Perspectives on Organoids: Reducing the Use of Animal Models in Research

Introduction

Animal models are animals that are used in research particularly for aiding in understanding diseases and to test possible treatments. They are used to prevent any harm that could possibly happen to humans, for example if a treatment has an undesirable effect. Although animal models have been a useful tool in research, there are several drawbacks including the ethical issues involved with experimentation on living animals. There has been a demand for a more humane and more suitable model that can be used to replace or at least reduce the use of animal models in research. A breakthrough in the scientific community is the development of organoids. An organoid is a smaller and simpler version of an organ that has been grown in vitro three-dimensionally. Organoids have proved to be a strong candidate for disease modelling, but they also have shortcomings of their own. There are several areas of study that require the use of disease models. These include oncology which is the area of medicine concerning cancer. Other areas are neurology which deals with the nervous system and hepatology which is the study of diseases affecting the liver, gallbladder, biliary tree and pancreas.

Oncology

According to the World Health Organisation, the second leading cause of death is cancer and the prevalence of cancer is expected to rise in the future. Therefore, it is crucial to improve treatments and to develop more successful treatments. 406, 038 entries of clinical trial data from January 1, 200 to October 31, 2015 were analysed and it was found that the overall success rate of phase I – III clinical trials for cancer treatments was only 3.4%. Lack of efficacy accounted for over half of failures^[1]. This shows that there is a need for improvements in drug screening methods for cancer. Animal models are currently being used and organoids are another option for disease modelling.

The most common types of animal models are genetically modified models (GEM) and patient derived xenografts (PDX), which involves implanting tissue or cells from a patient's tumour into an immune-deficient mouse. These two models are often used in disease research and for the pre-clinical testing of drugs. They are very useful in observing pharmacodynamics and pharmacokinetics. GEM can aid in the development of molecular targeting drugs but aren't a reliable model for displaying the diversity of patient populations. PDX have traits much more similar to the tumour that they were derived from than two-dimensional cell lines, but genetic changes can begin to build up after several passages^[2]. Due to PDX involving immune-deficient mice, they cannot be used in research for immunotherapy. GEM that are also mice have many differences to the human immune system, so they are not a useful model either. Humanised mice have been designed to allow for investigation of immunotherapy. These are immune-deficient mice that have had haematopoietic cells or tissues inserted which allows them to express human genes³. All of the animal models described above require a lot of time and money to produce making them unideal for high-throughput drug screening.

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Cancer stem-like cell (CSC) organoids can be used in cancer research. Like PDX, they have very similar traits to the original tissue that they were derived from, but they have the advantage of not collecting as many genetic alterations over several passages^[3]. Another favourable characteristic of CSC organoids is their ability to be easily used in an ex vivo PDX systems. There are some downsides associated with these organoids, however, as they require a lot of skill to create and the materials required to do so are not as accessible as other models. They are also less plentiful. These organoids are generated by turning tissue into single cells, but this creates a high level of anoikis. The cancer tissue-originated spheroid (CTOS) method has been devised to overcome this problem as the cells are never singular when using this method. This results in a much lower rate of cell death^[4]. Unlike animal models, CSC organoids are very useful in studying the diversity between patients' drug responses. Relationships between genome alterations specific to patients and the drug effects seen in organoids have also been observed^[5]. CSC organoids are mainly used in research for personalised medicine rather than drug developments due to their lowthroughput in drug screening. Organoids generated with the CTOS method have also displayed high fluctuations in drug sensitivity which allows for the further evaluation of the variations in cancer^[6]. The CTOS method allows for high-throughput drug screening and is also beneficial in the pre-clinical testing of drugs^[7].

Neurology

Animal models are very useful in researching the mutated genes that are associated with certain brain diseases, however, they fall short in their ability to study complex polygenic neurological disorders or diseases with unknown gene defects. Organoids can be used to evaluate the mechanisms of neurological diseases that cannot be studied using animal models.

Microcephaly is a condition characterised by a smaller brain than considered normal. Animal models have been unsuccessful in their ability to recreate the same features of this disease that have been observed in humans. Brain organoids that were generated from a patient that had a mutation in CDK5RAP2 demonstrated that organoids were a suitable model. RNA interference in these organoids recapitulated the phenotype of the disease and overexpression of CDK5RAP2 resulted in a reduction in the phenotype, showing that CDK5RAP2 levels were somewhat responsible for the disease^[8]. Macrocephaly is on the other end of the spectrum, characterised by a larger brain than considered normal. Using CRISPR to delete the phosphatase and tensin homolog (PTEN) gene led to a significant expansion of radial glia and intermediate progenitor cells in human and mouse brain organoids. However, only the human brain organoids had a high occurrence of surface folding. The lack of surface folding in the mouse brain organoids may be due to variations between humans and mice in areas such as signalling regulation^[9]. This further shows that human brain organoids have an advantage over animal models in researching phenotypes that are only found in humans and the mechanisms behind the phenotypes.

Miller-Dieker syndrome (MDS) is a condition in which patients display lissencephaly, an abnormally smooth brain with fewer folds and grooves than considered normal. It is caused by a deletion of chromosome 17p13.3. Research on MDS in the past was conducted on postmortem human brains and animal models but this was not very useful as it is typical of mice to have smoother brains than humans. An experiment using human brain organoids discovered a miotic defect in outer radial glia, which are not usually found in mice, that are necessary for neocortical expansion in humans^[10]. This experiment is an example of how human brain organoids can be used in place of animal models when structural differences between species is an issue.

Alzheimer disease (AD) is the most common form of dementia and results in difficulties with memory, thinking and behaviour. Extracellular deposition of misfolded amyloid-β (Aβ) consisting of plaques and intracellular tangles is the primary trait of this disease. Human brain organoids that were administered β- and γ- secretase inhibitors displayed a major depletion in Aβ and tau pathology which has never been observed in animal models^[11].

Similar experiments can aid in pre-clinical drug developments for neurogenerative diseases^[12].

Hepatology

Animal models have proved as a useful tool in the study of hepatology but there is a lot of areas where they have not been able aid in. They are not particularly helpful in recreating the complicated structure of the liver and for researching its metabolic functions. Organoids can be used to better display the structure and therefore be of great benefit in researching the functions of the liver.

Malaria is a disease caused by Plasmodium parasites which reproduce in the liver of the host. Humanised mice have been used in the study of malaria and viruses that affect the liver, but these models do not display the complexity of the hepatocytes sufficiently, are very expensive and not easily accessible. Due to these reasons, high-throughput drug screening requires an alternative model. Differentiated liver organoids have proved to be a suitable model as they still possess innate immune responses and the cell polarity of hepatocytes. This also makes them useful to investigate hepatitis B^[13] and hepatitis C^[14] infections as the entry and cell-to-cell transmission of these infections are possible. Hepatitis is an inflammation of the liver^[15].

Conclusion

Animal models and organoids both have their own advantages and disadvantages, however, it appears that organoids are generally the better suited disease model for research. Organoids seem to have the same benefits as animal models as well as succeed in other areas where animal models do not such as their ability to be used for high-throughput screening and their ability to allow the evaluation of heterogeneity in patient populations. This makes them a very useful tool for drug developments including personalised medicine. Organoids are still making many advances and with the use of emerging bioprocessing technologies they are a very strong candidate for disease modelling.

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