‘Making Precision Cut Tissue Slices for Ex Vivo Assay Services’ webinar was given in partnership with Precisionary Instruments.
Abby: Good morning everybody and welcome to our Precisionary Webinar. We are partnering with Visikol and we have 2 wonderful speakers to talk to us about precision cut liver slices. And so I wanted to do a quick introduction and hand the baton off to them.
So Ian McCloud here is a Director of Operations at Visikol Inc. It is a BICO Company. He earned his Bachelors of Science from Rutgers University and has worked at Visikol for 3.5 years. And then Mr. McCloud is the head of the Ex Vivo and Manufacturing Department, as well as being the Lab Director of General Laboratory Operations. His team developed the precision cut liver slice protocol which they are going to talk about today, and also the logistical support system which Visikol offers to its clients. Visikol has worked closely with Precisionary Instruments to integrate the Compresstome as the key tool in the PLCS Assay Workflow. And so we are very very excited to welcome him. In addition we also have Griffin Ferrara here and he is going to talk a little bit about Visikol at the beginning and introduce you to their company. He is the Business Development lead, and without much further ado I am going to hand it over to Ian and Griffin. Thank you so much, and a quick note for all of you who are attending, Please feel free to use the chat box to ask questions or add comments. We will have a 10-15 minute Q&A Session at the very end, so hang on until then and we will be sure to get your Q&A asked and answered.
Alright Ian and Griffin it’s all yours.
Griffin: Excellent. Thank you Abby for that great introduction and hello everyone my name is Griffin Ferrara and along side Ian today, and in partnership with Precisionary we are going to talk to you a little bit about our precision cut liver slices, what exactly that is and what that assay provides. As well as using the Precisionary Compresstome in that workflow. So you can go to the next slide, Ian.
Griffin: So, before we jump into it I want to explain just a little bit about our company and who we are. Visikol is a contract research services company focused on leveraging advanced imaging, 3D cell culture assays and digital pathology to accelerate the drug discovery and development process. Our research services can be divided into 2 key areas, which are our Advanced Imaging and Digital Pathology Services which includes Multiplex tissue imaging, confocal and light sheet, and digital pathology. Our other main service area is Advanced Cell culture Assay Services which include 3D Cell Culture Assays, Ex Vivo Assays which we will be talking about today, as well as high content imaging. And just for a more general overview of who we are, we are a US based lab in Hampton NJ, about an hour outside of New York. We have a worldwide customer base that ranges from startups to small, medium, and big pharmaceutical companies, as well as prestigious academic institutions. We have an expertise in drug discovery, assay design, oncology, immunology, inflammation, and liver disease-this is where a majority of our projects come from. And then in terms of our unique technology and patents, we have a number of these that help with all of our workflows and really help us to provide clients with the best information possible. Ian, you can go to the next slide.
Griffin: And so, as a whole the way we work with clients is whether you’re sending us tissues or slides or potential therapeutics or data sets, we are going to leverage all of our key tools to make sure that what we are providing you is the most insightful information. So whether that is our advanced cell culture models, our AI machine learning and image analysis, or any of our advanced imaging tools, our goal is to be as insightful as possible to really answer Researchers’ questions to help deliver better therapeutics and ultimately better patient outcome. As a whole we are a part of the BICO company, we are one of 12 daughter companies under the BICO umbrella. It’s been a really exciting summer for us we have access to all of the great BICO resources and it’s been really helpful for us to provide better outcomes to all our clients.
And so with that, I will pass it over to Ian to kind of walk you through the Precision Cut Liver Slice Assay.
Ian: Sure thing Griffin and thank you for the introduction Abby my name is Ian and I am going to talk to you guys today about the Precision Cut Liver Slices Assay and I think an important place to start when explaining why the PCLS assay is such a powerful tool is to explain how we help our clients choose which models and assays are best suited for their research needs.
So we start with
- Biology: We decide what model is most appropriate. This isn’t as easy as it might sound, it’s not as simple as ‘human’. Done. Is this a Toxicology Assay, where all we need to know is does the compound kill the cells? Is it a Migration Assay, where we need to see in 4 dimensions? Is the compounding getting where we need it to go or is the compound causing cells to behave and move in in a beneficial way like we want them too? All these questions are significant and costs are a big part of this determination, as is relevancy, repeatability, and validation.
