From absolute in vitro to complete in vivo models
The various models used in research ranging from in silico to in vivo models. In most cases, these components are used in tandem and more than one model is used at any stage of research. However, in vitro models are the first ones that are used and based on the results obtained, the most promising studies are extended for in vivo testing. The 3D cell cultures and organoid cultures bridge the gap between absolute in vitro and total in vivo models.
Research in biology involves the use of several model systems for characterization of molecules, testing safety and efficacy of drugs and elucidating the morphological and physiological parameters of the system. The various research models that have been extensively used in drug development studies are in-silco, in-vitro, ex-vivo, in-vivo and clinical trials.
When the outcomes of a research are to be extrapolated to human benefit, it becomes essential to involve a myriad of testing methods and test models. The requirements of an ideal test system are yet to be met by any of the available models, nevertheless existing testing models form a linked chain that cushions the transition between testing compounds in the laboratory to testing in actual human patients. With the advance of technology, nowadays researchers are able to computationally test the suitability of test molecules using computer generated models (termed as in-silico research). Through in-silico models, targets and leads are discovered and validated as potential drug interventions. Gene function analysis, chemoinformatics for giving rise to compound libraries and estimation of drug-likeness are some techniques which are performed prior to any laboratory work.
Once in-silico analyses have been performed, the molecules are tested in-vitro in a laboratory setting. Testing here involves the employment of various assays that serve to functionally quantify the effects observed prior to and after an intervention. Traditionally, once in-vitro tests yielded positive results, the molecules would be tested on animal models. Prior to direct administration of molecules into live animals for testing, they are tested in ex-vivo models. The model organism is first rendered unfit by artificially damaging the organ, to mimic the conditions which will be relieved by the molecule under study. The organs harvested may either be benefitted therapeutically by the lead or may be used to detect the toxicology profiles of the lead molecule.
Once ex-vivo organ testing received positive results, the molecules are tested on live animal models. Even with the obvious ethical issues surrounding animal testing, it remains an indispensable strategy due to the physiological and anatomical similarities between animals and humans. The complex physiological associations present in humans are mimicked in animal models like rats, mice, guinea pigs, dogs, rabbits, and monkeys. Another similarity observed is that the pathophysiology of infection is quite similar. This similarity is observed in disease states such as diabetes also, prompting the development of animal models.
But tests in in-vitro conditions are not sufficient for claiming safety of the test molecules. Testing it in- vitro conditions only often resulted in the molecules showcasing a different pharmacological profile than the one documented. This discrepancy is observed between animal and human models also, owing to the vast variations in the genetic makeup of animals across species. This manifests as a gap between successful lead detection and successful therapeutic application. As a counter to this disability, 3D cell cultures and organoid models were developed. These systems are more complex and closer to the in-vivo microenvironments than conventional in- vitro systems.
3D cell cultures, serve to provide more accurate information regarding human physiology and pharmacodynamics of drugs. The reason for a higher reliability while dealing with 3D cultures, is due to the resemblance of the cultured cells' architecture to actual physiologic environments. The freedom of growth spanning the third dimension allows cells to take up specific spatial orientations. This allows intracellular signaling and receptor organization which ultimately leads to variations in signal transduction. The effects of this can be seen in downstream processes including gene expression. Cells cultured in 3D scaffolds take up a variety of shapes, the most commonly observed shape being spheroids. Even among spheroids, the structural parameters of spheroids vary based on the underlying cell type. These 3D models have been extensively expanded using cancer cell lines. Allowing cancer cells to grow in 3 dimension allows cellular organization resembling in-vivo microenvironments closer than any monolayer culture could achieve. Hence these cultures are being inculcated within drug discovery and pathophysiology research extensively.
A newer research strategy involves the use of organoids to test molecules. Organoids have made it possible to study disease systems of humans in great detail. These are basically stem cell based or healthy tissue based 3D cultures that contain the major cell types of the organ in question. The cells are also allowed to localize and orient themselves within the scaffold, giving rise to an organization akin to that seen in-vivo. A major advantage with organoids is its histological similarities to in-vivo tissues. To establish organoids, adult cells are first de-differentiated into induced pluripotent stem cells, which undergo expansion and organization to give rise to organoids. Hence they are also ideal to track developmental paths.
Given these advantages, it is more prudent to establish workflow in 3D cultures and organoids before indulging in animal testing. The use of 3D cultures is more widespread because of advantages like availability of cells and lack of pre-processing into iPSCs. For cancer research 3D cell cultures have become irreplaceable. The morphology of cancer cell lines observed in 3D cultures is a very good indicator of the processes that occur in-vivo. Hence, oncology and treatment strategies are being researched in 3D cultures over conventional monolayers.
1.Brogi S, Ramalho TC, Kuca K, Medina-Franco JL and Valko M (2020) Editorial: In silico Methods for Drug Design and Discovery. Front. Chem. 8:612. doi: 10.3389/fchem.2020.00612
2.Barré-Sinoussi F, Montagutelli X. Animal models are essential to biological research: issues and perspectives. Future Sci OA. 2015;1(4):FSO63. doi :10.4155/fso.15.63
3.Kim, J., Koo, BK. & Knoblich, J.A. Human organoids: model systems for human biology and medicine. Nat Rev Mol Cell Biol 21,2020, 571–584. doi : 10.1038/s41580-020-0259-3
4. Mirabelli P, Coppola L, Salvatore M. Cancer Cell Lines Are Useful Model Systems for Medical Research. Cancers. 2019; 11(8):1098. doi : 10.3390/cancers11081098
5. Jean-Pierre Gillet, Sudhir Varma, Michael M. Gottesman, The Clinical Relevance of Cancer Cell Lines, JNCI: Journal of the National Cancer Institute, Volume 105, Issue 7, 3 April 2013, Pages 452–458. doi : 10.1093/jnci/djt007
6. Edmondson R, Broglie JJ, Adcock AF, Yang L. Three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors. Assay Drug Dev Technol. 2014;12(4):207-218. doi:10.1089/adt.2014.573
7. Kapałczyńska M, Kolenda T, Przybyła W, et al. 2D and 3D cell cultures - a comparison of different types of cancer cell cultures. Arch Med Sci. 2018;14(4):910-919. doi:10.5114/aoms.2016.63743
Author contact details:
Abinaya Ramesh, Devaki Muralidharan and Soumika Ghosal are pursuing their BSc in Biomedical Sciences specializing in Human Genetics at the Department of Human Genetics, Faculty of Biomedical Science, Sri Ramachandra Institute of Higher Education and Research, Chennai – 600116, India. Dr. Maddaly Ravi is a Professor in the mentioned Department.
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