3d-research-application-ersources-showcase

Learn more about the complexities of 3D cell culturing and analysis. Use our many tools to gain a better understanding of how to get started or continue into more advanced methods of researching organoids and spheroids. On this page you will find useful application notes, technical training documents, R&D scientific posters previously presented at conferences, how-to guides and scientific presentations.

Use the search function below to look up your cell type, area of research, products, or application and see for yourself how Thermo Fisher scientists are using them for 3D modeling and research on organoids and spheroids.

Title Topic Resource
Primary Human Hepatocyte 3D Spheroids for Studying Hepatic Function and Drug Toxicity (ISSCR Symposia 2019) Hepatocyte, Spheroid, ADME, Nunclon, metabolism Scientific poster
Evaluating antibody-mediated cellular cytotoxicity and potency of antibody-drug conjugates within three-dimensional tumor models A549, NK Cells, Epithelial, Spheroid, Nunclon, Cx7, Cancer, Tumor Scientific poster
New and improved cellular health evaluation of 2D and 3D cellular models using microplate reader assays (SLAS 2019) HTS, High Throughput Screening, Spheroid, cancer, tumor, CX7, Varioskan Scientific poster
Matching refractive index of mounting media improves axial resolution and image quality in 3D biological samples Mammary, Neuro, Cortical Scientific poster
Analysis of cellular processes within 3D cell models using fluorescence microscopy HeLa, A549, Nunclon, CX7 Scientific poster
In-vitro evaluation of primary animal hepatic function using a 3D-spheroid culture Hepatocyte, Animal, Spheroid, ADME, Nunclon, metabolism Scientific poster
Optimization of HepaRG Workflow for Use in 3D Spheroid Models HepaRG, Spheroid, ADME, Nunclon, metabolism Scientific poster
Primary Human Hepatocytes 3D in vitro Culture Model for Studying Hepatic Function Hepatocyte, Spheroid, ADME, Nunclon, metabolism Scientific poster
In Vitro Evaluation of Hepatic Function Using a Primary Human Hepatocyte 3D Spheroid Culture System Hepatocyte, Spheroid, Collagen, ADME, Nunclon Application note
In vitro evaluation of hepatic gene expression using a Gibco 3D spheroid culture system Hepatocyte, Spheroid, ADME, Nunclon, metabolism, Gene Expression Application note
3D spheroid culture as a tool for studying drug metabolism Hepatocyte, Spheroid, ADME, Nunclon, Drug metabolism Application note
Differentiation of pluripotent stem cells into neural organoids Neuro, Stem Cell, iPSC, Organoid, Geltrex, Nunclon, DMEM Application note

Review our complete offering of scientific posters and application notes focused on the culture and analysis of 3D models, organoids and spheroids below. Get started researching in a new dimension.


Scientific posters

Primary human hepatocyte 3D spheroids for studying hepatic function and drug toxicity (ISSCR Symposia 2019)

Primary human hepatocyte 3D spheroids for studying hepatic function and drug toxicity (ISSCR Symposia 2019)

Primary Human Hepatocyte (PHH) culture provides the closest in vitro model to human liver that can produce a metabolic profile of a given drug very similar to that found in vivo. Hence, PHH culture is the gold standard for studying the in vitro hepatic biology, liver function, and drug induced hepatotoxicity. The conventional 2-dimensional (2D) PHH culture is limited by de-differentiation and rapidly loss of hepatic specific functions. Therefore, there is a need for more robust in vitro models that reflects in vivo liver biology with better culture longevity. Recently, 3-dimensional (3D) in vitro models for hepatocytes have gained a lot of attention for their ability to recapitulate the hepatic function and greater longevity. We have developed an easy-to-assemble in vitro PHH 3D-spheroid model. Our initial work shows that PHH can assemble into spheroids using Nunclon Sphera super low attachment 96-well U-bottom plates and standard centrifugation method within 5 days of seeding. As part of our future work, we are investigating the possibility of introducing nonparenchymal liver cells like Kupffer and Stellate cells to the spheroid system to assess feasibility of creating various liver disease models.

