Applications

IncuCyte® Tumor Spheroid Assays for Live-Cell Analysis

Introduction

What are tumor spheroids?

Spheroids, or tumor cell aggregates, are more representative of in vivo conditions than cell monolayers, and tumor cells in these microenvironments exhibit several physiological traits including relevant morphology, increased cell survival, and a hypoxic core.

Growing body of evidence suggests that more relevant and translational observations can be made compared to 2D monolayer models, notably in the cancer biology and hepatotoxicity area. Though three-dimensional tumor cell culture has been shown to mimic the physiological cancer situation more closely than simple two-dimensional cell monolayers, most currently available three-dimensional techniques for generating and quantifying spheroids are time consuming, laborious, costly and/or lack reproducibility.

Request A DEMO or more info    See all applications    Download the Tumor Spheroid Brightfield Analysis Application Note


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Introducing IncuCyte® Tumor Spheroid Assays

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.

tumor spheroids video

Continuous monitoring of spheroid growth and cytotoxicity with the IncuCyte® Live-Cell Analysis System and IncuCyte® Cytotox reagent. Label-free SKOV-3 human ovarian adenocarcinoma cells treated with and without 1µM Camptothecin, imaged in brightfield and green fluorescence over 10 days.

ULA Spheroid Assay Quick Protocol

spheroid protocol

Spheroid formation protocol adapted from Vinci et al. BMC Biology 2012,10:29.


Download the protocol — IncuCyte Tumor Spheroid Protocol For Cell Health Reagents

Download the detailed protocol — IncuCyte Tumor Spheroid Protocol for Fluorescent Label

Key Advantages

Key Advantages of IncuCyte® Tumor Spheroid Assays

While your spheroids are growing undisturbed inside your tissue culture incubator for days or weeks, IncuCyte tumor spheroid assays offer the following advantages:

Derive more physiologically relevant information

  • Cost effective, automated, label-free monitoring of growth and morphology of spheroids to derive more physiologically relevant information about your cells  Read more below

Reveal cellular changes over time

  • Streamlined quantification of cell death over time, utilizing targeted reagents (e.g. IncuCyte® Cytotox or Annexin) to reveal cellular changes from treatment Read more below

Real-time measurements with reagents and tools

  • Real-time spheroid viability measurements, pairing the power of IncuCyte® NucLight or CytoLight fluorescent proteins and purpose-built analysis tools Read more below

Generate consistent and reproducible data

  • Consistent and reproducible data for pharmacological analysis — easy to achieve with robust experimental protocols  Read more below


Cost effective, automated, label-free monitoring of growth and morphology of spheroids to derive more physiologically relevant information about your cells

spheroids figure 1

Monitor spheroid size over time as they grow inside your tissue culture incubator. Brightfield images show MDA-MB-231 breast cancer spheroids ± cytotoxic agent camptothecin (1 µM).  Vehicle treated spheroids increase in size while CMP treated spheroids remain compact.  Images taken automatically every 6h for quantification of brightfield area.

spheroids figure 2

Visualize spheroids with high definition phase optics to reveal morphological differences. High quality HD phase and corresponding BF images of spheroids formed from A549 and MDA-MB-231 cells (2,500 or 5000 cells per well respectively), 72-hours post seeding. Visualization of detailed phenotypic variation is observed in HD phase images. A549 cells present a loose aggregate morphology compared to the compact spheroid formed by MDA-MB-231 cells. Compaction of MDA-MB-231 aggregates into spheroids was achieved by the addition of 2.5% v/v Matrigel® post centrifugation. All images captured at 10x magnification.


Streamlined quantification of cell death over time, utilizing targeted reagents (e.g. IncuCyte® Cytotox or Annexin) to reveal cellular changes from treatment

spheroids figure 3

Establishing cytotoxic vs cytostatic mechanism of action by comparing Brightfield and Fluorescent readouts using IncuCyte® Cytotox reagent. Images show the green fluorescence within the masked brightfield area of SK-OV-3 spheroids at 10 days post-treatment.  The timecourse profiles of brightfield area show similar response to both drugs – the spheroid growth is inhibited as the drug concentrations increase.  The mean green intensity within the brightfield area (bottom row) shows a differential response to cytotoxic (camptothecin, left) and cytostatic (cycloheximide, middle) agents.  In the presence of camptothecin the cells die, yielding an increase in fluorescence intensity from the reporter (cytotox Green); cycloheximide and vehicle treated spheroids show only a nominal amount of cell death as expected.


Real-time spheroid viability measurements, pairing the power of IncuCyte® NucLight or CytoLight fluorescent proteins and purpose-built analysis tools

spheroids figure 4

Continuous monitoring of spheroids growth and cell health in IncuCyte Live-Cell Analysis System. SK-OV-3 human ovary carcinoma cells stably expressing nuclear restricted fluorescent protein. A time-dependent increase in fluorescence within the spheroid area defined by the brightfield mask is inhibited by the cytotoxic drug camptothecin (1 µM).


Consistent and reproducible data for pharmacological analysis — easy to achieve with robust experimental protocols

spheroids figure 5

Spheroid growth assay shows robustness and reproducibility. Deep view shows masked brightfield area of three spheroid types (lung carcinoma, fibrosarcoma, ovarian carcinoma) at four cell densities. The brightfield area plot indicates that the recommended seeding density (2500 cells/well) for each of these cell types yields a robust timecourse.

spheroids figure 6

Perform robust pharmacological analysis in physiologically relevant conditions. Spheroids are analyzed inside your tissue culture incubator without labels. Effect of CMP, CIS and OXA on growth of SKOV3 cells in a spheroid assay. SKOV3 cells were plated at a density of 5,000 cells per well and spheroid allowed to form (72-hours). Cells were then treated with serial compound dilutions and kinetics of spheroid growth were obtained. Plate-Graph shows the individual well Largest BF area (µm2) over time. Concentration response curves represent the Largest BF area (µm2) at 204-hours post-treatment. Data were collected over 240-hour period at 6-hour intervals. Each data point represents mean ±SEM, n=8.

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