Using Organoids for Disease Modeling

Generating organoids is a delicate procedure and requires adjustments and testing for the specific cells and applications used. Culturing organoids is a likewise sensitive process and the specific requirements of the cells and the organoids needs to be evaluated.

The successful generation and long-term culture of organoids depends on many factors, including:

• Cell type (stem cells? From which organ?)
• Culture conditions (culture conditions, medium, ECMs used ?)
• Cell challenges (Low viability, organoids that disassemble, etc.)
• What assay is planned after the generation of the organoids?

Since this information is not outlined in this question, we wanted to share some helpful technical documents on the generation and culture of organoids for a period longer than 24 hours:

Application Notes:

• A Novel Method for Generating Single, Intestinal Organoids for High Throughput Screening
• Culture of Mouse Intestinal Organoids in Corning® Matrigel® Matrix for Organoid Culture
• High Throughput Gene Expression Analysis of 3D Airway Organoids

In recent years, Corning® Matrigel® matrix has developed into a powerful new tool for organoid cultures in areas like basic research and drug discovery, with several protocols available to support this work. Please find below some technical document suggestions that support the creation of different types of organoids.

Citations on Corning® Matrigel® Matrix and Organoid Culture
A compilation of research article citations, where several organoid types are referenced.

Modeling Development and Disease with Organoids

In this review article, Hans Clevers gives an overview on several organoid types and how they are generated. Using this foundational information as a starting point, we would recommend conducting a specific literature search on specific organoid types to find the most appropriate protocol for the desired application.

Organoid Models and Applications

Visit the Corning Life Sciences organoid models and application web page to learn more about a variety of organoid types and to access relevant published landmark papers that discuss methodologies to generate organoids.

For more specific organoid resources:

Gastrointestinal organoids

Single Lgr5 Stem Cells Build Crypt–villus Structures in vitro Without a Mesenchymal Niche

Cerebral organoids

Cerebral Organoids Model Human Brain Development and Microcephaly

Prostate organoids
Identification of Multipotent Luminal Progenitor Cells in Human Prostate Organoid Cultures

Kidney Organoids

Directing Human Embryonic Stem Cell Differentiation Towards a Renal Lineage Generates a Self-Organizing Kidney

Ovarian organoids

Long-term organoid culture reveals enrichment of organoid-forming epithelial cells in the fimbrial portion of mouse fallopian tube

Liver and pancreas organoids
Culture and Establishment of Self-renewing Human and Mouse Adult Liver and Pancreas 3D Organoids and their Genetic Manipulation

There has been a growing interest to evaluate synthetic alternatives for MatrigelÒ matrix to culture organoids. Researchers have shown that alginates and functionalized polyethylene glycol (PEG) formulations can support human pluripotent stem cell-derived intestinal organoids as well as organ progenitors from healthy mouse intestinal tissue. However, the researcher will need to tune the stiffness and functionalize the synthetic hydrogels appropriately. It is important to control both the biochemical cues as well as the structural support to get successful organoid growth. Here are a few links that address these methodologies.

Synthetic Hydrogels for Human Intestinal Organoid Generation and Colonic Wound Repair

Nonadhesive Alginate Hydrogels Support Growth of Pluripotent Stem Cell-Derived Intestinal Organoids

Designer matrices for intestinal stem cell and organoid culture

In this context, it is also important to note that many of these synthetic options may not be as robust as Matrigel matrix in its ability to support organoid culture. For e.g. synthetic formulations may support organoid growth from healthy intestinal tissue but not patient-derived organoids; they may not support the various morphologies associated with organoid growth (budded vs. cystic forms). In those instances of pluripotent stem cell (PSC)-derived organoids, one still expands PSCs in Matrigel matrix prior to moving to a synthetic environment; therefore a total synthetic replacement for Matrigel matrix is yet to be identified.

Corning recently launched new Matrigel matrix for organoid culture, an optimized ECM that provides the biochemical cues, porosity and stiffness to culture intestinal organoids.

The nature of organoids provides an excellent basis for drug discovery. As referenced in the question, a large volume of organoids would be required to move organoids into a high throughput screening environment. Although there are still currently challenges in this area, including labor-intensive work and time, there are protocols that briefly introduce how organoids can be expanded.

Application Notes and Review Articles:

• A Novel Method for Generating Single, Intestinal Organoids for High Throughput Screening
• Generation of Cerebral Organoids from Human Pluripotent Stem Cells
• A Simple Bioreactor-Based Method to Generate Kidney Organoids from Pluripotent Stem Cells

For scalability, organoids can be plated in single well plates. When preparing organoids for a drug screen, an extra split in which organoids are processed 1:1 and plated again for 1-2 days canincrease the number of organoids significantly. This gives the organoids time to recover from the stress of disruptions, and let them reach the appropriated size for screening activities (20-70 µm). We  have not had good experience plating organoids for drug screening as single cells, since not all single cells are able to grow as organoids. During the manipulation of the organoids for drug screening, we recommend supplementing the washing medium with RhoKi 10 µm to reduce the stress that generates on organoids that are not surrounded by ECM. We do not recommend storing organoids on ice while collecting all material.

