Work at the Center is intended to meet Brazilian internal demand ranging from toxicity tests for pharmaceuticals and cosmetics as an alternative to animal testing to domestic production of dressings made with human cells, which cannot be imported.
A recent publication in the scientific journal Lab on a Chip described a physiological system in a device containing three organoids (liver, skin, and intestine) that can be exposed to both toxic and non-toxic substances and indicate how these organs would respond in the human body. The research resulted from a partnership between the Brazilian Center for Research in Energy and Materials (CNPEM) and the Brazilian cosmetics company Natura. Natura has already put this method developed in conjunction with CNPEM into practice in its testing routine for quality control and toxicity of the raw materials used in its products as an alternative to animal testing.
Ana Carolina Figueira, a researcher leading CNPEM’s Tissue and Microfluid Engineering Platform since 2020, explains that the organ-on-a-chip system is only about ten years old, and that the Center is a Brazilian pioneer in this technology which has emerged around the world for a variety of reasons. “On the one hand, there is strong international demand for alternative methods that can make good predictions about the effects of chemical compounds on the human body in research in various areas. And on the other hand, there is also domestic demand to develop this methodology in the country. Another example is regenerative medicine, which is where skin dressings come in, since development is still in very early stages, and although some countries in the Global North produce this type of treatment, they cannot be imported,” notes Figueira about the pressures and importance of tissue engineering in Brazil and explaining some of these reasons.
The statement comes on the heels of a federal resolution announced in February of this year prohibiting the use of animals in scientific research and in the development and control of personal hygiene products, cosmetics, and perfumes containing compounds whose safety and efficacy have already been proven. This measure is in line with the global trend, and while it is far from banning all forms of cosmetic testing involving animals, it shows the need for manufacturers to work together and move toward alternative solutions.
Today, the European Union has the strictest legislation of this type for the cosmetics sector. Animal testing involving ingredients or final products has been illegal in most procedures since 2013 in all member countries. In Brazil, the Brazilian Biosciences National Laboratory (LNBio) at CNPEM is one of only three Central Laboratories of RENAMA, the Brazilian National Network for Alternatives to Animal Testing. RENAMA is an initiative of the Ministry of Science, Technology and Innovation, which fosters the implementation, development, and validation of methods that do not utilize animals. Natura, under its own initiative, has rejected all types of animal testing since 2006 in the development of raw materials for its products as well as the ingredients it acquires from suppliers, and is also part of RENAMA as an Associated Laboratory.
Regenerative skin dressings are adhesive patches of biofabricated tissue made from skin cells (cuticle, epidermis, dermis, and hypodermis) and stem cells which act as a graft to restore the skin after injuries such as severe burns. These patches are not imported into Brazil for several reasons related to customs issues as well as the inherent characteristics of biological tissue. Additionally, CNPEM has been researching the development of bioprinted cardiac tissue for regeneration in people who have suffered heart attacks, which would involve the use of cells from the patient, considering the high rates of rejection for this organ. According to the project coordinator, making a flexible patch with the heart’s own tissue presents a challenge compared to fabricating skin.
How organs are cultured and integrated into chips
An assay was developed by CNPEM and Natura involving chips with chambers containing the equivalents of liver, skin, and intestinal barrier to test systemic toxicity when molecules are introduced orally and topically.
Artificial production of tissue and organs is an evolution of in vitro cell cultivation and has played a role the researcher Ana Carolina Figueira calls the “missing link” in a dilemma related to pre-clinical testing. “The in vitro model is human (involving human proteins and genes), but it is not systemic; in other words, there is no integration with the body’s organs as a whole, and for this reason research involving a certain medication often does well in in vitro testing but unfortunately does not progress to the subsequent stages. And animal models are systemic, but not human. That is to say, they are not good predictors of the human body’s response due to a lack of genetic proximity, even though they involve a real organism,” explains the researcher, who considers the engineering of tissues and organoids to be a more robust solution to this problem.
Organoids can be created manually or with a 3D printer for biological tissues (bioprinting) that uses a combination of various cells and substances like collagen, stem cells, and real cells for the desired organ as the “bio-ink” to print a miniature organ that mimics its real-life functions. When they are inserted into or biofabricated on chips, structures with separate chambers for each culture, the organoids are irrigated in a simulation of the bloodstream, which creates a more integrative environment that stabilizes the miniature system. When the same system contains more than one organ which are irrigated with the same circulatory fluid, the device is known as “human-on-a-chip.”
