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.
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.
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.
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
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
Reveal cellular changes over time
Real-time measurements with reagents and tools
Generate consistent and reproducible data
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.
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.
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.
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).
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.
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.
|IncuCyte® S3 Spheroid Software Module||1||9600-0019|
|IncuCyte® Cytotox Green Reagent||1||4633|
|IncuCyte® Cytotox Red Reagent||1||4632|
|IncuCyte® Annexin Red Apoptosis Reagent||1||4641|
|IncuCyte® Annexin Green Apoptosis Reagent||1||4642|
|IncuCyte® Nuclight Green Lentivirus (EF-1a Promoter, Puro selection) Nuclear Labeling Reagent||1||4624|
|IncuCyte® Nuclight Red Lentivirus (EF-1a Promoter, Puro selection) Nuclear Labeling Reagent||1||4625|
|IncuCyte® CytoLight Green Lentivirus (EF-1a Promoter, Puro selection) Cytoplasmic Labeling Reagent||1||4481|
|InCuCyte® CytoLight Red Lentivirus (EF-1a Promoter, Puro selection) Cytoplasmic Labeling Reagent||1||4482|