E56: In Vitro Vs. In Vivo

Mary:

I'm Mary Parker, and welcome to this episode of Eureka's Sounds of Science. One research area that has seen tremendous strides in the last few years is in vitro assays, organoids, organ-on-a-chip, and other technologies have improved to the point that it is past time to talk about how they can supplement or even replace some in vivo research.

To speak on this topic, I am joined by Anjli Venkateswaran, Director of Strategic Partnerships for Charles River. Welcome, Anjli.

Anjli:

Hello, Mary. Thank you for having me on the podcast today.

Mary:

Thank you so much for being here. I'm really excited to talk about this topic.

Mary:

So can you tell me, first, a little bit about your career in science?

Anjli:

Yeah, so moving way back in time, my graduate research focused on both in vitro and in vivo models of cancer. And I think I've spent, gosh, about the last 17 years at various life science and drug development organizations, both in the lab and commercial positions.

One of the really interesting things about my career evolution is I've had the opportunity to observe the evolution of in vitro models from simple, commercially available cell lines, which you can purchase from a repository like ATCC, to really complex 3D models that essentially recapitulate a huge chunk of the physiological state. Because keep in mind, with a lot of these cell lines, yeah, they're cheap, they're relatively easy to work with, but do they really represent the disease state? In most cases, no. Right?

Anjli:

And so you can get gorgeous data in the lab with these cell lines that are workhorses in many ways, but what happens is, and we've seen this all the time, is as these drug candidates, whether they're simple or very complex, as they progress through the drug development evolution, they fail. And there's a lot of talk in the industry about the high attrition rates of the drug pipeline, and one of the reasons this happens is that upfront, when you're doing a lot of the in vitro studies, you're looking at a mechanism of action. You're doing your basic foundational studies. You're not really using a relevant model to do your studies.

Anjli:

I'd like to actually just take a pause here and just kind of describe what the differences are, I guess, between in vitro, in vivo, ex vivo. Right?

Mary:

Oh yes, please. Please do.

Anjli:

Because these words get thrown around, even in common language where folks hear about this, and I'm not sure if everybody really understands the distinctions. And it's very important to understand the distinctions, right?

Mary:

Totally.

Anjli:

So starting with in vivo, simply put, if you actually look at what in vivo stands for, it's these are studies conducted in or on a complete living organism, and it actually comes from the Latin for, "In the living," meaning in vivo, as in you're doing something in a living organism. So basically, that would comprise your typical animal model. It would even comprise studies of first-in-human studies.

Anjli:

In contrast, in vitro is from the Latin for, I believe it translates to, "Within the glass," and I think glass refers to your typical lab equipment, plates or dishes or flasks or whatever. And it really, again, refers to studies done outside a living organism, typically in a lab setting. So a lot of the cell-based models that people have been using are typically classified as in vitro models.

Anjli:

Now, bridging your in vitro and in vivo, because there is a big difference, you have a concept called ex vivo. Ex vivo, essentially, it refers to experiments done outside an organism, but it uses samples of tissues from an organism. A great example of ex vivo studies are biopsies, tumor explants. So you basically, in the case of a biopsy, you're still taking the sample from an in vivo organism, but you are doing the experiment outside of that organism.

Anjli:

So that's a nice bridge and there's a ton of work going on in ex vivo studies, especially in oncology, where you can basically take a chunk of a cancer out of a patient and manipulate it and test it evaluate which therapies could potentially work on it, and that'll help guide and say, "Okay, for this particular patient, we need to use this drug combination." So it's a really good way of really driving personalized medicine forward.

Mary:

Yeah, totally. And I think, as you said earlier when we were talking about this topic, cancer is really good at growing. It's kind of its thing. So it's not too hard to make it grow in a dish as opposed to growing in its host person.

Anjli:

Yes and no. Yes. I mean, yes, most cancers actually, if you use the right culture conditions, they will grow. 

