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5 Steps to Creating 3D Cell Culture Models
What are 3D cell culture models?
3D cell culture models are in vitro multicellular structures designed to emulate tissue or organ like properties that better replicate the cellular environment in vivo, providing opportunities to produce more relevant results. Both organoid and spheroid models are being increasingly used in multiple areas such as neurobiology stem cell research, regenerative medicine, and cancer biology for the key insights they provide in translation research.
Better in vitro models for disease research
Whether you are just beginning your research with 3D cell culture models and need to learn more about organoids or spheroids or need appropriate tools to validate your in vitro models, we have outlined these five easy steps to support your progress.
Five-step workflow for organoid and 3D cell culture and analysis
Step 1: Source cells for the in vitro model
Choose cells that allow you to closely mimic the in vivo state and generate more relevant data
Creating relevant in vitro models starts with selecting the cell line that best represents the biology you are trying to reproduce. In some cases, this means starting with isolated primary cells to build predictive cell culture models for liver toxicity (hepatocytes) or skin toxicity (HUVEC or keratinocytes) to study drug absorption.
For cell types that are more difficult to obtain, such as neurons, it may be advisable to start with stem cells and leverage the 3D microenvironment to derive more complex cellular structures or “mini organs” that contain multiple cell types.
Figure 1. High content analysis of bile canaliculi formation in hepatic spheroids. HepG2 spheroids on day 14 (left) and hepatic spheroids on day 7 (right) were stained with CFDA and DAPI and imaged using Thermo Scientific CellInsight CX7 platform at 10x magnification. Hepatic spheroids show clear formation of the bile ducts in comparison to the HepG2 spheroids (used as a negative control).
Cryopreserved cells for 3D in vitro models
| Cell type | Tissue source | Products |
|---|---|---|
— |
Custom services
Our Custom Biology Cell Model Services can help you establish your 3D cell culture model. We offer you choices at every stage of your research and ability to support any project, no matter the complexity and size. Choose the combination of tools and services that best fits your lab resources and technical capabilities. Partner with us to extend your impact by utilizing our dedicated team of cell biology experts to provide a custom 3D cell culture model solution.
Some of our most popular services
- Stem cell modeling services
- Cell line engineering
- Profiling and screening services
- Cell model services (characterization, reprogramming & differentiation)
- Custom assays (cellular and biochemical)
Tips and tricks
- It is important to carefully consider your cell type when setting up your experiment
To understand more, watch this informational webinar - Regardless of the source of your cell lines, it is important to make sure they are tested for Mycoplasma contamination before you use them
Learn more
Step 2: Choose 3D cell support material
Selecting the right culture matrix is an important step in developing a successful culture system for organoids and spheroids
Extracellular matrices and 3D cultureware are used to easily transition from a 2D monolayer environment to 3D cell culture models. 3D cultureware systems are used for scale-up and full organoid model development. Porous membrane-based systems are advantageous when polarization of the epithelial cells is needed in cell differentiation and tissue formation.
Extracellular matrices
Whether you are just getting started in 3D or looking for more complex ways to apply 3D cell culture models, our extracellular matrices and 3D cultureware allow you to develop physiologically relevant 3D cell culture models by helping improve intracellular interactions. Trust Thermo Fisher Scientific, the most cited brand in 3D cell culture research, for consistent and cost-effective optimized media, supplements and matrices for most major cell types and applications.
Featured cell matrix products
Cultureware for 3D cell culture models
Typically, culture systems enable better control in the number and size of spheroids and organoids and are highly compatible with plate readers and HCA platforms. The Thermo Scientific Nunclon Sphera culture system was specifically designed to enable 3D cell culture (i.e., spheroid and organoid growth) with many options for plates, dishes, flasks and well inserts. The special design of our Nunclon Sphera allows cells to grow in suspension with almost no cell attachment to the cultureware, supporting the establishment of 3D spheroids and organoids when used either as a scaffold-free system or with our optimized Gibco scaffold-based substrates (ECM/matrices).
Figure 2. Minimal cell adhesion on Nunclon Sphera surface. Cell adhesion is much lower on the Nunclon Sphera surface than that on the cell culture-treated and non-treated controls for all cell types tested. Nunclon Sphera also shows lower adhesion than another brand of low–cell attachment surface (Brand C) for the differentiated U937 cells.
