Non-animal methods

Non-animal methods

Alternatives to animal experimentation : this expression, to which we prefer that of substitute methods, is used to designate research methods and technologies that do not use living or dead animals for this purpose.

Directive 2010/63/EU on the protection of animals used for scientific purposes sets as the ultimate objective the “total replacement of procedures applied to live animals […] as soon as this is scientifically possible” and underlines that the use of animals for such purposes should be considered only when there is no other method not involving the use of animals.

Methods and technologies, gathering under the designation non-animal research, are based, in their development and use, on the most advanced and innovative scientific knowledge.

Indeed, in its resolution TA(2021)0387 adopted on 16 September 2021, the European Parliament considers that “the panoply of experimental models not using animals is expanding and shows that it is possible to improve our understanding disease and accelerate the discovery of effective treatments”.

Based on the 2021 report (Dura, Gribaldo, Deceuninck), “Review of non-animal models in biomedical research — Neurodegenerative Diseases” of the Joint Research Center (JRC or Joint Research Center) of the European Commission which indicates “that a heavy dependence on animal experimentation can hinder progress in certain areas of disease research”, the European Parliament, still in its resolution of September 2021, not only mentions the bureaucratic obstacles which oppose the acceptance of these methods, but also mentions that their use is not properly enforced and that funding for their development remains insufficient.

Thus, there is still a long way to go… financial and regulatory levers must be activated, practices must be modified, in order to achieve the ultimate objective set more than a decade ago by Directive 2010/63/EU.

But let’s see a little more in detail what these non-animal methods are and their specificities.

The set of substitutive methods to animal experimentation in fact brings together a large number of scientific research tools and technologies offering other satisfactory means of research, more reliable for human health, offering better protection of the environment and without animal suffering.

The panoply of non-animal methods includes organ-on-chip technologies, imaging systems and computer simulations (so-called in silico methods), 3D cultures of human cells, 4D bio-printing, or adult stem cells of human origin, omics approaches, etc.

The particularity of these methods : the development of knowledge specific to our species and for some, specific to each individual.

The NAT (Non-Animal Technologies) database created by our German partners Ärzte gegen Tierversuche (Doctors Against Animal Experiment) is a unique project of its kind. The database started with 250 entries of procedures developed around the world, supplemented continuously. These are available in German and English, so that everyone can learn about these innovative research and technologies. The collection also covers many disciplines such as oncology, toxicology, drug development. This database also includes the research programs supported and developed by Pro Anima.

Below, an overview of the main substitutive technologies and methods that make and will make animal testing obsolete.

Organs on a chip (or organoids)

Clinical and applied research, toxicological tests

This in vitro technology consists of the reproduction on an electronic chip of human tissues and organs with their nerve and blood endings, thus modeling human physiology, but also its pathologies.

Designed from cells often cultured in 3D, and enabled by the convergence of tissue engineering and the development and manufacture of semiconductors, organs on a chip make use of several other technologies such as microfluidics and nano- sensors.

Allowing a concrete and applied mimicry of the human body, by the association of several organs-on-chip, these microphysiological models offer promising prospects in a growing number of fields, whether for toxicology studies, disease modeling and the development of treatments for “personalized medicine”.

TED TALK Boston - Wyss Institute (USA) - Body parts on a chip – Geraldine Hamilton, bioresearcher


The creation of organs in the laboratory has been and is one of the great challenges of modern science. A breakthrough made possible with stem cells.

Stem cells from adult humans

Clinical and applied research

In cell biology, a stem cell is a cell which is said to be undifferentiated in that it is capable both of generating specialized cells by cell differentiation and of self-renewing and maintaining itself in the body when needed.

Stem cells are the "mother cells" of all other cells.

Stem cells from adults are most often derived from human surgical waste destined for incineration. These include, for example, cells found in the skin and in fatty tissue, such as surgical waste from cosmetic procedures to get rid of fatty tissue on the abdomen or breast reduction. Their use does not pose an ethical problem, as long as the informed consent of the patient is obtained.

With technological advances, it is possible to program these stem cells, they are said to be pluripotent, that is to say that they can give rise to almost all the different types of cells in the body.

"The scientific feats rewarded by the 2012 Nobel Prize in Medicine (awarded to researcher Shinya Yamanaka) consists in taking practically any cell in an adult and genetically reprogramming it to make it pluripotent, that is to say capable of infinitely multiply and differentiate into all types of cells that make up an adult organism, such as an embryonic stem cell. These cells are called iPSC for induced pluripotent stem cells”. (INSERM website)

The iPSC has the capacity to produce any other specialized cell, which notably makes it possible to recreate any mini-organ. The organoid thus formed is controlled by adding substances to the nutrient medium.

