Tumor Spheroid Assays
Spheroids, or tumor cell aggregates, are more representative of in vivo conditions than cell monolayers, and tumor cells grown as spheroids exhibit several physiological traits including relevant morphology, increased cell survival, and a hypoxic core.
A 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. A simple and inexpensive model for solid tumors involves generating a single spheroid in a round bottom ULA plate.
Application
Incucyte® 3D Single Spheroid Assays
Effective analysis of 3D liquid-based multi-tumor spheroids can be challenging. Traditional plate reader assays lack key image-based analysis features such as morphological information and the ability to verify data within images. Conventional imaging systems are difficult to adapt for kinetic analysis of in vitro cultures due to several factors.
Key Limitations
- Incomplete data from missed imaging intervals
- Environmental fluctuations caused by repeated transport from incubator to imaging system and lengthy 3D image acquisition protocols outside the controlled environment, leading to temperature shifts and loss of oxygen and carbon dioxide regulation
- Time-consuming optimization of image acquisition parameters
- Complex image processing requiring expert operators to extract quantitative information
Solution
The Incucyte® CX3 Live-Cell Analysis System, equipped with spinning disk confocal imaging, enables researchers to acquire high-quality, time-lapse 3D images of single spheroids with minimal disturbance to the culture environment—supporting more detailed and consistent analysis over time. The Incucyte® 3D Single Spheroid Assay is designed for high-resolution, kinetic monitoring of individual tumor spheroid formation, growth, and viability in real time, directly within the incubator.
This assay utilizes ultra-low attachment (ULA) 96-well or 384-well plates to promote the self-assembly of a single, centralized spheroid per well, ensuring uniformity and reproducibility across experimental conditions. Researchers can combine fluorescent or brightfield imaging to quantify key parameters such as spheroid size, morphology, and response to treatment over time. For widefield fluorescence and brightfield imaging, this assay remains fully compatible on the Incucyte® SX1, S3, and SX5 systems.
View all Incucyte Applications
| Additional Products | Qty. | Cat. No |
| Incucyte® Spheroid Analysis Software Module (Module available for Incucyte® SX5, S3, SX1, and CX3 Live-Cell Analysis System.) | 1 module | 9600-0019 |
| Incucyte® CX3 Live-Cell Analysis System | 1 instrument | Explore the CX3 |
| Incucyte® SX5 Live-Cell Analysis System | 1 instrument | 4816 |
Key Advantages
Key Advantages of Incucyte® 3D Single Spheroid Assays
- Derive more physiologically relevant information - Quantify growth and investigate morphology of 3D single tumor spheroid cultures — inside your incubator.
- Reveal cellular changes over time in mono- or co-culture - Investigate mechanisms of action or immune modulation with real-time viability and toxicity measurements using non-perturbing reagents.
- Generate reproducible, quantitative data - Lab-tested protocols, high quality images, and unbiased analysis deliver robust data suitable for pharmacological analysis.
- Unlock your productivity - Automatically acquire, analyze and graph thousands of images from up to six 96/384-well plates in parallel and get to answers faster.
Derive More Physiologically Relevant Information
Figure 1. Monitor spheroid size over time as they grow undisturbed inside your tissue culture incubator.
Images display brightfield and confocal fluorescence U-87 MG breast cancer spheroids expressing Incucyte® Nuclight Orange 2.0 Lentivirus ± cytotoxic agent camptothecin (3µM). Incucyte® Cytotox Green Dye was used to quantify cell death within the spheroid. Brightfield area of the vehicle treated spheroids increase in size while CMP treated spheroids remain compact. Images taken automatically every 8h for quantification of brightfield area.
Figure 2. Reveal morphology with high quality HD phase, brightfield, and confocal images.
High quality HD phase, brightfield, and confocal fluorescence images of spheroids, acquired 72-hours post seeding, show differences in morphology in spheroids formed from Incucyte® Nuclight Orange expressing A549, MCF-7 and U87 MG cells. Easily distinguish between loose aggregate and compact spheroid morphologies in different cell types. All images captured at 10X magnification.
Reveal Cellular Changes Over Time in Mono- or Co-culture
Figure 3. Visualize cell health effects in single spheroid application.
