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HOME > Product category > Cell culture reagents > ~Interview of Pioneer in Organoid Research~ Prospects and Challenges of Organoid Culture Systems : From the Gastrointestinal Tract to Taste Buds

~Interview of Pioneer in Organoid Research~ Prospects and Challenges of Organoid Culture Systems : From the Gastrointestinal Tract to Taste Buds


Professor Ken Iwatsuki</strong></span><br>

Professor Ken Iwatsuki
Tokyo University of Agriculture


[Biography]
1994 B.S. School of Agricultural
Sciences, Nagoya University
1999 Ph.D. Graduate School of Agricultural and Life Sciences, The University of Tokyo
2000–2002 Tokyo Metropolitan Institute of Medical Science
2003–2007 Icahn School of Medicine at Mount Sinai
JSPS overseas research fellowships/instructor
2007–2013 Ajinomoto life science research institute, innovation research institute
2014–2019 Associate Professor, Tokyo University of Agriculture
2020–present Professor, Tokyo University of Agriculture

Professor Iwatsuki was born in Aichi Prefecture, Japan. After graduating from the Department of Agricultural Chemistry, Faculty of Agriculture, Nagoya University, Graduate School of Agricultural and Life Sciences, University of Tokyo, and working in Tokyo Metropolitan Institute of Medical Science, he started his new career as a taste researcher at Mount Sinai School of Medicine in the United States. After returning to Japan, he worked at the Innovation Research Institute of Ajinomoto Co.

In 2014, he started research on endoderm-derived sensor cells using organoid culture method. The research is being done at the Department of Food Safety and Health, Faculty of Applied Biological Sciences, Tokyo University of Agriculture, where he currently works.


Preface

After Sato et al. succeeded in constructing epithelial tissue organoids from Lgr5-positive stem cells in 2009 (Sato T. et al., Nature 2009), organoid research has made remarkable progress. Organoid research is applied to basic research such as embryology and the investigation of disease causes, but also drug discovery research such as drug testing, and regenerative medicine.

We visited professor Ken Iwatsuki’s laboratory at the Tokyo University of Agriculture and interviewed professor Iwatsuki about the prospects and challenges of his research using organoids.



1. Next generation 3D culture systems: organoids

―Could you please give us a brief introduction about organoid for a better understanding of organoids and organoid culture systems?





The term "organoid" is actually not a new word but it simply referred to as a 3D-structured culture. Recently, a 3D culture system that could maintain gastrointestinal epithelial stem cells, has been developed and named organoid culture system. In this system, stem cells of the target tissue are embedded in an extracellular matrix of cancer cells called Matrigel and supplemented by various growth factors including Wnt3a, R-spondin and Noggin. As the growth and differentiation of the cells can be observed in 3D structure that mimic organs in the body, the growing cell mass is called an organoid.

The characteristics of organoids we culture are as follows:

(1)Organoids have the potentials to be passaged like immortalized cell lines, but the proliferating cells are normal cells.
(2)One can obtain cells of interest by inducing cell differentiation.
(3)One could directly observe responses of chemicals from food and drug on epithelial cells without considering the effects from nerves, blood vessels, and connective tissues.
(4)Since organoid recapitulates tissue function in vivo, one can replace animal experiments with organoid experiments.


2. Growing interest in organoids from the food industry

―Recently, we have been receiving many inquiries about organoid culture system, its related products and protocols not only from pharmaceutical companies but also from the food and cosmetics industries. What is the possible reason for this?

There is a worldwide movement to reduce animal experiments from the viewpoint of animal ethics. This is the reason why in vitro culture systems such as organoid culture systems are becoming popular. Organoids can be both passaged and cryopreserved. In other words, they can be treated like other cell lines that are commonly used in experiments. In addition, since organoids are not transformed cells, they can reproduce the same functions as in vivo and are expected to be utilized in a variety of experiments.

―Which tissues are organoids being made from?

Organoids are currently established from the stomach, small intestine, large intestine, liver, pancreas, lungs, and taste buds and other organs. These organs are “endoderm-derived” organs. Of these, organoids from the small intestine are most frequently reported. Intestines have various functions such as hormone secretion, barrier function, food digestion, absorption, and various studies using organoids have been conducted. In our lab, we are focusing on hormone-secreting cells and tuft cells in the small intestine, which express taste signaling molecules found in the oral cavity. In addition to the small intestine, we also generated organoids from pancreas and taste buds and managed to differentiate them into various cell-types for function analysis.


3. Solving the trade-off problem between maintenance and differentiation of stem cells

―So you are saying that it is important not only to maintain organoids but also to obtain mature cells to perform functional analysis?

Yes, that is correct. In organoid culture systems, the cells that proliferate mainly are stem cells or progenitor cells and fully differentiated cells do not have the ability to proliferate. Therefore, we must find the right culture condition to both fulfill proliferation and differentiation. It is difficult to maintain this balance.


