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Devanjali Dutta, PhD

Postdoctoral researcher (Hans Clevers group), Hubrecht Institute, Netherlands

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Biography

Devanjali is currently pursuing her Postdoctoral research in the lab of Prof Hans Clevers (Netherlands), where she is using human tissue derived 3D organoid cultures to study host-microbiome interactions, infectious diseases and cancer. She received her PhD from University of Heidelberg (Germany) in 2015 from the lab of Prof Bruce Edgar. Her doctoral research involved the development of methods to isolate and profile rare cell types and tumor cells from the fruit fly midgut and the generation of transcriptome profiles of the cells of the adult Drosophila intestinal epithelium.

Learn about Devanjali’s research

Title: Disease modeling and cancer therapy in stem cell-derived 3D organoid systems

Learning objectives

  • Organoids can be used for studying cancer and hereditary diseases, as well as in the examination of host cell–microorganism interactions
  • Patient-derived organoids may enable personalized medicine

While the significance of the microbiome is unprecedented, a thorough study to dissect the role of individual populations of the natural gut microbiome in healthy and diseased states is still lacking. Currently available in vitro models of the human intestine have certain limitations, e.g., lack of immune cells or microbiota. Therefore, systems closely mirroring the human intestine are needed to gain a better understanding of intestinal dysfunction, host–pathogen interactions and for the development of more effective therapies for cancer. It is important to establish a patient derived, fast and efficient system which would mimic the in vivo architecture in humans and permit precise studies on interactions of the gut microbes with the epithelium and the immune cells. The in vitro 3D “mini-gut” system developed in the Clevers lab displays many important functions of the normal intestinal epithelium. Along with the developed toolbox for the analysis of these organoids (including FACS-based cell sorting, confocal imaging, RNA Sequencing, mass-spec proteomics and CRISPR-Cas9), it has proven to serve as a powerful system to investigate regulatory and pathological mechanisms of the intestinal epithelium on a molecular level.

To this end, we are developing a triple co-culture system of epithelial cells, immune cells and microbes to better understand the functional role of the gut microbiota, enabling personalized healthcare for the benefit of patients suffering from GI tract diseases. It might be possible in the future to determine the microbiome composition of cancer patients and provide personalized immunotherapy drug treatment.

Watch the webinar

Presenter:  Devanjali Dutta, PhD

00:00 – Slide 1

Alexis Corrales:  Hello, everyone, and welcome to today’s webinar, Disease Modeling and Cancer Therapy in Stem-Cell Derived 3D Organoid Systems. I am Alexis Corrales of LabRoots and I’ll be your moderator for today’s event.

Today’s educational web seminar is presented by LabRoots and brought to you by Thermo Fisher Scientific.  To learn more about our sponsor, please visit thermofisher.com.  Now let’s get started.  (00:30)

Before we begin, I would like to remind everyone that this event is interactive.  We encourage you to participate by submitting as many questions as you want, at any time you want during the presentation.  To do so, simply type them into the Ask a Question box and click Send.  We’ll answer as many questions as we have time for at the end of the presentation.  If you have trouble seeing or hearing the presentation, click on the Support tab found at the top right of the presentation window or report your problem by clicking on the Ask a Question box located on the far left of your screen.  (01:04)

This presentation is educational and thus offers continuing education credits.  Please click on the Continuing Education Credits tab located at the top right of the presentation window and follow the process to obtain your credits.  (01:18)

I now present today’s speaker, Dr. Devanjali Dutta, postdoctoral researcher, Hubrecht Institute, Hans Clevers Lab. Dr. Dutta is currently pursuing her postdoctoral research in the lab of Professor Hans Clevers, Netherlands, where she is using human tissue-derived 3D organoids cultures to study host-microbiome interactions, infectious diseases and cancer. For a complete biography on our speaker, please visit the Biography tab at the top of your screen.

Dr. Dutta, you may now begin your presentation.

Devanjali Dutta: Thank you so much for the introduction and thank you all for joining me for this webinar.  I am a postdoc in Hans Clevers’ group and today I will be talking about how the 3D organoid system can be used for disease modeling as well as cancer therapy.

02:06 – Slide 2

So what exactly are organoids? So I mean, very broadly speaking, organoids are 3D stem cell tissue-derived structures or, as we call them, the mini-organs in a dish. These mimic the in vivo architecture of tissues structurally, functionally as well as genetically. And because of these features, the organoids provide really powerful means for ex vivo modeling as an in vitro model system for stem cell research, and also in developmental biology. (02:37) Stem cell organoids can be developed from two different kinds of stem cells and, very broadly, again, they can be either pluripotent stem cell-derived organoids, which are basically embryonal stem cells or induced pluripotent stem cell-derived organoids; and the adult stem cell-derived organoids. And I’ll be talking about these a bit more in detail in the coming slides.