3D and 4D bio-printing

Applied and fundamental research

3D printing is a technique that makes it possible to produce a real object from a computer file and material. This material stacked in successive layers creates volume. By combining cell biology and physical engineering, the artificial design and three-dimensional printing of a range of human tissue models becomes possible. Human tissue becomes the material to be “printed”. This technology offers tissues with the appropriate architecture and entirely composed of human cells and bio-ink. The structures obtained, living tissues and organs, represent an opportunity to progress for tissue engineering, regenerative medicine, pharmacokinetics and more generally biology research. Skin, liver, bone and heart tissue models have already been developed.

With the maturation phase of the printed fabrics, we can speak of 4D printing. This is the phase during which the assembled cells will evolve and interact together to form a coherent and viable tissue. During the post-printing process within a bioreactor, the tissues undergo rapid maturation, in particular through the development of vascularization and innervation at several levels, increasing the resistance and mechanical integrity of the tissues for a transplantation. Placed in an incubator, the tissues grow until they form a coherent tissue. This phase begins approximately 48 hours after printing and can last several weeks depending on the size of the fabric.

This is for example the case of the 4D BioDISC project of the company Bioregate which is interested in the 4D bio-printing of an intervertebral disc model (IVD) with material gradients in order to study disc degeneration. IVD degeneration is one of the main causes of low back pain, a painful, disabling condition with considerable socioeconomic impact. Conventional treatments are only symptomatic and new bio-inspired therapies are needed to counteract disc degeneration.

Bio-simulation or bio-modeling

Clinical and basic research

Bio-simulation is the dynamic computer modeling of biological systems and human physiology. Combining theoretical and computational chemistry, physics and biology, biosimulation encompasses any application of mathematical and computational techniques to chemical, biological and related problems and systems of interest.

Unlike molecular interaction maps which, by their static nature, only give a limited overview of the behavior of the biological system, especially when conditions change (evolution of the disease, effect of chemical substances, etc.), computer modeling allows a finer and more prospective approach by the integration, through simulations, of silico perturbations and analyses, of dynamic properties.

Relying on a very large amount of data, with the potential to reduce drug development time and costs, computer bio-simulation is also used and promoted in the field of pharmacology. The European Union provides funding for networks of excellence (BioSim) with the aim of structuring efforts to develop simulation models for the design, selection and testing of drugs.

Simulation of neurological mechanisms

Applied research

Despite the many constructions of digital models of the human brain allowing a better understanding of the functional and organizational mechanisms of this organ and a glimpse of the complexity of neuronal interconnections, the modeling of the whole brain remains a challenge both technically and in terms of the analysis and mobilization of the processing capacities of supercomputers. However, the modeling of brain regions as it is already developed is relevant, allowing an interpretable description of neuronal mechanisms that can be coupled and enriched with data from brain imaging systems.

Thus, “the customization of simulations is one of the most exciting aspects of this type of research. It is indeed possible to shape cerebral models using structural data acquired empirically, after having analyzed the brain of a given individual by MRI, for example. In other words, the shape of a person's brain and the connectivity of their neurons can be used to shape a brain network that is unique to them” (The Conversation, Médecine personnalisée : modéliser le cerveau pour mieux soigner - Personalized Medicine: Modeling the Brain for Better Healing -, March 2021)

The development of simulators of neurological mechanisms is therefore very interesting in cases such as epilepsy or to better understand the cognitive deficits observed in Alzheimer's disease and schizophrenia.

In the case of epilepsy, 600,000 people in France suffer from this neurological disorder which includes around fifty epileptic diseases (or epileptic syndromes) and which mainly affects children and the elderly (INSERM figures, 2018). About 30% of patients do not respond to current treatments and the success rate of brain surgery for epilepsy is not improving, stuck at around 50%. Animal models do not provide satisfactory answers.


In 2013, a first computer simulator of the biological mechanisms of epilepsy was validated in the laboratory by Rhenovia Pharma. Modeling the fundamental biological mechanisms that generate the transmission signal between brain cells, the device made possible to simulate the dysfunctions leading to an epileptic seizure. This R&D tool opens up new perspectives for antiepileptic treatments and for identifying the pro-convulsive risks of future drugs. In 2018, the Epinov project was launched. Labeled RHU (hospital-university research), the project focuses on the use of large-scale modeling of the epileptogenic networks of epileptic patients. This innovative approach aims to better guide surgical strategies. Indeed, it is a question of providing doctors with a tool capable of modeling the patient's brain in 3D as well as the epileptogenic zone superimposed above; this, in order to identify more easily the brain area in question, to ensure the success of the operation and to optimize personalized treatments.

Imaging systems and multimodal images

Clinical and basic research

Medical imaging is an essential element in clinical research, the study of diseases and the development of new treatments. Many imaging techniques have been developed. Among the various systems, magnetoencephalography (MEG) and electroencephalogram (EEG), for example, are non-invasive technologies that measure the activity of the human brain by capturing its magnetic field.

