Dr Adrian Biddle, Senior Lecturer, Queen Mary University of London

Date: 15th September 2021

Dr Adrian Biddle, is a Senior Lecturer at the Blizard Institute Queen Mary University of London. Nomthandazo Ziba is completing a master's degree in pharmacology at Coventry University. This transcript has been edited for clarity and brevity.

Dr Adrian Biddle.

Photograph of Dr Adrian Biddle, credit Biddle. Dr Biddle is Senior Lecturer in Animal Replacement Science within the Centre for Cell Biology and Cutaneous Research at the Blizard Institute Queen Mary University of London. His research focuses on cancer stem cells in oral cancer and developing human disease models for studying tumour spread and therapeutic resistance.

Nomthandazo

What is your background?

Adrian

I've been involved for some time now in animal replacement research and also with organisations like Animal free Research UK and the National Centre for the three Rs (Replacement, Reduction and Refinement). They fund animal replacement research, and the National Centre for the 3Rs also funds refinement research. One of the most important things is really making people aware and making them think about how they do their research. For example, I know now that when people submit an application to the home office for an animal licence to be able to do animal research, they have to write quite detailed notes on how they've considered replacing, reducing and refining the experiment. So you'll see that, in the UK, even projects that involve the use of animals should be observing the three R's right from the start.

I think there has been a huge shift in attitudes on the use of animals in research. Over time replacement of animals has really become embedded in the research culture, particularly in the UK. And across Europe as well there's really much greater consideration now about how we can replace animal research, for example, with the organ on a chip models that we're going to discuss today.

On the refinement side there is also a lot of discussion about how people can reduce the number of animals in their experiments by better planning. Because, of course, if you have too few, you have to keep doing the experiment with more animals, and if you have too many, then you're using animals you wouldn't otherwise need to use. Then on the refinement side, there's a lot of emphasis now on animal welfare, and making sure that the techniques used in animal experiments are the most humane, the most refined. There's one interesting study from Professor Jane Hurst in Liverpool that got a lot of attention where they found that if you use the standard way of picking up mice by the tail it’s quite stressful for them (Gouveia, Hurst). They found that if they use a tube, a bit like a toilet roll, and let them run into the tube and move them like that the mice were actually less stressed. They got better experimental outcomes because less stressed mice gave more reproducible outcomes. So that was interesting on the refinement front. Of course, my main focus has been on animal replacement, which we're going to talk about today.

Nomthandazo

I think that's quite interesting when you talk about lifting mice because I remember last semester, I was doing this other project about a new cancer drug. And they were saying that when it comes to doing the test with the mice, they have to look at their reaction whether they are stressed or they are puffy and stuff like that to see if they're reacting adversely to the drug. So I think it does really play an effect if the mice are being taken care of quite well, the results also then translate to that.

Adrian

I think that's very important. It's becoming increasingly clear that keeping mice in a laboratory setting is stressful for them anyway and it actually has quite big effects on the outcomes of experiments.

Nomthandazo

How does the collaboration between Queen Mary University and Animal Free Research work?

Adrian

We've worked together for quite some time now. Animal Free Research UK is an organisation who funds research into animal replacement. They want to replace animals in medical research. They focus on raising money to fund research into animal replacement technologies and also to raise awareness with other organisations, for example within the government, to increase awareness about replacement technologies and promote their adoption. Back in 2016, we were very fortunate that Animal Free Research UK gave us a large grant for the Animal Replacement Centre at Queen Mary University London. It was the first animal replacement centre in the UK funded by Animal Free Research UK. So myself and Professor Mike Philpott and some other members of our team, are funded through this very, very generous grant from Animal Free Research UK and their supporters, specifically with the aim of replacing animals in cancer research. Our research is in head and neck cancer, and skin cancer. The funding is to develop technologies for replacing animals in those areas.

Nomthandazo

Do you just collaborate with them when they give you funding to try and look for other alternative methods?