Cell culture plates, inserts, and dishes for 3D cell culture models
The Thermo Scientific Nunc cell culture inserts are made up of a porous and unique structure that allows the attached surface of your cells to be simultaneously exposed to growth media and to the variable condition(s) your experiment may entail. Even more, the carrier plate option makes it easy to carry out these procedures with precision and efficiency.
Tips and tricks
- A few ways to control the size of spheroids include: adjusting the initial cell seeding density, avoiding culture vessels with large surface areas, such as a T flask or a large petri dish, or using a more confined physical space to promote spheroid formation, such as a low-attachment microplate (e.g., Nunclon Sphera 96-well U bottom plate).
- Geltrex matrix is a soluble form of basement membrane extracted from murine Engelbreth-Holm-Swarm (EHS) tumors and can be used as a replacement for products like Matrigel matrix. Learn more about Geltrex basement membrane extract.
Step 3: Select cell culture media and supplements
3D culture systems can be modified and developed to suit specialized cell types like stem cells or cancer cell lines
Growing 3D cell culture models is a commitment in time and resources, and you need reassurance that your investment is going to give you the biology you anticipate. Gibco media and reagents are widely used in the growth, differentiation, and maturation of 3D cell culture models.
Recombinant growth factors and cytokines can help fine-tune your culture system to generate the 3D cell model you need. Gibco Peprotech recombinant proteins undergo rigorous testing and quality controls to support robust cell culture models.
Available 3D cell culture media and supplements
| Cell type | Media | Supplements |
|---|---|---|
Pluripotent stem cells | ||
Skin cells | ||
Cancer spheroids | ||
Hepatic cells | ||
Brain cells | ||
| Tumoroids |
Tips and tricks
- Check your media requirements—half or full media changes may be required for long-term culture or differentiation protocols
- When preparing to aspirate media from a well, tilting the microplate at an angle will prevent the pipette tip from touching the bottom of the well where the spheroids are settled.
- Thermo Scientific Wellwash Versa Microplate Washer can be programmed to quickly, safely, and consistently exchange media without sample loss
- Recombinant proteins for organoid culture can be used to develop these more sophisticated 3D cell culture models
Learn more
Step 4: Monitor and visualize spheroid growth
Visual and quantitative analysis of 3D cell culture models
Confirming that 3D cell structures are developing and maintaining the appropriate morphology is paramount to establishing a 3D in vitro model with the correct cellular and biochemical makeup. Methods to monitor 3D cell cultures include measurement of cell count, cluster size, and growth patterns over time in multi-well plate formats. These measurements can be achieved with technologies, such as microscopic imaging and high-content analysis.
Brightfield imaging can easily establish the diameter of spheroids and organoids, and this data can be used to calculate volume and estimate the number of cells in the 3D clusters. Quantification by fluorescence imaging of cells in a 3D cell culture model can be more difficult due to the dense cellular organization of a cluster of cells. As 3D clusters get larger, they become opaquer, so seeing within the spheroid is not always possible. Clearing agents and antifade mounting agents can help to visualize 3D cultures while maintaining the morphology of the cells, making them amenable to techniques like immunofluorescence (IF), immunocytochemistry (ICC), and immunohistochemistry (IHC).
Figure 3. Spheroids growing in Nunclon Sphera low attachment plate. A549 cells plated at 5K/well on a Nunclon Sphera 96-well plate and incubated 24 hours. Automatically imaged with 10X objective using brightfield illumination on a CellInsight CX7 LZR High Content Analysis system.
Figure 4. Fluorescence imaging results of spheroids following 3D culture clearing. A549 spheroids with clearing (left) HeLa spheroids with clearing (right) using CytoVista 3D Culture Clearing Agent, designed to promote optical transparency and enable visualization inside thick samples of fixed cells.