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Evaluating antibody-mediated cellular cytotoxicity and potency of antibody-drug conjugates within three-dimensional tumor models (SITC 2018)

Evaluating antibody-mediated cellular cytotoxicity and potency of antibody-drug conjugates within three-dimensional tumor models (SITC 2018)

Three dimensional tumor spheroids provide biochemical conditions that closely resemble the tumor microenvironment in an intact organism. Noninvasive approaches such as fluorescence microscopy are highly advantageous as they allow for the study of these 3D systems. Here we investigate the penetration and potency of natural killer cells, cytotoxic T cells, and antibody-drug conjugates in three-dimensional models of breast and lung cancer

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New and improved cellular health evaluation of 2D and 3D cellular models using microplate reader assays (SLAS 2019)

New and improved cellular health evaluation of 2D and 3D cellular models using microplate reader assays (SLAS 2019)

Researchers in drug discovery and cancer research rely on reproducible biological assays to guide medicinal chemistry programs. A current goal in the cancer research field is to explore and expand the use of microplate readers and assay systems into the realm of 3D cellular models for drug discovery in anticancer research, specifically aimed at modelling solid tumors to screen compounds and confirm their activity. In this study, we screened multiple therapeutic drugs using different HTS assays to establish drug dose response curves and to understand the diversity in cell health assays. Despite the lack of guidelines and enabling technologies to study 3D cellular models, we establish the different impact therapeutics have on 2D versus 3D cellular models using existing cellular assay

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Matching refractive index of mounting media improves axial resolution and image quality in 3D biological samples

Matching refractive index of mounting media improves axial resolution and image quality in 3D biological samples

Here we use laser scanning confocal microscopy to demonstrate the deleterious effects of mismatched refractive index in conventional mounting medium by measuring the point spread function (PSF) of subdiffraction-sized fluorescent microspheres. We then compare these results with specimens prepared in Invitrogen  ProLong  Glass Antifade Mountant, which features an optimized refractive index of 1.52. Our data indicate that at depths beyond 20 µm, an RImismatched mounting medium suffers significant losses in axial resolution, whereas the RI-matched mountant maintains optimal axial resolution throughout, enabling up to 75% improvement in axial resolution at focal depths of 100 µm and beyond. By eliminating unwanted refraction, ProLong Glass Antifade Mountant also maximizes the light reaching the objective lens, thus providing higher sensitivity and a brighter image when imaging thick biological samples, such as spheroid cultures that mimic tumor microenvironments. Combined with protection from photobleaching in the visible and near-infrared spectra, the optimized refractive index of ProLong Glass Antifade Mountant measurably increases fidelity and image quality for bright, highresolution 3D analysis of any cell or tissue type

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Analysis of cellular processes within 3D cell models using fluorescence microscopy

Analysis of cellular processes within 3D cell models using fluorescence microscopy

High attrition rates in drug development and lack of translation relevance can be traced back to a lack of physiological relevance when performing target identification, lead identification and optimization. Whilst more physiologically relevant, animal studies are time consuming and expensive. Therefore a niche exists for experimental models that span the gap between in vitro cell-based assays and in vivo animal studies. These systems would mimic the in vivo setting while recapitulating the ease of manipulation of a cell based assay. Traditional two dimensional cell culture models lack physiologically relevant environmental conditions. Their whole physical and biochemical setting is drastically different. For this reason researchers have been turning to three dimensional organoid and tumor spheroid systems. In this systems cells benefit from cell to cell and cell to ECM contacts. Moreover the cells exist in a more biochemically relevant state with gradients through the 3D system existing, primarily these are oxygen, nutrients and metabolites. Functionally different zones also exist within the three dimensional models with apoptotic or necrotic regions observed in one location (typically at the core) while zones of proliferative cells can be detected along the periphery. These conditions closely resemble the macro environment surrounding the cells in an intact organism. Noninvasive approaches such as fluorescence microscopy are highly advantageous as they allow for the study of these three dimensional systems. We describe the application of a suite of fluorescent biosensors in combination with automated fluorescence microscopy for the high throughput, quantitative analysis of 3D cell models. Data will be shown quantifying the induction of apoptosis in spheroid models in combination with either mitochondrial membrane potential measurements or the sequestration of therapeutic antibodies labeled with environmentally sensitive fluorescent dyes. Assessment of other pertinent cellular parameters such as viability, proliferation, cell cycle and ROS production will be demonstrated within 3D cell models. Finally, live-cell analysis of the penetration of activated immune cells into a tumor spheroid model will be shown.

