Numerous experimental models currently used in the preclinical arena can aid the research and development of new pharmaceutical compounds and Drug Discovery. These experimental models may range from basic high throughput computational models to more biologically significant animal models but all of them are conceived to better predict the behavior of a particular drug before interacting with the human body.
Attending to the support on which these models are based, they can be classified as:
- In silico
- In vitro
- Ex vivo
- In vivo
All of these expressions are taken from Latin and in this article, we will briefly analyze the differences among them.
Silicon plays a central part in conventional information processing, hence the name “in silico” for this computational model. Our current knowledge in information technology has been determinant for the discovery of new molecular entities. The use of modern molecular-modeling tools is helping us to improve drug design strategies to lower toxicity and improve drug stability. These in silico methods are relevant in limiting the broad number of compounds found in the initial phases of pharmacological research known as Drug Discovery and contribute to adding a rationale in the structural design of new drugs.
In vitro is a Latin expression meaning “in glass”. In vitro experiments have been historically conducted in glass test tubes and Petri dishes however, modern laboratory practices mandate the use of single-use plasticware to avoid cross-contamination. These experiments are often conducted on cells and sometimes using also bacteria. When the test subjects are cells, they are derived from living organisms and maintained in culture in special conditions that try to mimic physiological circumstances including temperature and sterility. There is a wide variety of cell types which in many cases can be amplified in plastic flasks and distributed in plates with hundreds of small wells for high throughput screening. Some cell types cannot adhere to plastic and are cultured in suspension, forming aggregates. Currently, there is an important shift towards using three-dimensional (3D) cell systems where the higher cell-cell interaction adds another layer of complexity in cellular communication.
Furthermore, advanced in vitro systems such as the organ-on-a-chip technology, offer the next level of complexity introducing microfluidic channels that reproduce blood and/or airflow just as in the human body.
The expression “in vitro” appears 18 times more frequently than the expression “ex vivo” in the scientific literature. This is probably why the less common term “ex vivo” might create some confusion. Ex vivo means “outside of a living body” and that implies that tissues are isolated from an animal, taken to the laboratory, and maintained until their use as a research model.
The main difference between in vitro and ex vivo assays is that the former is simply a cell system established in a cell culture laboratory whereas the latter is a tissue not created artificially but directly taken from a living organism. The level of cellular complexity is therefore superior in an ex vivo system and implies a minimal alteration of the organism’s natural conditions.
Some ex vivo assays are simple and just use lymphocytes extracted from whole blood but others involve the use of tissue slices from the liver or the brain which retain the original cytoarchitecture and intercellular connections. The culture and maintenance of these complex tissues are known as organotypic cultures. There are other ex vivo studies where whole organs might be used. This is the case of bovine corneas used for testing ocular corrosives under the eye irritation test OECD 437.
As it can be seen, ex vivo systems represent a model between in vitro and in vivo that can be very resourceful for more specialized studies.
Finally, the use of animal models is the last step in the complexity of biological systems for research. In vivo assays offer a scenario of a whole organism having inherent biological interactions and with high homology to humans. This is why the use of animal models is highly relevant to predict for example the toxicity of a compound before entering into clinical phases. However, there is a growing ethical concern about the abuse in the use of animals for research and there are policies such as the 3Rs policy that advocate its correct use. The 3Rs stand for Replacement, Reduction, and Refinement of animals meaning that they need to be replaced whenever possible, reduce their number and minimize the pain, suffering, and distress exercised during the experimental phase.
Overall, there is a need to implement alternatives to animal models that rely on the exploitation of advanced in vitro and ex vivo systems. Moreover, the ideal assay would take advantage of the benefits offered by in vitro testing including the high throughput capacity and economical advantage, and at the same time benefit from the complexity and reliability of an animal model. The Zebrafish is a vertebrate animal model with high genetic homology to humans that gathers all these benefits at once. Zebrafish larvae represent a robust cost-effective animal model with high throughput capacity and no ethical concerns since is not considered an in vivo assay when larvae are under five days postfertilization. Due to all these reasons, Zebrafish are picking up interest and finding their place within the Drug Discovery process.