5 steps to creating 3D cell models

Better models for disease research

Organoid and spheroid models are increasingly being used in neurobiology, stem cell research, regenerative medicine, and cancer biology. Sophisticated tissue engineering approaches can enable the advancement from 2D cell cultures to 3D cell models, bringing more physiological relevance to your experiment. Generating organoid and spheroid models can be technically challenging but once established can offer key insights for translational research projects, as they exhibit gene and protein expression signatures closer to those observed in vivo.

Whether you are just beginning your research in 3D cell modeling and need to learn more about organoids or spheroids or need appropriate tools to validate your models, we have outlined these 5 easy steps to assist you as you progress.

Download the free 3D product resource guide

Consider the 5 step workflow below to help improve your 3D cell culture and analysis.

Step 1: Source

Choose cell lines that allow you to closely mimic the in vivo state and generate more relevant data

2D cell models grown from immortalized cell lines have long served as the foundational tool for disease modeling and drug development. While these models are easy to grow and maintain, they typically lack the biological complexity and physiological relevance that are more commonly observed when using 3D models.

Creating relevant 3D models all starts with selecting the right 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 models for liver toxicity (hepatocytes) or skin models (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.

6-panel fluorescence images of stained HepG2 and hepatic cell spheroids

Figure 1. Evaluation 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).

Products to establish 3D cell cultures

Custom services

Our Custom Biology Cell Model Services can help enable you to reach your desired outcomes faster. 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 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.

Step 2: Support

Selecting the right culture matrix is an important first step in developing a successful culture system for organoids and spheroids

Extracellular matrices and 3D cultureware are used to more easily transition from a 2D monolayer environment to 3D models. 3D cultureware systems are used for scale up and full organ development. Porous membrane-based systems are advantageous when polarization of the epithelial cells are needed in cell differentiation and tissue formation.

Extracellular matrices and 3D cultureware

Whether you are just getting started in 3D or looking for more complex ways to apply 3D models, our extracellular matrices and 3D cultureware allow you to develop more physiologically relevant 3D models by improve intracellular interactions, with less lot-to-lot variability than other products. Trust Thermo Fisher Scientific, the most cited brand in 3D research, for consistent and cost-effective optimized media, supplements and matrices for most major cell types and applications.

Featured matrices products

Geltrex hESC-Qualified Basement Membrane Matrix

Cultureware for 3D models

Nunclon Sphera plates and dishes

Typically, culture matrices 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 model culture (i.e., spheroid and organoid growth) with many options of plates, dishes and flasks. The special design of our Nunclon Sphera surface allows virtually 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).

Bar chart showing cell attachment on a typical cell culture-treated surface, a nontreated culture surface, and Nunclon Sphera surface in Vero, A549 and U937 cells
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 significantly lower adhesion than another brand of low cell attachment surface (Brand C) for the differentiated U937 cells.

Featured instruments for image based analysis of 3D cultures

Tips and tricks

  • A few ways to control the size of spheroids are to adjust the initial cell seeding densities, avoid culture vessels with large surface area such as a T flask or a large petri dish, or use a more confined physical space to promote spheroid formation, such as a round bottom microplate (e.g., 96-well U bottom plate).
  • Geltrex matrix is a 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.

Step 3: Culture

Your 3D culture system can be modified and developed to suit specialized cell types like stem cells or cancer cell lines

Growing 3D models is a large investment in time and resources, and you need reassurance that your investment is going to give you the 3D models that you anticipate. In addition to providing the appropriate matrices and culture vessels, we offer the necessary Gibco basal, stem cell, and specialized media to effectively grow your 3D model. Gibco media and reagents are cited in numerous organoid and spheroid peer-reviewed articles, indicating their wide use in the growth, differentiation and maturation of cellular 3D models. The appropriate supplements and media are critical for growing and in some cases differentiating the cells within the model. Additionally, Gibco growth factors are designed to give you high biological activity, high purity, and proven compatibility.

Featured products

Tips and tricks

  • 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

Learn more

STEP 4: Monitor & Analyze

Visual and quantitative analysis of 3D cell models

Confirming that 3D cell structures are maintaining the appropriate morphology is paramount to establishing a 3D model with the correct cellular and biochemical makeup. Success in 3D model research settings relies on such technologies to enable accurate quantification of cell counts, size and growth patterns over time, in multi-well plate formats. Furthermore, significant technological advancements have made HCS methods amenable to high throughput assays, through the rapid acquisition of z-stacks from multicellular structures.

Fluorescent readout quantitation of all cells in a 3D culture can be more difficult due to the inherent cellular organization of a cluster of cells. As 3D clusters get larger and thicker, they become more opaque, and seeing within the spheroid is not always possible as current techniques are biased toward the periphery of the cluster. Clearing agents and antifade mountants can help to visualize 3D cultures while maintaining the morphology of the cell, and compatibility with techniques like immunofluorescence (IF), immunocytochemistry (ICC), and immunohistochemistry (IHC).