(02:58)

But just to give you an overview of how many kinds of organoids have been developed already, here you see a panel of organoids, so from the adult stem cells we have isolated and developed intestinal organoids, liver organoids, ovarian organoids, lung organoids; whereas from the pluripotent stem cells, again, liver, stomach, even kidney organoids have been developed. Brain organoids have been developed which have been used recently for Zika virus infection studies, and also other sensory organoids.

03:34 – Slide 3

So what exactly or how are these pluripotent stem cell-derived organoids developed? So this is a schematic showing how they are actually generated. So the pluripotent stem cells are first grown in lab as embryoid bodies or the spheroids, and these are pluripotent in nature. Basically, they are derived from master cells, as we call them, because these can actually differentiate and form any kind of cell type. That is the idea behind the pluripotent stem cell.

(04:04)

So when these are grown as embryoid bodies or spheroids, the tissue cultures, depending on what kind of signals are provided to them, like differentiation signals, they can be differentiated into different kinds of tissue. And these, when they are embedded with the Matrigel in 3D, these then form the specialized organoids.

04:23 – Slide 4

Whereas the adult stem cell organoids are somewhat more defined because they are already isolated from the tissue of origin, for example in this case, what I’m showing you is like an intestinal biopsy and what we do is we get a biopsy, we mince the tissue into small pieces and then ideally I either isolate the crypts from the intestines or we kind of make them into single cells which are then plated in 3D in Matrigel, which is an extracellular matrix, and within two to three days, we already see these organoids growing.

(05:00)

And most of the work that I show today is based on the adult stem cell-derived organoids, because these are the organoids that we use in our lab, in the Clevers lab, for all the studies.

05:10 – Slide 5

So what are these organoids exactly? So, these organoids that I’m talking about, the adult tissue-derived organoids, are mostly epithelial in origin. They do not have any mesenchymal niche per se and they are grown in an extracellular matrix which provides them with a lot of factors to help them grow. This can be either Matrigel, the basement membrane extract, or recently we have also used a synthetic hydrogel, which is essentially polyethylene glycol, and this is important because both Matrigel and BME are not very much defined because they are isolated from the sarcoma cells of the mouse, whereas if there is a synthetic hydrogel which is more defined in characteristics then the studies are also way more defined. (05:59)

And one more important aspect of these organoids are that they can be kept in culture for extended periods of times, or sometimes—or most of them actually have been grown for more than a year in culture, and they maintain all their genetic structure or genetic basis as well as the structure and all the functions. (06:17)

And here is a picture which basically shows you how, from a single cell at day zero, those cells then divide, form these small organoids and by day 13, it forms—the full organoid is formed, with basolateral as well as apical polarity. So it is important also to know that organoids have polarity, mostly the interior or the lumen side of the organoids are apical in nature, whereas the outside is more basolateral in nature. (06:49)

So next, we will see a video, which basically will give you an idea of how these organoids are grown. These organoids, as I showed, start—they basically start as single cells and in culture, they grow into these big organoids, and then they need to be sheared using trypsin and then again plated with Matrigel so that they grow and can be expanded for long periods.

(07:16) – Slide 6

[VIDEO PLAYS]

(08:05) – Slide 5

So what you saw just now is how the organoids are grown and expanded in culture, right. So what is also again important to know is that the organoids are really like a mini-organ. So when they are grown in expansion media, these, or the EM, we call them as EM, so these essentially have a stem cell like characteristics and that is why they expand in cultures. That’s how you see when you trypsinize them and then you again plate them and they keep growing. (08:33)

And if you want to really differentiate them into a certain cell type, then you have to put them in differentiation media. This is by either withdrawing some factors, like for example in the case of intestinal stem cell organoids, you have to withdraw the Wnt so that they kind of become more differentiated in characteristics, and so on for different organoid cultures.

(08:52) – Slide 7

So we call them as expansion media and differentiation media, two different kinds of media. And here what I’m showing you is when you put these organoids in differentiation media, they kind of form different cell types, here in (MUG @ 09:07) and alkaline phosphatase are the enterocytes, mucin-producing goblet cells and so on. (09:15)

So the organoid technology, there are lots of applications of organoid technology. First application is biobanking of patient-derived organoids. So biobanking is basically like a bank. So you can go there and you have like a panel of organoids derived from patient material of different individuals, and these can be used for drug testing or for disease modelling. (09:39)

We have also used tumor and cystic fibrosis organoids to test different drugs. And also, we can do host-microbe interaction studies using these organoids, and I’ll be talking mostly about disease modelling of cancer along with a little bit of host-microbe interaction studies and how also biobanking of patient-derived organoids is being done and how all of these together can be brought into a co-culture system.