Culture of tissue and organs: cells are mixed with biomaterials to form bio-ink. This bio-ink is put into the bioprinter or manually injected into molds to make the 3D cultures, which then undergo a period of maturation and differentiation in a controlled chamber.
Bioprinter in action, producing 3D cardiac tissue
Besides pre-clinical testing with organs-on-a-chip and human-on-a-chip and the studies to develop regenerative skin dressings, CNPEM researchers involved with this relatively new and promising line of academic investigation have also been advancing in their studies on the effects of chronic diseases and viral infections in tissues, such as fatty liver disease and the impacts of the yellow fever virus on the liver, which shows the versatility of this method.
Furthermore, a biobank is being established, a bank of human cells stored in a dedicated structure that will be able to supply all the material needed to create these cultures and also offer this material to the entire scientific community.
Next steps
In the recently published study, the group of researchers from CNPEM and Natura undertook a delicate process to validate the organ assays, calibrating them between the normal functions for the organ in question and functions in response to abnormal situations. This was done, respectively, through tests showing that the mini-systems are capable of producing the typical molecules for that organ (such as bile in the liver, for example) and by exposure to compounds known to be toxic or carcinogenic (such as formaldehyde) and which cause harmful effects as they would on real organ tissue. According to the researchers, this procedure confirms that the models are good representatives of their equivalent organ, since they respond in the same way under normal conditions and when exposed to toxicity, which enhances prediction when product ingredients are tested.
Kelen Fabíola Arroteia, who coordinated the study on the Natura side, explains that the three organs used (skin, intestinal lining, and liver) correspond to two of the three main ways substances enter the body: oral and topical absorption. She says that the tests are important because of their power to safely permit higher doses of raw materials, depending on the results, which ultimately can add value to ingredients from the Amazon’s biodiversity and for the communities involved. As for absorption of molecules dissipated in the air, the third main way substances enter the body, CNPEM has also been developing bioprinting of lung tissue, a project which is still underway.
Researcher Ana Carolina Figueira reinforces that the broad range of possibilities offered by tissue engineering technology is directly dependent on a multidisciplinary environment, since in order to characterize and validate the organ assays and to develop chip prototypes connections are required between various knowledge areas like electron microscopy, microtomography, mass spectrometry, and genetic expression testing: “you need to gather researchers who understand tissue engineering, microfluid engineering, biology…. not just the material.” The need for multidisciplinarity can be seen as a challenge to progress in this line of research. But institutions like CNPEM foster this environment and consider this type of exchange a founding principle through the presence of various laboratories dedicated to different topics but which are close to one another and interact constantly and are open to external researchers and partnerships with the public and private sectors.
Researchers from the Brazilian Center for Research in Energy and Materials (CNPEM) analyze a human-on-a-chip device with organoid cultures
About CNPEM
A sophisticated and effervescent environment for research and development, unique in Brazil and present in few scientific centers in the world, the Brazilian Center for Research in Energy and Materials (CNPEM) is a private non-profit organization, under the supervision of the Ministry of Science, Technology and Innovation (MCTI). The Center operates four National Laboratories and is the birthplace of the most complex project in Brazilian science – Sirius – one of the most advanced synchrotron light sources in the world. CNPEM brings together highly specialized multi-thematic teams, globally competitive laboratory infrastructures open to the scientific community, strategic lines of investigation, innovative projects in partnership with the productive sector and training of researchers and students. The Center is an environment driven by the search for solutions with impact in the areas of Health, Energy and Renewable Materials, Agro-environment, and Quantum Technologies. As of 2022, with the support of the Ministry of Education (MEC), CNPEM expanded its activities with the opening of the Ilum School of Science. The interdisciplinary higher course in Science, Technology and Innovation adopts innovative proposals with the aim of offering excellent, free, full-time training with immersion in the CNPEM research environment. Through the CNPEM 360 Platform, it is possible to explore, in a virtual and immersive way, the main environments and activities of the Center, visit: https://pages.cnpem.br/cnpem360/