Reagents for imaging of 3D cultures
| Product | Benefits |
|---|---|
Clearing Reagents for 3D Tissue, Organoid, and Spheroid Imaging | CytoVista reagent helps provide clarity in fluorescently labeled 3D cultures (including spheroid cores) of samples that are up to 1,000 microns in diameter. |
| ProLong Glass Antifade Mountant | ProLong Glass Antifade Mountant is a glycerol-based, hard-setting, ready-to-use specimen mounting agent that can be applied directly to fluorescently labeled cells or tissue samples on microscope slides or coverslips. ProLong Glass mountant reduces the spherical aberration by having refractive index of 1.52 after curing, which helps enable the best resolution for up to 150 µm thick specimens. |
Tools for image-based analysis of 3D cell culture models
Invitrogen imaging technologies help ensure clear visualization and analysis of your 3D cell culture model system.
| Product | Benefits |
|---|---|
EVOS cell imaging systems allow you to visualize, assess, and document live 3D cell cultures via high-resolution microscopy. | |
CellInsight high content screening systems can be used to rapidly image, identify, and analyze fluorescently labeled spheroids and other 3D cell clusters in a multiwell plate format. | |
Multi-dimensional analysis software enables deep analysis of cellular and subcellular structures to extract meaningful metrics about your 3D cell culture model. |
Tips and tricks
- Spheroids more than 300 µm in diameter can have a necrotic core and can be difficult to image and analyze.
- Leverage clearing reagents such as the CytoVista 3D Cell Culture Clearing Reagent to analyze your whole 3D cell culture model from the outer layers to the core.
Step 5: Characterize and assay genotype and phenotype
Molecular and cellular characterization of spheroids and 3D cell culture models
Visualization of 3D cell culture models and assessing their health are important to ensure that growth conditions are correct, but researchers must go a step further. New 3D in vitro models need to have their gene expression profiles, phenotypic markers, and organelle function analyzed to help ensure they resemble the microanatomy of the tissue or organ under investigation. Antibodies can be used to characterize the cell line or primary cells, from which spheroids are developed. Monitoring mRNA expression level in the developing cell culture model can help to identify cell types or changes in gene expression pattern that occur under certain treatment conditions. Mitochondrial health, cytotoxicity, reactive oxygen species levels, and apoptosis, are measured using live-cell functional probes.
Assay products
Thermo Fisher Scientific offers the necessary tools to validate 3D cell culture models. TaqMan® Assay probes and QuantStudio Real Time PCR are easy, fast ways to monitor gene expression. Our inclusive portfolio of validated antibodies and immunoassays can be used to detect intracellular, membrane bound or secreted protein markers. Invitrogen fluorescence reagents support high-content screening and analysis of a wide variety of cellular phenotypic properties.
Figure 5. T cell-mediated killing of A549 spheroids. Activated T cells (isolated from PBMC using Dynabeads magnetic beads coated with CD3/CD28), or non-activated T cells were added to A549 spheroid cells at various concentrations. A549 cells were analyzed for levels of caspase activity to determine the amount of apoptotic death. HeLa cells treated with activated T cells showed increased levels of caspase activity, while those treated with non-activated T cells showed decreased levels of caspase activity. Prior to co-culturing, T cells were stained with CellTracker Deep Red dye (purple dye). Apoptosis (cell death) was measured using CellEvent Caspase-3/7 Green Detection Reagent. Images were collected on a CellInsight CX7 LZR HCA instrument in confocal mode. Bright field images were used to determine spheroid area.
Reagents and systems for 3D cell culture model characterization
| Product | Benefits |
|---|---|
When used in combination with TaqMan Assay probes, QuantStudio real-time PCR instruments are a fast, easy way to monitor gene expression levels in your developing organoids. | |
Applied Biosystems and Invitrogen reagents and assays support the validation of 3D cell culture models to help ensure physiological relevance. Cell health, viability, apoptosis, cytotoxicity, mitochondrial health, DNA damage, and reactive oxygen species are just a few approaches to measuring cellular health in live cells. | |
Primary antibodies recognize specific cellular markers and can serve as useful tools to characterize cell lines. Invitrogen antibodies are available for a wide selection of targets, range of host reactivity and applications. | |
CellInsight high content screening systems can be used to rapidly image, identify, and analyze fluorescently labeled spheroids and other 3D cell clusters in a multi-well plate format. | |
From single-analyte identification using ELISA to multiplexed formats with ProcartaPlex assays, Invitrogen immunoassays comprise a suite of options for biomarker identification, quantification and analysis. | |
Compatible with a wide range of Invitrogen cell health assays, and capable of five different measurement modalities including absorbance, fluorescence intensity, luminescence, AlphaScreen or AlphaLISA, and time resolved fluorescence. |
Tips and tricks
- When performing image-based characterization, using a laser-based confocal system typically results in an improved signal-to-noise ratio and helps enable clear and rapid visualization of any 3D cell culture model system.