However, keep in mind, while they do grow, they also can and do change. And one of the huge areas of research is the concept of tumor microenvironment. And you'll see it as an abbreviation of TME. And there's so much work being done, especially in the space of immuno-oncology because typically, the tumor doesn't live in isolation. It's heavily dependent on what's around it, what's the environment around it. So the basic concept is if you make that environment inhospitable, either you put in a ton of immune cells that keep attacking the tumor, or you make the environment very difficult, inflammatory, make it very difficult for the tumor to survive, it can and will die.

And people use so many different methods. Heck, they use viruses, they use engineered T cells. People have heard of CAR Ts, of course. So there's many different ways of basically making the tumor microenvironment a tough place for the tumor to grow in.

Yeah. That's, I think, one of the key elements of cancer researchers. One is, of course, directly attacking the tumor, and there's a lot of work going on in that space, but it's also really focusing on the environment around the tumor to basically make the tumor die at the end of day.

Mary:

We actually had a podcast several episodes ago about the tumor microenvironment and another on CAR T. So if people are interested in more explanation on that, feel free to go back and check out those podcasts.

But anyway, moving right along. Can you give me an example of a new kind of in vitro technology that's really cool?

Anjli:

Yeah, I mean, gosh, there's a lot to choose from. There really is, you know? And I think organoids has really come a long way. I mean, organoids were described many, many years ago. Like all new technologies, organoids took time, right? Because they're tough to create, they're tough to grow.

For me, what I think is really cool is neuronal organoids.Neuroscience is such a challenging space for drug development. You don't have very good in vitro models because neurons are not that easy to culture and grow in a lab.

And the in vivo models, yes, there are some disease models, and are again, the workhorses of R&D. However, they don't fully recapitulate what's happening in the disease state. And you layer on the complexity of neurodegenerative diseases like Alzheimer's and Parkinson's and frontotemporal dementia, it gets really hard to model the disease state pre-clinically. And keep in mind, a rodent brain or central nervous system is very different from a humans. I mean, one option is for some people to use large animal models, but then there's ethical issues, there's cost issues. So that becomes another barrier to overcome.

I think one alternative model that's coming a long way is the concept of 3D neuronal tissues. Now, neuronal organoids or 3D neuronal tissues, they're very complex and they're very challenging to develop because brain development is tricky. And we really fully don't know the entire cascade of development because it typically requires the timely engagement of multiple signaling cascades and things like that. And similar to other organoids, the extracellular environment is critical.

There's so much work going on in this space. And one area that I find really cool is the concept of developing these extracellular matrices for neuronal organoids. And the reason I think it's so cool is because it brings together different bodies of science. Matrices have been around for a while. Matrigel is probably one of the most well-known matrix that's used in cell culture. But increasingly, scientists are developing other novel biomaterials, and they're doing a lot of really interesting tweaks, not just in the composition, but also in the elasticity, in the rigidity, in the composition. It's just incredible what they're doing.

What I find really cool is that this requires multiple knowledge areas to come together. So you have the biology, you've got the material engineering component, and you have the physics. So you've got all these amazing branches of science collaborating together. And in fact, there are companies out there, typically startup companies, that bring these groups together. You have physicists collaborating with material engineers, collaborating with your pure neurobiologists, and they're solving these incredibly complex problems through lenses that we've never really looked at.

Mary:

I love it.

Anjli:

I think that's incredible.

Mary:

Yeah, absolutely. And I also personally love it when physicists get involved. I don't know. It seems more sci-fi-y to me when they do.

Anjli:

And they bring whole new concepts, right?

Mary:

Absolutely, yeah.

Anjli:

They bring ways of thinking about things where you are like, "Oh gosh, I didn't even think of that. Oh, that's such a great idea." You know?

Mary:

Yeah, totally.

Anjli:

I'll give you an example. Apparently, a big deal about these matrices is the rigidity. Do they give? Are they flexible? Sometimes you need to have more flexible matrices, sometimes you don't. What's the diffusion characteristic? So if you put, say, a growth factor in this matrix, does it diffuse nicely? Does it diffuse slowly so it reaches the organoids?

