Story | 05/23/2024 12:08:56 | 13 min Read time

Can we free animals from cages and bottles?

There is a unanimous push towards animal welfare and a more human relevant science. While testing on animals in EU has decreased in recent years thanks to policy push (3R’s policy, 2010), the use of animal-derived chemicals and pharmaceuticals is not even quantified. Why are these animals invisible when turned into reagents in bottles? Where does the shoe pinch and what could be done? 

Regulatory push to avoid animal use

In the fall 2023, a group of representatives for academia, industry, animal welfare and regulating bodies gathered in Helsinki to discuss the use of animals in scientific research and regulatory testing. 

The venue was the Biofore House, the head office of the big wood-based materials company UPM, and the host was UPM’s start-up subsidiary UPM Biomedicals that innovates and manufactures birch nanocellulose for life sciences and clinical use. 

The workshop marked the 20th anniversary of Fincopa, the Finnish National Consensus Platform for Alternatives to animal testing. The topic for the work-shoppers was replacing animals in experiments within the regulatory testing and scientific research. 

Already in the opening remarks it became apparent that the solid push for the so-called 3R’s i.e. replacement, reduction, and refinement of animal experiments during recent two decades has brought meagre results. 

“Animal testing has not decreased as much as the public thinks. When 12 million test animals were used in Europe five years ago, now the number is maybe 10-11 million”, said Tuula Heinonen, one of the keynote speakers and former head of FICAM*, Finnish Center for Alternative Methods. 

Why are animals still needed?

There are two major areas, one related to new drug development and the other one to safety of other chemicals to which humans are exposed. 

In pharma, new drugs will undergo a long and an expensive development route covering pre-clinical efficacy, safety and pharmacokinetics and then clinical efficacy and safety with high quality material and formulation. Starting from many potential molecules to mitigate effects of a disease. First hundreds of thousands of molecules are tested against potential disease targets in silico (in a computer model), followed by in vitro tests (in plasticware in laboratory), then comes in vivo (in living organisms, such as animals), then three clinical trial phases (in human) and finally seeking regulatory approval. Without mandatory (animal) tests, a new drug does not receive a marketing authorization in EU. In USA, animal studies are no longer a requirement for FDA license for biosimilar or interchangeable products.2

 

Chemicals safety is the other major reason. The ever-expanding use of chemicals in everyday life, and the ever-tightening regulations regarding the safety of these. In layman’s terms, the average person is steadily being exposed to more and more chemicals daily (in various products including biocides, agrochemicals, food, feed ingredients, preservatives, cosmetics), and the effects of these chemicals have raised concerns, for example on human hormonal and neurological development. Therefore, there is a greater demand and need for toxicological tests, ensuring these chemicals are safe in everyday use for humans and to the environment.

In cosmetics, use of animals for testing is banned, so why are these animal-based tests not replaced in pharma and other areas yet? 

Cosmetics is the only field, where tests on animals were banned in EU in 2004. Since 2013, EU Cosmetics Regulation prohibits testing of finished cosmetic products and ingredients on animals and also prohibits marketing of cosmetics that do not adhere to the ban. In pharma, there is regulation that aims to reduce their use in EU (3R’s principle: Replacement, Reduction, and Refinement of Animal Testing first set in 2010) and newly in US (FDA Modernization Act 2.0 set in force in 2022). Other areas, such as chemicals, environment, food, feed are bound to follow. 

Professor Heinonen and one of her mentees Hanna Vuorenpää, postdoctoral Research Fellow in Adult Stem Cell Group (Tampere University), quickly point to the main reason for the slow progress in reducing animal testing in biomedical research, drug development and chemical safety assessments. Plenty of methods have been developed, but only a few have been validated.

A myriad of human cell and tissue culture-based methods have been developed to complement or replace animal testing, but only a few have been validated for official regulatory use although the benefits of using human biology instead of animal biology seem obvious. 

Validation is the necessary step towards ensuring the safe development and provision of new chemicals and medicines for public use. In essence, validation means demonstrating that a cell or tissue culture-based model consistently produces relevant, expected, and desired results in tests, and the test is repeatable and reproducible. Validation requires expertise and rigour, and it takes time and money.  

“Pharma uses a lot of human cells in drug discovery, indeed for the most part, but for their own purposes. It's a competitive advantage. When these companies come up with a good cell-based model, they don't necessarily put it out for validation by the regulatory bodies. The secret sauce is kept to themselves”, Heinonen explained. The same applies with the other chemical sectors.  

Whereas industry keeps its innovations close to vest, academic incentives for validation are not there. 

“From an academic point of view, validation is not rewarding. The goal of researchers is to produce new knowledge and publish it. The more publications, the better the researcher. Validation takes a long time, and you get one publication at most. It is not scientifically meritorious,” Vuorenpää said. 

In Finland, FICAM was the trailblazer of validation of in vitro models (e.g. cells growing in plasticware such as Petri dishes or well-plates) in the spirit of the EU Commission's Animal Experiment Act (EU Directive 2010/63). The act, however, is promotional, not obligatory, and has resulted in nominal but not substantial funding for validation, in FICAM’s case it was 200 000 euros yearly. 

“Almost too tight to mention. You can do one or two validations,” Heinonen says and is quick to add that money is not the only challenge. “There are only a few people in Finland who know how to do validations. A basic scientist can't do that, can't even think that way.” 