Learn more
Step 1: Source cells for the in vitro model
Choose cells that allow you to closely mimic the in vivo state and generate more relevant data
Creating relevant in vitro models starts with selecting the cell line that best represents the biology you are trying to reproduce. In some cases, this means starting with isolated primary cells to build predictive cell culture models for liver toxicity (hepatocytes) or skin toxicity (HUVEC or keratinocytes) to study drug absorption.
For cell types that are more difficult to obtain, such as neurons, it may be advisable to start with stem cells and leverage the 3D microenvironment to derive more complex cellular structures or “mini organs” that contain multiple cell types.
Figure 1. High content analysis of bile canaliculi formation in hepatic spheroids. HepG2 spheroids on day 14 (left) and hepatic spheroids on day 7 (right) were stained with CFDA and DAPI and imaged using Thermo Scientific CellInsight CX7 platform at 10x magnification. Hepatic spheroids show clear formation of the bile ducts in comparison to the HepG2 spheroids (used as a negative control).
Cryopreserved cells for 3D in vitro models
| Cell type | Tissue source | Products |
|---|---|---|
— |
Custom services
Our Custom Biology Cell Model Services can help you establish your 3D cell culture model. We offer you choices at every stage of your research and ability to support any project, no matter the complexity and size. Choose the combination of tools and services that best fits your lab resources and technical capabilities. Partner with us to extend your impact by utilizing our dedicated team of cell biology experts to provide a custom 3D cell culture model solution.
Some of our most popular services
- Stem cell modeling services
- Cell line engineering
- Profiling and screening services
- Cell model services (characterization, reprogramming & differentiation)
- Custom assays (cellular and biochemical)
Tips and tricks
- It is important to carefully consider your cell type when setting up your experiment
To understand more, watch this informational webinar - Regardless of the source of your cell lines, it is important to make sure they are tested for Mycoplasma contamination before you use them
Learn more
Step 2: Choose 3D cell support material
Selecting the right culture matrix is an important step in developing a successful culture system for organoids and spheroids
Extracellular matrices and 3D cultureware are used to easily transition from a 2D monolayer environment to 3D cell culture models. 3D cultureware systems are used for scale-up and full organoid model development. Porous membrane-based systems are advantageous when polarization of the epithelial cells is needed in cell differentiation and tissue formation.
Extracellular matrices
Whether you are just getting started in 3D or looking for more complex ways to apply 3D cell culture models, our extracellular matrices and 3D cultureware allow you to develop physiologically relevant 3D cell culture models by helping improve intracellular interactions. Trust Thermo Fisher Scientific, the most cited brand in 3D cell culture research, for consistent and cost-effective optimized media, supplements and matrices for most major cell types and applications.
Featured cell matrix products
Cultureware for 3D cell culture models
Typically, culture systems enable better control in the number and size of spheroids and organoids and are highly compatible with plate readers and HCA platforms. The Thermo Scientific Nunclon Sphera culture system was specifically designed to enable 3D cell culture (i.e., spheroid and organoid growth) with many options for plates, dishes, flasks and well inserts. The special design of our Nunclon Sphera allows cells to grow in suspension with almost no cell attachment to the cultureware, supporting the establishment of 3D spheroids and organoids when used either as a scaffold-free system or with our optimized Gibco scaffold-based substrates (ECM/matrices).
Figure 2. Minimal cell adhesion on Nunclon Sphera surface. Cell adhesion is much lower on the Nunclon Sphera surface than that on the cell culture-treated and non-treated controls for all cell types tested. Nunclon Sphera also shows lower adhesion than another brand of low–cell attachment surface (Brand C) for the differentiated U937 cells.
Cell culture plates, inserts, and dishes for 3D cell culture models
The Thermo Scientific Nunc cell culture inserts are made up of a porous and unique structure that allows the attached surface of your cells to be simultaneously exposed to growth media and to the variable condition(s) your experiment may entail. Even more, the carrier plate option makes it easy to carry out these procedures with precision and efficiency.
