The considerable advances in biotechnology, microengineering, microfluidics, informatics, computing power and their multidisciplinary cooperation, have allowed the development of New Approach Methodologies (NAMs), inaccessible only a few years ago. Although this is a relatively new field, born with the emergence of the ongoing paradigm shift for toxicology of the 21st century (1), NAMs already allow to study human conditions and diseases in a human-based perspective, that is, focused on human biology and in physiologically relevant conditions, playing an important role in toxicological and biomedical research (2, 3). NAMs have already proven to be better than animal models in predicting human responses (4-9). If promoted and applied with an integrated approach, human-based NAMs allow on the one hand the development of safer and more effective drugs and vaccines for the human species and on the other hand the reduction and replacement of animals employed in biomedical and toxicology research.
A list of major NAMs:
Advanced imaging/scanning techniques
There is a wide range of scanning technologies that can reveal processes in vivo, non-invasively, in humans. Images produced at very high resolution are particularly useful in the study of the brain, neurodegenerative diseases such as Parkinson’s disease and Alzheimer’s. Examples include Magnetic Resonance Imaging (MRI) functional magnetic resonance imaging (fMRI), computerized axial tomography (CT) with three-dimensional reconstruction, tomography and positron emission (PET). These technologies, which can be combined with each other or with other techniques, allow to study in a non-invasive way not only the anatomical structures but also the biochemical composition and metabolism of the various organs. Application examples:
- Brain study: Watch video
- Alzheimer’s disease: study of the association between diet, metabolic syndrome and Alzheimer’s (10)
- Cancer biology (11)
Human tissues reconstituted in vitro / Tissue engineering
Human tissues can also be reconstituted in vitro from individual cell types, aiming to reproduce the original architecture of in vivo tissue. Thanks to recent developments in 3D printing techniques, it is possible to reconstitution different human tissues with a high level of accuracy, for scientific research. Examples of tissues reconstituted in 3D are the epidermis, corneal, oral and gingival epithelium, vaginal and respiratory epithelium. Tissue engineering is the science that studies the possibility of regenerating organs and tissues of the human body. Although originally born for therapeutic purposes (regeneration/replacement of damaged tissues or organs), in recent years it is becoming of great importance in the development of human-based experimental models for research. Several tissues or systems such as human lymph node or joint have already been created in the laboratory.
- Models of respiratory diseases, including tumors, infections, allergies: http://www.epithelix.com/products/
Human tissues and organs
Raw materials of human origin can be obtained and used in various ways, from post-mortem donation to in vivo (e.g. DNA, blood). Intact slices of human tissue, ethically obtained from patients undergoing surgery or biopsies, can be kept in the laboratory so that they preserve their function. Tumor biopsies, for example, can be used to see if a drug has tied to the intended molecular target. Comparing donated, healthy and sick organs can provide important information about pathological processes. Stem cells of human origin also have a huge advantage in research.
Induced pluripotent stem cells (iPSCs)
Pluripotent stem cells are “immature” cells that can differentiate into the different cell types that make up the organism. Given that this type of cell is normally inside the embryo at the early stages of development, until not many years ago, it would have been necessary to destroy human embryos in order to obtain human pluripotent stem cells; this greatly limited its use for ethical issues. Thanks to the findings of the team of Prof. Yamanaka of Kyoto University (Japan, 2007), today iPSCs can be obtained directly from the patient’s cells, for example those of the skin dermis and differentiated into a large amount of cell types of the human body (such as neuronal, pancreatic, cardiac and liver cells), without having to resort to embryos. Research is often limited by difficulty in accessing patients and the reduced availability of fresh tissues. iPSCs can be used as sources for the indefinite production of all those cell types otherwise inaccessible or limited survival.In addition, with iPSCs it is possible to do something that has never been possible in the history of biomedical research: to obtain virtually all cell types from the same patient. Because iPSCs have the same genes and mutations as the patients they come from, researchers can use iPSCs to recreate diseases in the lab and study how a patient’s genetics and environmental conditions contribute to his disease, opening the door to a new era in personalized medicine.