(10:07) – Slide 8

So how does the organoid system help in personalized therapy or personalized cancer treatment? The first benefit of having organoids is that most of the cell lines that have been used till date are cancerous, but we do not really have a baseline or a healthy tissue. But, for organoids, what we do is when we get patient material, we isolate the tissue from the healthy part of the tissue as well as the cancerous part, and as a result, we have organoids of the same patient from the cancerous tissue as well as the healthy tissue. (10:41)

So these organoids are all genetically characterized, so we basically can go back and correlate which kinds of mutations are present and how these organoids are responding to drug therapy.

(10:53) – Slide 9

And as said, they can be cryopreserved and also expanded and kept in culture for extended periods of time. They can be used again and again for various disease testing and drug testing. (11:08)

How do these help actually in drug—I mean, figuring out what kind of drug is actually good for a certain patient? So here in this picture, you see, for example, we have three drugs: Drug A, Drug B and Drug C. Out of this, when we tested all these drugs on the patient-derived organoids as well as the cancer organoids, Drug A kills both the healthy as well as the cancerous organoid. However, Drug B and C selectively killed the cancer organoid, which shows us kind of that this is a good target or these two drugs can potentially be drugs which can be used for therapy. (11:52)

But one thing that obviously we all know is that the drugs which mostly fail the clinical trial phase is because they cause liver toxicity. And so we again bring in the liver organoids at this stage and if we test these drugs, for example, from our first screen we know Drug B and Drug C were good or potential drugs for this patient, and then we check for liver toxicity and here we see that while Drug B doesn’t cause any liver toxicity, Drug C does. And so we come to the conclusion that for this specific patient, Drug B is probably the best combination or the best drug that can be provided. (12:33)

So the living biobanks of cancer organoids have been developed. The first living biobank that was developed in our lab was the colorectal cancer biobank, and recently we have also developed a breast cancer organoid biobank, and this was developed from 150 patient samples, and this list shows all the different types of cancers which have been created as biobanks and which can be obtained, and also different tests can be performed on these.

(13:20) – Slide 10

Recently in our lab, we have also developed a new system which is the lung organoid system, and we call them as the human airway organoids, and these can be used for both cancer as well as cystic fibrosis modelling. These carry all the cell types, the basal cells, the club cells and the ciliated cells, and we are also in the process of developing a lung cancer biobank which will also be available for testing different kinds of drugs in the future. (13:40)

So till now, I’ve been talking about how we isolate the cancerous tissue and then develop them into organoids, but we can also do it the other way round. Basically, we can model cancer using healthy organoids but making mutations in them.

(13:57) – Slide 11

So this was in a paper published in 2015 where we used CRISPR/Cas9 to induce mutations in the healthy organoids and we could follow these different, different mutations, so basically a single mutation was first created and then these mutated organoids were selected, then a second mutation was added and so on. And it was found that while a single mutated—single gene-mutated organoid did not really lead to metastasis, whereas a quadruple mutant which had four mutations did lead to metastasis and was much more malignant in nature.

(14:34) – Slide 12

And so this is another aspect of cancer modelling which can be used or useful in labs for cancer modelling.

But when we talk about like the human body, as I said, these organoids are mostly epithelial in nature, we should not forget that our body actually has the gut that is epithelial cells, the microbes and also the immune cells. So if we really want to recapitulate the in vivo environment, we need to add immune cells as well as the microbes into this. (15:06)

And so the idea is to have a triple co-culture system, and the recent branch of cancer therapy, which is very much in use today, is cancer immunotherapy and the 2018 Nobel Prize went to Dr. Allison and Dr. Tasuku for their work on immunotherapy.

(15:30) – Slide 13

Immunotherapy is essentially modelling or kind of inducing your own immune cells to target the cancer tissue, and we think that with the triple co-culture system of organoids, immune cells as well as the microbiome, we might be able to help this field of immunotherapy, and how we plan to do that is what I will explain now. (15:50)

So this is a model of how we think the triple co-culture system should look like. The first, with the organoids, we plan to add the immune cells isolated from the same donor, so basically the immune cells from the same patient from whom we have isolated the organoids—tissue for the organoids, and then add the bacteria or the commensal bacteria into the organoid lumen so as to have all three components together. (16:18)

And this idea was tested recently in collaboration with another lab, where it was shown that the tumor-reactive T-cells when they are cultured with tumor cells and healthy cells, the T-cells which were, when they are cultured together basically, they react with the tumor organoids and they lead to the death of the tumor organoids. However, they do not cause any death in the healthy organoids.

