Reproducing the Tumor Microenvironment in 3D Culture Models

In conventional 2D culture platforms, tumor cells are grown in monolayers on non-biological rigid surfaces in excess culture medium, producing hyperoxygenated and hyper-nourished cells with unrestricted and nonphysiological proliferation characteristics. As a consequence, drug screens in 2D platforms primarily identify agents that target a uniform population of proliferative cells and overestimate drug efficacy.

A more realistic setting for drug screening in vitro involves recreating the physiologic heterogeneity inherent to a 3D tumor structure and providing a microenvironment similar to in the in vivo situation, which includes key interactions between the tumor and the extracellular matrix (ECM).

  • ECM provides physical and functional support for cell survival, expansion and tissue integrity
  • ECM deposited by tumor cells produces a physical barrier to drug penetration and distribution
  • Tumor cells modulate the ECM through the release of growth factors and factors that facilitate tumor cell migration and invasion

Scaffold-based 3D spheroid models, in which tumor cell aggregates are grown in ECM scaffolds, such as Matrigel®, recapitulate 3D physiologic growth and interactions of tumor cells with the microenvironment. Tumor cells grown in 3D scaffold-based culture form cell-cell and cell-matrix interactions, via cell-cell junctions and biochemical and biomolecular signaling pathways. Co-culture of different cell types, such as cancer cells and fibroblasts, can be employed to investigate interactions between cell populations.

  • Use of ECM mimics tumor microenvironment
  • Cells never come in contact with a non-biological surface
  • Suitable for co-culture and patient-derived cells


Introducing Incucyte® 3D Multi-Tumor Spheroid Assays

Effective analysis of 3D scaffold-based multi-tumor spheroids can be challenging. Traditional plate reader assays 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 due to various factors:

  • 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 Multi-Tumor Spheroid Assays offer an integrated turnkey solution to automatically track and quantify tumor spheroid formation, growth and health in real time inside your tissue culture incubator.

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Key Advantages

Derive More Physiologically Relevant Information

Quantify label-free growth and investigate morphology of 3D multi-tumor spheroid cultures on Matrigel® – inside your incubator

Figure 1. Monitor spheroid number and size over time while spheroids grow undisturbed inside your tissue culture incubator. MCF7 cells plated on a layer of Matrigel® and treated with and without 1µM Camptothecin, imaged over 7 days.

Figure 2. Reveal morphology with high quality HD phase and DF Brightfield images. Easily distinguish between round and stellate morphologies as exemplified here in MCF7 and MDA-MB-231 spheroids. All images captured at 10x magnification.

Reveal Cellular Changes Over Time

Investigate mechanisms of action with real-time multiplexed viability and toxicity measurements using non-perturbing reagents.

Figure 3. Establish cytotoxic vs cytostatic mechanism of action with kinetic, multiplexed growth and health measurements. A549-NucLight Red cells (2K cells per well) were seeded in the presence of Incucyte Annexin V Green reagent (1%) and spheroids were allowed to form  for 3 days. Spheroids treated with a range of CMP or CHX concentrations, or vehicle control, were imaged every 6 h for 7 days. Brightfield (top row), Incucyte Nuclight Red (nuclear-restricted FP indicating viability, red fluorescence, middle row), and Incucyte Annexin Green (apoptosis marker, green fluorescence, bottom row) images are compared at day 4 post-treatment. A loss of total integrated red intensity, lack of growth and a simultaneous increase in Annexin V green mean fluorescence intensity was observed in CMP treated spheroids. Despite CHX inhibiting spheroid growth, RFP expression remained high, while little or no increase in Annexin V fluorescence was observed, suggesting minimal cell death as expected for a known cytostatic drug such as CHX.

Figure 4. Derive EC50s to confirm mechanisms of action. Both CMP (cytotoxic) and CHX (cytostatic) drugs caused a concentration-dependent inhibition of spheroid growth (Total BF time courses, not shown). CMP EC50 using AUC from 0-7 days after treatment shows concentration-dependent loss of viability and increase in apoptosis, while CHX EC50s show little change in viability and no increase in apoptosis.

Generate Reproducible, Quantitative Data

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

Figure 5. Incucyte’s lab-tested Multi-Tumor Spheroid Protocol reduces time spent troubleshooting 3D cell culture techniques and eliminates the need for a trial-and-error approach to obtain images suitable for quantitative analysis.

Figure 6. Incucyte’s proprietary image acquisition technique, DF Brightfield for 3D Cultures, generates high contrast, extended depth of focus images when used in conjunction with our Multi-Tumor Spheroid protocol. Subsequent segmentation and analysis with Incucyte’s proprietary image processing algorithms results in robust data free from operator bias.

Figure 7. Perform robust, reproducible pharmacological analysis in physiologically relevant conditions. MCF-7-NucLight Red spheroids were allowed to form for 3 days prior to 7 day treatment  with known cytotoxic compounds. Time-course plate views enable rapid visualization of treatment effects on both spheroid size (Total BF Area)  and viability (red FLU Intensity within BF Boundary). Concentration response curves represent area under curve (AUC) analysis of the time-course data 0-7 days post-treatment. All compounds caused a concentration dependent inhibition of growth and viability with rank order of potency CMP > CHX > OXA.

Unlock Your Productivity

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

Figure 8. Guided interface is easy to use for even first-time users. Automated image acquisition and analysis tools provide a ‘set up and walk away’ experience. View images remotely to monitor experimental progress and analyze in real time for rapid decision-making.

Ordering Information

Ordering Information



Cat. No.

Incucyte® Spheroid Analysis Software Module

1 module


Incucyte® Nuclight Green Lentivirus (EF-1a Promoter, Puro selection) Nuclear Labeling Reagent

1 vial (0.2 mL)


Incucyte® Nuclight Red Lentivirus (EF-1a Promoter, Puro selection) Nuclear Labeling Reagent

1 vial (0.2 mL)


Incucyte® CytoLight Green Lentivirus (EF-1a Promoter, Puro selection) Cytoplasmic Labeling Reagent

1 vial (0.6 mL)


Incucyte® CytoLight Red Lentivirus (EF-1a Promoter, Puro selection) Cytoplasmic Labeling Reagent

1 vial (0.6 mL)


Incucyte® Caspase-3/7 Green Apoptosis Reagent

One vial (20 µL)


Incucyte® Annexin V Red Reagent

One vial (100 tests)


Incucyte® Annexin V Green Reagent

One vial (100 tests)


Incucyte® Cytotox Red Reagent

Five vials (5 µL)


Incucyte® Cytotox Green Reagent

Five vials (5 µL)



Literature and Documentation

Application Note

Validation of Real-Time, Live-Cell Assays for 3D Multi-Spheroids Formed on Bio-Matrices

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Application Note

Real-Time Live-Cell Analysis of Multi-Spheroid, Co-Cultured, 3D Tumor Assays

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Incucyte® Multi-Spheroid Assay Protocol

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Incucyte® Single-Spheroid Assay Protocol

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Poster: 3D Spheroid Growth and Shrinkage Assays

Simplifying High Throughput 3D Spheroid Growth and Shrinkage Assays Using Live Content Imaging

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AACR 2018 Poster

Development and optimization of matrigel-based multi-spheroid 3D tumor assays using real-time live-cell analysis

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EACR 2018 Poster

Development of multi-spheroid co-culture 3D tumor assays using real-time live-cell analysis

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Related Applications


Spheroid Growth

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Proliferation Assays

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cytotoxicity assays

Cytotoxicity Assays

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