Most of the organoids require a morphogen called Wnt3a, but since highly bio-active Wnt3a is not commercially available, researchers have to prepare Wnt3a conditioned medium (Wnt3a CM) by themselves. The activity of Wnt3a in the culture supernatant had to be measured for each lot and the quality control was also difficult due to the influence of FBS, which is necessary for Wnt3a production. These issues required considerable efforts, and still a need for Wnt3a with stable activity. Recently, Medical Biological Laboratory (MBL) Inc. has released a supplement medium containing Wnt3a called Afamin/Wnt3a CM, and when we introduced it into a monkey gastrointestinal organoid culture system, we found that it successfully supported both cell proliferation and differentiation. In particular, the efficiency of cell differentiation was better than that of conventional methods, and many differentiated cells were obtained. Afamin is a molecule that has been reported to bind to and solubilize Wnt3a, thereby maintaining its activity (PMID: 26902720). In addition, Afamin/Wnt3a CM does not contain any serum components, making it suitable for cell culture for future transplantation because it reduces variation in cell culture condition caused by FBS. In our experimental system, we have achieved similar or better culture results with a smaller amount of Afamin/Wnt3a CM as compared with Wnt3a CM. We normally use MBL's Afamin/Wnt3a CM because of its higher differentiation efficiency. We are currently investigating the molecular background of why the differentiation efficiency is higher than conventional Wnt3a CM.
CM(Conditioned Medium)

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Afamin/Wnt3a CM

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4. About "taste bud organoids"

―Thank you so much for the information and we are glad that our product is helpful in your research. We heard that you have also succeeded in establishing organoids from taste buds using Afamin/Wnt3a CM. Can you tell us more about taste organoids and their future applications?

Photograph of taste bud organoids cultured by Professor. Iwatsuki

Photograph of taste bud organoids cultured by Professor. Iwatsuki

We are one of the first research group that successfully established taste bud organoids in the world. The strength of our taste bud organoid is that it could express taste receptors and can respond to various taste substances. At first, we produced taste bud organoids from mice and recently we have also succeeded in producing taste bud organoids from monkeys, which are primates like humans. We are now trying to produce human taste bud organoids. We aim to contribute to scientific and medical society through various collaborative research projects in the future. Specifically, we are considering a screening system for taste substances using primate organoids. Furthermore, we hope to develop taste organoids that can be applied to regenerative medicine. As you know, many elderly people lose taste sensitivity. We think that this is just like the phenomenon of hair turning gray or falling out. We believe in the future that taste organoids will be good donor cells for transplantation.


5. Challenges of organoid culture

―This is so wonderful. We are really looking forward to seeing the organoid to be further used in regenerative medicine. By the way, I have heard that the method of establishment and quality of organoids differ from facility to facility. What are your thoughts on how to ensure that each research facility can conduct stable experiments in the future?

In order to obtain experimental results with good reproducibility, we should define standardized methods for organoid generation, preparation of reagents and evaluation are necessary. Particularly, we should pay more attention on using reagents of biological origin such as BSA and FBS. Since BSA and FBS will often determine success or failure of organoid culture due to difference among the Lot, so we usually check the quality of these reagents when using a new Lot. This process is quite time-consuming and nerve-wracking. Therefore, we were searching for the method that does not require usage of BSA or FBS.

Also, from a viewpoint of animal welfare and effective usage of limited animal resources, we think it would help many researchers to generate organoid libraries for each animal species and organ. It would be nice if the system could be established for sharing organoids at any time like cell line.

Actual monkey small intestinal organoids were shown during the interview!

Actual monkey small intestinal organoids were shown during the interview!


―What are other challenges do you face in handling organoids? And what are the problems of widely using organoids as an alternative to animal experiments?

First of all, organoid culture is expensive. The reason is that Matrigel and cytokines are expensive. Matrigel, in particular, is a reagent derived from cancer cells and there are differences among batches. There are no other gels that can replace it at this moment, so we have no choice. Since many cytokines are also expensive, we are making homemade cytokines which we must evaluate every time, because the activity may vary depending on the batch.

Public awareness of organoid culture is also important. In our hand, the endoderm-derived organoids which we culture do not change their properties even after repeated passages. Once we have established organoids, we can increase the number of organoids in large quantities, freeze them, and use them for research when necessary. In other words, there is no need to get tissues each time from animals. In this sense, it is very similar to cell lines derived from cancer cells.

―We fully understand the potential of organoids for use in drug discovery, but also showing good potential in the food and cosmetics industries, and even in regenerative medicine. Thank you so much for the introduction of organoid and sharing your wonderful research results with us today.

Thanks for visiting us, too. Please come and talk to us again to see our new progress in the research.


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Stable organoid culture with serum-free medium becomes possible!


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