Microscopy image of individual spheroids growing in wells of a 96-well Nunclon Sphera plate

Figure 3. Spheroids growing in Nunclon Sphera 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 HCA.

2 panel fluorescence image of A549 and HeLa spheroids showing two-color staining
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 to provide superior optical transparency enabling 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 to 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 mountant 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 um thick specimens.

Instruments for image-based analysis of 3D models

Invitrogen imaging technologies help ensure clear and rapid visualization of any 3D cell model system.

Product Benefits

EVOS Cell Imaging Systems

EVOS cell imaging systems allow you to image and analyze 3D cell models.

Cellnsight HCA systems

Can be used to identify the fluorescence properties of single 3D clusters within a culture of clusters in multiwell plate formats.

Tips and tricks

  • Spheroids more than 300uM 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 model from the outer layers to the core.

Step 5: Characterize and assay

Molecular characterization and applications of 3D spheroid models

Visualization of 3D models and assessing their health are important to ensuring that growth conditions are correct, but researchers must go a step further. Developing 3D models need to be have their gene expression profiles, phenotypic markers, and organelle function analyzed to ensure they resemble the micro anatomy 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 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 functional probes to determine successful outcomes in cancer research experiments

Assay products

Thermo Fisher Scientific provides researchers with the necessary tools to validate 3D 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 within your model system. Invitrogen functional assays can be used in the assessment of parameters such as mitochondrial health, cytotoxicity, reactive oxygen species, and apoptosis, to name a few.

Fluorescence images and graphed results showing caspase activity as a function of activated T cell treatment

Figure 5. T cell–mediated killing of A549 spheroids. Activated T cells (from PBMC using Dynabeads magnetic beads coated with CD3/CD28), and inactivated T cells were added to A549 spheroid cells at various concentrations. A549 cells were analyzed for levels of caspase activity to determine amount of apoptotic death. HeLa cells treated with activated T cells showed increased levels of caspase activity, while those treated with inactivated 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.

Featured products

Product Benefits

TaqMan® Assays and QuantStudio Real Time PCR

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.

Fluorescence-based reagents

Applied Biosystems and Invitrogen reagents and assays support the validation of 3D models to 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.

Antibodies

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 HCA systems

Useful for the identification of fluorescence properties of individual 3D clusters within a culture of clusters in multi-well plate formats.

Immunoassays

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.

Varioskan LUX microplate readers

Compatible with a wide range of Invitrogen cell health assays, and capable of 5 different measurement technologies, including absorbance, fluorescence intensity, luminescence, AlphaScreen or AlphaLISA, and time resolved fluorescence.

Step 1: Source

Choose cell lines that allow you to closely mimic the in vivo state and generate more relevant data

2D cell models grown from immortalized cell lines have long served as the foundational tool for disease modeling and drug development. While these models are easy to grow and maintain, they typically lack the biological complexity and physiological relevance that are more commonly observed when using 3D models.

Creating relevant 3D models all starts with selecting the right 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 models for liver toxicity (hepatocytes) or skin models (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.

6-panel fluorescence images of stained HepG2 and hepatic cell spheroids

Figure 1. Evaluation 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).

Products to establish 3D cell cultures

Custom services

Our Custom Biology Cell Model Services can help enable you to reach your desired outcomes faster. 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 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.

Step 2: Support

Selecting the right culture matrix is an important first step in developing a successful culture system for organoids and spheroids

Extracellular matrices and 3D cultureware are used to more easily transition from a 2D monolayer environment to 3D models. 3D cultureware systems are used for scale up and full organ development. Porous membrane-based systems are advantageous when polarization of the epithelial cells are needed in cell differentiation and tissue formation.

Extracellular matrices and 3D cultureware

Whether you are just getting started in 3D or looking for more complex ways to apply 3D models, our extracellular matrices and 3D cultureware allow you to develop more physiologically relevant 3D models by improve intracellular interactions, with less lot-to-lot variability than other products. Trust Thermo Fisher Scientific, the most cited brand in 3D research, for consistent and cost-effective optimized media, supplements and matrices for most major cell types and applications.

Featured matrices products

Geltrex hESC-Qualified Basement Membrane Matrix

Cultureware for 3D models

Nunclon Sphera plates and dishes

Typically, culture matrices 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 model culture (i.e., spheroid and organoid growth) with many options of plates, dishes and flasks. The special design of our Nunclon Sphera surface allows virtually 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).

Bar chart showing cell attachment on a typical cell culture-treated surface, a nontreated culture surface, and Nunclon Sphera surface in Vero, A549 and U937 cells
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 significantly lower adhesion than another brand of low cell attachment surface (Brand C) for the differentiated U937 cells.