From whole animals to human body-on-a-chip

 

Today, there are many cell-based models that can shorten drug development time and especially avoiding costly failure of the drug later, more expensive stages. In addition these models are eligible to be used in safety testing of all chemicals used in various products, such as biocides, agrochemicals, food and feed ingredients and preservatives and cosmetics. The models are improved in a number of ways, cells that are directly from patient biopsies resemble better real patients, automation avoids human error, tailored mix of nutrients are mixed for specific cells. Unfortunately, this also means that one model does not fit all purposes.  

In past decade developments included Organ-on-Chip platforms or microphysiological systems, where multiple cell types are cultivated in a 3D structure with added sensor technology for oxygen measurement or electrical recording, for example. This way, neuronal activity can be recorded on a mini-EEG level or a brain stroke can be simulated by depriving the cells of oxygen. 

The most developed and complex in vitro model platform have cells growing in clumps (called spheroids) forming human mini organs (called organoids), and channels between them that model the flow of blood with a nourishing fluid. So you can think of mini brains, mini hearts, mini guts and mini livers connected to form a mini human for testing. 

“These not only model a human biology, but you get down to the individual level. You take a cell from a patient, take it back to the stem cell level, differentiate cells and you can see how your own cells react. This is the big advantage of these models, personalised medicine and personalised toxicology,” said Vuorenpää. 

"As the cell-based models get better than animal models, they still are expected to be proven against animal data, which is frustrating. Why validate good cell model with bad animal data? All-in-all, by definition cell-based models should surpass the animal models in all three key measures: they should be reproducible, be repeatable and be relevant to the intended purpose," Johana Kuncova-Kallio, director of UPM Biomedicals, explained.

Freeing animals from bottles 

Many of the advanced models indeed replace animal testing (animals are freed from cages), but they still may contain the invisible or hidden animals that are needed to be sacrificed for production of different chemicals. When researchers grab a bottle of a reagent, they often do not think, how many animals were needed to produce it.

“When implementing 3R’s, the focus tends to be on visible animals used in in vivo experiments. However, a great number of animals are used in producing antibodies, sourcing foetal bovine serum for cell nutrition and importantly for extracellular matrix, the material in which cells are being cultivated in assays. Many researchers are blissfully unaware that the nice and clean bottle contains liquids for which animals had to be sacrificed,” Kuncova-Kallio, said. 

This is where the host of the anniversary workshop, UPM Biomedicals, steps to the plate. It has a range of products, GrowDex® and GrowInk™, an animal-free hydrogel made of birch pulp, that offers an optically transparent and inert 3D environment for cell culture. 

“We need to provide cells a three-dimensional environment. It is important that this environment can now be animal-free,“ concluded Vuorenpää. 

Hydrogels for 3D cell culture, organ-on-a-chip models, high throughput screening, drug release studies and more, carries a huge hidden benefit that circles back to the theme of the Fincopa anniversary workshop, the goal of reducing the use of animals. 

According to an EU study1, many researchers are not even aware of alternatives to animals that come as liquids in bottles or vials1. These animals remain invisible to the eye as well as to the conscience of the researchers. There is, however, light at the end of the tunnel. Researchers, who are developing new products, such as cell therapies or other new types of therapies, need to ensure their products are approved for use in human by EMA or FDA. Therefore, they must aim to avoid use of animal-derived materials along the whole workflow to avoid difficulty in regulatory admission of their product. These trail blazer researchers pave the way for others.

According to estimates, the number of these invisible animal offered on the altar of science, may significantly outnumber the actual test animals. It is estimated that one million animals are used in the EU alone for antibody production3 and up to 1.8 million unborn calves are killed worldwide to produce FBS (Fetal Bovine Serum) every year.

 
 

One widely used ‘animal in a bottle’ product for cell cultivation is a matrix isolated from the tumour of a mouse. For a common application, an average of 5 millilitres of animal-derived matrix is needed. That, in essence, is one mouse. 

The same can be done with 3,3 millilitres of nanofibrillar cellulose (NFC), consisting of wood and purified water. GrowDex hydrogel has been found to support cell growth and differentiation with over 150 different assay protocols available. 

”Advanced cell models provide an answer in the long run, when validated. In the meantime, animal free materials provide a quick and significant step towards the goal pursued by all,” states Kuncova-Kallio. 

Saving animals makes a lot of business sense, too. One cubic meter of fresh birch could replace 1.5 million mice.

Animal-free cell cultivation, if anything, is value added. 

 

*FICAM, founded in 2008 under the auspices of Tampere University, has recently transformed into FHAIVE, Finnish Hub for Development and Validation of Integrated Approaches, reflecting integrating advanced in vitro models with toxicogenomics and AI-enabled advanced data modelling.

  1. M. Cassotta, J.J. Bartnicka, F. Pistollato, S. Parvatam, T. Weber, V. D’Alessandro, L. Ferreira Bastos, S. Coecke, A worldwide survey on the use of animal-derived materials and reagents in scientific experimentation, Engineering in Life Sciences, 24.6.2022
  2. FDA Modernization Act 2.0,  https://www.congress.gov/bill/117th-congress/senate-bill/5002, passed 29.9.2022
  3. Joint Research Centre (European Commission) (2020). EURL ECVAM Recommendation on Non-Animal-Derived Antibodies, EUR 30185 EN https://www.nc3rs.org.uk/3rs-resources/replacing-animal-derived-antibodies-animal-free-affinity-reagents 

Additional information: 
Drug development in Finnish: Lääketutkimus ja tuotekehitys - Lääketeollisuus ry

About ban on animal testing in cosmetics in EU: Ban on animal testing - European Commission

NC3Rs: Replacing animal-derived antibodies with animal-free affinity reagents

 
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