Cellular Imaging and Analysis for 3D Cell Culture Applications and Assays
Development of more complex, biologically relevant, and predictive cell-based assays for compound screening is a primary challenge in drug discovery. The integration of three-dimensional (3D) assay models is becoming more widespread to drive translational biology. Higher complexity cell models have gained popularity because they better mimic in vivo environments and responses to drug treatment. Specifically, 3D cell cultures offer the advantage of closely recapitulating aspects of human tissues including the architecture, cell organization, cell-cell and cell-matrix interactions, and more physiologically-relevant diffusion characteristics. Utilization of 3D cellular assays adds value to research and screening campaigns, spanning the translational gap between 2D cell cultures and whole-animal models. By reproducing important parameters of the in vivo environment, 3D models can provide unique insight into the behavior of stem cells and developing tissues in vitro.
3D cell imaging and analysis workflow
While the development of quantitative assays using 3D cell cultures has become an attractive investigative tool to understand complex biology, challenging 3D cell image acquisition and analysis workflows have hindered wider adoption by the screening community. High-content, high-throughput tools for microscopy offer innovative and automated techniques for evaluating this complex biology. After the 3D cell culture is grown or transferred to an imaging microplate, cells can be treated with compounds and stained with the marker of choice. For acquisition, the ImageXpress® automated imaging systems and our cellular imaging and analysis software provide the capability to screen and analyze these models within a single, integrated interface to dramatically reduce time to discovery.
Steps used for a typical 3D cell culture assay:
3D cell culture imaging and analysis techniques
Our imaging technology continues to evolve for easier implementation of more physiologically-relevant models. Learn more about how our technology can help scale-up 3D cell culture from small-scale assays to high-throughput screening.
Acquire images for 3D assays
In recent years, there have been significant advances in automated microscopy and imaging for delivering more predictive, physiologically-relevant measurements for drug discovery and environmental toxicity applications. Implementation of more complex assays and 3D models requires high resolution to capture publication-quality images and data.
Confocal imaging provides a high signal-to-noise ratio by reducing out-of-focus light for crisper images to yield more cellular detail even within a thick sample. Watch the video to learn about high-throughput techniques on how to acquire images in 3D using the ImageXpress Micro Confocal system.
Analyze images for 3D applications
There are different types of image sets that may be generated even though your sample is 3D. A single plane may be sufficient or multiple planes may be acquired. When multiple planes are acquired, it’s useful to be able to analyze them individually or to analyze them after combining them into a single 2D projection. Ultimately, analysis of the entire 3D image may be the goal of the experiment. Watch the video to learn more about techniques for analyzing images from multiple z-planes as a 3D volume using MetaXpress software.
Advantages of 3D Cell Models
Three-dimensional (3D) cell models are more physiologically relevant than two-dimensional cell cultures, and they more closely represent the tissue microenvironments, cell-to-cell interactions, and biological processes that occur in vivo. Now you can generate more predictive data using high-content imaging (HCI) systems like the ImageXpress system. By integrating both high-content image acquisition and analysis software like MetaXpress® software with the 3D Analysis Module, the ImageXpress system can be used for in-depth analysis, visualization, and assessment of 3D structures. This single interface will enable you to meet 3D acquisition and analysis challenges without compromise to throughput or data quality, giving you confidence in your discoveries.
Learn more about 3D cell model application and assays:
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3D neural spheroids
To accelerate the development of effective and safe drugs, there is an increasing need for more complex, biologically relevant, and predictive cell-based assays for drug discovery and toxicology screening.
We show that functional and morphological assays using 3D neural spheroids formed with human iPSC-derived cells can be used for evaluation of drug candidates and neurotoxicity assessment.
3D neural spheroids
To accelerate the development of effective and safe drugs, there is an increasing need for more complex, biologically relevant, and predictive cell-based assays for drug discovery and toxicology screening.
We show that functional and morphological assays using 3D neural spheroids formed with human iPSC-derived cells can be used for evaluation of drug candidates and neurotoxicity assessment.
Acquire and analyze 3D images like a pro
There has been significant progress in the development of 3D models and techniques during the last few years. Methods include biodegradable scaffolds, organ-on-a chip structures, or self-assembled organoids. Recently, simple, automated workflows suitable for high-content imaging have become popular.
