Methods to replace animal models

Two-dimensional culture/2D model is a culture system that allows to grow cells in monolayers on a substrate of interest, which can be glass, plastic, or plastic coated by filaments from extracellular matrix e.g. fibronectin. Many cell types can be efficiently cultured and co-cultured in 2D systems. Cell suspension culture – can be considered a subtype of 2D culture for single cells that do not adhere to the surface and prefer to be cultivated in a suspension.

2D culture is one of the most widely used models in the world, being a relatively straight-forward technique that works for many cell types. It owes a big part of its popularity to the ease of culture and availability of hundreds of different immortalized cancer cell lines.

Advantages

Its advantage is the simplicity of using these cells, allowing easy growth, manipulation, treatments, observations, sorting and quantifying live cells in a scaled-down environment. 2D culture is a base for many established cell-based assays that predict an effect of a drug or a chemical on a certain cell type (e.g skin). Due to the fact, that cells are grown in monolayers, they caneasily be studied in a microscope and subjected to automated microscopy e.g. for drug development (e.g. cytotoxicity, genetic toxicity studies) or studied with other optical techniques.

Disadvantages

The planar growth of 2D cultures does not recreate to the full extent native conditions in which cells grow in a tissue, which is three-dimensional. Another important aspect is a lack of flow – cells grow in the same growth media in a flask, and media is only exchanged every few days. Some cell types do not grow well in this scaled-down environment, for example liver cells.

How to get started

Established cell lines for cell culture can be bought from ATCC American Type Culture Collection (a non-profit global resource centre) or can be isolated from patient tissues (primary cultures, with proper ethical approval and consent from the patient). The cells can be immortalised for prolonged use (this will allow to culture cells under longer time).

Three-dimensional culture/3D model is a culture system that allows layered growth of cells, often with the help of a scaffold, to better mimic tissue organization. Scaffolds are structural supports that often consist of proteins from extracellular matrix (e.g. fibronectin) or hydrogels. Components of the scaffolds can be of animal-origin (with batch variation) or completely animal-free (like alginate).

Many cell types can be efficiently cultured and co-cultured in a 3D system. In 3D environment cells usually display a different morphology than in the classic 2D culture: they tend to aggregate more as a result of increased cell-to-cell contact, whereas in the 2D culture systems cells usually are more stretched and have less contact points.

The 3D system is a great tool to mimic a more complex, organ-like, environment. It takes advantage of cells spontaneous self-organisation and differentiation capacity that leads to assembly of spheroid structures and even mini organs, organoids (e.g. from embryonic stem cells, iPS cells, or somatic cells).

Another subtype of a 3D culture model is advanced organotypic models (or assembloids, reconstructed tissue models) – these are models of tissues assembled through differentiation or co-culture of two or more cell types or organoids that can further be assembled to mimic organotypic physiology, such as presence of specialized cell-to-surface contacts e.g. cell-to-air.

Organoids are mini-organ structures formed by self-assembled and self-differentiated embryonic or iPS cells with accurate microanatomy and functionality. Organoids can be established for many organs e.g. liver, lung, brain, intestine, stomach, kidney and heart.

Moreover, organoids can be established straight from the patient tissues – for example tumour organoids. Depending on tissue type, organoids can be used in many applications. They can for example be used for drug testing, developmental studies, and toxicity assessment on various human organs, as well as screening of human developmental and perinatal toxicity.

Spheroids are self-assembled structures that arise when culturing or co-culturing cells in 3D environment, with less complex structure than organoids. They can be used for toxicity, drug screening and to monitor cell responses in a 3D environment, where cells have different exposure to toxins and nutrients, dependent on their position in the three-dimensional tissue.

Advanced organotypic models can be established by culture or co-culture of different type of cells assembled to mimic tissue layers or tissue interactions of a human organ, e.g. skin or lung, or a process, e.g. infection. Examples of such models are advanced lung models, infection models and so on.

Advantages

A main advantage of a 3D culture is that cells experience an environment that allows growth and cell-cell interactions in all directions, which is not possible in an 2D cultures. The 3D structure facilitates self-organisation and differentiation of cells into small organ-like or tissue-like structures. These structures have the potential to replace animal testing e.g. when it comes to study development and drug toxicity.

Since they are derived from human cells, they may also produce more human relevant data than what can be acquired from animal models. The 3D-structures can also be effectively used for cells that do not function well in 2D environment e.g. liver cells.

Disadvantages

Likewise, the complexity of these cultures can also be a disadvantage. To cultivate a more complex structures like organoids one has to follow complex and long protocols, with addition of expensive growth factors, and the process of differentiation often lasts for weeks and can lead to a high variation. In addition, cells growing in clump-like spheroid structures are more difficult to study than 2D models using standard microscopy settings and may require clearing protocols for efficient imaging.

Another important aspect is that spheroids/organoids can only grow to 1-2 mm in size after which efficient exchange of nutrients and gases gets limited to the core of the cell structure. This is because these structures lack vascularization. Use of a bioreactor that effectively stirs the organoids may help in efficient exchange with the culture media.

