Seen from above, this device looks like an old cassette tape. The difference is that instead of having two holes, it can have three or four, each of which holds reconstructive human tissues: skin, intestines, and liver. A fluid with nutrients and oxygen circulating between the orifices simulates blood circulation, making the tissues function as interconnected mini-organs.
Tested in other countries by cosmetics and pharmaceutical companies to assess the toxicity of their products under development, the device, known as human-on-a-chip or body-on-a-chip (BoC) is gaining ground in Brazil. The 3D-printing technique (see Pesquisa FAPESP issue nº 276), employed to prepare skin and intestinal tissues (liver tissue is still produced manually), has also been applied experimentally for other purposes (see table).
“We apply the test-ready ingredient to reconstituted skin and evaluate its toxicity, simulating human body function,” explains biologist Juliana Lago, a researcher in preclinical assessment for cosmetics giant Natura, which adopted this technology in the first half of 2023.
Imported from a German corporation, BoC joins other techniques used since 2006 to substitute the safety and efficacy testing of beauty, personal hygiene, and perfume products on animals, prohibited in March 2023 by the Brazilian Animal Experimentation Control Board (CONCEA) of the Ministry of Science, Technology, and Innovation (MCTI) (see Pesquisa FAPESP issue nº 245).
Léo Ramos Chaves / Pesquisa Fapesp
Manually reconstructed liver tissueLéo Ramos Chaves / Pesquisa FapespIn addition to indicating any harm caused by external agents, the tissues filling the chip cavities reproduce certain functions of the organs themselves. “The mini-liver produces bile [a yellow-green fluid that facilitates the absorption of fats and vitamins] and carries out all the processes of detoxification [outer layer] and release of mucus [a white or yellowish fluid that facilitates the elimination of feces],” describes biologist Ana Carolina Figueira, of the Brazilian Biosciences National Laboratory (LNBio), an arm of the Brazilian Center for Research in Energy and Materials (CNPEM) in Campinas, São Paulo State. Figueira coordinated the project, integrating use of the chip with the other tissues, in collaboration with Natura.
In 2023, CNPEM licensed the production technology and sale rights of liver and intestinal tissues for startup 3D Biotechnology Solutions (3DBS), also based in Campinas. In exchange, as well as paying royalties, the company helped to refine the process for producing intestinal tissue using 3D bioprinting, and shared the human skin production method.
Production of artificial tissues
At 3DBS, the intestinal tissue is made from cells purchased from the Rio de Janeiro Cell Bank (BCRJ), with the skin produced from human cells isolated from tissues after phimosis surgery [removal of excess foreskin from the penis] performed on children at a hospital in Santa Bárbara D’Oeste, in the interior of São Paulo State. “Cells discarded after surgeries on children rapidly produce type-I collagen, a protein that we need to give the skin strength and elasticity,” explains Ana Luiza Millás, the company’s research director.
Léo Ramos Chaves / Pesquisa Fapesp
Human skin produced by bioprintingLéo Ramos Chaves / Pesquisa FapespA solution with different types of cells is the raw material used in the so-called bioprinters, which create three-dimensional structures with live cells, molecules, and biocompatible materials. In this case, instead of the plastic material injected by a conventional 3D printer to create an object, a syringe discharges cells mixed with a collagen solution, for example, onto a transparent tray with internal compartments, such as those used to make ice in the freezer. A computer sends information on the dimensions and format of the tissue to be constructed layer-by-layer to the device.
The reconstructed intestinal tissue forms a circular layer of 12 millimeters (mm) in diameter inside the plate compartments, and is then kept in an incubator at 37 degrees Celsius (ºC) for 21 days. During this time, the cells differentiate to form the intestinal lining, which absorbs nutrients and produces mucus. When ready, the tissues can be used within up to a week.
