Hello, welcome to today's webinar. It's a collaborative webinar between Promega and UPM Biomedicals. My name is Simon Moe, I'm an Associate Product Marketing Manager at Promega. The title of today's webinar is A multi Asset Strategy for Monitoring Long Term health in a 3D liver model. Before we begin, I would like to cover a few housekeeping items on your screen. There are multiple windows, all of which are movable and resizable, so feel free to move them around to get the most out of your desktop space. We also have a few ways to be interactable during this webinar. You can submit a question at any time during the webinar. We will answer these during the live Q&A session at the end. There's also a resource library on your screen with a list of helpful materials, which also includes a copy of today's presentations. Feel free to download any of those resources or bookmark them as links that you may find useful. After the presentation, there will also be a survey. I would highly encourage you to fill that out. It'd be really helpful to us to continue to produce really great webinars in the future. And then we'd also encourage you to share this webinar with anyone that you think would be interested to get the word out so that they can learn a little bit more. And now to introduce our featured presentation, speakers have a little bile for each of them. The first one is Doctor Jonathan Sheared. He's an application scientist with UPM Biomedicals working with the Grodex Hydrogels for 3D cell culture and is really passionate about its use for the replacement of animal derived matrices. He previously worked at the University of Reading as a postdoc researcher with Darius Wadera, using the Grodex hydrogels for a wide range of cell culture applications and model developments. During his time at the Reading University, Jonathan wrote many application notes, tested and developed many cell culture protocols, as well as gained an appreciate appreciation for the technical handling of the Krodex hydrogels. He's now part of the Life Science team at UPM Biomedicals. Next we have Doctor Darren Haywood. He's Product Manager at Promega UK. He received his PhD in Neurobiology from the University of Bristol in 2003. His research was focused on investigating the involvement of signaling pathways involved in mediating neuronal cell death. Currently is a product Manager at Promega UK. He manages the cell health portfolio and has a particular interest in tools to monitor 3D cell death. With the demand for more physiologically relevant models, there comes a need for more accurate and sensitive cell based assays that can deliver reliable results in complex 3D micro tissues. Darren works to support customers who wish to investigate cell health in both 2D and 3D cultures, and our third speaker today is Doctor Johanna Lempe. She's a scientist at Life Science Applications for UPM Biomedicals, and she has a background in cancer and virus research. During her doctoral and postdoctoral projects, she worked with primary cancer samples and became interested in 3D cultures and in reducing the number of animals used in research. Johanna is now part of the product development team and uses her experience for developing models and Grotex hydrogels and combining them with a wide range of readout models. She aims to help scientists obtain more physiologically relevant results with precise and reproducible assays. So with that, I will turn over the stage to our first speaker. Brilliant. Thanks very much Simon for the introduction and hello everybody. It's great to be able to join today with the premiga team to present and to talk about the the Gradex hydrogels. So I'm Jonathan Sheared, an application scientist with the team and I'll talk to you today about UPM Biomedicals, the Gradex hydrogels, their properties and a couple of examples of liver culture applications prior to handing over to Darren. So UPM Biomedicals is a part of the UPM Cumulate Company. UPM stands for United Paper Mills and it's a forestry based industry company. UPN Biomedicals is a start up area within that where we have now been actually going for 10 years with the the products and just to talk about the the products. So we manufacture nanocellulose from Birch trees. So we take the Birch wood, we break that down into wood chips that gets processed into cellulose and further processed into nano cellulose, nano frivolous cellulose. And from that, we then have the products which I'll talk about and you can use those for different cell culture applications. So really you've only got two ingredients within the product. So the product range that we provide, from the left you can see the Gradex, the hydrogels for 3D cell culture, which are the Gradex range. They have Gradex, Gradex T and Gradex A. Today, Johanna will be talking about the use of Gradex for the different assays. And paired with that, you have grades. That's a cellulase enzyme that can digest the Gradex and release the cells and it can also be used for the Gradex T and Gradex A. We also have bio inks for 3D bioprinting. So if you needed a more viscous version of the hydrogels, then that is the range that you can use and those can also be used for cell culture. We have a wind dressing product which is also on