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

Key Advantages of Multi-Spheroid Assays

Generate Reproducible, Quantitative Data

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

Quick Guide: Multi-Spheroids on a Layer of Matrigel®

Figure 1. Incucyte®’s lab-tested multi-spheroid protocols reduce time spent troubleshooting 3D cell culture techniques and eliminate the need for trial-and-error approaches to obtain images suitable for quantitative analysis.

Derive More Physiologically Relevant Information

Quantify label-free growth and investigate morphology of 3D multi-tumor spheroid cultures on a layer or embedded in Matrigel® – as they remain undisturbed, inside your incubator.


MS on a Layer of Matrigel® (10x magnification)


MS on a Layer of Matrigel® (10x magnification)

MS Embedded in Matrigel® on a Base (4x magnification)

MS Embedded in Matrigel® on a Base (4x magnification)

Figure 2 (videos). Monitor multi-spheroid growth and reveal differential spheroid morphologies over time. MCF-7 and MDA-MB-231 cells were seeded (1K cells/well) and allowed to form multi-spheroids (3 days) on a layer of Matrigel® or embedded in Matrigel® on a base. Time-lapse videos monitoring spheroid growth over time (0 – 7 days post formation) demonstrate morphological differences between round (MCF-7) and stellate (MDA-MB-231) multi-spheroids.

Figure 3. Quantify cell-type dependent label-free growth profiles using real time analysis. MCF-7 and MDA-MB-231 cells were embedded in Matrigel® (1K cells/well) on a base in flat bottom 96-well plates and allowed to form multi-spheroids for 3 days. Segmented (yellow outline) DF-Brightfield images and corresponding time-courses illustrate cell type specific kinetic growth profiles and demonstrate the inhibitory effects of camptothecin on spheroid growth.

Reveal Cellular Changes Over Time

Investigate mechanisms of action with real-time multiplexed viability and toxicity measurements using non-perturbing reagents*.
*Fluorescence image acquisition only available using the Multi-Spheroid on a Layer of Matrigel® model.

figure 4

Figure 4. Establish cytotoxic vs cytostatic mechanisms of action with kinetic, multiplexed growth and health measurements. A549-Nuclight Red cells (2K cells/well) were seeded in the presence of Incucyte® Annexin V Green Dye (1%) and spheroids allowed to form for 3 days. Spheroids were treated with a range of camptothecin (CMP) or cycloheximide (CHX) concentrations and imaged for an additional 6 days. (A) Brightfield (BF, top row), Incucyte® Nuclight Red Dye (nuclear-restricted FP indicating viability, red fluorescence, middle row), and Incucyte® Annexin Green Dye (apoptosis marker, green fluorescence, bottom row) images are compared 4 days post-treatment. (B) A lack of growth (BF Area time-course), loss of total red intensity within the BF boundary (red fluorescence time-course) and a simultaneous increase in the mean green fluorescence intensity (green fluorescence time-course) was observed in CMP treated spheroids. Despite CHX inhibiting spheroid growth, RFP expression remained high, while minimal increase in green fluorescence, suggesting minimal cell death was observed as expected for a known cytostatic agent. CMP EC50 using AUC data from 0 - 6 days post treatment shows concentration-dependent loss of viability and increase in apoptosis, while CHX EC50‘s show little change in viability and no increase in apoptosis, supporting expected mechanisms of action. 

Figure 5. Perform robust, reproducible pharmacological analysis in physiologically relevant conditions. MCF-7-Nuclight red multi-spheroids were formed over 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.

Conduct Biologically Relevant Co-culture Assays 

Incorporate additional cell types to recapitulate the tumour microenvironment and investigate cellular changes over time.

Figure 6. Visualize and quantify the impact of stromal cells on tumor multi-spheroid morphology and assess immune cell-mediated toxicity within tumor multi-spheroids. (A) MDA-MB-231 cells were seeded in flat bottom 96-well plates on a bed of Matrigel® in mono or co-culture with NHDFs (1:1 ratio, 1K cells/well for each) and multi-spheroids allowed to form (3 days). Incucyte® DF-Brightfield (DF-BF) images compare mono and co-culture conditions over 3 days (6 days post cell seeding). Note the temporal impact of NHDFs on spheroid morphology. (B) BT474 cells stably expressing cytoplasmic restricted GFP were seeded (1K cells/well) on a base of Matrigel® and allowed to form multi-spheroids (3 days) prior to the addition of freshly isolated PBMCs (E:T, 5:1) and Herceptin. Incucyte® DF-BF and fluorescence images (7 days) compare the effect of Herceptin on spheroid proliferation in the absence (top panel) and presence (bottom panel) of PBMCs (Brightfield outline mask shown in yellow). Note the loss of fluorescence intensity in the presence of PBMCs. Time-course shows a Herceptin concentration-dependent decrease in green fluorescence, indicating a reduction in viable target cells. Concentration response curves to Herceptin show sensitivity differences between HER2-positive and HER2- negative multi-spheroids (BT-474 vs MCF7 respectively).

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® Embedded Multi-Spheroid Assay Protocol

For Quantifying Growth and Death of Multi-Spheroids Embedded in Matrigel® Label-Free

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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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Organoid Culture QC

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Organoid Assay

Organoid Assay

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