The second installment of the “What’s it all about series?” aims to tackle the broad utilization of in vitro three-dimensional (3D) culture models, termed organoids. There are numerous definitions for what constitutes an organoid, and just as many applications for their use. Though sometimes hard to define, the use of 3D in vitro cultures has become more of a standard as cell biologists accept that they are a more physiologically relevant system than traditional 2D cultures (see the advantages reviewed here). I aim to leave you with a jumping-off point for using these techniques in your own research and/or providing you with the necessary information to be able to hold that all too important networking conversation about this trending technique being used across cell biology.

What are organoids?

Fig. 1 Cervical cancer cell spheroid from Scott Wilkinson.

The rise in popularity of this technique comes from the recently accepted view that cells do not behave in historic 2D cultures as they do in vivo. Rather, 3D culture models present a more physiologically relevant approximation of the in vivo environment. Organoids represent cells grown in specific 3D environments to create mini, simplified organs that retain some physiological function. The terms “organoid,” “3D culture,” or “spheroid” are used somewhat interchangeably depending on what cell type is used, ranging from established cell lines (Fig. 1), stem cells, or primary tissue samples. In a recent review, pioneers in organoid research  Marina Simian and Mina Bissell, discuss the definitions of organoids and the historical timeline leading to the widespread popularity of these techniques.

How do you generate organoids?

Part of what makes organoids so hard to define is the vast range of structures that are recapitulated in  in vitro 3D cultures. Scientists have been able to create many functional epithelial organoids, along with various types of gastrointestinal, brain, kidney organoids, etc. The range of successful organoid models has been comprehensively covered in multiple reviews, see a review out of Nick Barker’s lab with corresponding publications and a review from Giuseppe Remuzzi’s lab discussing their biomedical research applications. Adding to the complexity of this technique is the fact that there is no universal method for creating an organoid. The field started by culturing established cancer cell lines in 3D, usually embedding them in collagen or matrigel. This provided a different context to assay physiological function, and now elaborate 3D spheroid cultures from various cell lines have been used for drug discovery and biosensor applications. More recently, 3D culture techniques have expanded immensely, allowing for the creation of more organ-like models (more commonly referred to as organoids instead of spheroids) from two general starting points: primary tissue, either harvested or biopsied, or stem cells, either embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). Primary tissue, once harvested, is dissociated, either to functional regenerating units or down to single cells enriched in niche stem cells. These are then implanted in the specific 3D environment for that model. Stem cells, alternatively, are differentiated into specific cell types and then implanted in the specific 3D environment once mature enough. Multiple factors for the 3D environment specific to each organoid model must be taken into account. These can broadly cover the type of extracellular matrix (ECM) used, the cocktail of signaling factors needed for maintenance and growth, and the use of differentiation factors for stem cell-based models. Another major variation in many protocols where investigators are particularly interested in cell-cell interactions is how many cell types are present and their number relative to one another.  With so many factors to consider, it is a genuine marvel that so many organoid model methodologies have been utilized successfully.

What’s happening in the field now and where is it going?

Fig 2 Data presented from www.ncbi.nih.gov/pubmed

Since the late 1990s 3D culture has been gradually gaining popularity in publications with a more recent spike in the term “organoids” since 2012 (Fig. 2). Thus far we have discussed the basis for using organoids as an in vivo-like model, but what are the real applications of organoids? What is everyone using them for? The answer to this is as broad and wide-ranging as the technique itself.

Organoids have almost unlimited possibilities for basic scientific research. Everything we have learned from the extensive history of 2D cell culture can now be tested in 3D. Earlier this year, organoids earned their own special issue in Development, where their applications for studying morphogenesis and organogenesis were showcased.  In addition to studying normal development and normal physiological function, organoids allow for better disease modeling as it takes these 3D environments into account. Organoids are being used now to model many types of cancer, cystic fibrosis, autism, and microcephaly. Not only do organoids take 3D environments into account, they do this for the first time for many human tissues in the lab. Despite researchers best efforts to learn as much as they can from model organisms, there are still differences when looking at human disease specifically. Organoids allow for human-specific disease modeling in near in vivo environments. Better disease modeling means a better base of understanding on which to build clinical applications. Scott Wilkinson (see expertise below) describes what he sees for the future of organoids in the lab:

“although the techniques are relatively novel and challenging, and require additional time, resources, and funds to perform, the benefits of studying cell biology in a 3D environment far outweigh any costs associated with it. As we move forward with initial results from organoid studies across a multitude of diseases and basic cell biological understanding, the acceptance within the scientific community will continue to grow. As we move toward the future, I see an environment where 3D organoid cultures become as common as western blotting and remain an integral component of basic cell biology research for many years.”