Anjli:

Think about it. You're almost creating an entire organ system outside the body, so you've got to, to a large extent, reproduce what's going on inside the body. And that's a really tall order.

Mary:

Absolutely. And it also, like you said, depends on the complexity of the organ that you're trying to study. If you're trying to study, I guess, the simplest organ might be skin, that might be kind of easy. But something like the brain is at the opposite end of the spectrum.

That's a whole separate podcast in itself.

Anjli:

Indeed, indeed.

Mary:

You brought up economics, so I'm curious. Can you explain why in vitro studies are kind of having a moment, like the technology's improving, but people are also getting more interested. Why is that?

Anjli:

Yeah, definitely. I think in vitro models are definitely having a renaissance, and I think there's a few contributing factors. One is that the technology platforms that are being used are improving by leaps and bounds. And what is really heartening to see is the progression of this research. You start seeing, okay, improved ways of building organoids. You see these advances that come from these labs and companies on improved matrices, increased improvement in organ chip technologies, and more importantly, how these technologies are no longer just confined to labs. They're actually going out into a more commercial application. So that, I think, is one of the big contributing factors.

At the end of the day, if you want a specific area of science to really go mainstream, you've got to have the data, and you've got to have a body of work to support it because scientists by nature are skeptical, as they should be. So they need to see the data, they need to believe, and really evaluate the data and believe in it in order for a technology platform to be accepted widely.

And the other thing is, of course, which we should never overlook, is also the development of the readouts. Because you can develop a really cool model system that works beautifully, but you've got to make sure that you have the right readouts in place. And that's when the whole wide world of biomarkers comes in. Clinically relevant readouts, right? I mean, biomarkers, I think you can have a discussion for centuries on how important those are, and how difficult they can be, especially in a clinical setting. And they have to be robust.

Mary:

Especially for a new drug. I mean, you don't know exactly what it does yet.

Anjli:

Indeed. Exactly. 

Mary:

So as your role is also Director of Strategic Partnerships, you should be able to handle this next one. The startup market for in vitro technologies is growing rapidly. So what's up with that?

Anjli:

Oh my. I mean, it's such a fast-growing space. It's incredible. But what the macro trends are showing in the market is that people are noticing.

Typically, when VC firms, they're always at the forefront of scouting out new technologies. They're looking for the next big thing, and they've got extremely diverse and brilliant people on staff, and they're noticed in vitro models. Over the past few years, several companies have been launched with really nice funding.

I mean, one example that comes to mind is a company called Xilis, spelled X-I-L-I-S, but it's pronounced, "Xilas." These guys have developed a technology called MicroOrganoSphere Technology, and again, it's very organoid-based to study different diseases. And they raised $70 million. $70 million dollars.

Mary:

Whoa.

Anjli:

In Series A funding, which is very high for your typical Series A.

And then you've got another company called Bit Bio. They're based in the UK. They focus on iPSC derived cell lines, and they raised over $100 million in Series B funding. I believe it was last year. So again, the fact that these companies are being launched with such excellent funding backing them suggests to me that the market is only going to keep growing and growing, and that the investment community is willing to take a chance on these technologies because they really do believe that these technologies have a lot of potential.

Mary:

Yeah, definitely. Well, you mentioned earlier that scientists can be a little skeptical and conservative, but that's kind of nothing compared with scientific regulators. So go into the sort of FDA regulatory challenge. Where is that at in terms of in vitro studies right now?

Anjli:

Yeah, you're absolutely right. I mean, I would say the FDA is probably one of the most conservative organizations, as they should be because they're under intense pressure to make sure that the drugs that they do approve, the therapies they do approve are extremely safe and work across multiple population segments. I feel sometimes they're in a tough spot, and I do feel that sometimes they get more than their fair share of criticism.

But the caveat here to think about is, with the FDA, I think as these therapies become more complex ... Now historically, I think, gosh, about a decade ago, you had the bulk of approvals were small molecules. So these were defined chemical entities. You knew what the structure was, you knew exactly what was in there, right down to the last atom. So that was a huge chunk of the pipeline. And then over time, as we all know, there's been this absolute tsunami of monoclonal antibodies. There's interest in gene therapy, cell therapy, there's just these modalities, of course oligos. These modalities are just getting more and more complex.

