Introduction

As medical research advances thew need for new models of study arises, many new areas of research will require researchers to observe the microscopic world in greater detail. The rise of stem cell research has led to the development of several cell-based cultures, the most recent of these developments are stem cell derived organoids. Organoids are 3-dimensional structures derived from stem cells that simulate the structure and functions of various organs. With the development of these structures a wave of new potential research avenues has been opened in medical and veterinary research as well as further applications in other areas of biological science potentially down the line. There is also potential to reduced preexisting ethical problems with medical research that comes from live specimen testing. With organoids only a small samples of stem cells need to be taken from a subject and a vast supply of ethical test models can be developed. This allows for more personalised research tailored to specific species or even individual requirements. The purpose of this essay is to look into currant research into organoid technologies and the applications both possible and potential. In addition to this it shall look into potential problems with organoid technology such as ethical considerations that comes from a technology that is derived from living tissues especially when it comes from human tissue.

Background on organoids

Organoids are 3D structures derived from stem cells or progenitor cells; they can recreate important aspects of 3D anatomy as well as simulate basic tissue function on a much smaller scale. These living structures can be cultivated in vitro or in vivo as well as in chimeric animal cultures. They can be produced through at least 3 types of cells:

• Induced pluripotent stem cells (IPSC)

• Embryonic stem cells

• Adult stem cells

Organoids are allowing for ground breaking developments in numerous fields such as personalised medicine, regenerative medicine and reproductive medicine. One study used cerebroids (brain tissue derived organoids) to show neurodevelopmental changes caused by the zika virus. It has been shown that it could be possible to create tumoroids from cancer cells to help create more personalised cancer treatments for patients. In regenerative medicines organoids could help create grafts for patients such as burn victims (Stoekle. H. C, et al, 2021). Traditionally animal testing and cell culture models were the main way to do drug testing, these however have their limitations. For instance, animals will respond differently to pathogens as well as the obvious ethical concerns. Cell cultures in their own regards cannot properly replicate organ functions and have low success rates. 3D cell cultures are more sensitive to drugs due to organisation of surface receptors also cells are often in different stages of development similar to cells in vivo conditions. Petri dish replace the cellular monolayer with gelatinous scaffolds that will replace ECM, the culture can also attach to the bottom of the dish and assemble 3D morphology. It is also possible to facilitate co-cultivation of different cell lineages creating a more accurate representation of in vivo conditions. One benefit pluripotent stem cell have is they can be differentiated into almost any cell type present in a living organism. With the main downside being the time taken to generate the organoids from PSCs (Sakalem. M, et al, 2020).

Prior suggested precursors to organoids are differentiating human stem cells in 2D within a 3D matrix and bioprinting human cells, and culturing cells in a microfluidic device (aka organ on a chip). There are a number of biological phenomena that are specific to humans and are not able to be reproduced in animal models. An example of this is of course the human brain which is far more complex than anything in the various model organisms used in research. This is owed to unique developmental events and mechanisms such as the neurons in the human cortex which arise from a cell type not present in most animals and only minutely present in others. Humans also develop much more slowly than other organisms. Certain drugs such as ibuprofen warfarin which are beneficial in the correct dosages in humans but is toxic to rats even in small doses. The process of organoid formation involves three crucial steps:

1. Key signalling pathways that regulate development patterns are either activated or inhibited.

2. Media formulations that allow proper terminal differentiation of cell type are developed.

3. Cultures are grown that allow their expression in three dimensions.

A lot of early organoid development research were created using mouse stem cells as a model organism. In recent years however organoid technology helped develop vaccines for covid 19 at a much faster rate by replicating organs that are most affected by the virus (Kim. J, et al, 2020). One of the main interests for IPSCs for humans and agricultural species lies in obtaining cells with properties similar to ESCs without going through derivation from embryos. The concept of somatic programming is to induce a cocktail of genes including transcription factors involved in the control of a cell’s pluripotency into asomatic thus reprograming the cells (Pain. B, 2021). Adult stem cells derived stem cells represent a simplified version of IPSC organoids and consist mainly of epithelial cell types of endodermal origin. But they can however be generated without IPSC reprograming, while ADSC derived are used for mimicking certain organs, their use for ectodermal and mesodermal derived structures are limited (Gabriel. V, et al, 2024).