Tips and tricks
- A few ways to control the size of spheroids include: adjusting the initial cell seeding density, avoiding culture vessels with large surface areas, such as a T flask or a large petri dish, or using a more confined physical space to promote spheroid formation, such as a low-attachment microplate (e.g., Nunclon Sphera 96-well U bottom plate).
- Geltrex matrix is a soluble form of basement membrane extracted from murine Engelbreth-Holm-Swarm (EHS) tumors and can be used as a replacement for products like Matrigel matrix. Learn more about Geltrex basement membrane extract.
Step 3: Select cell culture media and supplements
3D culture systems can be modified and developed to suit specialized cell types like stem cells or cancer cell lines
Growing 3D cell culture models is a commitment in time and resources, and you need reassurance that your investment is going to give you the biology you anticipate. Gibco media and reagents are widely used in the growth, differentiation, and maturation of 3D cell culture models.
Recombinant growth factors and cytokines can help fine-tune your culture system to generate the 3D cell model you need. Gibco Peprotech recombinant proteins undergo rigorous testing and quality controls to support robust cell culture models.
Available 3D cell culture media and supplements
| Cell type | Media | Supplements |
|---|---|---|
Pluripotent stem cells | ||
Skin cells | ||
Cancer spheroids | ||
Hepatic cells | ||
Brain cells | ||
| Tumoroids |
Tips and tricks
- Check your media requirements—half or full media changes may be required for long-term culture or differentiation protocols
- When preparing to aspirate media from a well, tilting the microplate at an angle will prevent the pipette tip from touching the bottom of the well where the spheroids are settled.
- Thermo Scientific Wellwash Versa Microplate Washer can be programmed to quickly, safely, and consistently exchange media without sample loss
- Recombinant proteins for organoid culture can be used to develop these more sophisticated 3D cell culture models
Learn more
Step 4: Monitor and visualize spheroid growth
Visual and quantitative analysis of 3D cell culture models
Confirming that 3D cell structures are developing and maintaining the appropriate morphology is paramount to establishing a 3D in vitro model with the correct cellular and biochemical makeup. Methods to monitor 3D cell cultures include measurement of cell count, cluster size, and growth patterns over time in multi-well plate formats. These measurements can be achieved with technologies, such as microscopic imaging and high-content analysis.
Brightfield imaging can easily establish the diameter of spheroids and organoids, and this data can be used to calculate volume and estimate the number of cells in the 3D clusters. Quantification by fluorescence imaging of cells in a 3D cell culture model can be more difficult due to the dense cellular organization of a cluster of cells. As 3D clusters get larger, they become opaquer, so seeing within the spheroid is not always possible. Clearing agents and antifade mounting agents can help to visualize 3D cultures while maintaining the morphology of the cells, making them amenable to techniques like immunofluorescence (IF), immunocytochemistry (ICC), and immunohistochemistry (IHC).
Figure 3. Spheroids growing in Nunclon Sphera low attachment plate. A549 cells plated at 5K/well on a Nunclon Sphera 96-well plate and incubated 24 hours. Automatically imaged with 10X objective using brightfield illumination on a CellInsight CX7 LZR High Content Analysis system.
Figure 4. Fluorescence imaging results of spheroids following 3D culture clearing. A549 spheroids with clearing (left) HeLa spheroids with clearing (right) using CytoVista 3D Culture Clearing Agent, designed to promote optical transparency and enable visualization inside thick samples of fixed cells.
Reagents for imaging of 3D cultures
| Product | Benefits |
|---|---|
Clearing Reagents for 3D Tissue, Organoid, and Spheroid Imaging | CytoVista reagent helps provide clarity in fluorescently labeled 3D cultures (including spheroid cores) of samples that are up to 1,000 microns in diameter. |
| ProLong Glass Antifade Mountant | ProLong Glass Antifade Mountant is a glycerol-based, hard-setting, ready-to-use specimen mounting agent that can be applied directly to fluorescently labeled cells or tissue samples on microscope slides or coverslips. ProLong Glass mountant reduces the spherical aberration by having refractive index of 1.52 after curing, which helps enable the best resolution for up to 150 µm thick specimens. |
Tools for image-based analysis of 3D cell culture models
Invitrogen imaging technologies help ensure clear visualization and analysis of your 3D cell culture model system.