So the FDA has had the really difficult task of keeping up and establishing regulations for these complex modalities.

But one thing I will say is, historically, the FDA has been resistant to approve an investigative new drug or IND applications that had a heavy reliance on in vitro models because I think their opinion was, "Yeah, these may not be representative of the human state or the human disease state," which I would say they were, right. But that's changing.

And I think one of the key developments, which has been applauded by, I think, the scientific community is the recent passing of the FDA Modernization Act. And what this does is this gives drug developers the option to use proven non-animal test methods. So they basically, they took the wording from the original act, they took the word animal, and that was what the word was. It was animal. You could only use animal models in your testing. They took that word out and they replaced it with language that says, "As long as the non-animal test ..." It can be pretty much any model, as long as it's proven, it's reliable, robust. Meaning the proof of validation is on the researcher, but they don't have to only use animal models.

So if you have to use animal models, that's fine, but use it thoughtfully. Try to see if you can ... And I think this will probably flow right into the conversation around the three Rs-

Mary:

Absolutely, yep. I was just going to say.

Anjli:

Use it thoughtfully. Use it in a way that makes sense, not because it's like, "Okay, well, let's just try in vivos, let's see how this drug works an in vivo setting." Right?

Mary:

Right.

Anjli:

But I think coming back to the FDA, what this has done is I've seen an evolution in the FDA's thinking, and this, I would say, is extremely visible in the rare disease space.

I had the opportunity to hear one of the former FDA Commissioners, Dr. Scott Gottlieb, talk at a conference. And initially when I went to sit down, I was like, "Oh, boy. Is this going to be really boring?" But it was one of the best presentations I'd ever heard. And he was talking with such passion and enthusiasm about how the FDA really wants to support, the agency wants to support these rare disease drug developments, accelerate them because in a lot of cases, as you know Mary, patients are waiting. And a lot of these patients, unfortunately, are children, and they don't have the time. Their time will run out very quickly for them. So there's always this insane clock ticking. And the FDA, I was so glad to see the FDA acknowledge that.

And really, I think there's a lot of times they've engaged with the patients, with the caregivers of the patients, with the researchers, with the clinicians, to basically accelerate therapies for rare diseases.

So again, that's an example, I think, of how nicely the FDA has evolved. I mean, it's not perfect, but again, I think they're in a little bit of a tough spot. They've got to do this tightrope between being nimble and agile and completely plugged into the patient needs while making sure that the appropriate processes, that there's no shortcuts, there's no cutting corners in the research.

I mean, the other example, of course, you can think with COVID-19 vaccines. The breakneck speed at which those vaccines were developed and tested, and the FDA, I think really did an amazing job reviewing all those data and basically making sure that these vaccines were okay, ready, were fine for the patient population.

And so I think these are couple of examples, I think, where I see the FDA is evolving, but as I said, they've got a tightrope act to do, which is not an easy place to be.

Mary:

Yeah, it's true. And no one ever accused a government agency of being nimble and agile and fast-paced.

Anjli:

Right, exactly. It's a tough position for them. It's hard.

Mary:

It is. It's very ... Yep.

Going back into what we were talking about before about the three Rs, how does in vitro technology affect the three Rs?

Anjli:

I mean, if you look at the three Rs: replacement, reduction, refinement. And if you think about the first two, replacement and reduction, and that's where I think these in vitro models have such an absolutely huge impact. Because as in vitro model ... Relevant, keep in mind, I'm always going to go back to the word relevant. As long as these models are relevant and there's increased adoption of these relevant models in the drug development community, that has a huge impact on the replacement and reduction of the use of animal models.

But there is a fascinating new paper that I came across that came out of a company called Emulate Bio. I believe it's a relatively recent paper. It was published in Nature Communications Medicine. So if people want to look it up it's, I believe, I want to say December 2022, Nature Communications Medicine. And the first author is Ewart, E-W-A-R-T.