History of organoid development

The first example of human pluripotent stem cells was established in 1998, before that it was done in 1981 with mice. In 2006 mouse fibroblast cells were reproduced into PSCs, these all helped pave the way for organoid development. The term organoid was first coined in 1946 and was then later used in the 1960s by developmental biologists. However, these early organoids were limited and could not self-renew, it was not until 2009 after the advent of stem cell research, that organoid research began to become truly viable (Tang. X. Y, et al, 2022). Pigs have always been used as a model for studying human gastro-intestinal diseases due to them sharing many physiological and anatomical characteristics with humans. As such early studies using organoids used pig stem cells to produce them. Pig based organoids were first successfully developed in 2013 with distinct crypt-villi structures and epithelial cells such as enterocytes, progenitor cells and goblet cells, they were later used to test for lentivirus infection. In 2017 bovine enteriods were cultivated long-term using intesticult organoid growth medium. Horse organoids have been cultivated in 3D Matrigel

supplemented with recombinant human R-spondin and wnt3a and noggin (Haq. I, et al, 2021).

In some literature organoids have been divided into two categories, organoids and spheroids:

• Organoids will be classified as those grown in cultures of PSCs or directly from tissue with self-renewal capabilities.

• Spheroids are obtained from single celled clones or aggregates of cell lines; spheroids will show similarity to the original tissue.

Spheroids have been used to describe an intermediate stage of organoid development before 3D organoid cultures. One limitation observed in organoids is that in some organoid models have lacked some important cell components. These are typically those found in regular organs, one observed area lacking is vasculature, though organoids transplanted into mice have shown signs of vascularisation (Augustyniak. J, et al, 2019).

Possibilities for medical research

Organoids can mimic brain tissues which can help with different neurological studies:

• Organoids based on the hippocampus could help with studies into conditions such as Alzheimer’s disease and schizophrenia, however there is still some challenge in this area.

• The striatum could assist in studying conditions such as Huntington’s disease and addiction.

There is demand not only for brain-based organoids but also organoids of other complex organ structures in the body. Due to their complexity, it took several decades to create a working procedure to create more complex models such as retinal organoids. However, we now have organoids that replicate 3D retinal structures which could be used to investigate eye development and visualisation functions. Organoids can not only be used to help develop new drugs but also to help test the toxicity at different dosages. Organoids could even be a step towards a new form of transplant therapy by facilitating autologous transplantation therapy including helping transplant cerebral tissue into brain damaged patients (Tang. X. Y, et al, 2022). Organoids could also be used to study various microbiota whether pathogenic or otherwise to gain a better understanding of interactions that go on in the human microbiome (Pain. B, 2021). The gastrointestinal line plays a key role in homeostasis and maintenance of organism health, it is a complex structure composed of numerous specialised cells. It performs numerous functions including: nutrient uptake, electrolyte absorption, hormone secretion and some innate immunity. As such enteriods are an important area for potential medical research due to the numerous functions it has which can be linked to numerous conditions (Haq. I, et al, 2021).

Future animal-based organoids could be used to test potential adverse effects of various drugs on different species (Pain. B, 2021). The one health initiative is based on the sharing of knowledge between human and veterinary medicine to further stimulate biomedical research and improve clinical and para-clinical care (Gabriel. V, et al, 2024). Organoids are unfortunately not ideal to study inflammatory responses due to the lack of immune cells in their culture (Augustyniak. J, et al, 2019). Livestock organoids have been used for research into numerous pathogens over the years since their development. Organoids derived from both human and animal sources are excellent tools for monitoring pathogen’s ability to enter host cells. Intracellular replication and propagation as well as how pathogens can exit host cells can be monitored in particular the roles of specific cell types involved in this process (Beaumont. M, et al, 2021).