- End points: What data is sufficient? More isn’t always better. We can generate Terabytes upon Terabytes of data, but if it doesn’t identify the key variables and how we can influence them then you’re not going to be able to answer any of the research questions required to move forward. So, we offer tons of endpoints like RNA-Seq, qPCR, viability assays, multiple immune-labeling, traditional histopathology, and others on demand.
- Reporting: Is the data communicated in a useful and actionable way? This one kind of goes without saying, but if overlooked can completely negate an otherwise well-executed experiment. We make sure our clients are fully understanding of the results of our work with well-formatted data and figures, a clean report, and a debriefing conference call and as many follow-ups as required to make sure our clients are satisfied that their next steps are going to be a clear transition from the project that we’ve just completed with them.
Ian: This slide is a useful visualization for the way that we think about the costs and the benefits of the different models we use. You can see the case of 2D cell cultures, where throughput is the highest and the cost is the lowest, but of course the relevancy is also lower as a single cell monolayer isn’t going to behave like a three-dimensional human organ system. Next, we have our 3D cell culture models which are probably the best balance of the 3 factors- they have a moderate throughput, a moderate price, and they have a significantly improved relevancy over the simple 2D, but they’re still not an in vivo human organ. Lastly, in red, we have our ex vivo models which is a broader category that PCLS fits into. Ex vivo has a higher cost and a significantly lower throughput, but it has the best relevancy out of animal models.
Ian: Here is just another visual representation of the spectrum of relevancy, with 2D as being the least relevant but the highest throughput and ex vivo PCLS being the most relevant. So let’s focus on those PCLS models and what makes them so in vivo relevant. Well, they’re actually live human tissue-how much more relevant can you get? We produce these non-transplant- sorry- we procure these non-transplantable human organs with less than 6 hours of cold ischemic time, or time outside the body, and then we process them into uniform quarter millimeter slices. We culture them in special 24-well plates, where you can treat them with whatever therapeutics the clients require. After a set culture time we can collect the slices and the media for different, all kinds of different, end point analysis.
Ian: Before I dive into the nitty-gritty of the PCLS protocol itself, I just want to take a second to highlight the need for this model. One of the most pressing diseases facing the modern world is fibrosis and liver fibrosis, particularly non-alcoholic fatty liver disease, has a rapidly growing prevalence around the globe. There is no FDA approved drug to treat NAFTAL or NASH, but many Pharma companies are putting a lot of effort into developing these treatments.
Ian: And beyond just screening for compounds and treating liver diseases there is a much broader concern that all drugs have to be tested to prove that they aren’t an acute liver toxic compound. You can get really far and think you’re really onto something and have a bit of a red herring, thinking that you have a great compound on your hands, only to find out that it would kill the patient; the medicine is worse than the disease, if you will. People want to be able to fail fast. Over 99% of drugs fail before they get to phase 3 trials, so for a relatively low cost but very high relevancy model that’s going to be really attractive to the kind of researchers who are nearing the pre-clinical stage and don’t want to get let down later on by some toxicological oversight.
Ian: So, the PCLS model. I’m going to go over what the assay is, how the Compresstome VF-310 is used, and then 3 examples of different kind of materials that it can handle; normal human liver, psoriatic human liver, and diseased induced mouse livers.
Ian: So, the assay is based on a standard 24-well plate. Each well is going to be filled with one of these Transwells. A Transwell is a little plastic insert cup with a permeable membrane at the bottom. Once the liver is received and processed each Transwell is then loaded with a single 250 micron thick liver slice. Each well is going to have enough cell culture media to keep the cells alive for at least a 24-hour period and may, or may not, have treatment compound in it. Once the plate is full with all the slices and the media it’s loaded into the incubator for a set time or the time points that we have previously established with our clients. At these points, we’ll either then give them fresh media, we could re-dose them with additional compound, or we could end the trial for that particular slice by collecting the slices and collecting the media for subsequent endpoint analysis. The main endpoints that we do offer in-house are going to be RNA-seq, qPCR analysis, ELISAs, Immuno-labeling on high content imagers. We have a highly multiplex histopathology panel that we can apply. HPLC and LCMS analysis for pharmacokinetics, and of course what other endpoint is most important to our clients. If they come to us with a specific objective in mind we’ll make sure to adapt the protocol to incorporate that. We can also send the samples back to the client for their own analysis, we would snap freeze the tissue samples or the media in liquid nitrogen and we can ship them on dry ice to whatever in-house facility our clients want to conduct the end points of their research for. Turn around for our reports are generally 2-3 weeks from the end of a culture cycle.