As soon as it leaves the bioprinter, the skin cell solution needs 10 days in the incubator to acquire its final form of small, pinkish gelatinous discs of about 6 mm in diameter. “In this period the cells form five layers in the dermo-epidermal model, known as full human skin. Another simpler model, known as reconstructed human epidermis (RHE), has only the epidermal layer, and is used for cosmetics safety and efficacy testing,” says Millás who has been researching human tissue reconstruction since 2010, initially with an end to creating skin for regenerative medicine. During her doctorate at the University of Campinas (UNICAMP), supported by FAPESP, she worked with a substance extracted from the diesel tree (Copaifera langsdorffii), native to Brazil, which, when incorporated into ultrafine fibers, can be used as a three-dimensional cutaneous substitute (see Pesquisa FAPESP issue nº 226).
New directions in the research led to skin production methods using bioprinting, developed with specialists from the University of São Paulo (USP) and Natura, and described in a March 2019 article in the publication International Journal of Advances in Medical Biotechnology.
Léo Ramos Chaves / Pesquisa Fapesp
Ana Luiza Millás of 3DBS operates a bioprinter; in close-up, the syringe depositing the cell solution into the compartments of a trayLéo Ramos Chaves / Pesquisa Fapesp“Initially we were producing larger skin masses, with double the diameter, but corporations and research centers prefer smaller tissues in lower quantities and at lower cost for toxicology testing,” says biologist Gabriela Gastaldi, a researcher at 3DBS.
The liver tissues are still produced manually using cells imported from overseas and from the Rio de Janeiro bank, steeped in an agarose gel and placed in molds with 81 orifices. After five days in the incubator, the cells bond to form circular cell aggregates known as spheroids, some 300 micrometers (µm) in diameter and visible to the naked eye.
With the sale of these tissues by the company beginning in 2022, 80% of turnover comes from the bioprinters and electrospinning equipment, produced since 2018 at the 3DBS workshop in São Paulo. In wider Brazil, 3DBS also distributes the chips and pumps that circulate the nutrients, manufactured since 2019 by German corporation Tissue-Use, whom 3 DBS represents in the country. “We are invested in the growth of tissue and chip use in view of the need to standardize toxicity tests and other possible applications in the early stages of emergence,” observes business administrator Pedro Massaguer, strategy and innovation director.
At the National Service for Industrial Training (SENAI) Manufacturing and Technology Integrated Campus (CIMATEC) in Salvador, Bahia State, materials engineer Josiane Barbosa uses a 3DBS bioprinter to test different formulations of meat produced from bovine or vegetable protein cells. “Bioprinting facilitates reproduction of products with the required dimensions and geometry. This also helps with cell adhesion, given the layered arrangement, which is more difficult to achieve using manual techniques,” she says.
At the beginning of October, the Brazilian Agricultural Research Corporation (EMBRAPA) Genetic Resources and Biotechnology wing, in Brasília, transformed vegetable-based ingredients such as soy flour, fava beans, and chickpeas into fish-fillet substitutes. If successful, this research may result in new foods, primarily geared toward the vegetarian and vegan markets.
And there are other advances in this area. In a study published in Science Advances in October, Brazilian and American researchers reported the development of skin tissue with similar structures to hair follicles by means of bioprinting. If it advances, this technique may provide cells capable of helping to treat wounds or to perform grafts, given that it is the follicle base cells that initiate their healing.
Project
1. Validation of 3DBSkin full-thickness bioprinted human-skin-equivalent model (nº 21/06621-3); Grant Mechanism Innovative Research in Small Businesses (PIPE); Principal Investigator Gabriela Gomes Cardoso Gastaldi (3DBS); Investment R$453,435.08.
2. Development of bioactive scaffolds incorporated with vegetable oils for skin tissue regeneration using electrospinning technology (nº 12/09110-0); Grant Mechanism Doctoral Fellowship; Supervisor Edison Bittencourt (UNICAMP); Beneficiary Ana Luiza Garcia Massaguer Millás; Investment R$135,828.23.
Scientific articles
MILLÁS, A. et al. Approaches to the development of 3d bioprinted skin models: The case of Natura Cosmetics. International Journal of Advances in Medical Biotechnology. Vol. 2, no. 1. Mar. 2019.
CATARINO, C. M. et al. Incorporation of hair follicles in 3D bioprinted models of human skin. Science Advances. Vol. 9, no. 41. Oct. 2023.