the market called Fibdex. We also provide the hydrogel for OEM, which means that it can be included in the product that you might want to create. And we're very excited that this year we will be releasing A clinical hydrogel for inpatient use, which is also just a nanocellulose based product. So what can you use these products for? As you can see here, many different cell types have been cultured in these great X hydrogels and in many different application areas. So today we're highlighting the culture of liver cells and for a sort of screening assay heading towards high throughput screening. So I hope that you're looking forward to that. Just to go over the key material properties, the Gradex hydrogels, what we'd like to highlight is specifically the sheer thinning property that means that the hydrogel is in, in its rest state. The fibers within the hydrogel are interconnected, They are in suspension and then when you apply force the hydrogel changes its properties where it's sheer thins it becomes more like liquid as a force is applied. So when you are perpetting for example, or extruding the hydrogel from the syringe, it actually starts to the fibers align and flow in the direction that they're being forced and you can embed the cells and that allows the cells to be carried throughout. As you can see on the right with an ACM image that is mesochymal stem cell embedded within the grade XT. You can see that they have, they really are quite happy in there. They've got a lot of ECM and anchor points and so on. Something else to highlight them with the hydrogels is the animal free nature. So when we manufacture the nanocellulose as I showed you earlier is it is only manufactured with nanocellulose and water only. So it is ultra pure, there is no endotoxins and no animal derived DNA or proteins or RNA or anything like that. So therefore it does not behave the same as animal derived hydrogels. It stays in its gel form when you are using it and therefore we would class it as room temperature stable. So that means that you can handle it at room temperature. You don't have to cool it down for it to be in its liquid handling state. And on the screen you can see that is a perpetting robot that is seeding some cells which are embedded within grade X from A through into a 384 well plate. So you can see that all of this can be done very easily at at room temperature. So that's all part of the shear thinning material properties. So now to go on to the hydrogels, this is actually with the grade X range they get supplied in a syringe. That's because of the viscosity and the differences between these are, as you can see from left to right. I'll just go through them briefly. Grodix, which we're talking about today, we class that as a native hydrogel and the fibers are not charged, it's neutral and charged and neutral pH, so 1.5% weight volume. And you can easily image cells within this with bright field and fluorescence. And you can retrieve the cells with the Grodase cellulase enzyme. And then we have the Grad XT and the Grad XA. Those are both anionic. They have a negative charge on the fiber and that's gained during the process of manufacturing. And some cells prefer the Grad XT and others prefer Grad X. But many different cells also will grow within both. We actually will share any protocols with you if if you if you need them. And both the Grad XT and Grad XA are great for bright field imaging and fluorescence as well. And you can use the Grad aids, but please connect with us and I can give you some hints and tips on how to how to do that. So now, how might you like to set up your essay? What I'm showing here is several different options. Johanna will be talking about how the cells were embedded within grade X, so you can prepare a solution of the Gradex hydrogel with your cells embedded, and they can grow and proliferate and form spheroids and so on through the culture time. Alternatively, going clockwise, you can see the spheroid in the bottom of the well where a spirit has been preformed, and you can simply add Gradex or Gradex tea on top of that as a covering. So that helps to, for example, stop a spirit from wobbling around the well. If you're using it in a in a high throughput fast imaging system for example, you can also use it as a wet or a dry coating and so you can dehydrate it onto the surface. You can use it with transfer inserts within the inside and on the underside of the transfer insert, and you can also use it in that setup for migration assays, as you can see as well on the bottom on the bottom row. So there's many different ways that it's been used, and because of the room temperature stability, it makes it very easy to to handle and to set up. This is just an overview that I'd like to show, demonstrating the process of how you would set up your essay. And you can do this both manually in a smaller volume as we're demonstrating here with an Efendorf, or alternatively in a larger volume with A50 mil Falcon tube. And the main steps are that you would have your media your diluent and you could use something such as just basic DMM or RPMI to have a stock and then you would add your gradex mix that thoroughly. That needs to be done for about 90 seconds to make it nice and homogeneous. And