Organoids used in precision medicine

Through organoids precision medicine aims to increase cost effectiveness and improve risk /benefit ratios of therapies by more precisely targeting treatments to individual patients. In 2016 this was demonstrated by generating more personalised treatments for people with cystic fibrosis. A big part of organoid research are biobanks which facilitate research aimed at areas such as drug research and disease modelling as well as enabling large scale data sharing and analysis (Lensink. M. A, et al, 2020). There is a great deal of individual genetic diversity within humans compared to most animals owing to no inbreeding humans, as a result more personal cellular models would be of great use for medical research. It is a hope that organoids will help with the development of CRISPR technologies to help with genetic disorders (Kim. J, et al, 2020). Organoids can be used to model hormonal responses in patients to certain stimuli allowing for more easily predictable results for patients on different treatments (Gabriel. V, et al, 2024).

Use of organoids in cancer research

At present there are over 100 types of tumours that have been identified each with different localisations and consist of different types of tumour cells. On top of this their markers can vary due to individual location, stage of disease and various host genetic factors. Cells and matrixes are required to create tumoroids, the matrix plays a role in the structural support of the cell structure. Matrigel is widely used as a matrix and cells necessary can be obtained directly from tumour tissue. Tumour derived IPSCs can aid cancer stem cell research such as being used to observe associations between genotype and responses to anti-cancer drugs. As well as observe the effects of a specific cancer genotype or specific driver mutation, IPSCs can be obtained from patients with familial cancer syndrome. Organoids and spheroids can be used to create individual response profiles for patients in areas such as chemosensitivity, this is an important step in developing precision medicine. This method could help create a predictive platform for therapeutic agents for cancer patients as well as highlight potential toxicity of new drug combinations (Gilazieva. Z, et al, 2020).

Cell cultures grown as 3D organoids show more resistance to chemotherapy measures that their monolayer counterparts, this shows that 3D cultures will show are more accurate reaction of an ailing person (Sakalem. M, et al, 2020). Generating tumoroids alongside organoids developed from healthy patient tissue could develop more efficient antitumor treatments to target specific tumour cells (Pain. B, 2021).

Ethical considerations

As with any new science new ethical considerations have risen up around organoid technology for numerous areas. Firstly, the use of using human tissue to create organoids implies the prior existence of biobanks storing various samples of human tissue for research purposes. This leads to concern for the form of consent necessary to collect required samples for such research from patients. As well as how much information about what tissue samples will be used for, needs to be given to potential donors (Stoekle. H. C, et al, 2021). The 3 Rs (replacement, reduction and refinement) has long been a major part of ethics in scientific research especially were research involving live specimens are concerned. In rodent models for instance, since rodents don’t often develop multifactorial diseases being modelled in studies, they are being used in. therefore they must have a nonetiological insult to the organ to mimic the disease as it would manifest in humans. The use of organoids as a model in disease research rather than rodent would reduce the need for such procedures (Gabriel. V, et al, 2024).

Discussions on brain organoids have engaged with ethical concerns regarding sentience and the possibility of researchers pursuing advanced brain models. Intestinal and gastro enteriods concerns are more about possibilities of embryonic development. A study in 2021 looked at patient opinions about organoid research and found broad support for the concept. Particularly for medical research, however there was concern particularly about the commercialization of the technology. There is specifically a concern for profit driven motivations for organoids technology. Many also did not endorse any ethical duty of patients to participate in research directly. One area participant was concerned about were brain and gonadal organoids which were viewed as morally distinct from other organoids. This is mostly due to concerns regarding development of consciousness and what is considered human. Donor consent was also seen as a major issue by those interviewed with routine research oversight. Aswell as having accurate information available for potential donors. It is also vital people understand the currant limitations of organoid research as not to have unrealistic expectations for their currant capabilities (Bollinger. J, et al, 2021).

On the flip side further, organoid development could help reduce need for animal and live human testing in medical research (Stoekle. H. C, et al, 2021). One area of consideration for patients is lack of control of what their tissue is used for or whether their samples can be truly anonymous. It has been put forward that more active patient participation is required to remove these ethical concerns in the future. This will ensure precision medicine and organoid research can be a sustainable area of medicine in the future (Lensink. M. A, et al, 2020). Somatic reprograming technology was first developed in 2006 and greatly advanced the field of stem cells research, they allowed for the development of induced pluripotent stem cells (IPSCs) which share properties with ESCs without many of the ethical issues. These were initially created using mouse models but soon where being developed from non- human primates, rabbits, livestock, dogs, cats and even snow leopards allowing for more cruelty free research. However, within these models are difficulties validating the status of reprogramed cells because of their developmental status being rarely tested (Pain. B, 2021). Use of livestock derived organoids could be a good middle ground between ethical issues surrounding live animal testing and issues surrounding use of human tissue and long-term storage of human genetic material (Haq. I, et al, 2021). Another study in 2024 states that concerns for organoids include combining with other technologies such as gene editing, creating chimeras, transplantation as well as commercialisation of human derived technology (Gabriel. V, et al, 2024).