Multimodal medical imaging is the fusion of multimode images allowing the matching of several heterogeneous data, the visualization of the results, their interpretation by increasing the quality and the relevance of the complementarity of the merged data. The analysis of multimodal images is developed in particular for the personalization of the treatment plan. Multimodal medical imaging has become a very powerful tool in the diagnosis and management of human diseases.

In the case of the study of diseases and mental disorders for example or of certain cancers, imaging systems and the fusion of multimode images are completely appropriate and more relevant than the invasive procedures on animals whose the usefulness of experiments remains limited because of the impossibility of creating in animals the complex set of symptoms and responses observed in humans.



Toxicogenomics is the application of genomic technologies to study the impact of chemical, environmental, pharmaceutical and radiation substances and agents on the structure and function of the genome, in the short, medium and long term.

This discipline provides information on variations in genes coding for proteins and metabolites involved in various functions of the body. It makes it possible to study the reaction of cells after direct contact with a substance in order to know its potential danger, the dose-effect relationship and the mode of action, or even for the development of a biomarker and the evaluation of relevance to humans. Indeed, used at the preliminary stage (in screening), this method makes possible to select and sort the molecules.

Practiced from human cells grown in the laboratory and less expensive than tests based on animal experimentation, toxicogenomics received approval from the European Parliament in June 2007. Benefiting from massive investments in the United States and Japan, toxicogenomics makes it possible to support national and international measures and more effective integrated approaches in the assessment of toxic risks for human health and the preservation of the environment.

Research in a blood test

Applied research

The blood test makes it possible to search for markers that explore the functioning of the body and of different organs. Researchers propose to assess the toxicity of a drug directly from the blood sample of the patient concerned. This technique is part of personalized medicine, through the direct analysis of what is happening in the patient concerned, and in particular the interactions with other drugs. Research from a blood sample can complement a virtual identity card of the patient's biological profile.

Research based on a blood test can also take place within the framework of specific studies in order to answer a scientific question. The blood samples are then grouped into a sample. Participants in this type of research are volunteers who must confirm their informed consent. They are supervised, informed of the aims of the study, as well as the possible risks and benefits.

Synthetic human and animal models

Clinical and surgical, human and veterinary training

Synthetic models reproduce human and animal anatomy very accurately, including muscles, tendons, veins, arteries, nerves and individual organs. Indeed, made from complex composites, the models reproduce the mechanical, physicochemical and thermal properties of the living tissues concerned. Models can bleed and breathe, use hundreds of replaceable muscles, bones, organs and vessels.

Work on these synthetic model technologies were launched in 1993 at the University of Florida (USA). The materials and models developed since can replace the use of animals in the study of medical devices, clinical training and surgical simulation, both in the human and veterinary fields, but also in the evaluation of consumer products and tests. ballistic, as offered by Syndaver.

Within some veterinary schools, students are also offered simulation platforms including these synthetic models, virtual reality, role-playing games, and workshops such as at Vet Agro Sup; this, with the aim of “increasing awareness of animal welfare and subsequently avoiding any unnecessary suffering for living animals”.

Virtual simulation tables and interfaces

Clinical and surgical, human and veterinary training

The technological evolution within education is gradually giving rise to new training practices and methods through simulation devices intended for both human and veterinary medicine students. Indeed, medical training, and surgical training in particular, is based on learning theory and putting it into practice, in some cases on animals, particularly such as pigs in surgery, before being continued by clinical experience acquired through direct contact of students with patients.

In 2016, in France, more than 34,000 animals were killed for educational purposes. Therefore, these simulation devices have a large number of advantages for students, by performing dissections, manipulations and operations without using living beings (Virginia Polytechnic Institute and State University).

The history of computing and virtual reality in medical education began with the proposal of a human-computer graphical interface called Sketchpad, proposed by Ivan Sutherland in the 1960s. Advances in this area accelerated in from the 1980s, with the development of computers. In the 1990s, a number of medical schools began to pay more attention to simulation as a teaching method, as in Brazil. Dissemination of courses such as Advanced Cardiovascular Life Support (ACLS) and Advanced Trauma Life Support (ATLS) have been a major factor in spreading the use of simulation devices to faculties and professional groups.

Virtual tables with hyper-realistic rendering obtained using data from medical imaging merged with anatomical data have also been developed. Offering a real 3D digital anatomical library, these devices are enriched by collections of clinical scanners and hundreds of scenarios in order to understand the complexity of real cases in a wide variety of environments. These virtual patients with time-changing symptoms based on physiological algorithms, provide relevant immersive simulation experiences as offered by the BodyInteract device.