Billions of dollars are spent annually on the Discovery and Development of promising new Drugs to treat human diseases. Pre-clinical Drug Discovery and Development aims to identify one or more candidate molecules with sufficient evidence of biological activity at a disease-relevant target in order to advance from in vitro or in vivo testing to human clinical trials.

In this blog post, we will explore what does in vitro means, what is in vitro testing and when are in vitro studies preferred to in vivo ones.


In Vitro Definition and Meaning


The etymological origins of in vivo and in vitro come from Latin. In vitro meaning refers to “in the glass.” In vitro testing doesn’t require live subjects; studies and assays are performed on organic material in a controlled laboratory setting. Generally, in vitro studies refer to work performed with cells, tissues, or other biological components isolated from the living organism of interest.


The ultimate goal of both in vivo and in vitro studies is to provide as much information as possible on how the novel drug will target and affect human health in a predictive way.


In vivo means “in the whole body, " which requires live test subjects. Murine models are the most common pharmaceutical and biomedical research test subjects.


In vivo models provide a clear understanding of how a whole organism is affected by a treatment. In particular, they primarily focus on the pharmacokinetics of a compound in a living organism, focusing on Drug metabolism, safety, and efficacy. Before an experimental drug is studied in vivo, its mechanism of action needs to be thoroughly evaluated on in vitro studies. In vitro models provide a starting point for researchers to gather insights into how a compound acts at the cellular level, focusing on a specific cellular or tissue phenotype.


In vitro testing is a gateway to more expensive and evolved in vivo testing. Indeed, in vivo, models involve significant challenges, specifically the costs of animal care and welfare (also in terms of ethical costs), the compliance and regulatory issues facing in vivo examinations, and the general overhead such assays incur. However, they give more valuable information, as they are complex organisms with all the cells, organs, and systems working together. This is more similar to humans, the final target of all studies.


In vitro testing offers several advantages over in vivo research, including time reduction, ease of use, cost-effectiveness, the possibility to provide high-throughput screenings (HTS), and a higher level of scalability. The Drug Discovery process must be characterized by an efficient and scalable approach from the early stages to avoid wasting resources during the subsequent in vivo phases.


In vitro testing examples


During Drug Discovery and Development processes, even the most promising candidates can fail when translating from pre-clinical into clinical trials. The parameters usually resulting in late lead failure are efficacy, unpredicted toxicity, and human safety incidents in clinical trials, which translate into extremely costly investments of funds and resources, ultimately detrimental to human health and financial resources. 


Using pre-clinical models to reliably predict clinical outcomes is crucial as resources, time, and costs rise with each Drug Discovery and Development process phase. As a result, in recent years, there has been an increasing interest in New Alternative Models (NAMs) for innovative in vitro testing as they can help in the selection of the most promising candidates at the optimum discovery stage, accomplishing the 3R Principle of Reducing, Replacing and Refining the use of animals in research.


In vitro testing has been developed to study several aspects of drug pharmacokinetics and pharmacodynamics. These include absorption, metabolic stability, the potential for inhibition and induction of CYP enzymes, and metabolite profiling, distribution, and excretion. Absorption assays, for example, investigate gut penetration of drugs, an important parameter in producing orally active therapeutics. Cell-based assays are typical examples of in vitro models. Nevertheless, other in vitro methodologies are available for Drug Discovery and Development, and many are highly innovative. Organ-on-a-chip devices are three-dimensional, microfluidic devices that combine cell culture with biomedical engineering to simulate the physiological environment of an entire organ in vitro. 


However, there is an in vitro new alternative model that not only resembles a physiological system but is a full-fledged, complete physiological organism, including many in vivo models’ advantages: the Zebrafish New Alternative Model (NAM) for Drug Discovery and Development. 


2010 European Commission Directive declared, "Some early life-stage animals are not considered experimental animals until they start feeding independently once they leave the chorion.” Zebrafish embryos, under 5-6 days post-fertilization (dpf), meet these conditions and are considered NAMs, conserving at the same time all the advantages of the in vitro and the in vivo studies, as well as their highly predictive results.


Zebrafish share more than 70% of their genome with humans, while more than 80% of human disease genes are conserved. This model can allow us to understand the mechanism of action of a novel Drug within a living whole-animal system, monitor its responses over time, during disease progression and resolution, and evaluate drug-induced adverse effects with high predictiveness. Their embryos are transparent, and this unique characteristic makes them easy to observe in their natural state, avoiding invasive procedures. Fluorescent proteins and colored dyes can allow the direct observation of internal body parts intended for study, a characteristic notably suited for phenotypic screenings. 


Zebrafish assays are more cost and time-effective than other in vivo options, as well as easier to house, maintain, and breed. Moreover, it accomplishes the 3Rs principle of Reducing, Replacing, and Refining the use of animals in research. In a nutshell, Zebrafish combines some of the advantages of in vitro testing with the best characteristics of in vivo models, making it a leading NAM for Drug Discovery and Development.




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Dash SN, Patnaik L. Flight for fish in drug discovery: a review of zebrafish-based screening of molecules. Biol Lett. 2023;19(8):20220541.

Patton EE, Zon LI, Langenau DM. Zebrafish disease models in drug discovery: from preclinical modelling to clinical trials. Nat Rev Drug Discov. 2021;20(8):611-628