An organoid is an in vitro cellular cluster in 3D, a simplified and small-scale version of an organ, which summarizes its architecture and function in the most salient aspects. An organoid has a complex multicellular structure where cells, when subjected to adequate biochemical stimuli in vitro, differentiate, self-assemble and organize themselves into tissues, summaring what happens in the embryo in the first weeks of development. They may be used in:- study of the regulatory mechanism of organogenesis;- mode
lling of human disorders: a) infectious diseases; b) hereditary diseases; c) neoplasms.- toxicity and pharmacological efficacy tests; -personalized medicine;
Several types of organoids have been developed, including brain organoids. Organoids can be produced from induced pluripotent stem cells (iPSCs), obtained from patients.
Modular multicompartimental fluidic bioreactors
They are advanced in vitro systems that thanks to the presence of a fluid circuit and a peristaltic pump allow the dynamic interaction between cultures and co-cell cultures, housed in chambers or modules, connected to each other. Each module represents an organ of the human body and by connecting the modules to each other in series or in parallel through a fluidic circuit that mimes the blood flow it is possible to model the interaction between organs and systems similar to what happens in vivo.
The concept of a microphysiological system (organs on chips, organoids on chips, human body on chips) has recently evolved and is described as a system of cell cultures, each representing a tissue or organ that interact with each other at different levels on a microchip, through a microfluidic circuit, under strictly controlled conditions. Thanks to the integrated sensors, it is possible to monitor cellular responses to mechanical or chemical stimuli in real time with more precise control of the cellular environment than conventional methods. The system allows to mimic the interactions between cells, tissues (organ on a chip) and even different organs and systems (human on a chip), and to provide the appropriate mechanical, structural and biochemical stimuli, reproducing at all levels what happens in vivo. The use of cells taken from the individual patient for the assembly of customized human on a chip is one of the greatest promises for the near future of medicine.
Omics sciences deal with the study of pools of biological molecules (e.g. nucleic acids, proteins, enzymes) in certain biological samples (e.g. serum, urine, liquor, saliva, tissues). They analyse, as a whole: (a) DNA genes (genomics) and their functions (functional genomics); (b) DNA transcribeds, i.e. RNA (transcribing); (c) proteins (proteomics); (d) metabolites within an organism (metabolomics). They also study the interactions between these molecules (interattomics) and between these molecules and environmental factors (expelosomal), nutrients (nutrigenomics), epigenetic factors (epigenomics), etc. The purpose of this holistic approach is to be able to understand by operating with integrative approaches, higher-level operational principles, which overall define the biology of systems. This is in order to be able to answer more complicated hierarchical biological questions (for example, pathogenetics, natural history or therapeutic success and prognosis of a disease). They use techniques of comparative genetic analysis (array-CGH) or variations in the number of copies of certain DNA traits (CNV) or DNA sequencing, cytometry, single cell analysis techniques, mass spectrometry and computational methods that analyze data from tens, hundreds or thousands of molecules/samples.
Computational and read-across methods
Human systems, from individual organs to the whole body, can be simulated using highly sophisticated computer programs. These are created using data obtained from people. Computer simulations have been developed, for example, to predict the behavior of a drug in the digestive system. These simulations are likely to predict such effects in humans more accurately than animal models and in a much more efficient way.
The read-across uses relevant information on similar substances (“basic”) to predict the properties of “target” substances. If the read-across is applied correctly, experimental tests can be reduced as there is no need to test each target substance.
One of the most important characteristics of a drug candidate is its pharmacokinetic profile, that is, the way in which the drug is absorbed, distributed, metabolized, excreted by the human organism. Unfortunately, pharmacokinetic data provided by traditional preclinical models, whether in vitro or in vivo (animal models) are often not reliable, as they are not relevant to human biology. Not by chance, the biggest cause of failure in drug development is attributed to the inability to obtain pharmacokinetic data relevant to humans at an early stage. Too low concentrations of the drug at the target organ level, for too short a time, can cause ineffectiveness while too high concentrations, for too long, could induce toxic effects.