Acquire and analyze 3D images like a pro
There has been significant progress in the development of 3D models and techniques during the last few years. Methods include biodegradable scaffolds, organ-on-a chip structures, or self-assembled organoids. Recently, simple, automated workflows suitable for high-content imaging have become popular.
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Capture True 3D Cell Culture Insights
Developing more complex, biologically relevant, and predictive cell-based assays for compound screening is a primary challenge in drug discovery.
This ebook highlights the 3D cell culture imaging and analysis advantage and includes a few 3D cell model applications as well as tips and techniques for optimizing your 3D assay workflow.
Capture True 3D Cell Culture Insights
Developing more complex, biologically relevant, and predictive cell-based assays for compound screening is a primary challenge in drug discovery.
This ebook highlights the 3D cell culture imaging and analysis advantage and includes a few 3D cell model applications as well as tips and techniques for optimizing your 3D assay workflow.
Cardiotoxicity
Live-cell imaging and 3D models provide greater predictivity for compound ecacy and toxicity. We present methods for the formation of 3D spheroids derived from human iPSC-derived cells.
Cardiotoxicity
Live-cell imaging and 3D models provide greater predictivity for compound ecacy and toxicity. We present methods for the formation of 3D spheroids derived from human iPSC-derived cells.
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Cells in Extracellular Matrices
One common way of culturing cells in three dimensional space is through the use of extracellular matrix-based hydrogels, such as Matrigel. Cells are grown in an extracellular matrix (ECM) to mimic an in vivo environment. Differences between Matrigel and 2D cell cultures can be readily seen by their different cell morphologies, cell polarity, and/or gene expression. Hydrogels can also enable studies on cell migration and 3D structure formation, such as endothelial cell tube formation in angiogenesis studies.
Cells in Extracellular Matrices
One common way of culturing cells in three dimensional space is through the use of extracellular matrix-based hydrogels, such as Matrigel. Cells are grown in an extracellular matrix (ECM) to mimic an in vivo environment. Differences between Matrigel and 2D cell cultures can be readily seen by their different cell morphologies, cell polarity, and/or gene expression. Hydrogels can also enable studies on cell migration and 3D structure formation, such as endothelial cell tube formation in angiogenesis studies.
Organoid technology / Organ-on-a-Chip
Organoid technology, like organ-on-a-chip emulates organ physiology though co-culture of cells in a supportive 3D matrix and use of microfluidic channels to perfuse nutrients or compounds over the resulting cellular structures. It is rapidly gaining popularity as a biologically relevant screening model for new drugs and toxicity.
- 3D image analysis and characterization of angiogenesis in organ-on-a-chip model
- Video: Physiologically-Relevant Tissue Models Using a High-Throughput Organ-on-a-Chip Platform
- High-content assay for morphological characterization of 3D neuronal networks in a microfluidic platform
- Webinar: Developing high-throughput organ-on-a-chip tissue models for drug discovery using high-content imaging
- Poster: Water immersion objectives for automated high-content imaging to improve precision and quality of complex biological assays
Organoid technology / Organ-on-a-Chip
Organoid technology, like organ-on-a-chip emulates organ physiology though co-culture of cells in a supportive 3D matrix and use of microfluidic channels to perfuse nutrients or compounds over the resulting cellular structures. It is rapidly gaining popularity as a biologically relevant screening model for new drugs and toxicity.
- 3D image analysis and characterization of angiogenesis in organ-on-a-chip model
- Video: Physiologically-Relevant Tissue Models Using a High-Throughput Organ-on-a-Chip Platform
- High-content assay for morphological characterization of 3D neuronal networks in a microfluidic platform
- Webinar: Developing high-throughput organ-on-a-chip tissue models for drug discovery using high-content imaging
- Poster: Water immersion objectives for automated high-content imaging to improve precision and quality of complex biological assays
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Organoid vs. Spheroid
The terms spheroid and organoid are often used interchangeably. Both cell models are 3D multi-cellular structures that better mimic the in vivo environment as compared to their 2D cell counterparts. Organoids typically consist of a co-culture of cells which demonstrate a higher order of self-assembly to allow for an even better representation of complex in vivo cell responses and interactions. In contrast, spheroids have a more simplified 3D structure making them amenable to high reproducibility and scaling. Functionally, both model types are used as better in vitro models for capturing more physiologically relevant data.