(16:38) – Slide 13

And this is a picture showing the same. On the left, you see healthy organoids which were exposed to autologous T-cells, and this is in the presence of a green-fluorescent caspase probe, so essentially all the cells, if there is cell death, it will light up as green. And on the left are the control cells along with the immune cells.

(17:04) – Slide 14

There was no cell death, whereas when there are tumor organoids and the T-cells are added, a green-fluorescent signal was observed, basically showing that this co-culture of immune cell with the tumor organoids is very much efficient in recapitulating the in vivo architecture and also the in vivo functioning.

(17:33) – Slide 15

Now, at the same time, what we were also developing is how can we get the microbes into the organoids, because the microbes, as I said, are present on the apical side and for us, the intestinal organoids, mostly the apical side, is on the interior part. And so we have developed this new technique which is basically microinjection of the microbes into the organoid lumen, and here you see an organoid which is injected with microbes, and we added green dye so that we can visualize and see which organoids have been injected or not. And below is an example of fluorescently labelled bacteria injected into the organoid lumen. And as you see, this is visible not only three hours later but also for extended periods of time.

(18:17) – Slide 16

So when we wanted to check if our co-culture of bacteria and organoids is actually functional or not, what we did was we first tested a pathogenic bacteria and we injected shigella, which is a pathogenic bacteria known to induce cell death in vivo. And what you see here is when we injected the control organoids with normal PBS, there was no effect. However, when we injected them with the pathogenic bacteria, there was a massive cell death and a complete destruction of the organoid architecture. This was proof of concept that indeed, a co-culture of organoids with the microbes would work.

(19:04) – Slide 17

We extended our study, and of course it was important for us to also somehow let the anerobic bacteria also survive in the organoid lumen and for that, we have now set up conditions by which we can grow these anerobic bacteria inside the organoid lumen for extended periods of time, and here you see commonly forming assays for different bacteria and the fact that they actually grow both in expansion and differentiation media; they are able to survive in the organoid lumen. (19:33)

As again a proof of concept, what we did was we added the immune cells, and here in the picture you see on the left a control organoid without any virus and surrounded by the—surrounding the organoid are the immune cells.

(19:50) – Slide 18

Whereas on the infected panel, you’ll see in green the virus which was added to the organoid. And as soon as the virus was added, you’ll see a massive influx of the immune cells towards the infected organoid. (20:04)

And so this was a proof of concept of like a first triple co-culture model where we brought in the epithelial organoids, a microbe and the immune cells.

(20:15) – Slide 19

And now, right now what we are developing in lab is a co-culture, as I said, of the tumor organoids, blood lymphocytes and the commensal microbes.

(20:22) – Slide 20

So I’ll quickly summarize what I told you today. So organoids are mini-organs in a dish, can be developed from pluripotent stem cells or adult stem cells. Organoids have been established from multiple organs which include the intestine, the kidney, the brain, liver, stomach, pancreas, ovaries and lung. Organoids can be used for multiple clinical applications including disease modeling, for drug screening, for various host-microbe interaction studies and for regenerative therapy. (21:00)

We can also manipulate genes or perform gene editing using CRISPR/Cas9. As I showed, you can sequentially mutate different genes, and this will again enable disease modeling and targeted gene therapy.

(21:13) – Slide 21

We have now been able to show that a co-culture of tumor organoids along with T-cells leads to tumor-reactive T-cells. Also, co-cultures of microbes with the organoids have shown that there is a complex interplay between the microbes and the epithelium, and we can do this for not just like one tissue but for brain, stomach and intestine and various different tissues. (21:48)

And finally, I think what is very important is development of a triple co-culture system which will help us figure out how exactly, what exactly goes on inside the body. And I think that will be the future of organ-in-a-dish.

With this I would like to take questions. Thank you so much. (22:09)

Question: Thank you, Dr. Dutta, for that informative presentation. We will now start the live Q&A portion of the webinar. If you have a question you'd like to ask, please do so now. Just click on the Answer a Question box located on the far left of your screen and we’ll answer as many of your questions as we have time for. (22:30)

So, let’s get started. Our first question is: are the organoids developed in the Clevers Lab available to other researchers on request?

Devanjali Dutta: Yes, absolutely. So we have a foundation called Hubrecht Organoid Technology where we have a large collection of these living biobanks.