In addition to experiments in the lab, the field is starting to look at the clinical applications of organoids, particularly for their possibilities in personalized medicine. Since deriving patient-specific iPSCs is now a blood draw away, patient-specific organoids could provide the means for personalized drug testing or specialized tissue sources. Ryan Conder (see expertise below) imagines the possibilities organoid technology may facilitate,

The most immediate direction is precision medical aspects of treatment screening. Currently, with any patient diagnosis of a specific disease, a treatment is applied based on previous collective outcome. Organoids provide the opportunity to ask the question of what treatment is best for a specific patient and test these treatment options in vitro. Proof of principle experiments have been performed in the Netherlands on cystic fibrosis patients and novel treatments that had no precedent were found that provided the best option. This approach is now being scaled up, with Dutch insurance companies funding screening of all cystic fibrosis patients. This commitment will hopefully lead to a paradigm shift in other countries and in more diseases, where a greater focus on diagnosis will inevitably lead to more successful treatments and better a better quality of life for those patients. Also, proof-of-principle transplantation experiments have been performed in mice demonstrating seamless integration of the donor intestine into a recipient with intestinal damage. These results raise hopes that, with the ability to perform gene editing in the patient’s organoid cultures, through transplantation, innovative treatments for tissue-specific diseases may be possible.”

The studies cited by Conder showcase the incredible potential of organoid technology, but as always with new possibilities and technological advancements, come new concerns. Bredenoord, Clevers, and Knoblich discuss in a recent article how organoid technology may contribute to the existing ethical debates surrounding stem cell research and the use of animal models. What’s clear is that organoid research will spark more discussion in the future, as it becomes a gold standard technique in cell biology.

Want to use organoids in your own research?

With the growing popularity of organoid research, many tools and resources have emerged to guide scientists in their experimental pursuits. R&D systems provides a large range of organoid specific culture reagents but has little protocol aids. Millipore Sigma provides a nice overall review of the techniques paired with their culture reagents. STEMCELL Technologies has an organoid information hub and provides an intestinal organoid training class, as well as culture reagents and media for many types of organoids. Trevigen also provides a one-stop-shop virtual organoid research laboratory. In addition to these commercial resources, a non-profit organization called The HUB has been founded based on the work of Hans Clevers, another pioneer in organoid technology. Their website provides educational resources and outlines their living biobank project on patient-specific cancer organoids.

Need some advice?

Thinking of using organoids in your research? Take some advice from these experts who have had success using this powerful technique in a range of contexts.

Ryan Conder – Senior scientist at STEMCELL working on research and development of products for intestinal organoid culture

Wun Chey Sin – Research associate in the Naus Lab working on modeling glioblastoma with human iPSC derived neural organoids

Scott Wilkinson – Postdoctoral fellow who used cancer organoids in his PhD work to study cancer cell invasion in the Marcus lab

Q&A

What are pitfalls to avoid when using organoids in the lab?

Conder – As with any novel technology, researchers have to be sure to remember organoids are just that: a technology. The potential this particular technology has to advance research and facilitate the promise of precision medicine is great but still needs to be addressed in the form of well-designed scientific questions. It is tempting to try to work this culture system into a research plan, knowing that the data produced will be novel and exciting, but research still must be performed to provide answers to unknown questions and advance science accordingly.

Wilkinson – With our assays using lung cancer cell lines, optimizing spheroid formation takes quite some time. Common pitfalls we would encounter would be the cells failing to come together to form a spheroid, the cell-cell contacts not being tight enough so that when we moved the spheroid to collagen it would fall apart, Trying to ensure that the spheroid is embedded in the 3D matrix and not floating on top or sitting on the bottom of the plate, etc.

What did you struggle with the most in getting your techniques off the ground?

Wilkinson – Identifying optimal growth conditions is challenging. When first beginning, we tried a variety of different methods for spheroid formation and growth and spent many months working to optimize the conditions. Fortunately, once you’re able to optimize for one line, others generally follow the same pattern. The process of embedding the spheroid in a 3D matrix can also be very challenging, as you’re working with a liquid matrix that will solidify in a short time. Optimizing all these conditions took our lab around 6 months, at which point we were able to expand the technique to encompass the entire lab.

Sin – There was no published protocol when we started. We had to optimize every step and condition ourselves. It is a challenge to have a proper mix of the right cell type. And even if the cell type is the same, the developmental stage of the cells are influenced by their environment. So, it is important to characterize the organoid with markers that are pre-validated in an intact brain (or whatever system you’re working on).

What essential advice would you give to someone just starting out?

Sin – To start cheap, play with mouse neural stem cells to gain technical experience in co-culturing glioma stem cells with normal neural tissue (which will be derived from iPSC ultimately).

Conder – Spend the majority of the time on the experimental design. Unlike extremely well characterized cell lines, each organoid culture will be unique to the patient or mouse donor.  Make sure to understand what the data generated by the experiment is going to be used for and that the proper controls are in place to be confident in the results.

Wilkinson – Patience. The technique is very powerful and provides one of the closest attempts to replicate an in vivo system using in vitro methods, but the process of developing and optimizing the assay can be very challenging and requires a lot of patience. I would strongly recommend seeking out advice from others who have done it before, especially in person if possible, so they can provide advice and guidance as you move forward with it.

The views and opinions expressed in this blog are the views of the author(s) and do not represent the official policy or position of ASCB.

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Amanda Haage

Amanda Haage is a postdoctoral fellow in Guy Tanentzapf’s Lab at the University of British Columbia in Vancouver, Canada. Here she investigates how cell adhesion to the extracellular matrix regulates animal development. She previously received her PhD in 2014 from Iowa State University in Ian Schneider’s Lab where she studied how extracellular mechanics regulates cancer cell motility. Twitter: @mandy_ridd and Email: mandy.ridd@gmail.com