quantify tumor spheroid formation, growth and death in real time and in 96- and 384-well formats — inside your tissue culture incubator.

Learn more     Watch the video

Multi Spheroids

Incucyte Assays for 3D Scaffold-Based Tumor Models (Multi-Spheroid)

An integrated turnkey solution to automatically track and quantify tumor spheroid formation, growth and health in real time inside your tissue culture incubator.

Learn more    Watch the video

Incucyte 3D Tumor Spheroid Key Advantages


Derive more physiologically relevant information

Quantify label-free growth and investigate morphology of 3D tumor spheroid cultures – while your spheroids are growing undisturbed inside your incubator


Reveal cellular changes over time

Investigate mechanisms of action with real-time viability measurements using non-perturbing reagents


Generate reproducible, quantitative data

Lab-tested protocols, high quality images, and unbiased analysis deliver robust data suitable for pharmacological analysis


Unlock your productivity

Automatically acquire, analyze, and graph thousands of images from up to six 96/384-well plates in parallel and get to answers faster

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