(22:51) – Slide 22

And we have biobanks for cancer organoids from colon, prostate, lung, pancreas and breast cancer as well as from cystic fibrosis patients. And all these organoids are characterized by genome sequencing, expression profiling and also the sensitivity of known drugs have been established and these databases linking the genetic and transcriptional information to the drug response is known. So yes, absolutely. So if somebody wishes to use these organoids, they need to write to the Hubrecht Organoid Technology and further processing will be done from there onwards. (23:29)

Question: Now our next question is a two-part question. Do you feed the bacteria and what are their carbon sources?

Devanjali Dutta: Feed the bacteria when we inject them? That's what I understand. So the way these bacteria are grown, so it depends. So the anerobic bacteria or the pathogenic bacteria.  So these are basically grown on the plates, like the LB plates or the black agar plates in the case of anerobic bacteria. That’s how we grow them. (24:00)

And just before injecting, we put them into capillaries, and we put them in capillaries and we suspend them in the media itself or the broth that they were grown in. And yes, so all the components which are actually used for growing the bacteria in general are also included in the liquid that we inject, and definitely that’s why we do feed the bacteria somewhat when we inject them. (24:25)

Once they go inside the bacteria, I think they take up sources from the media components and also from, yes, whatever we have added to the media for growing the organoid.

Question: Now, are there studies showing that normal tissue-derived organoids can undergo neoplastic transformation in vitro? (24:49)

Devanjali Dutta: So we have been able to show that, so when we isolate the tissue and grow them as normal healthy organoids and we keep them in culture for at least 26-27 passages and then we again check them for SNP, by SNP analysis and whole genome sequencing and we have never—there might be small aberrations but there's no significant development of any mutation or any kind of aberration in the organoids. (25:18)

So, I mean I think, I don’t—I am not sure of any study which has completely and only focused on this, but our results, from our results in the lab, we consistently see no such thing happening.

Question: In the context of tumor 3D cell culture, how are organoids different from spheroids?

Devanjali Dutta: So this is, so technically, spheroids are also somewhat 3D in nature. I think the way they are grown is already different because in the 3D organoids, we grow them in a Matrigel drop and we provide them in different factors. (25:56)

Spheroids also are 3D and the only difference I think, the major difference is also about the fact that the spheroids are isolated, and they cannot be isolated from healthy human tissue, whereas organoids can be. And that already puts organoids a bit at a higher pedestal because I think for any kind of drug testing, you really need to have a healthy and a tumorous tissue, whereas from at least till now, till date, I don’t think they have been able to grow the normal spheroid, normal healthy tissue spheroids. (26:27)

Question: Now Dr. Dutta, our next question. Have you tried culturing organoids in scaffolds instead of Matrigel and do you have any preference?

Devanjali Dutta: Yes, absolutely. So there was a paper from our lab and also from Matthias Lütolf’s lab from EPFL where we tested designer matrices as we call them, and these are essentially polyethylene glycol. And these were shown to really help the growth of the organoid and it was also shown that different elasticity of the matrices matters in terms of stem cell proliferation and also for the differentiation. (27:10)

And yes, so these are being developed, so these are called polyethylene glycol hydrogel, and these, we are definitely looking forward to these because right now, the extracellular matrix that is used is the Matrigel or basement membrane extract, and these are isolated from the sarcoma of mice and so they are actually isolated from mice, and it’s not very defined of course because it varies from batch to batch. And if we can have these designer matrices or synthetic hydrogels as we call them, then it will be really way more defined and we’ll know what exactly are the factors, importantly, and we are developing that along with the Lütolf Lab right now, yes. (27:53)

Question: Most of the organoids are made on Matrigel. However, in the case of pancreatic cancer, there are lots of collagen around the tumor cells. Do you really feel that Matrigel alone can reflect the real physiological situation? In our hands, when the tumor cells grow in collagen or in Matrigel, they have very different morphology. (28:17)

Devanjali Dutta: Okay, so I am not an expert in pancreatic cancer but I can say for sure that the people who are doing this research in our lab definitely use Matrigel or basement membrane extract, and we have never used collagen so I will not be able to say how different they are morphologically in that sense. But we definitely compare it to the tissue of the pancreatic tissue from which the organoids were derived and they definitely look like the tissue of origin. (28:46)

So I think it’s important to compare these two and I am, we are right now developing monolayers, where I think monolayers we use collagen, but then of course monolayers look very different from 3D organoids in general. So because collagen is, in our lab at least, used for making monolayers and not 3D organoids. (29:07)

But yes, what I can say about pancreatic cancer is the organoids look very similar to the cancer of origin, yes.

Question:   We are getting so many good questions today, so just as a reminder for our audience, any questions that we do not have time for today will be answered via the contact information that you provided at the time of registration.