Higher throughput for drug screening can be achieved by studying organoid in HTS microplates (e.g. 384-well plates) coated with Corning Matrigel matrix, using the “sandwich” method or embedded method (refer to Corning CLS Guideline for use SPC-356255-G). You can also refer to the article, “Assay Establishment and Validation of a High-Throughput Screening Platform for Three-Dimensional Patient-Derived Colon Cancer Organoid Cultures”, which reports the establishment and validation of a high-throughput screening platform in a 384-well format for 3D organoid culture derived from colon cancer patients.

Normally, 3 days it is very short time, but it also depends on the nature of your original materials (mouse or human hepatocytes) and whether after the isolation the hepatocytes were plated as single cells or clumps of cells. In the case of single human hepatocyte cells it will require more than a week to start observing hepatocyte organoids.

Under conventional 2D monolayer culture conditions, primary human hepatocytes (PHHs) rapidly lose their hepatic phenotypes whereas PHHs cultured in 3D culture can maintain the viability and functions for several weeks.

To support the 3D liver spheroid model in drug discovery and development studies, Corning offers 3D spheroid-qualified primary human hepatocytes using Corning spheroid microplates. Using the protocol described in the Application Note: 3D Primary Human Hepatocytes (PHH) Spheroids Demonstrate Increased Sensitivity to Drug-induced Liver Injury in Comparison to 2D PHH Monolayer Culture, PHHs form small cell clusters, larger cell aggregates, and then spheroids in 6 to 7 days. Once formed, PHH spheroids remain stable over 4 weeks as shown by morphology and size measurement.

Based on this finding, the generation of spheroids can take up to 7 days.

However, those spheroids were generated from primary human hepatocytes. Organoids can be generated from, for example, isolated duct cells. In “Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation”, a protocol for the generation of organoids is described in which the isolated duct cells are cultured in Corning® Matrigel® matrix after which organoids become visible after about 7 days in culture.

For both approaches, the formation of the spheroid/organoid can take up to 7 days. We, therefore, would recommend keeping the cells in culture and reevaluate the presence of organoids at day 7.

To support organoid growth, we recommend refreshing media every 2-3 days. Some of the compounds used are not very stable after several days at 37°C. The observation that organoids die right after medium refreshment suggests that a revision or optimization to the media formulation is needed. You also may need to optimize the final concentration of reagents, since the death of the organoids is unexpected if these formulations are all correct.

Although the generation of organoids from cancer cells seems contradictory, researchers now are focusing on this approach to study the heterogeneity found in cancers as well as for the development of personalized medicine. Cancer organoids can recapitulate organ function and express organ specific markers, as demonstrated in the following paper: Patient-derived lung cancer organoids as in vitro cancer models for therapeutic screening.

Several papers are available in which the generation of organoids from cancer biopsy of patients are described. For example: Cancer Sample Biobanking at the Next Level: Combining Tissue With Living Cell Repositories to Promote Precision Medicine.

Information on the generation of organoids from spheroids is, probably due to the nature of spheroids, more limited. However, it has been described that “intestinal” spheroids can form into organoids in the following paper, A process engineering approach to increase organoid yield.

Cryopreservation of organoids becomes more and more important to improve organoid-based therapy and for acquiring large numbers of cells. Please find here an article in which the cryopreservation of intestinal organoids either undissociated or dissociated is described: Long-term culture-induced phenotypic difference and efficient cryopreservation of small intestinal organoids by treatment timing of Rho kinase inhibitor.

Interestingly, protocols to freeze can differ when using mouse or human organoids. Here is a protocol handbook that describes organoid culture including those for cryopreservation of human and mouse organoids.

Additionally, we have observed a significant increase in cell survival after thawing of organoids, after being processed as described in the Tuveson lab protocol, step 1 to a small size (between 30-40 µm), organoids are plated in 55% extracellular matrix (ECM) drops for 1-2 days. With this plating step, we let organoids recover from the stress of disruption, but also activate the proliferations state right before freezing. After 1-3 days (depends on the human organoid model, for instance after 1 day some organoids are visually increasing in size (colon), while others need three days (lung or breast). We collect them from the ECM drop and freeze them following the steps indicated in step 3 onwards. We have observed that the presence of some ECM in the pellet does not have a negative impact in the survival of the organoids, so extra washes to remove ECM are not required. We recommend aspirating the ECM free of organoids from the pellet.