Compare brightfield and fluorescent readouts using Incucyte® Annexin V Green Dye. Confocal images show fluorescence (Annexin V Green and Nuclight Orange) within masked BF area of A549 spheroids 4 days post-treatment with cytotoxic compounds. Time course profiles of brightfield area show cytotoxic response to drugs at three-fold dilutions, while spheroid growth is inhibited as drug concentrations increase. Mean Green Intensity measured within Brightfield Boundary (bottom row) shows a differential response to compounds.
Figure 4. Continually monitor Spheroids and Multiplex Fluorescent Reagents using the Incucyte® CX3 Live Cell Analysis System.
U87 MG spheroids, stably expressing the Incucyte® Cell Cycle Green/Orange Lentivirus, were treated with Fluorouracil (5-FU) and Cisplatin. Confocal microscopy was employed to image the spheroids over a 96-hour period in the presence of the Incucyte® Annexin NIR Dye. Treatment with 5-FU resulted in an increase in orange mean fluorescence, indicative of an elevated number of cells in the G1 phase. In contrast, Cisplatin induced cell death at higher concentrations, as evidenced by the Annexin NIR response. At lower concentrations, Cisplatin caused a transient increase in Green Mean Fluorescence at early time points, suggesting alterations in cell cycle dynamics.
Generate Reproducible, Quantitative Data
Figure 5. Incucyte® lab-tested and validated Single Tumor Spheroid Protocol is easy to follow.
Reduce time spent troubleshooting 3D cell culture techniques and eliminate the need for a trial-and-error approach to obtain images suitable for quantitative analysis.
Figure 6. Spheroid growth assay shows robustness and reproducibility.
Incucyte® VesselView shows masked brightfield area of three spheroid types (lung carcinoma, fibrosarcoma, ovarian carcinoma) at four cell densities. Histogram compares the distribution frequency of the brightfield area (μm2) across all cell types plated at 2,500 cells per well at this time point. Variability plot analysis shows the Largest Brightfield Object Area of individual wells at 240 hours. Time-course plots represent the differential temporal profile of the Largest Brightfield Object Area metric (μm2) across the cell types. Data were collected over a 240-hour period at 6-hour intervals, all images captured at 10X magnification.
Figure 7. Perform robust pharmacological analysis in physiologically relevant conditions.
Effect of camptothecin (CMP), cisplatin (CIS) and oxaliplatin (OXA) on growth of SKOV-3 cells in a spheroid assay performed inside a tissue culture incubator and without labels. SKOV-3 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. Microplate Graph shows the individual well Largest Brightfield (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.
Unlock Your Productivity
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
Additional Products
| Product | Qty. | Cat. No. |
|---|---|---|
| Incucyte® Nuclight Red Lentivirus (puro) 2.0 | 0.6 mL | BA-04887 |
| Incucyte® Nuclight Green Lentivirus (puro) 2.0 | 0.6 mL | BA-04888 |
| Incucyte® Nuclight Red Lentivirus (bleo) 2.0 | 0.6 mL | BA-04889 |
| Incucyte® Nuclight Green Lentivirus (bleo) 2.0 | 0.6 mL | BA-04890 |
| Incucyte® Nuclight Orange Lentivirus (puro) 2.0 | 0.6 mL | BA-04891 |
| Incucyte® Caspase-3/7 Green Dye | One vial (20 µL) | 4440 |
| Incucyte® Caspase 3/7 Red Dye | One vial (20 µL) | 4704 |
| Incucyte® Annexin V Red Dye | One vial (100 tests) | 4641 |
| Incucyte® Annexin V Green Dye | One vial (100 tests) | 4642 |
| Incucyte® Annexin V Orange Dye | One vial (100 tests) | 4759 |
| Incucyte® Annexin V NIR Dye | One vial (100 tests) | 4768 |
| Incucyte® Cytotox Red Reagent | Five vials (5 µL) | 4632 |
| Incucyte® Cytotox Green Reagent | Five vials (5 µL) | 4633 |
| Incucyte® Cytotox NIR Dye | One vial (100 µL) | 4846 |