So let’s continue with our next question. How many bacteria are you using together? (29:37)

Devanjali Dutta: So that's the thing. So ideally, what we plan to do is, so we've started with injecting one bacterial specie at a time because the idea was to really see what each individual bacteria, commensal bacteria, and how it does to the epithelium and how the epithelium and the bacteria interact. (29:55)

And so now, we have been able to show that single bacterial species definitely grow and now we are trying to include multiple combinations and we are looking at the literature to see which kind of commensals kind of help each other or potentiate each other, and we are trying to do it in different combinations now. (30:15)

And I think eventually, when I talk about the triple co-culture system, it would have to include probably the whole microbiome, whatever, I mean if not whole, most of the species of the microbiome which are isolated from the patient fecal matter. Yes, so this is where we are right now but yes, I think it’s the long way to go definitely. We will have to compare lots of different combinations to come to a conclusion. (30:42)

Question: Now in your organoid immune co-culture, do you have to activate the immune cells and stimulate them?

Devanjali Dutta: Yes, definitely. So what we do is, you have to, so the paper which I mentioned in my talk, so what they do is they activate the immune cells so that they kind of become responsive, by using antigens. And then they are placed in co-culture with the tumor cells. And various studies have been done to check if it is causing some kind of unnatural activation of T-cells but it doesn’t because still the T-cells definitely attack only the tumor cells and not the normal organoids. So yes, we do artificially have, or we have to activate the T-cells to get the population of T-cells which actually are tumor-responsive. (31:38)

Question: Dr. Dutta, our next question. Do all organoids have an apical interior and a basolateral exterior?

Devanjali Dutta: So all organoids definitely have polarity, very specific polarity, but so right now, I have spoken more about the small intestinal organoids, which are more hollow in nature where the apical side is towards the interior, as I mentioned, and the basolateral side is on the exterior. (32:05)

But some organoids definitely grow as solid ball-like structures, or we call them the compact organoids, and for them, the polarity might be different. But definitely the polarity is there; it’s just that it’s not always on the interior or the exterior.

Question: I am wondering about the apical/basal polarity of the epithelial cells in organoids. Since the nutrients available in culture are on the outside of the organoid, would a nutrient transporter be localized basally, thus suggesting that organoids are not exactly polarized as in vivo? (32:45)

Devanjali Dutta: So the nutrients available… So I think the organoids, the way they grow, the nutrients are actually available, they can actually go through because it’s not really a very solid structure, right? So there is some kind of passage of the nutrients or of the media into the interior also. So they do pass through a bit. And so I don’t think anybody has checked that. It’s a very good question. I don’t think anybody has really checked if it’s completely, exactly like how it is in vivo. But I do feel that, I mean at least functionally, we do see a very distinct polarity of apical and basolateral side, yes. (33:30)

Question: And our next question: for a co-culture system, does the ratio of immune cells and organoid matter?

Devanjali Dutta: I think yes, because definitely an overcrowding of immune cells, we have to always maintain, any kind of co-culture, you have to maintain a balance between the cell type and—because the media also has to be supplemented for both the immune cells and for the organoids per se. So yes, definitely it should not be overcrowded because you also should be able to really see the movement of the immune cells towards the organoid. And so yes, I think the number does matter or the ratio does matter. (34:11)

Question: In triple co-culture, did you try or envisage other cells than immune cells—nerve cells for example?

Devanjali Dutta: Nerve cells we haven’t done yet, no. Unfortunately, we haven’t tried that but it will be very interesting to do that. Right now, what we have done right now is isolating the PBMCs from the blood fraction and we culture those. We isolate the T-cells from them and also culture all the PBMCs together and then we put them along with the organoids to grow. But yes, it will be very interesting to put other cell types also, definitely. (34:54)

Question: Dr. Dutta, how long can microbes grow in organoids and is it possible to culture them long-term?

Devanjali Dutta: So this is again a question of what kind of microbe we are talking about. So because for some pathogenic microbes, for example we have tried shigella, we also studied cryptosporidium, so these microbes are really pathogenic and so once you inject them, for shigella, in the case of shigella for example, the organoid architecture is completely disrupted in three days, or even less actually, whereas for commensal microbes, these can be grown for at least seven days easily, and then what we do is we passage these organoids and we have seen that these microbes can actually persist. So these have been grown for at least three to four weeks in our lab, yes. (35:46)

Question: Are the media components for healthy and tumor organoids the same?

Devanjali Dutta: No. Essential components are the same, for example we do have to add some growth factors for the cells to survive. But definitely, because in the case of, again, the most common type that we study, colorectal cancer, most of the colorectal cancer organoids are mutants for APC and there is a Wnt activation. So we can leave out Wnt from our condition media, and they are really happily growing without these external factors. (36:21)

But of course the basic, I think the basic factors are very similar but you can remove factors, and so there is a difference in the healthy and… You can of course grow them in the same media as healthy organoids, the cancer organoids, but you don’t really need to. You can remove some components, yes.

