3D cell culture can be successfully used as an alternative to laboratory animals, and as a cost effective and time-saving tissue culture technique, which also reduces the trial period for drug testing

Curtin, James

Tiwari, Brijesh K

Wanigasekara, Janith

English

Arrow@TUDublin

Technological University Dublin ARROW@TU Dublin Articles 2020-4 3D mammalian cell culture models in toxicology testing Janith Wanigasekara Technological University Dublin, janith.manoharawanigasekara@tudublin.ie Brijesh K. Tiwari Teagasc Food Research Centre James Curtin Technological University Dublin Follow this and additional works at: https://arrow.tudublin.ie/creaart  Part of the Cancer Biology Commons, and the Cell Biology Commons Recommended Citation Wanigasekara, J., Tiwari, B. and Curtin, J. (2020) 3D mammalian cell culture models in toxicology testing , Research magazine article. TRESEARCH | SPRING 2020 | VOLUME 15 : NUMBER 1 Available at: https://www.researchgate.net/publication/340569576_3D_mammalian_cell_culture_models_in_toxicology_testing. DOI: 10.21427/6sx4-wq76 This Article is brought to you for free and open access by ARROW@TU Dublin. It has been accepted for inclusion in Articles by an authorized administrator of ARROW@TU Dublin. For more information, please contact arrow.admin@tudublin.ie, aisling.coyne@tudublin.ie, gerard.connolly@tudublin.ie. This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 License Funder: Science Foundation Ireland (SFI) 26  Toxicology testing is performed to understand the adverse effects of drugs and chemical substances on humans and other living organisms. In the EU, the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) regulation (EC 1907/2006) applies a “no data, no market” rule, and responsibility has been placed on industry to manage the risks from new chemicals and to provide safety information on them.  What is 3D cell culture? 3D cell culture (Figure 1) describes a number of techniques to grow cells in three dimensions, such as in a spheroid, using an artificially created microenvironment. Cells in 3D cell culture have physiological cell-cell interactions and cell-extracellular matrix component interactions, which allow cells to grow in vitro in an environment that closely resembles in vivo conditions. For example, spheres possess a hypoxic (oxygen-deprived) core resembling solid tumours with cells at the centre of the sphere having extremely low oxygen and nutrient concentrations. Spheres also show complex transport dynamics by creating diffusion gradients of drugs, oxygen, nutrients and waste, and also show resistance and drugs/chemical substances show low potency as an in vivo condition. The neighbour geometry and cellular support found in 3D cell cultures can improve gene expression, cellular communication, migration, differentiation, survival and growth similar to in vivo tissues, which provide better representation for toxicological testing. Mixed-cell populations can also be cultured in 3D to closely model human tissues. Hence, 3D cell culture plays a vital role in measuring biological responses to new chemicals.  Why should I use it? Use of animals in toxicological research has been reduced due to the ethical concerns, expense, time consumption, and misleading results due to differences between human and animal physiology. The REACH regulation’s stated aim is: “to ensure a high level of protection of human health and the environment from effects of hazardous chemicals. It strives for a balance: to increase our understanding of the possible hazards of chemicals, while at the same time avoiding unnecessary testing on animals” (European Chemicals Agency, 2020). 3D cell culture is a much better technique, which supports the ‘3Rs’ of animal research (replacement, reduction and refinement) and the REACH recommendation to perform humane animal toxicology research. Conventional 2D cell cultures (Figure 1) are unable to detect organ-specific toxicity and have inadequate representation of cell migration, differentiation, signal transduction, survival and growth. A 2D cell culture does not reveal toxicological resistance (Figure 2), architecture as in vivo tissues, accurate depiction of cell polarisation or gene expression. It also provides unreliable predictions of in vivo drug efficiency and toxicity, which leads to low success rates in clinical trials. 3D cell culture can overcome the disadvantages of in vivo animal testing and conventional 2D cell culture by providing a more accurate platform for short- and long-term studies, demonstrating the long-term effects of the drugs.  