Featured instruments for image based analysis of 3D cultures

Tips and tricks

  • A few ways to control the size of spheroids are to adjust the initial cell seeding densities, avoid culture vessels with large surface area such as a T flask or a large petri dish, or use a more confined physical space to promote spheroid formation, such as a round bottom microplate (e.g., 96-well U bottom plate).
  • Geltrex matrix is a 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.

Step 3: Culture

Your 3D culture system can be modified and developed to suit specialized cell types like stem cells or cancer cell lines

Growing 3D models is a large investment in time and resources, and you need reassurance that your investment is going to give you the 3D models that you anticipate. In addition to providing the appropriate matrices and culture vessels, we offer the necessary Gibco basal, stem cell, and specialized media to effectively grow your 3D model. Gibco media and reagents are cited in numerous organoid and spheroid peer-reviewed articles, indicating their wide use in the growth, differentiation and maturation of cellular 3D models. The appropriate supplements and media are critical for growing and in some cases differentiating the cells within the model. Additionally, Gibco growth factors are designed to give you high biological activity, high purity, and proven compatibility.

Featured products

Tips and tricks

  • 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

Learn more

STEP 4: Monitor & Analyze

Visual and quantitative analysis of 3D cell models

Confirming that 3D cell structures are maintaining the appropriate morphology is paramount to establishing a 3D model with the correct cellular and biochemical makeup. Success in 3D model research settings relies on such technologies to enable accurate quantification of cell counts, size and growth patterns over time, in multi-well plate formats. Furthermore, significant technological advancements have made HCS methods amenable to high throughput assays, through the rapid acquisition of z-stacks from multicellular structures.

Fluorescent readout quantitation of all cells in a 3D culture can be more difficult due to the inherent cellular organization of a cluster of cells. As 3D clusters get larger and thicker, they become more opaque, and seeing within the spheroid is not always possible as current techniques are biased toward the periphery of the cluster. Clearing agents and antifade mountants can help to visualize 3D cultures while maintaining the morphology of the cell, and compatibility with techniques like immunofluorescence (IF), immunocytochemistry (ICC), and immunohistochemistry (IHC).

Microscopy image of individual spheroids growing in wells of a 96-well Nunclon Sphera plate

Figure 3. Spheroids growing in Nunclon Sphera 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 HCA.

2 panel fluorescence image of A549 and HeLa spheroids showing two-color staining
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 to provide superior optical transparency enabling 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 to 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 mountant 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 um thick specimens.

Instruments for image-based analysis of 3D models

Invitrogen imaging technologies help ensure clear and rapid visualization of any 3D cell model system.

Product Benefits

EVOS Cell Imaging Systems

EVOS cell imaging systems allow you to image and analyze 3D cell models.

Cellnsight HCA systems

Can be used to identify the fluorescence properties of single 3D clusters within a culture of clusters in multiwell plate formats.

Tips and tricks

  • Spheroids more than 300uM 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 model from the outer layers to the core.

Step 5: Characterize and assay

Molecular characterization and applications of 3D spheroid models

Visualization of 3D models and assessing their health are important to ensuring that growth conditions are correct, but researchers must go a step further. Developing 3D models need to be have their gene expression profiles, phenotypic markers, and organelle function analyzed to ensure they resemble the micro anatomy 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 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 functional probes to determine successful outcomes in cancer research experiments

Assay products

Thermo Fisher Scientific provides researchers with the necessary tools to validate 3D 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 within your model system. Invitrogen functional assays can be used in the assessment of parameters such as mitochondrial health, cytotoxicity, reactive oxygen species, and apoptosis, to name a few.

Fluorescence images and graphed results showing caspase activity as a function of activated T cell treatment

Figure 5. T cell–mediated killing of A549 spheroids. Activated T cells (from PBMC using Dynabeads magnetic beads coated with CD3/CD28), and inactivated T cells were added to A549 spheroid cells at various concentrations. A549 cells were analyzed for levels of caspase activity to determine amount of apoptotic death. HeLa cells treated with activated T cells showed increased levels of caspase activity, while those treated with inactivated 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.

Featured products

Product Benefits

TaqMan® Assays and QuantStudio Real Time PCR

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.

Fluorescence-based reagents

Applied Biosystems and Invitrogen reagents and assays support the validation of 3D models to 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.

Antibodies

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 HCA systems

Useful for the identification of fluorescence properties of individual 3D clusters within a culture of clusters in multi-well plate formats.

Immunoassays

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.

Varioskan LUX microplate readers

Compatible with a wide range of Invitrogen cell health assays, and capable of 5 different measurement technologies, including absorbance, fluorescence intensity, luminescence, AlphaScreen or AlphaLISA, and time resolved fluorescence.

One Cell Biology Product Handbook

One Cell Bio product handbook

We have simplified our portfolio to show you how our most popular products and services are used as single tools or integrated system solutions. This handbook is meant to expedite your search for the right cell biology product and give you an in-depth look at how we can help accelerate research.

Learn more