Adrian

That's right, they fund us. We also work closely with them in other ways. We do workshops for students and bring in some undergraduate students into our lab and from other labs around the country. Animal Free Research UK gives stipends to students to do summer projects. They encourage different groups with similar interests like ourselves, for example, in cancer, to put together a consortium to focus on different areas. And that's something which is quite exciting and ongoing. It can also add weight to raising awareness, such as working with the regulatory bodies who need to be aware of the new developments and how they could be incorporated into, for example, clinical testing.

Nomthandazo

Where do you think the organ-on-a-chip fits in, in terms of replacing the use of animals?

Adrian

I think it's going to be very important in several different ways. It's already replacing animals in basic scientific research. And there's great potential now for moving forward into more clinical facing research. We host the UK Organ-on-a-Chip Network with regular symposia where we invite keynote speakers and have people submit abstracts. That's a really nice way to get together to see the range of organ and chip research that's going on in the UK and more broadly and we've had some very interesting talks. We recently had a talk from Dr. Shannon Stott from Harvard Medical School who spoke about how she's using organ-on-a-chip technology to help test cancer patients using liquid biopsies. Rather than having to take a piece of the tumour, which can often be hidden in the body, you actually take some blood and test it for the presence of cancer cells.

There are a few microfluidic chip devices which can sort out cancer cells. They are not called ‘organ-on-a-chip’, but it's a similar technology. It picks out the cancer cells from the blood. And because cancer cells are very rare in the blood you can purify them out and then use that as a liquid biopsy to understand the characteristics of the patient's cancer. This will allow the treatment to be tailored to the patient. So that's one way the technology is being used clinically. Also the microfluidic chip would allow us to replace the practice of taking a piece of the patient's tumour and putting it into an animal like a mouse to see how it grows. The advantage with this method is you can purify the patient's cells directly rather than having to grow pieces of human tumour in a mouse.

People who present at these meetings are also looking at more basic medical research where we're trying to understand disease. A lot of this work is done using animals at the moment. For example, with my own work in cancer metastasis, where we look at how tumours spread, a huge amount of that work is performed using animals. People typically create genetically modified mice so they get cancer or actually inject human cancer cells into immunocompromised mice. This enables researchers to watch how the cancer spreads around the animal or how the human tumour grows in the mouse. We are now doing this in non-animal models, working with tissue cultures of human cells and other different systems such as an organ-on-a-chip.

Actually, it's been quite an eye opener to really see how, by collaborating with different people, engineers, and medical researchers, we can bring in a new technology, such as microfluidic chip technology, and start using it in the lab and then actually see it work. You have quantitative questions you want to answer and at the same time, by bringing in the technology, we are able to see what other questions we might be able to look at as well. Because often, by trying to answer some questions it allows you to throw in more questions. The great thing about the organ-on-a-chip technologies is you can control more of the variables than you can with animals.

Coming back to the organ-a-on-chip symposia that we run, there was a great recent talk from Professor Peter Friedl from the Netherlands. He has a very, very high profile in the cancer research community and he does a lot of work comparing both animal research and alternative methods. It was really interesting to see where he initially found things in vivo in animals that he's now able to research in vitro. One example is tumour invasion. When tumours move away from the primary tumour, they invade through the human body which we want to try to stop. When we look at that within the in vitro setting we worry about what kind of substance we should create for the tumour cells to invade through. By looking at that in vivo, in human specimens, and also in his animal models, Professor Friedel found that tumours come in a number of different environments. They are cutting through surfaces, some really long surfaces, they're moving through solid blocks of hydrogel. Now to be able to move through that, sometimes there are constraints, sometimes it's open, sometimes it's disorganised, and sometimes there's parallel fibres they can move along. Of course, in an animal you can't control any of that. You can observe what's happening, as he did. But actually a great opportunity is to take that into these alternative methods in vitro. With an organ-on-a-chip model you can actually create each environment so you can control it. We can, for example, look at how these cancer cells are going to move through aligned fibres, and this is what Professor Friedl did. So that's another great thing to highlight with these replacement methods is that you are not only trying to mimic what's happening in vivo, but you can actually control it. Whereas in animal models, you're limited to seeing what's happening within what you've got.

