In Vitro Approaches for Establishing 3D Cultures
2D monolayer cell cultures have been a mainstay of drug discovery and development for several decades, but they’re not ideal for some applications. For example, they’re less useful in modeling diseases where the cellular microenvironment plays a vital role, such as cancer. Therefore, scientists have been working for many years to develop 3D culture systems which better recapitulate in vivo cellular response, including predicting drug response.
Various options now exist to establish 3D cell cultures. Selecting the right method for each study depends on the nature and source of the cells, purpose of the experiment, and research question(s).
Scaffold-Based vs. Scaffold-Free Cultures
In general, 3D cell culture systems can be broadly categorized as scaffold-based or scaffold-free systems, each with their own pros and cons:
- Scaffold-based systems use natural or synthetic materials as a support for seeded cells to aggregate, proliferate, and migrate, ultimately generating a 3D structure.
- Scaffold-free systems rely on encouraging the self-aggregation of cells via specialized culture plates or physical parameters that prevent cell attachment.
Scaffold-free systems may be preferred for 3D cultures since they have no exogenous structures to block drug or growth factor delivery. However, 3D structures forming on scaffold-free systems tend to be irregular in size, and some cell types don’t aggregate at all without a scaffold present.
Scaffold-based systems allow greater control over design and architecture of the forming microstructure. The support matrix used can also be functionalized to better reproduce the cellular environment in vivo. It’s important to note that the scaffold itself can adsorb the test compound, which needs to be factored into pharmacology studies.
Two of the most prominent 3D cellular structures used are organoids (most frequently grown on a scaffold-based system), and spheroids (grown on scaffold-free systems).
Organoid 3D Cultures
Organoids are an in vitro model of human development and disease. They’re commonly thought of as miniature versions of organs, and they often display a very accurate microanatomy. Organoids have been established for many organ types, including (but not limited to) the small and large intestine, lung, brain, liver, stomach, kidney, and retina.
Organoids are typically derived from a single adult stem cell (aSC) or embryonic stem cell (ESC). They can also be generated from induced pluripotent stem cells (iPSCs), such as skin or blood cells that have been reprogrammed into an embryonic-like pluripotent state.
To successfully establish organoids, careful consideration must be given to the different available protocols, which vary depending on the nature of the stem cells used. For instance, while iPSC- and ESC-based organoids leverage endogenous developmental processes, aSCs must be coerced to form organoids. This has been successfully achieved by the Clevers lab for many organ types by manipulating the culture environment through supplementation with organ-specific cocktails of growth factors. These growth factors model the stem cell niche environment during physiological tissue self-renewal or damage repair.
Regardless of the type of stem cell used for organoid establishment, the common link is the use of stem cells capable of self-renewing and differentiating into multiple lineages in vitro. This results in a multicellular 3D structure made up of different cell lineages that reflect the important structural and functional properties of organs.
Spheroid 3D Cultures
Spheroids are 3D cultures consisting of cell aggregates generated from a single cell type or from a multicellular mixture of cells. Spheroids are established from immortalized cell lines, primary cells, or fragments of human tissue. Low-adhesion culture conditions are used to promote cell self-aggregation into sphere-shaped 3D structures.
When a multicellular mixture of cells of different origin are used to develop spheroids, the result is an ‘organotypic culture’, suggesting the reproduction of the multicellular aspect of a tissue in vivo.
Generally, spheroids contain layers of cells, with some exposed on the surface and others buried within the sphere. Spheroids are heterogenous and commonly include proliferating, non-proliferating, and necrotic cells, which can be well oxygenated or hypoxic.
What are the Differences Between Organoids and Spheroids?
In general, there are two major differences:
- Nature of the driving force for their development: Whereas internal developmental processes drive organoid formation, spheroids develop primarily via cell-to-cell adhesion.