Question: Now when are large animal model or human clinical trials expected? (36:46)

Devanjali Dutta: Very soon, actually. So I think for the human clinical trials, we are, for cystic fibrosis—not the cancer but for cystic fibrosis—we have already tested drugs and we have been able to make organoids from the patient and prescribe them the medicine which was actually most efficient for them. This has been already done. But for cancer, I would say we are looking at four to five years, and it should definitely be in clinical trials by then. (37:23)

Question: Now our next question is a two-part question. How is drug testing performed on 3D organoids and are the number of cells in each organoid the same? If not, then how does one account for the difference in number across organoids?

Devanjali Dutta: Yes, that’s one interesting thing that we observe in organoids all the time, that even if we inject, we see the same number of organoids—or sorry, cells—when we are forming the organoids at the first day, they grow differently and the organoid size varies from drop to drop. (37:58)

So for drug testing, what we do is before we actually perform the test, we shear them or kind of make them a single cell and then we place them in the 384-well plates or 96-well plate, whichever is being used, and we let them grow for two to three days so that they become mini-organoids, and that is when we do the drug testing. And of course when we plate them, before the drug testing, we make sure that the same number of cells have been plated. So this is how, right now, we are performing all our experiments and we use a machine called Multidrop which makes sure that the same number is distributed. (38:35)

Question: How do you manage bacteria growth—shigella is a fast-growing bacteria—after microinjection? And do you add some extracellular antibiotics such as gentamycin?

Devanjali Dutta: Actually, no. We grow them without any antibiotics because these bacteria, of course, would not like antibiotic in the media. So we have, we always have a control and when we inject, we have the injected bacteria. And we grow them without any Pen-Strep, so ideally the organoid media has penicillin, streptomycin and primocin. But when we are growing these organoids with bacteria, we leave out every kind of antibiotic from the media. (39:23)

Question: Now, do you keep the cytokines in the media for organoid immune co-culture?

Devanjali Dutta: Do we keep the cytokines in the media? We do. We do. They are the same and the media, because I think some of the cytokines are very essential for the growth and so we do keep the organoid culture media the same and then we supplement some which would help the growth of the immune cells. (39:48)

Question: Now, Dr. Dutta, our next question. Do you have any idea of the number of cells inside an organoid, like maybe even a rough number?

Devanjali Dutta: That’s, I think it’s very variable to be honest, because, so for if we see the small intestinal organoids, there are some which are really small which would be a few thousand or, yes, I would say 1,000-2,000, very small organoids. But the bigger ones would have 10,000-15,000 cells in one organoid. (40:19)

So it’s really variable because if you see picture of organoids how, when we publish, it’s really tough to say that. And these are for small intestinal organoids. Even between, within the same kind of organoid, but within individual, between different individuals we see different sizes and the way they grow are very different. So we cannot really say there’s a higher number really. (40:46)

Question: Now, when will organoid genetically modified intestinal stem cell transplantation or clinical trials be expected?

Devanjali Dutta: I think this is being tried already because we have been able to use CRISPR/Cas9 to—so for, again, cystic fibrosis, we could show that the mutation could be reversed using CRSIPR/Cas9 and also for human cancers, the research is ongoing. I think it will, again, be a few more years to go to actually reach a place where we can do clinical trials but it’s in the future, I would say again, three to four years to go. (41:28)

Question: How long are organoids differentiated in differentiation media?

Devanjali Dutta: This again is quite variable. So the one thing I think which always stands out is that there is a lot of variability from organoid to organoid. So for example when we enculture small intestinal organoids, we grow them, we make them as organoids in EM or expansion media for seven days and then we add differentiation media, which is without the Wnt and like the nicotinamide for example, and we let them grow for seven days. So for small intestines, we differentiate them for seven days and we see that most of the cells then differentiate towards an enterocyte or enteroendocrine state.

For liver organoids for example, it’s very different. Liver organoids, there are two different kinds that we have. One is of ductal origin or the cholangiocyte-like, and now we have recently developed a hepatocyte-like organoid. And these actually take way longer. They range from I would say ten days to sometimes almost four weeks. (42:35)

Question: Now, how many organoids per tumor should you grow to represent the whole mutation spectrum of a tumor?