Nomthandazo

I think it's also important because at the end of the day, animals are not human. So the results don't necessarily translate quite well. And that's why they say that 90% of the drugs end up failing after animal studies.

Adrian

Yes, that's right and there's a lot of reasons for that. One important reason that springs to my mind is the incredible heterogeneity of humans. We're all very different from each other and we all have very different biologies. And of course, that can't be modelled in animals because the animals that are used, for example, the mice that are used are inbred deliberately so they all give the same outcome. And the issue with that is that often outcomes that are very successful in these mice will not work in humans or will only work in a very small number of humans.

So, understanding the differences between people and how we can develop drugs that work for the greatest proportion of people is very important. For that you'll need organ-on-a-chip models where we can actually put in patient material from different patients, and look at the differences. There are publications from five to seven years ago, probably longer, where they are taking tumours out of patients and growing them in a dish and looking at how they respond to drugs and trying to understand why patients respond differently. But of course, with these new organ chip models, we can increase the sophistication of that by measuring more variables. So rather than just growing the cells in a dish and seeing whether they grow or not, and of course, they are plastic dishes, it is very unlike the conditions inside the human body, you start to create conditions in the in vitro replacement models which are more like the insides of the human body.

Coming to cancer research here, because this is my passion, I think that will be very important. Because again, there are big disadvantages to patient derived xenograft models, where human tumours are put into mice. These are incredibly challenging because you're working with animals, and they're running around and at times the mice get ill. You also have to have cages of hundreds of mice just to look at maybe one patient's outcome or for a small number of patients. So that's expensive, and it's increasingly unviable. And I think from that point of view, as well as the ethical point of view, once you replace animals, being able to do this in vitro, it will be much more efficient and we'll get more consistent outcomes.

So putting patient material into these more accurate representations of the human environment is really important. Again, with cancer we have a microenvironment of the organ in which a tumour sits. We look at head neck cancer in the oral cavity in my lab, where it is on the tongue and the inside of the mouth. And within that you have blood vessels and the fibroblasts, the cells that sit within the matrix around the tumour, and you have different immune cells infiltrating. Of course all of this will affect how the tumour will behave and how it will respond to drugs. So we want to try and start modelling that. There's been huge advances in that area recently as well and how we can put all that together in vitro. One of my colleagues at Queen Mary, Professor Fran Balkwill, who works on ovarian cancer has had some really great successes in recreating the ovarian cancer environment in a dish. She looked at how ovarian cancer spreads to the main metastatic site for ovarian cancer in humans. Her lab actually deconstructed human samples to look at what is the makeup of this tissue in humans and put it all back together again in a dish. So they can really accurately include the immune cells, and the other types of cells which are there, and then understand how the tumour cells behave in that environment. So I think these sorts of victories, advances, within cancer research are really important.

Nomthandazo

You've already touched a bit on how the organ-on-a-chip is going to have an effect on the clinical research process. Can you maybe expand on that.

Adrian

In terms of clinical research putting human tissue into big groups of mice to do testing, I think that's going to be very unreliable. I think a much better, more efficient method will be to use these organ-on-chip methods. There has been clinical research that has been going on for a long time using basic tissue culture methods. And I think by introducing these OOC methods, we'll be able to really increase the sophistication of what we can do in this clinical research, yes, and increase the efficiency and the outcomes that we can measure.

Nomthandazo

Does Animal Free Research fund different organisations?

Adrian

Yes, that's right. Animal Free Research UK funds projects through a number of different routes.

Nomthandazo

Are there any organ-on-a-chip projects that they have funded?

Adrian

We do some organ-on-a-chip projects, some of that is covered by Animal Free Research UK funding.

Nomthandazo

Do you think regulators are doing enough to ensure that there are alternative methods before approving animal research?

