Webinar: Methods and Reagents for Culturing and Characterizing Organoids and Cancer Spheroids

Hi, my name is Xi Lu, and with me today is Tahmina Naqvi. We are from the stem cells R&D group at Bio-Techne. Today we are going to be discussing how we culture and characterize organoids and spheroids.

One of the benefits of conducting organoid research at Bio-Techne is having readily available access to a vast array of high-quality reagents including cytokines and small molecules that we make in house. We also provide multiple instrumentations, including Simple Wes, Luminex, Proteome Profilers, and RNAscope™ to characterize our models.

Tahmina and I are located at Bio-Techne headquarters in Minneapolis, but we collaborate extensively with our global Bio-Techne colleagues by sharing methods, data, and the latest scientific advances.

In our lab, we work with both human adult stem cells and induced pluripotent stem cells. These cells are the starting point for modeling processes at all organizational levels of the body, from single cells to tissues and organs. But the focus of our talk today will be on spheroids and organoids.

Organoids, such as the iPSC-derived brain organoids in this video, are essentially self-organizing mini organs and ideally would contain multiple cell types and functions that are found in the organ from which they are derived. Compared to organoids, spheroids are much simpler and more reductionist. But both models would provide growth cues that are more reminiscent to what is found in vivo. Because of this, cells in 3-D models may behave more like how they would in the body in terms of expansion, differentiation, metabolic profiles, as well as gene and protein expression, compared to 2-D models. Unfortunately, the caveat is that these 3-D models are much more complex, laborious, and take longer to establish.

Typically, organoids are derived from either iPSCs or adult stem cells. iPSC organoids may provide access to more tissue and disease types as they can differentiate to any cell within the adult body. They are also amenable to gene edits, for example, using Bio-Techne’s TcBuster gene delivery platform. Because iPSC models cannot fully recapitulate age or older organs, we may find limited applications for certain degenerative diseases. Adult stem cell-derived organoids overcome some of these shortcomings and are critical for personalized medicine, biobanking, and drug discovery. They are also extremely valuable for studying host-pathogen interactions.

Here is an example of a typical workflow for organoids. Stem cells are isolated either as single cells or as an embryoid body, and then they are encapsulated in an extracellular matrix and placed in the culture plate. After polymerization of the matrix, media with soluble factors may be then added to take the cells to the next step. This next step could be either expansion, differentiation, or imaging and analysis.

While synthetic and defined matrices for different organ systems are being developed, the most widely used ECM is still the basement membrane extract, or BME. This is derived from an EHS mouse tumor. While BME is considered an undefined mixture, as it can contain thousands of different proteins, we have engineered our Cultrex™ BMEs for consistency, as you can see here, in the expression levels of major structural proteins, such as Laminin, Entactin, and Collagen IV in BME. Bio-Techne offers multiple BME products, with and without phenol red, as well as reduced growth factors, depending on your design application. Most of the data that we are presenting here today was gathered using Cultrex UltiMatrix.

In addition, selecting the right matrix can provide physical signaling and is essential to cultivate organoids in media with appropriate soluble cues for expansion or each stage of differentiation. To improve experimental reproducibility, it is critical to have media and supplements with fully defined factors. Bio-Techne offers high quality CGMP and animal-free proteins, including BMP-4, Noggin, as well as various small molecules from Tocris. In addition, we developed animal-free media, such as our iPSC expansion media, to better facilitate the transition from translational to clinical research.

Using all of these different tools and methods, we have been able to culture multiple organoid and spheroid types, including liver, brain, colon, heart, and lung. And using these 3-D models, our collaborators, both within and external to Bio-Techne, as well as our customers, are conducting research into multiple pathologies, including cancer, infectious diseases, and other genetic diseases shown here. So now, my colleague, Tahmina, will show data from our 3-D workflows in terms of being able to successfully generate organoids, spheroids, and their applications.

At Bio-Techne, we have cultured a variety of organoids from different tissue sources. This slide shows the expansion of intestinal organoids in UltiMatrix. We tested organoid formation efficiency from biopsy samples from different regions of small and large intestines, such as fetal duodenal, ileum, ascending, descending, and trans colon. The lower panel shows the differentiation of human descending colon organoids into highly folded structures, which express chromogranin, a marker for enteroendocrine cells, and E-Cadherin, which creates a key role in the homeostasis of LGR5+ intestinal stem cells.

This slide shows the staining of day six ileum organoids, with calcein, a live-cell permeant. The presence of all cells green indicates that organoids grow and expand well in these matrices with high viability.

Another important organoid type that we have tested is lung. The figure here shows day 52 lung organoids in culture. ICC profile of these lung organoids show all the major cell types of large airway epithelium such as mucin-secreting goblet cells, the KRT5-expressing basal cells that line the basement membranes, CC10-expressing non-ciliated club cells, and PDPN-expressing alveolar cells.

In addition to these markers, we also observe acetyl tubulin-expressing ciliated cells that promote mucus motility through movement of their apical cilia. These are in fat-active cilia, as you can see in the movie. In addition to ICC analysis, we also performed RNAscope fluorescent multiplex assays to visualize distinct populations of cells. RNAscope is a method of in situ hybridization, which allows this user to simultaneously visualize up to 12 different RNA targets per cell in sample. Using RNAscope, we compared the expression of KRT5 and SCGB1As in lung organoids, isolated at different stages of development. At day 14, there is a diffused expression of KRT5 and very little expression of SCGB1A, but by day 33, the KRT5 expression becomes localized toward basal lamina and there is an increased expression of SCGB1A.