Novel uses for animal derived organoids

Organoids derived from exotic species can be important for studying the species including their unique physiologies and disease pathogenesis. It also allows for studying endangered species more thoroughly without risking further reducing their numbers. Antivenom production largely depends on venom milking, a somewhat time-consuming process which requires an experienced herpetologist. In 2020 a snake derived organoid mimicking venom glands was able to produce crude venom along with its biological activity. This study also marked the first organoid developed from a reptile species (Gabriel. V, et al, 2024). IPSCs have been shown to generate mammary organoids derived from bovine cells which express common breast tissue, luminal and basal markers including oestrogen receptors and were even able to produce milk proteins. Mammary glands have a peculiar development during different stages of animal life and thus can be difficult to mimic. ASC derived organoids are more popular for animal-based models and can be characterised by specific stem cell and differentiated cell markers. Culture conditions for organoids will need to be optimised for each species independently (Augustyniak. J, et al, 2019). The study of animal intestinal epithelium has major implications for the agricultural sector such as improvements to feed efficiency. As well as veterinary research, colonoids and enteriods can provide a good model to individually study each species. one benefit of farm animal derived organoids is that they can be successfully recovered after cryopreservation in freezing mediums, as well long-term storage in liquid nitrogen. There are problems transporting tissue samples from slaughter houses to cell culture laboratories and developing from intestinal epithelial crypts from fresh tissue is not always feasible. With this in mind tissue samples will have to be cryopreserved right after being removed from the carcass. Though through this process unwanted bacterial and fungal infections developing are a concern (Beaumont. M, et al, 2021).

Conclusions

From the studies looked at in this essay it is clear that organoids can provide a massive boon to medical sciences. Organoids allow for real time monitoring of pathogens and can aid in developing new treatments based on looking at host pathogen interactions with the various types of organoids. The ability to create organoids from patient stem cells can allow for the development of personalised treatment plans. This could help developing CRISPR technologies and precision medicines which can make the medical process more efficient and cost effective by reducing amounts of dead-end treatments and removing potential for having to treat negative side effects of potential treatments. They could also help with cancer research through allowing to watch tumour growth and development in real time within a culture which could help researchers gain insights into how cancer develops in patients. Animal based research could become more ethical with the implementation of organoids reducing the need to perform invasive research on live specimens. This can be done by growing organoid cultures based on the required animal which can act as a model for the species. while this will not yet replace all forms of animal testing it will reduce a lot of unnecessary potential animal cruelty. However as with any new science there are some ethical considerations with organoid studies. Most concerns come from the idea of using of human genetic material without consent of patients and long-term storage of said material. Another concern is potential commercialisation of organoid technology as studies shown in this paper imply people find a profit driven attitude towards a human tissue-based science somewhat distasteful. There is also a great deal of potential development for organoid technology such as generating animal-based products through organoids, assist in organ grafts and regenerative medicine as well as assist in livestock research. As a whole, organoids present exiting new opportunities for medical science and hopefully will continue to be developed in the near future.

Glossary

CRISPR: a form of gene editing technology used for treating medical disorders.

Fibroblast: a type of cell that is responsible for producing collagen and extracellular matrixes which forms the structural framework for tissue.

Matrix: a substance used to keep cells in a native state and help facilitate communication between cells in a culture.

Mini organs: an older name for organoids.

Progenitor cells: a biological cell type that can differentiate into a range of specific cell types, they are similar to stem cells but cannot differentiate outside of their predefined range of cell types.

Striatum: a component of the brain that links motivation to motor movement as well as processing reward calculation, decision making and social interaction.

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