Adrian

The basic scientific research we do in universities doesn't involve regulators. A very important role for regulators with drug development research is when companies or organisations want to provide a case, for example, for putting a drug in humans in clinical trials. With all trials you have to convince the regulator there is a good enough case for that. And regulators will demand animal work to show the effects of the drug in animals for efficacy. For example, in cancer research, they put cancers into animals like mice to show if a drug cures cancer. Also they want toxicity studies in animals to check whether the drug is toxic before the drug goes into humans. I think there are great alternative technologies now available for this work which regulators are definitely engaging with. But the question of whether things are moving fast enough, and whether they take them on board fast enough I don't know because I don’t work on that side of things. I know that Animal Free Research UK and other organisations put a lot of effort into engaging with regulators to try and make sure that they do take up the technology. There have been some very interesting developments, for example, the Dutch government a few years ago passed a resolution where they were going to take up these new technologies as replacement of animal methods with the aim to fully replace animal methods. So that's encouraging and things are definitely moving in the right direction. But whether they are moving fast enough, I don't know.

Nomthandazo

How have biopharmaceutical companies responded to the organ-on-a-chip technology?

Adrian

Well, there's been a huge interest since we've started bringing some new chip technologies into my own group's research. I think the organ-on-a-chip sparks people's imaginations, because it's very visual. It makes you think of a human organ-on-a-chip and all the things you can do with that. And pharmaceutical companies are showing a lot of interest. As a university, we talk to companies about what we're doing and look for ways we might be able to work together. For a lot of companies the organ-on-a-chip work has consistently been at the front of what they're interested in looking at. There are a lot of initiatives, particularly through the UK organ-on-a-chip network and its hub at Queen Mary, where we are making efforts to engage with pharmaceutical companies to talk about how we can work together using replacement technology and they seem very receptive to that. I've been really pleased with what they want because their aim is to get drugs into the clinic as fast as possible. Although in some ways there's a huge inertia because animal models have been used for a long time so some people want to keep using them, because they are used to them, and that's what the regulator's want. Nevertheless, the pharmaceutical companies want to save money and get things going through more quickly so they are very open to looking at these technologies which is encouraging. I don't think we're there yet, in terms of fully replacing animals. I think we need to do a lot more to convince people. But I think we're making good progress in the right direction.

Nomthandazo

To what extent do you think the organ-on-a-chip is actually going to have an impact on reducing the use of animals?

Adrian

I think it is already having an impact. Certainly within university research, there are a lot of projects within our own organisation and within other organisations, which are using organ-on-a-chip which 10 years ago would have been done using animals. And in my own work, looking at tumour testing using organ-on-a-chip, there really weren’t very good technologies for doing that. You could look at human cell invasion in a 3D matrix, which has been going on for about 20 years, but you couldn't look at it in an integrated fashion at the different steps of how tumours spread, involving for example the vasculature and secondary sites.

So in university research animals have already been replaced. And that's probably also the case in the pharmaceutical sector, where there are now projects using organ-on-a-chip which would previously have used animals. So yes, replacement is definitely already happening. But how fast that's going to move, and at what point we will be able to fully replace animals, it's very difficult to know. But I think it's moving really quickly and as it moves it's going to pick up momentum as well. Because as we've always seen, these things start slowly, and then the momentum has really increased over the past decade. There is now a huge focus on it compared to 10 years ago. So not too far in the future, we may actually be able to be seriously thinking about total replacement. Technologies always start slowly and then as more people use them and more people develop them, there's more understanding about them and they improve really quickly. We've seen that in the case of genome sequencing. When the Human Genome Project started it took ages and cost vast amounts of money, and now you can sequence a human genome in no time at all for a tiny fraction of the cost. I think we'll see the same thing with these technologies in replacing animals. It'll get faster and faster.

Nomthandazo

What do you think are advantages and disadvantages of the technology?