Organoid vs. Spheroid
The terms spheroid and organoid are often used interchangeably. Both cell models are 3D multi-cellular structures that better mimic the in vivo environment as compared to their 2D cell counterparts. Organoids typically consist of a co-culture of cells which demonstrate a higher order of self-assembly to allow for an even better representation of complex in vivo cell responses and interactions. In contrast, spheroids have a more simplified 3D structure making them amenable to high reproducibility and scaling. Functionally, both model types are used as better in vitro models for capturing more physiologically relevant data.
Spheroids
Spheroids are multi-cellular 3D structures that mimic in vivo cell responses and interactions. They can be highly reproducible and to be scaled for high-content screening. Compared with adherent cells grown in 2D monolayers, 3D growth conditions are believed to more closely reflect the natural environment of cancer cells. Acquiring measurements from these larger structures involve acquiring images from different depths (z-planes) within the body of the spheroid and analyzing them in 3D, or collapsing the images into a single 2D stack before analysis.
- 3D Imaging of Cancer Cell Spheroids
- High-Throughput Confocal Imaging of Spheroids for Screening Cancer Therapeutics
- Functional and mechanistic neurotoxicity profiling using human iPSC-derived neural 3D cultures
- High-content 3D toxicity assay using iPSC-derived hepatocyte spheroids
- Acquire and analyze images of FUCCI spheroids
- Assay apoptosis in magnetically bioprinted HepG2 spheroids
Spheroids
Spheroids are multi-cellular 3D structures that mimic in vivo cell responses and interactions. They can be highly reproducible and to be scaled for high-content screening. Compared with adherent cells grown in 2D monolayers, 3D growth conditions are believed to more closely reflect the natural environment of cancer cells. Acquiring measurements from these larger structures involve acquiring images from different depths (z-planes) within the body of the spheroid and analyzing them in 3D, or collapsing the images into a single 2D stack before analysis.
- 3D Imaging of Cancer Cell Spheroids
- High-Throughput Confocal Imaging of Spheroids for Screening Cancer Therapeutics
- Functional and mechanistic neurotoxicity profiling using human iPSC-derived neural 3D cultures
- High-content 3D toxicity assay using iPSC-derived hepatocyte spheroids
- Acquire and analyze images of FUCCI spheroids
- Assay apoptosis in magnetically bioprinted HepG2 spheroids
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Stem Cells
Stem cells are an accessible source of primary-like cells which can be used in longer-term studies to measure targeted responses in specific cell types and 3D tissues. Complex assays and 3D induced pluripotent stem cell (iPSC)-derived cell models better represent tissue biology and cell interactions which make them more relevant for many toxicity and drug screening assays.
Stem Cells
Stem cells are an accessible source of primary-like cells which can be used in longer-term studies to measure targeted responses in specific cell types and 3D tissues. Complex assays and 3D induced pluripotent stem cell (iPSC)-derived cell models better represent tissue biology and cell interactions which make them more relevant for many toxicity and drug screening assays.
Whole Organisms
Model systems such as Drosophila, C. elegans, and zebrafish offer relevance to human biology, with the added advantage of genetic and whole-organism phenotypic screening. Zebrafish screening has gained favor as an alternative to mammalian screening due to reduced cost and space requirements, short generation times, and ease of genetic manipulation. Our zebrafish studies use the ImageXpress Micro System and MetaXpress software to set up and run automated imaging screens to monitor inhibition of angiogenesis, quantitate gene knockdown, and measure ototoxicity and neurotoxicity.
Whole Organisms
Model systems such as Drosophila, C. elegans, and zebrafish offer relevance to human biology, with the added advantage of genetic and whole-organism phenotypic screening. Zebrafish screening has gained favor as an alternative to mammalian screening due to reduced cost and space requirements, short generation times, and ease of genetic manipulation. Our zebrafish studies use the ImageXpress Micro System and MetaXpress software to set up and run automated imaging screens to monitor inhibition of angiogenesis, quantitate gene knockdown, and measure ototoxicity and neurotoxicity.
3D cell imaging and analysis workflow
The workflow of a 3D cell culture protocol is dependent on the type of cell model being cultured. The 3D cell imaging and analysis workflow depicted here is a generalized example of 3D spheroids in a 96- or 384-well flat or round-bottom microplate. Each step breaks down the process from cell culture to image analysis and highlights the instrumentation and tools needed to conduct the cell culture protocol including an automated high-content imaging system and cellular imaging and analysis software.
