Organoid: A Powerful Tool for Drug Testing and Discovery
Introduction
Drug discovery and development are divided into multiple steps, the ultimate goal being to demonstrate that a drug is both safe and effective for the patient’s disease.
To find a new drug, researchers typically need to understand disease processes and design a product to stop or reverse the effects of a disease.
Before testing a drug on humans, we need to find out potential harmful effects or toxicity. This is preclinical research.
To do so, researchers do need a model. A model is a way to reproduce/mimic a phenomenon we want to study. There are several tools including in vitro and in vivo models.
In vitro models refer to cellular models cultivated in controlled lab environments (temperature, pH…) like, for instance, cell lines.
In vivo models include animal models, mostly mice, but also rats, zebrafish, worms (C. elegans), and more.
Both these models have some utilities.
At in vitro models, we mostly work with a single cell line (cultured cell populations that can be maintained and grown in a laboratory environment) where it is much simpler to study and focus on a particular biological question.
In vivo models, for instance, allow us to inject a drug into a mouse and assess any biological parameter we want, even if this implies dissecting the animal and checking each single organ.
Unfortunately, models are just a superposition of hypothesis resulting in a
reflection of biological reality. We could say in biology, as in the field of statistics:
All models are wrong, but some are useful
George Box (1976)
For instance, cell lines can accumulate mutations over time, which might not represent the original tissue from which they were derived.
Besides, they do not have an organ-shaped organization and are only represented by one cell type, meaning they might not always directly translate to more complex living models.
This is where we want to validate findings in other systems, such as in vivo animal models.
Animal models also have their limitations because of the discrepancy between the species (mouse vs human for instance) and the disease we want to mimic. Indeed, many mouse models aim to create a disease with genetic alterations, surgery, environmental exposure, but are still not the same as the human disease.
Besides, animal models are far more costly and time-consuming than cell lines and also raise ethical concerns.
This is where a new model is becoming increasingly important in biology and drug discovery: Organoid.
Organoids are 3D structures derived from stem cells growing in specific culture conditions. These intestinal organoids recapitulate the structure, physiology, and function of the native organ. They provide a bridge between in vitro and in vivo experiments (sometime referred as ex-vivo) and represent a new generation of models that could gain relevance for human research (2).
We can obtain organoids from multiple organs (liver, lung, brain, kidney) (2), but this article focuses on applications on human intestinal organoid as I have some experience in it.
From stem cells, how to obtain organoids?
Historically, intestinal organoid technology has been developed first in mice (3) and later adapted for humans (4, 5).
First, extract stem cells! Intestinal stem cells are located at the bottom of intestinal crypts, which provide a particular environment containing all the mandatory factors to regulate stem cell proliferation, function, and survival (2, 4, 5). We can extract the crypts from intestinal tissues by scraping and dissecting the tissue followed by successive EDTA treatments to dissolve cell junctions.
This first step is very important, as a good and clean crypt extraction is key to obtain numerous and beautiful organoids.
This quality might be lower in stressed or inflammatory intestines (such as in inflammatory bowel diseases like Crohn’s disease). It is much simpler to use tissues from the colon compared to the ileum as the latter contains villi that do not contain stem cells and are just going to die, decreasing cell culture conditions.
I mainly worked on ileal inflamed tissue… lucky me.
Samples can come from surgical specimens in the scope of medical care, but also from biopsies, but the latter contains far less material to work with. Lastly, the tissue needs to be as fresh as possible.
Second, crypts are embedded in Matrigel (extracellular matrix) in a droplet. This Matrigel allows the cells to avoid anoikosis, a phenomenon where cells die shortly after losing contact with the cellular matrix. Many cells, when extracted from tissues, actually do not survive except in particular conditions.
Third, we need to recreate an environment similar to the bottom of the crypt which allows stem cells to proliferate. To do so, we add a cocktail of growth factors, inhibitory molecules, antibiotics (to avoid contamination), vitamins… This medium is refereed as stem medium and is the key for stem cells proliferation.
When I was young, I had to mix a dozen of these factors at particular concentrations to create this very specific culture medium. Now some companies actually provide ready-to-use culture media (for proliferation and differentiation).
After these last steps and some time, the magic will operate: Stem cells will proliferate and form organoids, the rest will die (figure 1).
(We need to redo the operation with a cell passage to get rid of cell debris and obtain only organoids and, of course, change the culture medium.)
That sounds like a lot of work, and it actually is…
Why do we use organoids when we can just use cell lines?
Organoids are a far more difficult technology to manage and involve accessibility to biological samples and higher costs and skill sets.
First, Both have self-renewal and proliferation ability, but only stem cells can differentiate into different cell types. For intestinal stem cells, we can obtain for instance: Enterocytes (absorb nutrients), Goblet cells (produce mucus), Paneth cells (produce antimicrobial peptides).
Second, stem cells only have the innate ability for intrinsic self-organization to form structures, whereas lab cells typically only grow into a single layer of cells. Intestinal organoids are round-shaped with the basolateral side (the surface adjacent to the base or away from the lumen) on the outside and the apical side (the surface facing the lumen or open space in the gut) inside (figure 1). After some time, organoids can even grow villi structures, closely resembling the architecture found in the native gut.