An experimental approach useful to overcome these problems is microdosing, which consists in the administration to healthy volunteers of extremely small doses, not pharmacologically active, of a given drug to establish its pharmacokinetic profile in humans. Microdosing is based on ultrasensitive analytical technologies capable of measuring infinitesimal quantities and concentrations (of the order of picogram or femtogram, i.e. one billionth of a gram and one millionth of a billionth of a gram!) drug and metabolites. The most widely used technologies for this purpose are liquid chromatography in combination with tandem mass spectrometry, accelerator ultrasensitive mass spectrometry, and positron emission tomography (PET).
These methodologies and approaches, integrated with each other and with epidemiology, are fundamental tools for the advancement of biomedical and toxicological research.
For further information:
1.Krewski D, Acosta D, Jr., Andersen M, Anderson H, Bailar JC, 3rd, Boekelheide K, et al. Toxicity testing in the 21st century: a vision and a strategy. Journal of toxicology and environmental health Part B, Critical reviews. 2010;13(2-4):51-138.
2.Langley GR, Adcock IM, Busquet F, Crofton KM, Csernok E, Giese C, et al. Towards a 21st-century roadmap for biomedical research and drug discovery: consensus report and recommendations. Drug Discov Today. 2017;22(2):327-39.
3.Busquet F, Hartung T, Pallocca G, Rovida C, Leist M. Harnessing the power of novel animal-free test methods for the development of COVID-19 drugs and vaccines. Archives of toxicology. 2020.
4.Barrile R, van der Meer AD, Park H, Fraser JP, Simic D, Teng F, et al. Organ-on-Chip Recapitulates Thrombosis Induced by an anti-CD154 Monoclonal Antibody: Translational Potential of Advanced Microengineered Systems. 2018;104(6):1240-8.
5.Hartung T. AI more accurate than animal testing for spotting toxic chemicals 2019 [Available from: https://theconversation.com/ai-more-accurate-than-animal-testing-for-spotting-toxic-chemicals-99708.
6.Goyal G, Long J, Ingber DE. Microenginered human lymphoid tissue on chip. Cancer Immunology Research. 2018;6(9 Supplements):A76.
7.Ahmed S, Bibby L, Dickinson A. Predicting adverse immune reactions to biopharmaceuticals using a human in-vitro skin explant test: a promising tool for biopharmaceutical R&D development. 2017.
8.Ahmed SS, Whritenour J, Ahmed MM, Bibby L, Darby L, Wang XN, et al. Evaluation of a human in vitro skin test for predicting drug hypersensitivity reactions. Toxicology and applied pharmacology. 2019;369:39-48.
9.Passini E, Britton OJ, Lu HR, Rohrbacher J, Hermans AN, Gallacher DJ, et al. Human In Silico Drug Trials Demonstrate Higher Accuracy than Animal Models in Predicting Clinical Pro-Arrhythmic Cardiotoxicity. 2017;8( 668).
10.Pistollato F, Cano SS, Elio I, Vergara MM, Giampieri F, Battino M. The Use of Neuroimaging to Assess Associations Among Diet, Nutrients, Metabolic Syndrome, and Alzheimer’s Disease. J Alzheimers Dis. 2015;48(2):303-18.
11.García-Figueiras R, Baleato-González S, Padhani AR, Luna-Alcalá A, Vallejo-Casas JA, Sala E, et al. How clinical imaging can assess cancer biology. Insights into Imaging. 2019;10(1):28.
12.Madden LR, Nguyen TV, Garcia-Mojica S, Shah V, Le AV, Peier A, et al. Bioprinted 3D Primary Human Intestinal Tissues Model Aspects of Native Physiology and ADME/Tox Functions. iScience. 2018;2:156-67.
13.Kraus T, Lubitz A, Schließer U, Giese C, Reuschel J, Brecht R, et al. Evaluation of a 3D Human Artificial Lymph Node as Test Model for the Assessment of Immunogenicity of Protein Aggregates. Journal of pharmaceutical sciences. 2019;108(7):2358-66.