When working with more complex organoid models, there are many important factors to consider including the extracellular matrix, fluid transportation, controlled medium exchange and spatial separation. For these more advanced models, the combined usage of extracellular matrices with a “organ on a chip” method may prove useful.  The chip serves as a “scaffold” which can be used to create compartments.

Please find here a few examples of organoids cultured on a chip:

• Gut-on-a-Chip microenvironment induces human intestinal cells to undergo villus differentiation
  Intestinal organoids: Use of a chip allows the culture to have an apical and basolateral side, allowing cells to show increased markers of intestinal cells.
• Interstitial flow regulates the angiogenic response and phenotype of endothelial cells in a 3D culture model
  Blood vessel formation: To date the formation of blood vessels has only been successfully demonstrated in 3D cell culture. In combination with a chip, the interstitial fluid flow, shear stress, and growth factor gradients could be mimicked.

This question is particularly difficult to answer as there are no “standard” 2D or 3D cell culture protocols in which the format, volumes and protocols are comparable. In general, cell culture products used for 2D are less advanced than products offered for 3D cell culture and therefore, the direct cost for 3D cell culture are typically more than for an experiment with a conventional monolayer culture. Additionally, within a few days, a simple monolayer of cells can be formed; the generation of 3D cell structures like organoids might take up to several days, but during this period no elaborate steps need to be conducted by the cell culture personnel.

However, there are certain applications where conventional 2D experiments will not lead to the same rich answers as experiments conducted in 3D cell culture, as cells grown in 3D more closely mimic in vivo behavior in tissues and organs. 3D cell culture environments create more biologically relevant models for drug discovery which may lead to more predictive results, higher success rates for drug compound testing, a faster path to market, and reduced development costs. If you are interested in setting up 3D cell culture and the generation of organoids in your lab, products and solutions are available that make starting with 3D cell culture easy, e.g. Corning spheroid plates or Corning Matrigel matrix for organoid culture.

Other resources on 3D Cell Culture:

Organoid Models

• All About Organoids e-book
• Explore Organoid Model Environments
• Citations on Corning® Matrigel® Matrix and Organoid Culture
• Working Successfully with Organoids

Spheroid Models

• Explore Spheroid Model Environments
• Citations on Corning Spheroid Microplates with Ultra-Low Attachment Surface
• Spheroid Culture Advancements

Tissue Models

• Explore Tissue Model Environments

At Hubrecht Organoid Technology, Corning Matrigel matrix has been by far the best extracellular matrix (ECM) supporting the expansion of the organoid models. To reduce variability generated by the presence of grow factors or other proteins/molecules that could impact organoid growth, we always work with growth factor-reduced and phenol red free versions of Matrigel matrix. We have looked for a synthetic alternative to biological extracellular matrices, but to date nothing we have tested supports the growth of organoids to a comparable level as Matrigel matrix.

To support the reproducibility and consistency essential for organoid research, Corning recently launched Matrigel matrix for organoid culture. This optimized formulation is verified to support growth and differentiation of organoids¹ with each lot measured for matrix stiffness, a property that supports organoid workflow. Matrigel matrix for organoid culture has been demonstrated to successfully grow organoids from both healthy and diseased cell origins². Each lot is qualified to form stable “3D dome” structures commonly used in organoid culture.

When expanding organoids under defined conditions, we recommend avoiding the use of serum. In all medium recipes developed by the lab of Hans Clevers, the presence of serum is found when the tissue-specific organoid model requires the presence of Wnt3a (i.e. human intestinal models). The Wnt3a conditioned medium contains 5% fetal bovine system (FBS) and it has been the most robust way to produce active Wnt3a. Luckily, alternative sources of serum-free Wnt3a are becoming available, such as Surrogate Wnt (Garcia lab, Stanford), making possible to expand organoids in the absence of serum.

Diffusion kinetics can regulate organoid size and health: upon reaching a certain size, organoids have arrested growth and develop a necrotic core. This process is possibly linked to a switch from a proliferative, stem-like state to a non-proliferative, differentiated state, as well as necrosis of the organoid core. Terminal differentiation can be a strategy to limit organoid size but preventing a necrotic core is crucial for organoid health as it could lead to premature differentiation. Perfusion/flow of nutrients around the organoids is a technology that has been discussed in scientific literature to assist with prevention of necrotic core. Organoid vascularization is an important criterion for long term culture; seeding endothelial cells within the system to allow for formation of blood vessels (neo-angiogenesis) as well as using bioprinting to engineer the architecture and placement of cells are important platforms that are being explored towards this objective. Included below are two links to helpful articles on the topic.