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In-vitro evaluation of primary animal hepatic function using a 3D-spheroid culture system

In-vitro evaluation of primary animal hepatic function using a 3D-spheroid culture system

Recent evidence suggests that culturing primary hepatocytes in 3-dimensional (3D) hepatic spheroids offers major advantages in providing longevity and increased physiological relevance in modeling liver metabolism. Dog (Beagle), Mouse (CD-1), and Rat (Sprague Dawley) hepatocytes from Thermo Fisher Scientific were plated for spheroid qualification. 3D hepatic spheroids were characterized in weeks 1-4 in which the following were assessed: cell viability (as indicated by ATP synthesis), albumin production, phase I metabolic activity, and gene expression. Our data indicate that primary Dog and Rodent hepatocytes formed 3D spheroids by Day 5, and moreover were viable and functional for up to 4 weeks. In conclusion, our data indicate that animal 3D spheroid cultures are an accurate and sustainable in vitro model of hepatocyte function that maintain hepatic functions for a longer period of time in comparison with traditional 2D cultures

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Optimization of HepaRG workflow for use in 3D spheroid models

Optimization of HepaRG workflow for use in 3D spheroid models

Growing evidence suggest that cells cultured as 3-dimensional (i.e., 3D) spheroids exhibit more in vivo-like cellular properties than conventional 2D monolayer cultures which underscores their potential as more physiologically relevant in vitro culture systems. The human hepatic cell line HepaRG, is a well-established model for studying drug metabolism and toxicity, and there have been numerous reports indicating its utility as a 3D model. However, these studies primarily recapitulate the standard monolayer (i.e., 2D) workflow, which is based on the dynamic temporal metabolic profile of cytochrome P450 (CYP) enzymes, to culture cells in 3D spheroids. Since gene expression profiles and the behavior of cells frequently differs in 2D and 3D cultures, we hypothesized that the commonly used HepaRG spheroid culture protocol has not been optimized. To address this, we compared the temporal profile of basal CYP3A4 activity in 2D and 3D HepaRG cultures to determine if cells may function differently between these culture formats. HepaRG monolayers were grown on Collagen I coated plates; 3D spheroids were formed in Nunclon Sphera 96-well U-bottom plates. Basal CYP3A4 activity in 2D and 3D culture was measured every 24 hours for 10 days and normalized to cell viability. Consistent with published data, our HepaRG cultures formed spheroids in 2-3 days of culture and the size of the spheroid was directly proportional to the number of cells seeded. Preliminary data confirmed that 2D HepaRG cultures had high CYP3A4 activity in the first 24 hours of culture with a subsequent reduction that slowly recovered to peak activity levels at day 6 of culture. Interestingly, these spheroid cultures have significantly higher basal CYP3A4 activity (~200 fold) everyday of culture compared to 2D cultures. During the aggregation phase of the spheroid culture (i.e., Day 1-2) CYP3A4 activity was elevated and reached peak levels at Day 3, when spheroids are fully formed. From Day 3-10, HepaRG spheroids showed relatively constant levels of CYP3A4 activity. In conclusion, these results suggest that the temporal profile of commonly studied metabolic enzymes is different between HepaRG monolayer and spheroid cultures. Moreover, the difference HepaRG spheroids peak activity levels suggest that the spheroid culture method offer more flexibility for experimental design with potentially shorter culture times.

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Primary human hepatocytes 3D in vitro culture model for studying hepatic function

Primary human hepatocytes 3D in vitro culture model for studying hepatic function

Primary Human Hepatocyte (PHH) culture provides the closest in vitro model to human liver that can produce a metabolic profile of a given drug very similar to that found in vivo. Hence, PHH culture is the gold standard for studying the in vitro hepatic biology, liver function, and drug induced hepatotoxicity. The conventional way of culturing PHH in 2-dimension (2D) has major pitfalls. The PHH rapidly de-differentiate and lose the hepatic specific functions in a week. Therefore, there is a need for more robust in vitro models that reflect in vivo liver biology more accurately and maintains the liver functions for a longer time. 3-dimensional (3D) hepatic in vitro models have gained a lot of attention for their ability to recapitulate the hepatic function and greater longevity. Recently we have developed an easy-to-assemble user-friendly in vitro Primary Human Hepatocyte (PHH) 3D-spheroid model. The 3D-hepatic spheroids are viable for at least 4 weeks in culture and remain phenotypically stable, retaining the hepatocyte-specific functions.

