What are spheroids?
Spheroids are three-dimensional (3D) cell aggregates that can mimic tissues and microtumors. In recent years, there has been significant progress in development of in vitro aggregates of tumor cells for use as models for in vivo tissue environments. When seeded into a well of a low-attachment round bottom microplate, these aggregates will form a discrete spheroid.
Spheroids are believed to mimic tumor behavior more effectively than regular two dimensional (2D) cell cultures because, much like tumors, they contain both surface-exposed and deeply buried cells, proliferating and non-proliferating cells, and a hypoxic center with a well-oxygenated outer layer of cells. Such 3D spheroid models are being successfully used in screening environments for better assessment of drug safety and identifying potential cancer therapeutics.

Z-stack going through a 250 µm depth of a spheroid. We are able to see cells in the center of the spheroid at this depth due to our specialized deep-tissue penetrating, confocal disk module.
The application of spheroids for cancer research and drug screening
One of the most significant objectives in cancer research is to understand tumor cell formation. Compared to 2D cell cultures, spheroids mimic solid tumors much more accurately. They help display the physiological changes that differentiate tumor cells from healthy cells. Multicellular tumor spheroid models give deeper insights into the tumor microenvironment, allowing researchers to visualize cell-to-cell interactions, how tumor cells absorb nutrients, and how they proliferate.
Spheroids are also ideal for preclinical drug screening and validation when monitoring cellular responses to therapeutic interventions. Specifically, they can reveal the tumor infiltration capacity (or incapacity) of drugs, as well as their inhibitory effects on metastasis.
Stem cell research is another significant application of spheroids, especially during the cultivation of embryonic (ESC) and neural stem cells (NSC). Recently, researchers cultured mesenchymal stem cells (MSCs) in spheroids and proved their regenerative and anti-inflammatory effects, which depicts potential for regenerative medicine and cell-based therapies.
Compared to other 3D cell culture methods, spheroids offer several advantages, including relevance to tumor biology, lower cost, less labor than animal models, reproducibility, ease of integration into high-throughput screening, and advanced imaging techniques.
Workflow for analyzing 3D spheroids for high-throughput screening
Imaging and analysis of spheroids are crucial to understanding the formation and behavior of tumor microenvironments. It can also aid in obtaining more insightful and realistic data from anti-cancer drug screening. Here we developed and optimized methods for the formation of 3D spheroids, as well as confocal imaging and analysis methods for high-throughput screening.
Spheroids can be grown in 96- or 384-well plates, treated with compounds, and stained with dyes that reveal the cellular processes and pathways at work. In some cases, spheroids can be imaged without washing. They may also be fixed if desired.
Culture spheroids – Cancer cells can be cultured directly in an ultra-low attachment (ULA), round bottom plate, or other labware to develop the typical morphology of a spheroid. Other labware allows one to grow multiple spheroids in a single well.
Treat with compounds – After spheroid formation, compounds at the desired concentrations are added into the wells, and then incubated for one to several days, depending on the mechanism being studied.
Stain for markers – After compound treatment is complete, stains are added directly to the media. Stains that require no washing can be used to avoid disturbing spheroids, but spheroids can be carefully washed, even using automation, if necessary.
Acquire spheroid images – Images within the body of the spheroid can be captured individually or as a Z-stack (multiple images taken at differing depths) using specialized imaging equipment.
Analyze cancer cells – Use cellular imaging analysis software to run quantitative analysis of the cell images to monitor the expression of different markers and to quantify biological readouts.
Application and assays
Cutting-edge 3D imaging and analysis methods are crucial to characterize, reproduce, and monitor spheroids. Such an intricate workflow can benefit greatly from high-content imaging and image analysis software. Learn more about our methods for spheroid research from the resources below.