Devanjali Dutta: This is a good question because I think this was really studied recently in our lab, in a paper where we could show that within the same tumor, there was a lot of heterogeneity. So ideally, we should grow as many organoids or—when we get the tissue, I would say we should mince it into as many pieces as possible and grow them into as many different lines as possible if we really want to do drug testing because it is really heterogeneous within the same—not even within the same patient but just really within the same tissue. A lot of heterogeneity was observed. So yes, I would say as many as possible from the same tissue resection. (43:22)

Question: Now is microinjection of organoids better than infecting monolayer? How does one account for MLI?

Devanjali Dutta: Yes, so we developed this microinjection for organoids because we wanted to really keep the 3D structure of the organoids intact and because we think the 3D structure really does mimic the in vivo architecture. And so I wouldn’t say this is better because definitely better when you can actually do it. For some organisms, you cannot really inject them or it’s not possible. (43:59)

So, or if you really have to maintain the MLI very strictly then I would go for monolayers. But in the microinjection procedure, we cannot control for the MLI but we control for the amount of microbes that we inject into each organoid so that each organoid has the same amount of microbe at the end.

Question: And it looks like we have time for one more question. (44:24)

Devanjali Dutta: Okay.

Question: How will the triple co-culture model help in immunotherapy?

Devanjali Dutta: Yes, so this is, I think, a dream that we have to use the organoid cultures to really study how these three things come together—the microbes and the immune cells and the epithelial organoids—because two recent studies have shown that even in humans, the responders, the responders to immunotherapy drugs, had a different microbiome as compared to the non-responders. (44:57)

And so there is a definite clue in there that the microbiome does play a role in immunotherapy drug response and so the idea would be if we can have the organoids from a single—of a patient—and then we can have the microbiome isolated from the patient and of course, like the immune cells from the same person. We basically mimic the person in a plate, if I can say so, and we will be able to test drugs and really recommend what kind of drug would work for that person specifically, because it’s not necessary that Drug A, as I mentioned again in my presentation, would work for everybody. So it’s very variable. (45:35)

So ideally, that is how I think it will be helpful to tackle cancer in general and especially it will also help immunotherapy, develop immunotherapy drugs.

Alexis Corrales: Thank you, Dr. Dutta. Do you have any final comments for our audience?

Devanjali Dutta: I would like to thank everybody for such great questions and for coming and listening to the webinar, and I had a great time and I hope I was able to make them a bit more aware about what organoids can do and excite them about organoids and the future.

Alexis Corrales: Thank you again, Dr. Dutta, for your time today and your important research. We would also like to thank LabRoots and our sponsor Thermo Fisher Scientific for underwriting today’s educational webcast. (46:20)

Before we go, I’d like to thank the audience for joining us today and for their interesting questions. Questions we did not have time for today and those submitted during the on-demand period will be addressed by the speaker via the contact information that you provided at the time of registration.

This webcast can be viewed on demand through April of 2019. LabRoots will alert you via email when it’s available for replay. We encourage you to share that email with your colleagues who may have missed today’s live event. Until next time, goodbye.

End Presentation:  46:55

Get to know Devanjali

Why did you choose cancer research? 
As a kid I lost my grandfather to cancer. That was my first introduction to the deadly disease. Since then I have always wanted to contribute to this field, in understanding the root cause of cancer and finding new drugs to tackle it. While cancer research has made tremendous progress, I feel there are many more unexplored avenues in personalized cancer therapies that still need to be improved. It is therefore my endeavor to contribute my bit by using organoid technology to tackle cancer on an individual-to-individual basis.

What motivates you to succeed in your field? 
The fact that what we do is going to help millions of people who suffer from the disease. It is a huge responsibility as well as a motivation to do our best.

What are your top 3 favorite things to do outside of the lab?
Adventure sports, painting, and travelling.

Describe yourself with 3 words:
Adventurous, curious, problem-solver.

If you could choose any other career what would it be? Why? 
I would love to be a travel blogger, as I love to explore new places and know new cultures and people.

What is your favorite day of your life thus far?
The day I got my PhD. My parents were there, and it was the best moment ever!

Is outreach/STEM important to you? Why?
Yes it is very important for me, as I believe every individual has the same basic rights to education and should be given equal opportunity irrespective of class, color, nationality or gender.

Favorite phrase?
Let’s do it!

Why did you become a scientist?
Being from a family of scientists, it was only natural for me to be in science as since childhood I have always marveled at the living world around us. Furthermore, having met and been trained under some of the best scientists of our generation, I always felt inspired to follow their footsteps to discover the unknown and add my little bit to society.

If you didn’t have to sleep, what would you do with the extra time?
Read more books, volunteer to walk dogs, and learn pottery.

What are some small things that make your day better?
A caramel cappuccino, good vibes, and good science.

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