How to get started

Choice of a 3D cell model is heavily dependent on the research question and which organ/tissue you want them to represent. This can include both disease models (e.g. infection disease model, 3D cancer model) and healthy tissue models (e.g. advanced lung model, reconstructed epidermis model, soft tissue model).

There are many different protocols developed by researchers and companies on how to establish 3D cell models. This may range from relatively easy 3D spheroid models to more complex multicellular organoid models. These protocols may need to be adapted to your particular need, and type of cell starting material you have (cancer cells, primary cells, iPSCs or embryonic stem cells). In general, low attachment culture plates facilitate 3D growth and organization, and often some type of hydrogel may be needed to simulate the extracellular matrix scaffold.

Mouse-derived Matrigel is a very popular hydrogel, but has the disadvantage that mouse-specific factors in the gel may interfere with your cells. Non-animal hydrogels are under development, such as alginate, originating from algae.

A drawback with 3D cultures is that they easily aggregate and lack vascularization. Therefore, it is needed to keep them in continuous movement which also more efficiently allows exchange of nutrients and gasses. To this end, a plate shaker, or better a spinning bioreactor, is needed to grow healthy and uniformed sized organoids.

Microphysiological systems – also known as organ on a chip – are basically miniature microfluidic devices for cell culture, where cells can be grown on membranes in a 3D environment that even closer mimics human tissue/organ physiology.

These devices recreate the microenvironment with the biomechanical forces experienced by organs, which include the organ’s interaction with a regulated flow of media or gasses, or both. Access to the flow is provided through a network of very small channels, often lined with for example endothelial cells, where fluid or air circulates (microfluidic).

Moreover, certain forces can be applied to the membranes on which cells are growing (e.g. expansion-shrinking) stimulating mechanical movement that is a part of natural environment of many cells, especially lung cells. Many cell types can be successfully cultured and co-cultured on organ-on-a-chip devices, for example lung, brain, liver, kidney, heart cells giving rise to the specific names lung-on-a-chip, liver-on-a-chip, brain-on-a-chip etc.

Organ-on-a-chip are great tools for recreating different tissue microenvironments such as the blood-brain-barrier, skin or the lung to study their function and response to drugs in a more controlled and precise manner than using conventional cell culturing or animal models. Thus, these devices can successfully circumvent the need for animal testing, by creating an optimal and controlled environment for accurate testing of substances on human tissue samples.

A series of devices that represent different organs can be joined into a network, creating a mini-organism connected via a microfluidic flow. Alternatively, different cell types from different organs can be cultured on a single chip, emulating an organism (body-on-a-chip). This has a great potential to emulate the human organism and study impact of chemicals, pharmaceutics or disease progression on several organs/tissues at the same time.

Advantages

Main advantage is a presence of the flow of nutrients through different tissue or organ compartments that better recreate the microenvironment and physiology of organs. Complex interactions such as cell-air, blood-brain can be studied relatively easy. Furthermore, many different cell types can be grown in this environment, event those that do not thrive in the simpler two-dimensional culture. The device can be equipped with many micro-sensors that allows to monitor, measure and control several different parameters automatically.

Disadvantages

Challenge lies in the design of these devices, since they need to reproduce the complexity of the tissue mimicking composition through co-culture of cell types of interest and exposure to flow. Since you introduce several new parameters, this also introduces new level of complexity when using and troubleshooting this kind of system. When it comes to combination of devices to imitate organ system, the difficulty lies in knowing how to link them and in which order to mimic a system of interest. In addition, these devices are not available for high throughput screenings yet.

How to get started

Researcher from the Wyss Institute were first with developing organ-on-a-chip models originate. Today these models are developed in many labs and you can find those in Europe through the European Organ-on-Chip Society. There are also several organ-on-a-chip devices that are commercially available. The field of microphysiological models is actively growing and more and more tissue-specific models are getting available.

The QSAR model is a model built to predict the function of a specific molecule. This model can help you find connection between molecular features of your molecule and the functional outcome it can have on the organism.

For example, features like chemical structure, molecular weight or distribution of the electrons can predict related functions like mutagenicity or skin sensitization. You can look for molecular functions you want to avoid or those you want to seek.

• The OECD QSAR Toolbox (oecd.org)

AOPs are not computational models, instead a systematic way of gathering knowledge about a molecule/group of molecules and the specific adverse outcome. AOP can help you determine a link between your molecular initiating event and an adverse outcome. For example, how a substance of interest can generate a sequence of molecular and cellular that lead to a toxic effect.

Moreover, AOPs can serve as a base for quantitative AOPs, i.e. computer models that can be useful for some application. OECD has initiated a knowledge base around AOPs to provide a platform to share, develop, and discuss AOPs.

• Adverse Outcome Pathways, Molecular Screening and Toxicogenomics (oecd.org)

PKPD models are dynamical models commonly used in drug development projects to design trials and to analyze outcoming data. PKPD models can help you describe the pharmacokinetic and pharmacodynamic properties of a drug (molecule). For example, how fast the drug is cleared out from the circulation, a binding site of the drug, drug-caused intracellular changes and changes in secretion patterns.

These properties of the drug will affect both the safety and the wanted outcome of the drug. FDA provides guidelines for the use of these models for regulatory purposes.