| Product | Benefits |
|---|---|
EVOS cell imaging systems allow you to visualize, assess, and document live 3D cell cultures via high-resolution microscopy. | |
CellInsight high content screening systems can be used to rapidly image, identify, and analyze fluorescently labeled spheroids and other 3D cell clusters in a multiwell plate format. | |
Multi-dimensional analysis software enables deep analysis of cellular and subcellular structures to extract meaningful metrics about your 3D cell culture model. |
Tips and tricks
- Spheroids more than 300 µm in diameter can have a necrotic core and can be difficult to image and analyze.
- Leverage clearing reagents such as the CytoVista 3D Cell Culture Clearing Reagent to analyze your whole 3D cell culture model from the outer layers to the core.
Step 5: Characterize and assay genotype and phenotype
Molecular and cellular characterization of spheroids and 3D cell culture models
Visualization of 3D cell culture models and assessing their health are important to ensure that growth conditions are correct, but researchers must go a step further. New 3D in vitro models need to have their gene expression profiles, phenotypic markers, and organelle function analyzed to help ensure they resemble the microanatomy of the tissue or organ under investigation. Antibodies can be used to characterize the cell line or primary cells, from which spheroids are developed. Monitoring mRNA expression level in the developing cell culture model can help to identify cell types or changes in gene expression pattern that occur under certain treatment conditions. Mitochondrial health, cytotoxicity, reactive oxygen species levels, and apoptosis, are measured using live-cell functional probes.
Assay products
Thermo Fisher Scientific offers the necessary tools to validate 3D cell culture models. TaqMan® Assay probes and QuantStudio Real Time PCR are easy, fast ways to monitor gene expression. Our inclusive portfolio of validated antibodies and immunoassays can be used to detect intracellular, membrane bound or secreted protein markers. Invitrogen fluorescence reagents support high-content screening and analysis of a wide variety of cellular phenotypic properties.
Figure 5. T cell-mediated killing of A549 spheroids. Activated T cells (isolated from PBMC using Dynabeads magnetic beads coated with CD3/CD28), or non-activated T cells were added to A549 spheroid cells at various concentrations. A549 cells were analyzed for levels of caspase activity to determine the amount of apoptotic death. HeLa cells treated with activated T cells showed increased levels of caspase activity, while those treated with non-activated T cells showed decreased levels of caspase activity. Prior to co-culturing, T cells were stained with CellTracker Deep Red dye (purple dye). Apoptosis (cell death) was measured using CellEvent Caspase-3/7 Green Detection Reagent. Images were collected on a CellInsight CX7 LZR HCA instrument in confocal mode. Bright field images were used to determine spheroid area.
Reagents and systems for 3D cell culture model characterization
| Product | Benefits |
|---|---|
When used in combination with TaqMan Assay probes, QuantStudio real-time PCR instruments are a fast, easy way to monitor gene expression levels in your developing organoids. | |
Applied Biosystems and Invitrogen reagents and assays support the validation of 3D cell culture models to help ensure physiological relevance. Cell health, viability, apoptosis, cytotoxicity, mitochondrial health, DNA damage, and reactive oxygen species are just a few approaches to measuring cellular health in live cells. | |
Primary antibodies recognize specific cellular markers and can serve as useful tools to characterize cell lines. Invitrogen antibodies are available for a wide selection of targets, range of host reactivity and applications. | |
CellInsight high content screening systems can be used to rapidly image, identify, and analyze fluorescently labeled spheroids and other 3D cell clusters in a multi-well plate format. | |
From single-analyte identification using ELISA to multiplexed formats with ProcartaPlex assays, Invitrogen immunoassays comprise a suite of options for biomarker identification, quantification and analysis. | |
Compatible with a wide range of Invitrogen cell health assays, and capable of five different measurement modalities including absorbance, fluorescence intensity, luminescence, AlphaScreen or AlphaLISA, and time resolved fluorescence. |
Tips and tricks
- When performing image-based characterization, using a laser-based confocal system typically results in an improved signal-to-noise ratio and helps enable clear and rapid visualization of any 3D cell culture model system.
Learn more
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