Adrian

What I have found from my own experience of bringing in different types of replacement technologies into my own group is that each technology has its own advantages and disadvantages. What we tend to do is we bring in technologies that are interesting, and then we quickly discover what their advantages are and what their disadvantages are. And then we can actually marry the different technologies together. So for example, in my own groups we have two different technologies we use a lot to look at tumour invasion. We have quite a large 3D gel plate, where we put the tumour cells in to invade through the gel. It’s very useful, but it's not very good for imaging for microscopy because it's too thick. It's also not great for separating different types of cells, because the cells are all mixed together in the middle. What it is really good for is getting cells back out again, and then we can analyse the cells using different techniques. So it's very good for that. The other method we have is the microfluidic chip method that we use in collaboration with engineers. Professor Julian Gautrot, who is an engineer in our university, developed some microfluidic chips which we use in my group. We found they're fantastic for imaging because they are very thin chips with little channels. In the central channel, you have a matrix, and you can put cells in the two side channels and watch how they come together and interact in that central matrix. It's fantastic because it’s in 3D, it's very thin and it's on a glass cover slip. It's brilliant for imaging. You can really see how the cells interact in great detail. It's also really good because you can create two compartments, one with one cell type and one with another cell type then let them come together so you can actually see these different cell types interact in real time. So it's fantastic for that. What it is not good for though, is getting lots of cells out because these chips are very thin glass chips and also there's very few cells in there. So if we want to look at how cells interact with each other in detail and or look at them through the microscope we use the chip. Then if we want to look at molecular pathways at the RNA level, for example by extracting the RNA, then we'll do a large gel to get the cells out.

At the moment, for us, it's about bringing different techniques into the lab, playing around with them, seeing what their advantages are, and then using them to answer a question. And, of course, we have various biological questions we want to answer about cancer. We're always looking to bring new technologies in and use them in different ways and to think about how they can work together to answer questions.

As we move forward into the future, you might see more and more technologies being developed and more integrated in a more formalised fashion within an organ-on-a-chip to do all of this. But at the moment, these models have advantages and disadvantages, but they're great because you can find out very quickly what they are and you can control the variables. This is unlike with an animal where you can't say I can use one mouse for this and a different type of mouse for that because you can't control what's happening inside the mouse. But in these you can control what happening, and you can bring together different models to use their strengths together.

Nomthandazo

How expensive are these chips?

Adrian

It depends. We made ours ourselves. Professor Julian Gautrot and his team in the engineering department developed the technique to make these chips. In my own group, I have a PhD student, Alice Scemama, who makes them for her project. And we have the equipment for making them within the Institute where we work. This makes them very cheap. All we pay for are the materials and reagents and our own time. It’s not a lot of money. If you were to buy them, there are quite a few different competing commercial systems. It can become quite expensive. Though, I'd expect that cost to drop sharply as these technologies become more routine. Then it'll become more accessible as well, you won't need to build them yourself. It depends on the complexity and what you're trying to do, it can be very expensive. But it can also be really cheap depending on what you are trying to do.

Nomthandazo

I know that one of the challenges is sourcing the cell culture is one of the expensive things that you use to put on the chip. Is that your experience as well?

Adrian

Sourcing the actual primary human cell types?

Nomthandazo

Yeah.

Adrian

That’s a really good question because a huge part of these models depends on what cells you put into them. You've got various options for cancer research. Again, coming back to what I do, we can use cancer cell lines, which are immortalised, these are cell lines which have been grown out from human tumours in a dish that have been growing for many years. These are great because they're a very plentiful resource, and they're cheap, and they're available. The main disadvantage is that they have been growing in a dish for a long time and that may have induced changes, so they may not accurately represent what's happening in a real human tumour. That being said, it also depends very much on the cell line you use. In our experience, using head and neck cancer cell lines, they do very accurately represent a lot of the characteristics of human tumour specimens, and they're a great resource because they're plentiful. You don't want to be working with a resource which is limited if you can help it, because then you're constrained in what you can do and that can be difficult.