How is it done? Different 3D culture techniques such as anchorage independent, anchorage dependent, hydrogels based and specialised culture platforms can be used. Anchorage-independent/scaffold-free techniques rely on non-adherent cell-to-cell aggregation to form spheroids, which show cell-cell interactions and secrete their own extracellular matrix.  These spheroids are formed without a physical support resulting  in consistency of shape and size, which provides better understanding about cellular cytotoxicity. Low-adhesion plates  FOOD TRESEARCH | SPRING 2020 | VOLUME 15 : NUMBER 13D mammalian cell culture models in toxicology testing  3D cell culture can be successfully used as an alternative to laboratory animals, and as a cost-effective and time-saving tissue culture technique, which also reduces the trial period for drug testing. 27are specialised culture plates with ultra-low attachment coating (polyhydroxyethylmethacrylate/agarose), which promote cell aggregation to form spheroids. Hanging drop plates are open bottomless wells, which promote the formation of droplets of media that provide space to form spheroids. In magnetic levitation, cells are preloaded with magnetic nanoparticles and external magnetic fields to provide non-adhesion, plate-like properties to form uniform tumourspheres. A rotational stirrer has a container to hold cells  and impeller, stirring continuously. Liquid flow prevents cell adhesion and distributes nutrition and oxygen uniformly to  form tumourspheres. The anchorage-dependent technique provides physical  support by using engineered porous membranes, polymeric fabric meshes called ‘scaffolds’; these scaffolds can be made of natural or synthetic components. This physical support can provide structures from simple mechanical up to extracellular matrix-like structures. Cells are embedded in extracellular components and are able to initiate cell-cell and cell-matrix interactions, and physical support for cell growth, adhesion and proliferation. Natural scaffolds have higher biocompatibility and lower toxicity when compared to synthetic polymers. Hydrogels can be defined as water-swollen networks of a polymer, which is a liquid at room temperature and forms a gel-like structure at 37°C. Cells can be embedded inside hydrogels and provide a similar microenvironment to an extracellular matrix. Animal- and plant-derived hydrogels can be used in 3D cell culture. Specialised 3D cell culture platforms, such as microfluidic devices, can equally distribute oxygen and nutrients, while removing waste to facilitate spheroid formation. Micro-patterned plates are micro-space, low-adhesion plates that promote the formation of spheres.  Conclusion 3D cell culture can be successfully used as an alternative to laboratory animals, as a cost-effective and time-saving tissue culture technique, and also to reduce trial periods for drug testing. Moreover, they can be used as an alternative to 2D cell culture, since they give an accurate outcome for toxicological testing. 3D cell culture reduces the gap between in vitro and in vivo drug testing and its effects at the clinical level.  Acknowledgment This study has been supported by Science Foundation Ireland (SFI) under grant number 17/CDA/4653.   Reference European Chemicals Agency. (2020). ‘Animal testing under REACH – ECHA)’. [Accessed February 23, 2020]. Available from: https://echa.europa.eu/animal-testing-under-reach.  Authors Janith Wanigasekara Doctoral researcher, Teagasc and School of Food Science and Environmental Health, Technological University Dublin  Brijesh Tiwari Research Officer, Teagasc Food Research Centre, Ashtown, Dublin 15 Correspondence: brijesh.tiwari@teagasc.ie  James Curtin Head of the School of Food Science and Environmental Health, Technological University Dublin FOODTRESEARCH | SPRING 2020 | VOLUME 15 : NUMBER 1FIGURE 1: 2D (left) and 3D (right) cell cultures of U373MG cells. FIGURE 2: Different concentrations of dimethyl sulfoxide (DMSO) cytotoxicity towards 3D and 2D cell culture.Log (concentration)Cell viability %150 100 50 0DMSO cytotoxicity assay for U373MG-1.0                -0.5               0.0                0.5                1.0                1.53D cells  2D cells

3D mammalian cell culture models in toxicology testing

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3D mammalian cell culture models in toxicology testing

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