Resources of Spheroids
Blog
How 3D Cell Models Will Shape the Future of Drug Discovery
How 3D Cell Models Will Shape the Future of Drug Discovery
Target discovery and drug development rely heavily on 2D cell and animal models to decipher efficacy and toxic effect of drug candidates. Yet, 90% of candidates fail to make it past…
Blog
A case study for assay-ready patient-derived organoids (PDOs) and high-throughput 3D imaging to advance drug discovery
A case study for assay-ready patient-derived organoids (PDOs) and high-throughput 3D imaging to advance drug discovery
Introduction – the problem. The average cost of bringing a new drug to the clinic is around $1 billion according to a study conducted by the London School of Economics in March 2020…
Application Note
Monitoring of T-cell invasions assay using a 3D spheroid model
Monitoring of T-cell invasions assay using a 3D spheroid model
T-cell therapies are designed to help our immune system eliminate cancer cells. Those include CAR T-cells (Chimeric Antigen Receptor engineered T-cells), tumor infiltrating lymphocytes (TIL…
Application Note
Imaging characterization of a 3D bioprinted ovarian cancer model
Imaging characterization of a 3D bioprinted ovarian cancer model
3D bioprinting is defined as the additive deposition of cells and biocompatible materials to build biologically functional 3D structure or artificial tissue models
Brochure
BioAssemblyBot 400 (BAB400): more than a bioprinter
BioAssemblyBot 400 (BAB400): more than a bioprinter
The BioAssemblyBot® 400 is an intelligent robot used by life scientists to build 3D model systems with increased throughput and precision, alleviating major concerns mentioned above.
Application Note
Automated dispensing, monitoring, and assay development of hydrogel-based cellular models
Automated dispensing, monitoring, and assay development of hydrogel-based cellular models
3D cellular models that more accurately represent various microenvironments are incredibly important for accurate drug screening and disease modeling.
eBook
Building complexity in organoid models
Building complexity in organoid models
Complex biological systems such as spheroids, organoids, and organ-on-a-chip are becoming more popular for disease modeling and drug screening as they better simulate organs and tissues comp…
Blog
Advanced technology for automated 3D biology workflows #SLASEurope2022
Advanced technology for automated 3D biology workflows #SLASEurope2022
SLAS Europe 2022, hosted numerous sessions packed with the latest research on emerging topics as well as sessions and panel discussions focused on how to build, and succeed, in a…
Application Note
Automated Monitoring of Development and Activity Analysis of iPSC-derived 3D Cerebral Organoids
Automated Monitoring of Development and Activity Analysis of iPSC-derived 3D Cerebral Organoids
The human brain is highly complex which makes it challenging to study both in vitro and in-model organisms. Cultured neurons do not sufficiently recapitulate the three-dimensional (3D)…
Application Note
AI enabled phenotypic analysis of label-free intestinal organoids
AI enabled phenotypic analysis of label-free intestinal organoids
The intestinal crypt system is an important part of the intestinal organoid where mature organoids have more complex and numerous crypt structures. Yet, due to their high turnover rate and…
Blog
How automated organoid cell cultures are developed, imaged, and analyzed
How automated organoid cell cultures are developed, imaged, and analyzed
3D cell models are becoming increasingly popular for studying complex biological effects, tissue functionality, and diseases. Their ability to self-organize and mimic…
Blog
Engineering Next-gen Organoids with Automated Lab Workflows at #SLAS2022
Engineering Next-gen Organoids with Automated Lab Workflows at #SLAS2022
SLAS2022, the Society for Lab Automation and Screening conference offered another exciting year for learning about innovative laboratory technologies. Whether you attended in-person…
Blog
Innovation at Molecular Devices: Updates in Automated, High-Content Imaging
Innovation at Molecular Devices: Updates in Automated, High-Content Imaging
From customer feedback to workflow improvements The path to understanding complex biological processes and diseases is paved with a lot of challenges. As the desired level of…
Blog
Stem cell science insights and breakthroughs presented at #ISSCR2021
Stem cell science insights and breakthroughs presented at #ISSCR2021
If you didn't get a chance to visit us at our poster sessions during ISSCR 2021, don’t fret. We've gathered all our sessions right here for you. The ISSCR Annual Meeting brought…
Publications