Ian: The main attraction here, the Compresstome VF-310. So, what makes it special? It’s because ex-planted liver is really tough to cut, and the uniformity of slices is critical. So, we’ve tried lots of different vibratomes, we’ve tried getting and modifying a microtome to enable viable tissue to be processed and none of them could handle even the most cooperative tissue samples. The tissue is too tough and it just can’t be held in place enough to get precise, or precisely cut slices, at the exacting conditions or thickness. The Compresstome was special though in that the sample can be loaded without damaging the tissue, and lowering its viability. So as the sample is advanced by extremely small distance, a really tightly controlled distance, fractions of a millimeter at a time, we can control it down to. It’s pushed through a nozzle just at the end that compresses the tissue from all sides simultaneously. This enables the tissue to be held in place so that when the blade comes in contact with it, the tissue can’t slide out from under it and it forces a much better and more controlled cut. Again, liver is a bit of an odd material, it holds extremely well to itself, but it has no support what-so-ever and it will just immediately get pushed away by anything coming in contact, even a seemingly sharp razor blade. So it’s really hard to get a blade that will catch instead of slip-I mean the tissues kind of can be slimy. The best part about this piece of equipment is that it is automated. Once you dial in your speed and your oscillation rates and the thickness of the slices you want there’s an automatic mode that you can put a switch into that will just keep the arm advancing for you until all of the sample is fully extended and all of the slices have been created from that core. We can usually have a full project’s worth of slices-that’s a few hundred slices, 500 slices or so, done in 2-3 hours. It’s pretty rapid too, if we put a few of these in series we can handle just about any of our client requirements with minimal cold ischemic time.
Ian: So, how do we use it? This is kind of a very brief run-through of the protocol itself, but I think it helps to visualize what the parts of the machine are and how it fits into our end-goal of creating these uniform slices. So, an organ is procured and grossed into slabs with a thickness of about 10 centimeters or so. It’s a little bit thinner than your index finger. The slabs are laid flat and we have these biopsy cores that are precisely five millimeters in diameter, and they are able to create cores hopefully of about 5 centimeters long, that as we generate them we store them in an ice-cold dish of UW solution. This is a specialized solution that was manufactured, it developed I believe many years ago as like the pre-eminent organ transfer solution that keeps live tissues in good shape. Then, each core is selected dried ,on one end and glued to the sample tube plunger. It’s then embedded in a liquid, low temperature liquid aggro-solution, and surrounded by that metal case you can see to the bottom right of the image. That is the plunger and the sample advanced casing. We then grab a cold block from the freezer and it immediately freezes, or solidifies the aggros, locking the sample in place and this is what allows for that funnel effect where pressure is applied at all ends-all sides-at the end of the nozzle as it’s extruded. The sample tube is attached to the Compresstome and the collection basin is filed with more ice cold UW solution, that is so as the slices are taken off of the core they land into a cold solution and are never exposed to air and it helps with viability very significantly. We attach a fresh razorblade to the cutting arm and the piston is advanced to make contact with the sample tube plunger-this is just getting everything right to the edge of the setup, before we press the ‘Go’ button. We have a generic idea of what the advancement speed and oscillation rates should be, about 4 and until we get-you know, each liver is slightly different, the material behaves differently and sometimes we have to adjust from there, but we find that that’s a very good starting place and you usually don’t have to venture too far in any direction before you start getting good, consistent slices. The first few cuts will likely fail as the Compresstome starts to really get into its groove and we dial into those measures. We’ll adjust oscillation rates, make it cut faster or we will slow down the speed of the blade so that there’s more time for the blade to contact the tissue. But, you know, one slice at a time, we’ll take it until we start getting consistently high-quality slices. Once we’ve gotten a few of those high-quality slices in a row, usually only takes 3 or 4 slices, we switch it over to automatic continuous slices and the Compresstome does the rest.