But what this paper showed, which absolutely was so amazing, is this company has developed a liver chip.

Now remember, when you put a drug into a person, or when you swallow, take a pill, the first place that goes is the liver. And so the liver is basically your clearinghouse, your metabolic engine for these drugs. So if a drug is very toxic and it's administered systemically, you have this concept of drug-induced liver injury, DILI.

So anyway, so typically these studies were done in vivo models, but now this paper from Emulate shows that the chips that were used are 100% specific, and I want to say 87 or something percent sensitive, so these chips could correctly identify which drugs had higher toxicity and which drugs had lower toxicity. And also what's even more interesting is that they didn't have any false positives where a drug that's not toxic was identified as toxic.

So that's, for me, those kind of studies are really those game changers for three Rs because if you have a legitimate alternative, an alternative that still gives you that high quality of data so you're not compromising on data quality, you're not compromising on data accuracy or sensitivity, because that's unacceptable, but if these models don't let you compromise, but they reduce the need for animal models? Wow, that's such a huge win.

Mary:

Yeah.

Anjli:

Right?

Mary:

Well, and you even mentioned the third, refinement. Back to your earlier point of the more from the in vitro data, the more refined your animal studies, if you still need them, will be because it won't just be give the rat the drug, does the rat die? Yes/No. It'll be you already have all this data, you know what it should do, you can be more specific with your observations of the animal.

Anjli:

Absolutely. Absolutely. And as I said previously, that's such a personal important point for me because again, thoughtful use of these animals is critical.

And I think these kind of initiatives really support the the responsibility, rather, that we all have in managing to the three Rs because it should not be a theoretical concept. It can be applied in every single drug development program. And it should be.

Mary:

Yeah. Well, wrapping up, what do you think is going to be the tipping point for making in vitro truly mainstream and able to replace animal models for some requirements with the FDA?

Anjli:

Yeah. I mean, as we've had a lot of discussion, there's so many positive developments in the space between the investors, the regulatory, the fast-moving technology advancements. I think these are all some of the critical parameters for increasing adoption of these in vitro models.

And I'd like to draw a parallel analogy to iPSCs. So iPSCs, Induced pluripotent stem cells, they were originally reported, I believe, back in 2006, so about, gosh, about 15, 16 years ago by an absolutely brilliant scientist named Shinya Yamanaka who won the Nobel Prize, and rightfully so, for the discovery. But the first few years, they were brutally hard to develop because they require a very complex cocktail of transcription factors, and Yamanaka's papers had a lot of the protocols, but it was very tricky to do. It was not like an easy, "Oh, I'll just copy. I'll just lift the protocol. I'll use the protocol that was published." There was a lot of tweaks and nuances and things like that.

Just put it really very simply, in other words, if you make a technology more accessible, more reproducible, and easier to use, people will try it. But if you have such a high barrier to entry where there's only a handful of people who know how to do this well, then it naturally would inhibit adoption.

So I think looking at things like iPSCs is a great path on how we expect additional in vitro models to grow. 

So it's on these early innovators, it's on these companies, and it's on these academic labs who are driving these platform technologies to really build the body of work and develop proof of concept data and move it forward to really show that a given in vitro model is really going to be a game changer.

Mary:

Yeah. Great. Oh, well, thank you so much for sharing your vast knowledge of this topic. I'm really pleased we could get a chance to talk about it. It's so interesting and cool.

Anjli:

No, Mary, it was a pleasure, Mary. I'm really glad we had this chance to talk. As you can tell, this is near and dear to my heart.

Mary:

Absolutely.

Anjli:

So I shall be watching, I will be observing and watching and hopefully supporting the advancement of in vitro models in the future.

Mary:

Yeah. Well, you're in a good position to do that here, so I'm glad for that.

Anjli:

Yes indeed.

Mary:

All right.

Anjli:

Thanks, Mary.

Mary:

Thank you so much.