Project Profile: Organoid Innovation Center
Project Profile: Organoid Innovation Center
Lab Manager speaks to Dan O’Connor, vice president, drug discovery, Molecular Devices, about the company’s Organoid Innovation Center in San Jose, CA. The center is 180 sq. ft., within a lar…
Publications
Organs-on-Chips: Expand the Boundaries of In Vitro Testing
Organs-on-Chips: Expand the Boundaries of In Vitro Testing
Humans began culturing animal cells and tissues as early as the late 19th century, when Wilhelm Roux first showed that chick embryos could be cultured in saline solution for a few days. Even…
Blog
Tips for running a successful live cell imaging experiment
Tips for running a successful live cell imaging experiment
There have been significant advancements in microscopy and camera technology, as well as advancements in technologies for labeling molecules of interest over the past decade. These…
Publications
Shaping the Future of Organoid Research
Shaping the Future of Organoid Research
Molecular Devices, a provider of innovative life science technology, recently unveiled a brand new, first-of-its-kind Organoid Innovation Center. Situated at the company’s global headquarter…
Publications
Disease Modeling with 3D Cell-Based Assays Using a Novel Flowchip System and High-Content Imaging
Disease Modeling with 3D Cell-Based Assays Using a Novel Flowchip System and High-Content Imaging
There is an increasing interest in using three-dimensional (3D) cell structures for modeling tumors, organs, and tissue to accelerate translational research. We describe here a novel automat…
Publications
Using 3D cancer cell models to push forward personalized medicine
Using 3D cancer cell models to push forward personalized medicine
Using 3D cancer cell models to push forward personalized medicine: an interview with Angeline Lim, PhD, of Molecular Devices Editor-in-Chief, Francesca Lake, speaks to Angeline Lim of Mole…
Publications
Disease Modeling with 3D Cell-Based Assays Using a Novel Flowchip System and High-Content Imaging
Disease Modeling with 3D Cell-Based Assays Using a Novel Flowchip System and High-Content Imaging
There is an increasing interest in using three-dimensional (3D) cell structures for modeling tumors, organs, and tissue to accelerate translational research. We describe here a novel automat…
Publications
Danaher’s SCIEX and Molecular Devices Businesses Debut New Products
Danaher’s SCIEX and Molecular Devices Businesses Debut New Products
It has been a busy beginning of the year for Danaher’s Life Sciences segment. In January, the business reported 2020 revenues of $10.6 billion, representing 47% of the total revenues of scie…
Application Note
Quantification of spheroid growth and morphology
Quantification of spheroid growth and morphology
There is a growing trend to develop screening assays using three-dimensional (3D) cell culture.
Application Note
High-throughput screening of 3D cell cultures with multiple high density scaffold-free spheroids for cancer toxicity studies
High-throughput screening of 3D cell cultures with multiple high density scaffold-free spheroids for cancer toxicity studies
3D spheroid models for cancer research are gaining popularity because they better mimic the in vivo tissue architecture, gene expression and metabolic profile of tumors compared to…
Publications
Improving acquisition and analysis of 3D cell model assays with water immersion objectives
Improving acquisition and analysis of 3D cell model assays with water immersion objectives
Sebastian Peck is a senior product manager for cellular imaging at Molecular Devices, one of the world's leading providers of high-performance bioanalytical measurement systems, software, an…
Publications
The workflow game-changer: combined technologies for efficiency
The workflow game-changer: combined technologies for efficiency
Microfluidic flowchip technology combined with three-dimensional (3D) imaging is a powerful duo poised to change the future of drug discovery and development. The integration of combined tec…
Publications
Neuroscience: bridging the gap between cell-based and human research
Neuroscience: bridging the gap between cell-based and human research
Neurological disorders impact up to a billion people around the world and appear to be on the rise [1]. In fact, those affecting the central and peripheral nervous systems – including stroke…
Application Note
Phenotypic characterization of neuroactive compound effects
Phenotypic characterization of neuroactive compound effects
The FLIPR Penta system is powered by a high-speed EMCCD camera and the new ScreenWorks Peak Pro 2 software. The system allows measurement and analysis of complex patterns of calcium…
