Nexcelom Bioscience
3D Tumor Spheroid

3D Tumor Spheroid Analysis

Automated Imaging and Analysis of 3D Tumor Spheroids, Cancer Stem Cells

The Celigo imaging cytometer has been developed to fully automate imaging and analysis of tumorspheres, embryoid bodies and cancer stem colonies. It has been used in the development of 3D tumor spheroid-based functional assays for target validation and drug evaluation.

Suspension cultures of tumor spheres, including mammospheres and neurospheres, can be rapidly and accurately analyzed using the Celigo Cell Imaging Cytometer. This automated morphometric analysis tool significantly reduces the time and effort needed to quantify key aspects of 3D spheres including size, growth, growth tracking over time, and response to chemotherapeutics. Analysis of tumor spheres generated from different cancer cell lines and primary cancer cells can be used to evaluate sphere-forming efficiency, tumorigenicity, and self-renewal of cancer stem/tumor-initiating cells.

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Tumor Sphere Analysis Benefits

  • Non-destructive quantification of live spheroids for correlation with malignant cell behavior
  • Whole-well imaging of suspension spheroids (96-well to 6-well)
  • Rapid data analysis (scan and analyze an entire 12-well plate in <15 min)
  • Analysis of sphere number, size, shape, and growth kinetics over time
  • Identify and count spheroids in flat or U-bottom wells
  • Eliminate cumbersome manual morphology measurements and improve data reproducibility
  • Monitor growth of spheroids over time
  • Screen numerous spheroid plates per day, 96- and 384- well plates
  • Scan an ultra-low adhesion 96-well plate in 8 minutes
  • Measure viability using dual-fluorescence assays
  • Multi-parameter analysis
Counts / Size / Short & Long Diameter / Est. Volume / Perimeter /Area / More . . .

spheroid generation
spheroid growth curve

Data courtesy of Dr. Maria Vinci, Cancer Research UK, Cancer Therapeutics Unit, The Institute of Cancer Research.

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Tumor Spheroid Growth Inhibition Assay

4-day old spheroids were treated with compounds for 72 hours. Drug-induced concentration-dependent growth inhibition was obtained.

  • Day 0-4: form spheroids
  • Day 4-7: drug treatment
  • Day 4-14: measure spheroid size

Experimental Setup

tumor spheroid growth setup

Drug Concentration


U 87-MG (giloblastoma) + 17-AGG


Vince et al. BMC Biology 2012, 10:29, March 2012

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Invasion into Matrigel Assay

  • Day 0-4: form spheroids
  • Day 4: add Matrigel to provide a semi-solid gel like matrix
  • Day 4-7: use Celigo imaging cytometer to determine the area occupied by individual cells or cell clusters

Experimental Setup

invasion into Matrigel setup
invasion into matrigel

U-87 MG + 17-AAG

invasion into matrigel data

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Two-fluorescence Viability Assay for 3D Tumor Spheroids

3D cultures were treated with multiple drugs (17-AAG, Paclitaxel, TMZ or Doxorubicin) and stained for viability after 21 days.

Live / dead stains used were Calcein AM (green), Propidium Iodide (red) to measure live and dead cells on days 4, 7, 10, 14, and 17. Plates containing spheroids were imaged on Celigo imaging cytometer.

  • 17-AAG decreased sphere size and caused the most significant cell death
  • Paclitaxel decreased sphere size but maintained a signifiicant number of live cells
  • Temozolomide caused no significant decrease in sphere size and did not cause cell death
  • Doxorubicin decreased sphere size and caused significant cell death

Experimental Setup

3D tumor spheroid fluorescence viability assay setup 3D tumor spheroids

Multidrug Viability 3D Assay

multidrug viability assay

Spheroid diameter (black) and total live fluorescent intensity (green) after drug treatment.

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