CULTURE SPHEROIDS
3D cell can be cultured directly in a ULA, round bottom plate to develop the typical spherical shape of a spheroid. Cells can also be grown in alternate labware like a petri dish, flask, or other microplate before transferring to an imaging microplate.
96- or 384- Well Plates
Most 3D cell cultures for high-content, high-throughput screening are run using 96- or 384-well microplates. ULA, U-shaped/round bottom microplates enable the spherical shape to form spheroids or spheroids can also be cultured in a flat-bottom microplate using hydrogels to simulate extracellular matrices (ECM). Optimized spheroid cell culture protocols for the ImageXpress® Micro Confocal High-Content Imaging System can use ULA, U-bottom 96- and 384-well plates with a one-step staining procedure that reduces assay time and minimizes variability.
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TREAT WITH COMPOUNDS
After spheroid formation, compounds are added into the wells, and then incubated for one to several days, depending on the mechanism being studied. Generally, shorter durations are used to study apoptosis and longer durations for multi-parameter cytotoxicity studies. For drug treatments requiring a longer duration, compounds are refreshed periodically during incubation. The concentration of compounds and the incubation period is dependent on the desired protocol.
STAIN FOR MARKERS
After compound treatment is completed, 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.
Assay Kits
Easy-to-use, robust assay kits for life science research, drug discovery and development, and bioassays. Our assay kits are optimized for use on our instruments. Screen more compounds earlier in drug discovery and enable characterization of a full concentration-response profile of test compounds for cell viability, cell proliferation, axiogensis, and more.
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High-content Imaging System
The ImageXpress Micro Confocal system is a unique confocal imaging solution capable of imaging more than a million wells a week. MetaXpress software enables acquisition of 3D z-stacks. Z-plane images can be saved individually or collapsed into a single 2D projection image using a mathematical algorithm.
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ACQUIRE 3D CELL IMAGE
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 3D CELL IMAGE
Use cellular imaging analysis software to run quantitative analysis of the cell images to assess and monitor how cells express against different markers and to quantify biological readouts.
High-content Imaging Software
MetaXpress High-Content Image Acquisition and Analysis Software is a multi-level analysis tool for a wide range of 2D and 3D applications optimized for the ImageXpress Micro Confocal system. The integrated acquisition and analysis application modules for 3D cell models simplify high-throughput quantification of 3D structures with volume, intensity, and distance measurements.
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Resources for 3D Cells
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…
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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…
Scientific Poster
3D imaging and analysis of angiogenesis in the organ-on-a-chip platform
3D imaging and analysis of angiogenesis in the organ-on-a-chip platform
Angiogenesis is the physiological process of formation and remodeling of new blood vessels and capillaries from pre-existing blood vessels.1 This can be achieved through endoth…
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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…
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Scientific Poster
AI-based analysis of complex biological phenotypes
AI-based analysis of complex biological phenotypes
Cell-based phenotypic assays have become an increasingly attractive alternative to traditional in vitro and in vivo testing in pharmaceutical drug development and toxicological safety ass…
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Application Note
3D image analysis and characterization of angiogenesis in organ-on-a-chip model
3D image analysis and characterization of angiogenesis in organ-on-a-chip model
Angiogenesis is the physiological process of formation and remodeling of new blood vessels and capillaries from pre-existing blood vessels. This can be achieved through endothelial…
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Scientific Poster
Multiplexed automated assays for neurotoxicity evaluation using induced pluripotent stem cell-derived neural 3D cell models
Multiplexed automated assays for neurotoxicity evaluation using induced pluripotent stem cell-derived neural 3D cell models
Cell-based phenotypic assays have become an increasingly attractive alternative to traditional in vitro and in vivo testing in pharmaceutical drug development and toxicological safety ass…
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Scientific Poster
Water immersion objectives for automated high-content imaging to improve precision and quality of complex biological assays
Water immersion objectives for automated high-content imaging to improve precision and quality of complex biological assays
The purpose of these studies was to determine if water immersion objectives, used to improve image quality in complex biological assays, could be used in a high-throughput environment.…
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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.