Ian: Okay, so, I want to kind of wrap it up, or tie it back in, by talking a little bit about the versatility of this assay and how we use the versatility of the Compresstome to enable that. Being able to load different states of tissues allows us to be able to offer multiple models.
First we have a normal, healthy, non-transplantable human liver. For whatever reason, something about the condition of the donor made it so that the liver could not be given to a recipient. These organs are still very useful for research. As I said before, we need to screen all drugs to be sure that they aren’t hepatotoxic, so there’s that, but also a healthy liver can be transformed into a diseased state that we then create as a model. We can turn healthy liver into fatty liver by incubating it in a free fatty acid enriched media, this will induce collagen deposits, basically laying down scar tissue. We can also use proteins like TGF Beta to mimic the actual signals that your body would produce when undergoing the advancement of this NASH disease state in, you know, normal case of disease development. The advantages to this model is it’s going to be much easier to produce healthy liver, or to procure healthy liver, and you can largely standardize the level of disease you induce-you have a controlled amount of FAA or TGF Data that you add in and that leads to a pretty uniformed disease state throughout whatever tissues you’re subjecting it to. But, the disadvantages here is that it’s just not the same thing as a naturally occurring disease progression, so whether it’s different metabolic pathways being activated, or whether it’s simply just the feeling of the researcher that they are not as directly translatable as they would like them to be, can be drawbacks that some clients are concerned about.
Ian: A diseased human liver, very tough to cut. It’s the best possible model though for a diseased liver because it is a diseased liver. It’s just the ultimate relevancy, and with all the endpoints that we can look at like H&E or Trichrome or Lipitox here, the multiplexed immune labeling and the RNA-Seq data researchers have great confidence that if they start to see a strong signal they’re really likely to be on to something as they continue down the drug development pipeline. The drawbacks are going to be, besides its processing difficulties, it can be very difficult to procure. A lot more difficult than you might expect. You also have to consider the degree of variants you will encounter between patients, even if it’s just the same disease people are different, even the way they get sick is different and so once diseased liver to the next can be pretty significantly different, and that’s something that we have to make sure that our clients are aware of and that they design their studies accordingly between different trials.
Ian: And then lastly, I just want to add the example of our mouse model. It’s cheaper than the human model, and it’s more standardized since mice are bred to be uniform. We also have a nearly unlimited supply, and it’s on-demand, if you will. But, of course, the disadvantages are that it’s a mouse, not a human. You can induce almost anything in a mouse, and you can cure almost anything in a mouse. But, that rarely translates 1:1 into human cases. Shorter life spans, different metabolic pathways, and ability to excrete things-it’s just not as easy as ‘if it works in a mouse it works in a person.’ But, there’s still advantages to this of course. There’s still a lot of conserved immuno-expressions, immuno-genetics, that do get preserved from mouse to humans. And so it can be a really attractive option for researchers who don’t think that they necessarily need the type of detailed data that a human model would give. But, of course, there’s also the development pathway for this disease is presumably not the same as it would be if we were to just feed mice extremely sugary and fattening food. It’s created by injecting mice with CCL4 directly into the liver, which is going to generate a really controlled and uniform fibrosis response. This again is kind of like inducing the disease with the human tissues, in that uniformity is good for screening but it’s you know, just not the same as the naturally occurring disease progression.
Ian: That is the overview of the PCLS assay, I hope you all enjoyed and learned a lot about the different models and the research conducted by Visikol and enabled by Precisionary Instrument’s Compresstome.
Abby: Wonderful! Thank you so much Ian and Griffin. I didn’t get any Q&A from the chat but I want to open up the floor to everybody. Feel free to unmute yourself to ask any questions for this webinar.
I had a quick question while we wait for things to roll in-which is, you know, after you make a slice how can you tell if it’s healthy or not? Are there specific liver stains that you would use to look for degrees of oxidation or enzyme activity?
Ian: Yes, absolutely. In post-culture we have many endpoints that will test for things like viability and general disease markers that we will stain for. In the moment during slices it’s much less clear the degree of viability of the tissue we’re looking at, especially when it’s more in the advanced disease state, it can look pretty, you know pretty rough shape. But, what it really comes down to is the quality of the slice-whether it’s a full circle, even in thickness from end to end versus if the tissue is very uncooperative, it will be either a crescent moon or it won’t be a full circle and that’s how we know that we are dealing with a difficult tissue that we need to kind of dial in the exact parameters of the Compresstome to handle.