Publications
3D Microscopy Keeps Getting Faster, Smarter, Leaner
3D Microscopy Keeps Getting Faster, Smarter, Leaner
With each passing day, continuous advancements are being made in the studies that cutting-edge instruments can perform and in the complexity of the biological samples that are being examined…
Publications
From 3D high throughput to simple automated imaging: Cellular imaging within reach of every laboratory
From 3D high throughput to simple automated imaging: Cellular imaging within reach of every laboratory
OUR ABILITY to image cells has come a long way since the pioneering days of Galileo Galilei and Antonie van Leeuwenhoek. The multiple imaging techniques available today range from simple lig…
Publications
What 3D imaging means for cancer drug discovery
What 3D imaging means for cancer drug discovery
Finding a cure for cancer has been called this century’s “moonshot.” As a senior scientist at Molecular Devices, I know first-hand that accelerating cancer drug discovery is a priority of no…
Publications
Making the move from 2D to 3D
Making the move from 2D to 3D
Jayne Hesley is a Senior Applications Scientist for Cellular Imaging at Molecular Devices, LLC. She has over 10 years’ experience developing cell-based applications using ImageXpress Micro h…
Publications
Revolutionizing early drug discovery for immuno-oncology and neurodegenerative disease modeling
Revolutionizing early drug discovery for immuno-oncology and neurodegenerative disease modeling
The development of 3D cellular models of disease has enabled researchers to better recapitulate in vivo cell environments. As 3D models become more prevalent in the drug discovery industry,…
Publications
2020 Society for Lab Automation and Screening International Conference & Exhibition: Laboratories Embrace Automation
2020 Society for Lab Automation and Screening International Conference & Exhibition: Laboratories Embrace Automation
The Society for Laboratory Automation and Screening (SLAS) held their annual conference January 25–29 in San Diego, California. The show hosts presentations, short courses and an exhibition…
Publications
LabTube Meets Molecular Devices & MIMETAS
LabTube Meets Molecular Devices & MIMETAS
Molecular Devices partners with MIMETAS to scale up organoid screening—helping researchers go from 2D to 3D phenotypic relevant organoid screening. At SLAS 2020, they featured the ImageXpres…
Publications
The importance of the cellular microenvironment in the development of more efficient drugs against complex diseases
The importance of the cellular microenvironment in the development of more efficient drugs against complex diseases
The biotechnological start-up is focused on in vitro preclinical research and development, with a specialization in automated cellular imaging: high-content analysis (HCA) and high-content s…
Publications
Microenvironment and 3D image analysis key in the development of more efficient drugs against complex diseases
Microenvironment and 3D image analysis key in the development of more efficient drugs against complex diseases
The biotechnological start-up is focused on in vitro preclinical research and development with a specialization in cellular imaging: high-content analysis (HCA) and high-content screening (H…
Application Note
Functional evaluation of neurotoxic and neuroactive compound effects in iPSC-derived 3D neural co-cultures
Functional evaluation of neurotoxic and neuroactive compound effects in iPSC-derived 3D neural co-cultures
There is increasing interest in using more complex, biologically-relevant, and predictive cell-based platforms for assay development and compound screening.
Publications
Harnessing the latest imaging technology to advance angiogenesis research
Harnessing the latest imaging technology to advance angiogenesis research
Ever since the discovery that tumors undergo angiogenesis in order to grow and survive, researchers have sought to better understand the detailed mechanism of new blood vessel formation. Tar…
Presentations
Functional and mechanistic neurotoxicity profiling using human iPSC-derived neural 3D cultures
Functional and mechanistic neurotoxicity profiling using human iPSC-derived neural 3D cultures
Expertise in human iPSC-derived neural and cardiac platforms; Focus on adapting platforms to HTS and producing assay-ready solutions to accelerate drug discovery; Neural platform: mocroBra…
Application Note
Assay apoptosis in magnetically bioprinted HepG2 spheroids
Assay apoptosis in magnetically bioprinted HepG2 spheroids
Learn how HepG2 cells can be bioprinted using a magnetic 3D cell culture system and imaged on the SpectraMax i3x plate reader with MiniMax 300 Imager.