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: mocro…
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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.
eBook
Acquire and analyze 3D images like a pro
Acquire and analyze 3D images like a pro
There has been significant progress in the development of 3D models and techniques during the last few years. Methods include biodegradable scaffolds, organ-on-a chip structures, or self-…
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Scientific Poster
Novel Imaging and Analysis of Zebrafish for High Throughput Screening
Novel Imaging and Analysis of Zebrafish for High Throughput Screening
Explore noval imaging & analysis of Zebrafish for high throughput screening, where Zebrafish Is an attractive model system for developing disease related assays for drug discovery.…
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Application Note
Measuring mitochondrial shape and distribution using the ImageXpress Micro High-Content Imaging System
Measuring mitochondrial shape and distribution using the ImageXpress Micro High-Content Imaging System
Using high-content imaging assays allow cells cultured and treated in microplates to be imaged alive without wash steps for real-time studies of cells in their most natural state, although…
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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…
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Application Note
High-content assay for morphological characterization of 3D neuronal networks in a microfluidic platform
High-content assay for morphological characterization of 3D neuronal networks in a microfluidic platform
Establishment of physiologically-relevant in vitro models is crucial to further understanding of the mechanisms of neurological diseases as well as targeted drug development. While iPSC-…
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Application Note
Automating the generation and analysis of 3D cell cultures in Matrigel®
Automating the generation and analysis of 3D cell cultures in Matrigel®
Researchers are turning to three-dimensional (3D) cell culture as a way to increase the biological relevance of their disease models and predictive value of drug studies. These 3D models…
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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…
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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…
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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…
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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…
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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…
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Application Note
Analyze cardiomyocyte spheroid contractions with SoftMax Pro Software
Analyze cardiomyocyte spheroid contractions with SoftMax Pro Software
Cell-based models for compound screening have become more complex in order to represent complexity of biological systems. Live-cell imaging and threedimensional models provide valuable…
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Application Note
High-throughput imaging assays using zebrafish, a model organism for human disease
High-throughput imaging assays using zebrafish, a model organism for human disease
Recently, zebrafish-based screening has gained favor as an alternative to mammalian screening due to cost, throughput and reduced ethical concerns. Zebrafish are a useful model for drug…
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Application Note
High-content screening of neuronal toxicity using iPSC-derived human neurons
High-content screening of neuronal toxicity using iPSC-derived human neurons
The nervous system is sensitive to the toxic effects of many chemical compounds, environmental agents and certain naturally occurring substances. Neurotoxicity can cause temporary or…
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Application Note
Multiplexed high-content hepatotoxicity assays using iPSC-derived hepatocytes
Multiplexed high-content hepatotoxicity assays using iPSC-derived hepatocytes
Drug-induced hepatotoxicity is an important cause for liver injury and acute liver failure. Thus highly predictive assays for safety and efficacy testing are crucial for improving drug…
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Scientific Poster
High-Content Assay for Morphological Characterization of 3D Neuronal Networks in a Microfluidic Platform
High-Content Assay for Morphological Characterization of 3D Neuronal Networks in a Microfluidic Platform
The absence of physiologically relevant in vitro models of the nervous system is an important limitation in using 3D cultures for assay development applicable for neurodegenerative diseas…
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Scientific Poster
In Vitro Assessment of Drug Effects on Human iPSC-Derived Cardiac Spheroid Cultures
In Vitro Assessment of Drug Effects on Human iPSC-Derived Cardiac Spheroid Cultures
for modeling developmental and tissue biology with the goal of accelerating translational research. Such 3D models can provide different perspectives from traditional 2D cultures on the r…
Videos & Webinars
Disease modeling in the 21st century: Automated organoid assays with 3D imaging
3D cell culture, tissue clearing, & high-content imaging in the quest of effective solutions to NAFLD
Transitioning high-content assays to 3D: Scientific opportunities and imaging challenges
Implementing 3D Neural Spheroids in Drug Discovery
Developing high-throughput organ-on-a-chip tissue models for drug discovery using high-content imaging
Solutions for High-Throughput 3D Acquisition using High-Content Imaging
Solutions for High-Content Analysis of 3D Images
Zebrafish Actin mask w FITC vessel mask
Angiogenesis Research: High-Content Imaging Systems Help Unlock the Full Potential of 3D Tissue Models
Physiologically-Relevant Tissue Models Using a High-Throughput Organ-on-a-Chip Platform