- Length of time 3D cultures can be maintained: Long term, in vitro expansion of cells in culture needs an immature stem cell population to replenish dying cells. Organoids are derived from, and maintain, a population of stem cells during in vitro culture, which guarantees their long term viability. This is achieved by optimizing culture growth conditions, such as providing a basement membrane matrix (i.e. Matrigel®), and adding a selection of agonists (e.g. Wnt and tyrosine kinase receptor) and inhibitors (e.g. bone morphogenetic protein/transforming growth factor-β).
Importantly, when organoids are passaged they retain the genetic features of the original organ over several generations. Therefore, when derived from several types of tissue they serve as “living biobanks” maintained ex vivo.
In contrast, long-term culture of tissue-derived spheroids is challenging, possibly due to inherent technical difficulties in extracting and maintaining viable cells.
Organoids and spheroids can be generated from a variety of healthy as well as diseased cell types and tissues, such as patient tumors. Tumor derived organoids and spheroids have been generated and extensively investigated for their use in drug discovery. However, there are some key differences in establishing the two from patient-derived tumors.
Patient-Derived Tumor Organoids and Spheroids
Patient-derived tumor organoids and spheroids (commonly referred to as tumorspheres) are relatively easy to establish and serve as robust models for drug discovery and development.
Patient-derived tumor organoids more faithfully recapitulate the complexities in cancer tissue, including the presence of both cancer stem cells (CSC) and their downstream more differentiated progeny. Some key features of patient-derived tumor organoids include:
- They more faithfully mimic the patient primary tumor with a more heterogeneous cell population compared to 2D monolayer and 3D spheroid cultures. Typically, classic 2D and 3D spheroid cultures become progressively enriched with immature cells that best withstand serial in vitro culture, losing similarity to the primary patient tumors.
- As well as development from patient-derived tumors, healthy tissue counterparts can also be generated. This allows side-by-side comparison of drug response between the tumor and healthy organoid from the same patient. This can help in predicting a personalized therapeutic window, among other uses.
- Patient-derived tumor organoids are more cost effective to develop than patient-derived xenograft (PDX) models, as they require less time and resources. In addition, organoids are much more amenable to high-throughput drug screens.
- They have been shown to efficiently predict patient drug response in the clinic.
One limitation of patient-derived tumor organoids is that they can lack the inter organ communication present in complex in vivo systems. This can affect both tumor growth and treatment response. Particularly important are immune cells recruited to the tumor microenvironment, especially with the latest advances in immunotherapeutics. Research efforts are currently ongoing to optimize co-culture protocols of organoids and immune cells.
Tumorspheres are anchorage-independent (floating) spheres that are regularly used to evaluate CSC-related characteristics in vitro. Tumorsphere key features include:
- They contain a high proportion of poorly differentiated cells with high replicative potential. This feature is reminiscent of CSC characteristics in vivo, making tumorspheres a good model for CSC expansion.
- They are clonal, simple to maintain, and easy to manipulate genetically. This makes them highly amenable to high-throughput drug screening (e.g. screening drugs that specifically target CSCs).
- Tumorspheres more accurately reflect clinical drug resistance profiles compared to 2D monolayer culture, however, their response profile can evolve over passages and therefore lose predictivity.
The high proportion of CSCs within a tumorsphere culture can also be considered a limitation, when specifically looking to mimic a heterogenous primary tumor. Tumorspheres often show little histological resemblance to the primary cancer they originated from.
3D cell culture systems better recapitulate in vivo cellular microenvironments which play an important role in determining cellular response to exogenous factors such as drugs. Organoids and spheroids are two of the most prominent 3D cellular structures and understanding the key differences between the two systems offers the best opportunity for selecting the right method.
Spheroids are the more traditional 3D system, better recapitulating in vivo features than 2D cultures and providing a simple and easy to use platform for evaluating CSC characteristics and targeting drugs.
If you need to move to a 3D system capturing more of an original organ physiological relevance then organoids are more applicable. Derived from a variety of stem cell types, organoids are amenable to long term in vitro culture from both healthy and disease tissue, providing a heterogenous culture of stem cells and differentiated progeny. Organoids can be expanded to derive living biobanks of tissues, and disease-specific organoids (such as patient tumor derived) have many drug discovery applications including prediction of patient response.