With cancer, you can work with fresh human specimens which we have done. But that's more tricky because you have to have a source from surgery. You have to actually have quite a logistical pipeline in place, including ethical approval and making sure that the patient has consented to the use of this tissue. The surgeon has to remember to call you and the theatre staff have to remember not to put the specimen in formalin, because you can't grow the cells out of it then. And then they have to guide you to the right place, you have to get access to the hospital. So there's a lot of complications, but with effort you can make it work.

More broadly, if you think about other organ-on-a-chip applications, there's a big drive to use primary human cells which come out of patients. It's always challenging getting good stocks of those and it can be very expensive. Rather than using cell lines, one technology which could be really useful is induced pluripotent stem cell (iPS) technology. It's been around for about 15 years now. It has the ability to reprogram cells from a person and then make any cell you want. That technology has really been driving a lot of elements in the organ-on-a-chip field rather than using primary cells. You can differentiate them into heart cells to make a heart on-a-chip. You can actually make patient specific iPS cells, if you want to tell if it's a particular genetic heart defect. For example, you could take skin cells, maybe from patients with this heart defect, reprogram them, and then turn them into heart cells. Then you have a heart-on-a-chip which is from the patient with the genetic condition. There's a huge amount of potential for combining induced pluripotent stem cells and organ on chip technology together for patient specific disease research.

Nomthandazo

What are the key challenges in taking the technology forward and where do you think the solutions might lie?

Adrian

One challenge is consistency and reproducibility and coming up with an agreed approach. At the moment, it's a little bit like the Wild West in some ways, with each group using their own approach. That's great at the start, because you want to be able to be innovative and be inventive and develop new technologies which can do different things and see what they can do. But there's going to be a challenge at some point where there's going to have to be a rationalisation of approaches, particularly in clinical research. So there has to be a rational and a consistent approach. Once the approaches have been rationalised and shown to work consistently and reproducibly, then it'll be much easier for others to take up the technologies as well. So that's one challenge.

Another challenge that has been talked about for quite a while now is multi-organ chips. How do you make things work together? For example, if you've got one method optimised for growing liver in the lab and you have another method optimised for growing kidney in the lab you want to be able to marry them together to have a liver kidney network. To some degree you have to start from scratch, because you haven’t come up with an environment that's going to work for both of them and it may be different. Actually, that's something which, even on the small scale of the microfluidic chips with the two different cell types that we look at, often you've got one condition on one side and another condition on the other side. Harmonising things is a challenge. It is a challenge finding conditions where you can start putting things together and growing multiple cell types, multiple tissue types together. The multi-organ chip has been a holy grail for the organ-on-a-chip field for some time. Also how do you put the microfluidics and larger scale fluidics together? Again it's got to be something which eventually can be rationalised, and established as a consistent repeatable technique, which can then be miniaturised so it's not huge so it can then be commercialised and used in lots of different labs around the world. It's not very useful if it only works in one person's lab. So those are some of the bigger picture challenges.

Nomthandazo

I know that there is a collaboration between Queen Mary and Emulate, are you involved in that?

Adrian

In our engineering department we have an Emulate lab through an agreement with Emulate. Actually this comes back to your question about the cost of the chips. Emulate chips are quite expensive and you have to grow them within a special incubator, which also adds to the cost. I think there's an agreement with Queen Mary where they can use the Emulate technology on a pay-per-go basis, rather than a single lab having to buy all the equipment. They can just go along and pay to use the equipment for a week. Obviously that’s cost-effective. I haven't used it myself yet. There have been some interesting projects using it. There's also been some seed funding from the UK Organ-on-a-chip Network for some people to start using the technology. They have these mechanical attributes where you can stretch them to mimic, for example, breathing in the lung, or other things.

Nomthandazo

Thank you very much. I appreciate you taking time out of your busy schedule to talk to me.

Reference

Gouveia, K, Hurst, JL (21 March 2017)'Optimising reliability of mouse performance in behavioural testing: The major role of non-aversive handling', Scientific Reports, 7.Back

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