Abby: Got it-and then for precision cut liver slices, when you start using them and you put them in your well plates, how long do you keep them around for for experimentation?
Ian: Yeah, it totally depends on what the client is looking for. So we’ve had instances where the client is interested in five minutes after culture. They asked us to try to do a dose and then a collection with a 5 minute time point. So it can be anywhere from just the immediate contact reaction. Our more traditional timeframes are going to be 4 hours, 24 hours, 48, 72, but we have had success in keeping these cultures for 168, or 7 days, is the longest that we’ve gotten our cultures to still show some degree of viability.
Abby: That’s amazing, and then you have to incubate them to keep them healthy right?
Ian: Absolutely. Every day we need to swap out and give them fresh media and we have a very tightly controlled incumbent setup with supplemental CO2 and, you know ,a temperature log to make sure we keep good 37 degrees Celsius, things like that.
Abby: Very nice. Well, I don’t have any more questions and there aren’t any more in our chat. I wanted to let everyone who is attending here know that if you think of other questions-Oh wait! We do have a question!
Question 1: Hello, nice talk! I was wondering what kind of aggros do you use to embed the tissue? I find it’s very floppy when I cut the tissue. It’s very tricky yes, I usually use 2% but I think it’s not enough.
Ian: That’s right, that’s right, we came across the same issue. We initially, um, it was suggested that we should try 3% and over time we have decided that 4% low temperature aggros works best for our purposes. It flowers pretty well, it’s definitely viscous, but it definitely can flow through a modified pipette tip. We just take a P1000 and we cut the tip off of one of the disposable microfiber tips, and it’s able to pull out of the water bath pretty well. We keep it in a water bath at around 40 degrees Celsius. That keeps it pretty well in its liquid state and it loads into the sample tube holder very effectively, and as long as we have that ice block or that cold metal block to immediately solidify it it works really well at the 4% concentration.
Question 1: 4%. Okay, great, thank you.
Abby: very nice. Anybody else? If not we can end right at 10:30, and people can have a quarter of an hour to enjoy the rest of their morning.
Question 2: How about one more question?
Abby: Hi there!
Question 2: My name is Thomas. Great talk, I’d be curious about the imaging part. How do you handle it, and what type of imaging do you provide?
Ian: Sure, sure. So, there are 2 directions we generally take our imaging, we can take it on the more traditional histopathology direction, where we will embed it in paraffin wax, we’ll go through the microtone process of generating slides and then we can do simple H&E or trichrome staining lipotoxic, a lot of the more standard histopath stain, and then we can put it on a slide scanner and do some Y-Field or Immuno-fluorescent imaging in a high-throughput way. We can go through many, many slides per day. That’s one direction that a lot of our researchers like to take it, it’s a fairly cheaper endpoint that a lot of researchers use it’s an easy add on to the larger experiments. But where we really find the most information can be gleamed is when we apply immuno-labels like Highly Multiplexed Immune Panels to a high content imager. So we will take much larger slices, perhaps even a full slice, a 250 micron slice, we will multiplex stain it and we will apply our Visikol clearing reagents in order to get a lower refractive index and allow for our high content imager usually a Confocal microscope to penetrate a little deeper in the tissue and we can get a full, you know with a series of Z steps, we can get a full Z stack, if you will, of all of the different images. We can recombine that with software and we can have a 3 dimensional image of any of the protein structures that we’ve labeled for, and we’ve done some really beautiful images there that have really told a compelling story about what’s going on with our clients’ therapeutics.
Abby: Oh great! Anybody else for a Q&A? If not, this session is recorded and will be available on the Precisionary website, there will be a tab called ‘Webinar’ and I think that Ian and Griffin are also going to work with Visikol to make it available on their website as well. And as always, don’t hesitate to contact us and reach us to us by our emails here if you have any further questions about either the Compresstome or Precision Cut Slices. And with that I am going to close up shop, and thank you so much everyone for attending and a huge, huge thanks to our speakers today Griffin and Ian from Visikol. I hope you guys have a great week!
Griffin: Thank you Abby.
Ian: Thank you Abby.