Application Note
Acquire and analyze images of FUCCI spheroids on the SpectraMax MiniMax cytometer
Acquire and analyze images of FUCCI spheroids on the SpectraMax MiniMax cytometer
Spheroids are small three-dimensional (3D) cellular microenvironments grown using a variety of specialized culture methods such as low-adhesion microplates. This 3D cell culture confers a…
Application Note
Assessment of drug effects on cardiomyocyte physiology using human iPSC-derived cardiac spheroids
Assessment of drug effects on cardiomyocyte physiology using human iPSC-derived cardiac spheroids
Cell models are becoming more complex in order to better mimic in vivo microenvironments and provide greater predictivity for compound efficacy and toxicity. There is an increasing interest…
Application Note
Multi-parameter imaging assay for measuring toxicity in a tumor model
Multi-parameter imaging assay for measuring toxicity in a tumor model
There is an increasing interest in using three-dimensional (3D) spheroids for modeling cancer and tissue biology to accelerate translational research. The goal of this study was to develop…
Application Note
3D analysis and morphometric characterization of compound effects on cancer spheroid cultures
3D analysis and morphometric characterization of compound effects on cancer spheroid cultures
Cellular transformation/tumorigenicity assays using cultures of cells in semi-solid media (soft agar or Matrigel) has been well established for cancer research1,2,5. The assay requires…
Application Note
High-content 3D toxicity assay using iPSC-derived hepatocyte spheroids
High-content 3D toxicity assay using iPSC-derived hepatocyte spheroids
There is increasing interest in exploring the use of three-dimensional (3D) spheroids for modeling developmental and tissue biology with the goal of accelerating translational research in…
Application Note
High-Throughput Confocal Imaging of Spheroids for Screening Cancer Therapeutics
High-Throughput Confocal Imaging of Spheroids for Screening Cancer Therapeutics
In recent years, there has been significant progress in development of in vitro aggregates of tumor cells for use as models for in vivo tissue environments. When seeded into a well of a…
Application Note
3D Imaging of Cancer Cell Spheroids
3D Imaging of Cancer Cell Spheroids
Many cancer cell lines will form spheroids if cultured on a favorable three dimensional (3D) matrix. These spheroids are believed to represent tumor physiology more closely than cells…
Publications
Phenotypic Assays for Characterizing Compound Effects on Induced Pluripotent Stem Cell-Derived Cardiac Spheroids
Phenotypic Assays for Characterizing Compound Effects on Induced Pluripotent Stem Cell-Derived Cardiac Spheroids
Development of more complex, biologically relevant, and predictive cell-based assays for compound screening is a major challenge in drug discovery. The focus of this study was to establish h…
Publications
Phenotypic Characterization of Toxic Compound Effects on Liver Spheroids Derived from iPSC Using Confocal Imaging and Three-Dimensional Image Analysis
Phenotypic Characterization of Toxic Compound Effects on Liver Spheroids Derived from iPSC Using Confocal Imaging and Three-Dimensional Image Analysis
Cell models are becoming more complex to better mimic the in vivo environment and provide greater predictivity for compound efficacy and toxicity. There is an increasing interest in explorin…
Publications
High-Content Assays for Characterizing the Viability and Morphology of 3D Cancer Spheroid Cultures
High-Content Assays for Characterizing the Viability and Morphology of 3D Cancer Spheroid Cultures
There is an increasing interest in using three-dimensional (3D) spheroids for modeling cancer and tissue biology to accelerate translation research. Development of higher throughput assays t…
Videos & Webinars

Spheroid Z-stack