The video here captures the growth and differentiation of liver organoids from day zero to day 14. Note the expansion in the size of liver organoids from day zero to nine. At day ten, the differentiation cues are given, resulting in the decrease of organoid size and increase in the organoid darkness, which is typical of differentiated cells.

Now, to characterize the organoid analysis, definitions were created using an incucyte organoid module. We focused on two analysis parameters. The first parameter is organoid object surface area, which effectively tracks the organoid growth in differentiation over a period of time. The second parameter tracks the changes in organoid darkness as they mature and differentiate in culture.

All the previous examples were from tissue-specific stem cells derived from biopsy samples. A second way of generating organoids is the use of induced pluripotent stem cells. Now, let’s take a look at some of the examples of iPSC-derived organoids. The slide here shows the use of Bio-Techne’s reagents for the in vitro maturation of iPSCs into intestinal organoids. As you can see, by day 13, the organoid formed highly convoluted epithelium surrounded by the mesenchyme. The fixed organoids display both absorptive and secretory functionalities, and around that time, the surrounding mesenchyme also starts to express mesenchyme-specific markers, such as vimentin, ectin, and desmin. The fact that mesenchyme differentiation coincides with the differentiation of the overlying epithelium indicates that maybe epithelial mesenchyme crosstalk is important in the development of iPSC-derived intestinal organoids.

The images here show iPSC-derived mesoderm differentiation into very nice beating cardiac organoids, expressing cardiac and endothelial-specific markers, such as troponin T and CD31. We were also able to observe regular contractions of cardiac organoids by fluorescent calcein flux.

And last but not least, iPSC-derived brain organoids are a powerful tool to study human brain development and disease modeling. Shown here is a day 62 brain organoid in UltiMatrix showing nice cortical enfoldings. Fixed organoids on the right show a cortical layer enriched in Pax6 progenitors and vimentin positive radial layer that extend processes out to outer apical surface of the cortex. So, in addition to organoids, the other 3-D systems that we have tested are spheroids, which are three-dimensional cell aggregates that can mimic tissues and microtumors.

HepG2 spheroids were used as a 3-D model for liver toxicity. HepG2 cells were cultured both as 2-D monolayers and as 3-D spheroids. Compared to their 2-D counterparts, the spheroids assume more in vivo-like morphology and function, as shown by greater expression of albumin and phase two drug metabolizing enzymes. A CometChip assay for DNA damage was performed to screen differences in drug responses in 2-D versus 3-D cultures. Certain drugs, in particular, drop ofloxacin, which is a broad-spectrum antibiotic, show high DNA damage, compared to 2-D cultures. The drug has long been withdrawn by the FDA due to its high risk of hepatotoxicity. Therefore, 3-D culture systems offer a more sensitive drug screening approach compared to a standard 2-D culture, as they are able to predict the efficacy, as well as toxicity, of therapeutic candidates in humans before drugs enter their clinical trials.

To evaluate and monitor immune cell killing, an antibody-dependent cell-mediated cytotoxicity assay was performed. These assays rely on three components: an effector cell, target cell, and a monoclonal antibody. We used Her-2A antibody to trigger natural killer cell-mediated antibody-dependent cellular cytotoxicity to SKOV3 spheroids. The assay was conducted in 96-well plates, where uniform-sized SKOV3 spheroids were formed in 2.5% UltiMatrix. To these spheroids, NK cells plus antibodies were added at different effector-to-target ratios. The time lapse movies show the killing of SKOV3 spheroids by NK plus antibody while the controlled spheroids continue to grow and expand in size.

Organoids, spheroids, and other 3-D models, such as assembloids, are really vital for physiologically relevant disease modelling. Bio-Techne is committed to supplying all of your 3-D organoid culture needs, including high quality GMP media, matrices, proteins, and other small molecules. We also provide multiple solutions for analysis of these models, including fluorescent probes, tissue-clearing solutions, as well as RNAscope.

Here are some links to additional information about organoids and 3-D cultures at Bio-Techne, as well as stem cells, and of the antibodies that we have used as well as our RNAscope technology. We also provide an organoid handbook for culturing and characterizing all of the organoids. You may also request Cultrex samples.

• Organoids and 3-D Culture
• Stem Cells
• Antibodies
• RNAscope Technology
• Organoid Culture Handbook
• Request a Cultrex BME Sample

Question # 1: Is there a transition step that is needed to culture iPSCs or organoids from other matrices?

Usually no transition is needed from our experience, however, it might be necessary to optimize the protein concentration of the BME when starting out with a specific tissue or cell type.

Question # 2: Are phenol red or antibiotics used in the production of Cultrex BMEs?

Yes, we do use gentamicin in the production of Cultrex BMEs. We also offer BME products with and without phenol red. Usually, unless explicitly indicated, the BMEs are not made with phenol red.

Question # 3: Is there autofluorescence present in gels derived from BMEs?

It is possible that there may be some autofluorescence, especially from matrices that contain phenol red. Certain fixation methods, such as glutaraldehyde, may also introduce significant autofluorescence, so typically we recommend the use of paraformaldehyde or reduce the levels of glutaraldehyde.

Question #4: What is the minimum protein concentration for creating gels?

When creating hydrogels, we usually recommend not going below four mg/mL, as the gel becomes too difficult to handle, they are very soft, and can collapse.

Question #5: Can plate types affect the success of BME gel formation?

Yes, they can, and we recommend the use of tissue-culture treated plates.