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Application notes

In vitro evaluation of hepatic function using a primary human hepatocyte 3D spheroid culture system

In vitro evaluation of hepatic function using a primary human hepatocyte 3D spheroid culture system

The conventional method of culturing primary human hepatocytes (PHH) in a 2-dimensional (2D) monolayer presents limitations in the study of hepatic biology, liver function, and drug-induced hepatotoxicity. Traditional 2D hepatocyte cultures dedifferentiate, resulting in the loss of specific hepatic function in approximately 5 days. We have developed and characterized a PHH 3-dimensional (3D) spheroid culture system that preserves hepatic function and promotes culture longevity. Gibco PHH can easily be assembled into a 3D spheroid culture in 5 days using Thermo Scientific Nunclon Sphera low-attachment U-bottom 96-well microplates and Gibco plating medium and plating supplements. The 3D spheroid hepatocyte culture requires a significantly lower number of cells than its 2D counterpart, allowing this system to better support high-throughput assays. Moreover, the PHH in the 3D spheroid culture are functionally viable for at least 3 weeks, enabling long-term studies of hepatocyte function.

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In vitro of hepatic gene expression using a Gibco 3D spheroid culture system

In vitro of hepatic gene expression using a Gibco 3D spheroid culture system

We have developed an easy-to-assemble, in vitro primary human hepatocyte (PHH) 3D spheroid model. In the previously released application note, we were able to prove that PHH 3D spheroid culture is a robust in vitro model that can survive 21 days in culture and improves hepatic function relative to 2D culture. To demonstrate this, we have analyzed the expression levels of various genes that play a role in human hepatocyte function, including ABC transporters and phase-1 P450 enzymes, in 3D hepatic spheroid cultures, and show that PHH 3D cultures can enable hepatic gene expression more efficiently than 2D hepatocyte cultures.

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3D spheroid culture as a tool for studying drug metabolism

3D spheroid culture as a tool for studying drug metabolism

The primary human hepatocyte (PHH) three-dimensional (3D) culture model developed by Thermo Fisher Scientific holds promise for disease modeling and functional studies. As the liver is the principal site of metabolism for most drugs, primary hepatocytes have become the most popular in vitro tool to evaluate hepatic drug metabolism. However, the efficiency of 3D hepatic spheroids for assessing drug metabolism is relatively unknown. One major difference between 3D hepatic culture and conventional two-dimensional (2D) culture is the number of cells per well. A single hepatic spheroid typically consists of 1,000 to 3,000 cells, whereas there are between 50,000 (96-well plate) and 400,000 (24-well plate) cells per well in a conventional 2D culture. Hence, it is expected that metabolites synthesized in 3D culture will be present at lower concentrations relative to those found in 2D culture. However, we have observed that relative gene expression levels for certain cytochrome P450 (CYP) enzymes are higher in 3D hepatic spheroids than in their 2D counterparts. As CYP proteins are critical phase I enzymes for drug metabolism, we measured CYP activities for 6 different enzymes with distinct substrates via high-resolution mass spectrometry, directly comparing the activities of 2D and 3D cultures. Our data demonstrate the utility of Gibco 3D hepatic spheroid models for studying drug metabolism.

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Differentiation of pluripotent stem cells into neural organoids

Differentiation of pluripotent stem cells into neural organoids

Recent advances in cell culture techniques have focused on creating 3-dimensional (3D) systems in an attempt to represent in vivo cell–cell relationships and microenvironments in vitro. Various tissue engineering technologies such as bioprinting, microfluidics, and organs-on-chips have been used successfully to generate 3D cultures. Remarkable progress has also been made utilizing adult and pluripotent stem cells (ASCs and PSCs) to generate 3D organ-like (i.e., organoid) cell models. PSC-based methods frequently start by aggregating cells in suspension culture to form clusters called embryoid bodies (EBs). Cells in these clusters are capable of differentiating into many types and can undergo self-organization and self-morphogenesis to create a complex cell model that better mimics the in vivo cell–cell interactions and microanatomy of a given tissue type. Some PSC-based approaches also require the encapsulation of cells within a natural or synthetic extracellular matrix (ECM)-like substrate. In all methods, the application of growth factors, small molecules, and other media supplements is used to guide the formation of organoid systems based on principles inferred from studies of embryogenesis and adult stem cell biology. There are now many published methods

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