Third, contrary to blood cells, some cells, including intestinal epithelial cells, actually do not survive when extracted from the tissue. They die shortly after losing contact with the cellular matrix, through a phenomenon called anoikosis. So, organoids might be the only option to work with some types of cells, especially in humans.
Fourth, fewer ethical concerns for animal experimentation!
There might be more reasons to use organoids! I will add a last one related to immunological use cases.
Organoids allow for work in autologous conditions (refers to cells, tissues, or organs sourced from the same individual in which they will be reimplanted) as we can use organoids and the immune cells from the same organism. This is a very attractive possibility as working with immune cells and tissues from another individual is a serious limitation to understand how the immune system could react to their own tissue.
Organoids are becoming increasingly important. As the imagination of researchers, use cases are endless. Organoids can offer a deeper understanding of human growth, disease mechanisms, organ development, stem cell biology, organoid cell therapies, and host-pathogen interactions. Moreover, patient-derived organoids for drug testing and toxicity assessments can lead to the development of personalized medicine.
Now that you are more familiar with organoids and I hope you are convinced of the significance of this technology, I will describe a use case.
Organoid : use case in inflammatory bowel disease
Disclaimer: This work was developed in my thesis laboratory.
Crohn’s disease is an inflammatory bowel disease characterize by inflammatory lesions on the intestine. The physiopathology is still unclear but involves immune cells function on predisposed individuals.
We aimed to study the function and interaction between these immune cells and intestinal epithelial cells (IEC) in patients. Our questions were:
How do immune cells interact with IEC?
Do immune cells in Crohn’s disease patients exhibit cytotoxicity toward IEC and destroy them?
By which mechanisms?
Do they produce inflammatory factors?
Do these results change in healthy individuals?
Organoids seem to be an attractive model for these questions because:
First, the basolateral side of the organoid is on the outside, where T cells would be located in the Matrigel, mimicking in vivo conditions.
Second, since we can use T cells and organoids from the same mucosa, this model offers the opportunity to work in autologous conditions, preventing allogenic reactions.
Third, immune cells are absent from intestinal organoid cultures but can be added and monitored in a controlled manner in co-culture models.
To do so, we culture together organoids and immune cells from the same individual in autologous condition. Finding the right culture conditions to allow both cell types to survive was essential for this experiment.
Next, we needed to assess the immune cells interaction with organoids, their activation, the release of inflammatory factors, and finally cell death.
These assessments could be achieved through various technologies, including:
- ELISA: To measure the release of inflammatory factors by immune cells in the culture medium.
- Flow cytometry: Activation of T cells can be assessed with activation or proliferation markers and death markers on IEC.
- Microscopy: To observe interactions/infiltration between organoids and immune cells and IEC death using markers such as Annexin V and caspases.
In my opinion one of the best assets of organoids is that you can spatially visualize structure and cells-cells interaction. In figure 3 panel B-C-D, you can clearly see a physical interaction between immune cells and the organoid. Beautiful, isn’t it?
Another significant aspect of this model is the ability to block mechanisms potentially involved in Crohn’s disease and observe if this results in protective effects on the IEC.
For instance, in our paper, we blocked molecules involved in interaction between immune cells and IEC (like anti-beta7 or etrolizumab) (6). Interestingly, we observed differences in cell infiltration, cell death, and inflammatory factors from patients compared to healthy individuals and with the utilization of blocking antibody. Check this article out if you want more info! (6)
This approach offers the opportunity to conduct drug testing in a model to determine if blocking molecules suspected to be involved in the disease could benefit patients.
Organoids represent a powerful model for preclinical research and drug screening. If results are favorable, the next step is to determine if the drug is safe and effective for the disease during clinical trials.
Very few drugs even reach the first steps of clinical trials, and fewer still receive approval for patient use.
However, organoids are a formidable tool for preclinical research and drug screening, potentially helping to discover strong candidates before starting clinical trials.
This also exemplifies how researchers continually find solutions to create the most relevant models possible to understand biology and design drugs that, after a long journey, could benefit patients.
Thank you for reading!
Feedback is welcome.
1. Hugo Bottois (2019) Thesis : Acquisition and regulation of effector T cell functions in Crohn’s disease. https://www.theses.fr/2019USPCC012
2. Barker, N. Adult intestinal stem cells: Critical drivers of epithelial homeostasis and regeneration. Nature Reviews Molecular Cell Biology (2014). doi:10.1038/nrm3721
3. Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, van Es JH, Abo A, Kujala P, Peters PJ, Clevers H. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459: 262–265, 2009. doi: 10.1038/nature07935.
4. Jung P, Sato T, Merlos-Suárez A, Barriga FM, Iglesias M, Rossell D, Auer H, Gallardo M, Blasco MA, Sancho E, Clevers H, Batlle E. Isolation and in vitro expansion of human colonic stem cells. Nat Med 17: 1225–1227, 2011. doi: 10.1038/nm.2470.
5. Sato T, Stange DE, Ferrante M, Vries RG, Van Es JH, Van den Brink S, Van Houdt WJ, Pronk A, Van Gorp J, Siersema PD, Clevers H. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology 141: 1762–1772, 2011. doi: 10.1053/j.gastro.2011.07.050.
6. Hammoudi N et al. (2022) “Autologous organoid co-culture model reveals T cell-driven epithelial cell death in Crohn’s Disease.” Front. Immunol. 13:1008456. doi: 10.3389/fimmu.2022.1008456.