then when you add your cells and they will stay in suspension, they will be in the three-dimensional space. In suspension they do not gravitate as some of the animal BME matrix and the before they get cross-linked the cells can gravitate down to the bottom but that doesn't happen with the cells in gradics. And when you've got your cells within the suspension then you can easily add that to the to the wells of the culture vessel that you are choosing and this can be from 96 through to 1536 well format and others. So, so a typical workflow and also what you might want to do with the with the cells, what I show on the bottom, you'll see there you might have your cells embedded within the hydrogel with your media phase on top. So you have this biphasic setup. In that top media phase you can do media exchange. You can also do treatments, something that's quite cool as you can easily turn over the plate and add drugs, for example with an echo dispenser. And so it really depends on the workflow that you you're doing. Then you can fix your cells whilst they're embedded, stain them and add them into an imaging system. Or else, as we've as we've shown, as we are showing today, do luminescence or fluorescence readouts. But it allows for a faster workflow. It's easy to stay in your cells and you don't have to worry about what stains you're using that they shouldn't be any cross reactivity with the ECM and for antibody binding for example. So this is just I wanted to give a bit of a history and example of what we've done previously with some of the liver cultures. Now these span from as far back as 2014. So over the last 10 years, liver cells have been successfully cultured within the Gradex hydrogels and this includes HEP G2, HIPAA RG primary human hepatocytes, Liverpool hepatocytes in many different formats. And what we've seen consistency consistently is albumin secretion from, for example, Hip G2 and the hip RRG over 4 and 25 days respectively, the Liverpool primary hepatocytes on the bottom and the orange graph. And you can see that there they formed the spheroids beforehand, then they cultured them for an extra 28 days, so that was a total of 35 days culture and they looked at the album and secretion of the time. And then we also had another project where we had to look at the format that you might choose to see the cells, whether it will be on top or embedded within and does it make a difference if you use AU bottom or a flat bottom? But what we saw was when the cells are embedded, they seem to prefer that and they had a better album and secretion between 7 and 14 days as we've highlighted with the boxes there. So really, really interesting you can see a lot of the stage as well in the application that's in the publications which we have supplied. We've also seen that the cells remain viable and they are they they will express H&F for Alpha S3A4 and they also the Sep 3A four is active. So you can see the live dead staining in the top left and that was compared to hyaluron and gelatin and and compared to 2D. So you can see that on the very far left the NFC is nano fibula cellulose, that's the greatest hydrogel. The cells were happy, they were viable and they were, they had good enzyme activity, HNF 4 gene expression and SIP enzyme gene expression as well. And then the the three A4 activity as you can see in the bottom right over 14 days within NFC hopefully you can see I've tried to highlight that with a green line and as I've said, you can see all of this within the application that's in publications or else please feel free to post a question in the Q&A box and I'll be happy to answer that as we go through the the rest of the presentations. So here I just wanted to give an example where the Nexus group from ETH Zurich were able to embed primary liver cancer open weight. So they were human hepatocellular carcinoma cells and they embedded them, they chose grade XT for this setup. They actually did test grade X as well and they embedded them within the grade XT directly seeded them into a 1536 well played format and then put that through a drug screening panel that they were having a look at with 1250 drug compounds. Actually because of COVID, they had the repeats only. They were only able to repeat them with a year as a gap, but that was great because it showed really good repeatability with a low Z factor, Z factor. And as you can see there, there was a really great response. So they've continued to use the Gradex for that. And actually the setup that they had helped to reduce the total cost of their setup as well. And just to go into that, my last slide is with a typical setup of Gradex, most users were used between 0.2 and 0.8% as an example. But what I've showed here is 0.2 and 0.5%. And for a for a 10 mil syringe of grade X, it's a 1.5% stock. If you did a 0.2 or a 0.5% dilution, that's just up to about a three fold dilution or A5 fold dilution around there. And you can see that in the different plate formats. For example, a 96 Watt plate you can get around 7.8 plates worth of and then I see those if you're using 0.2% or at 3.1 plates if you're using 0.5% Gradex and that just then we've tried to give your cost breakdown per well. So I hope that that's been a clear description of the Gradex hydrogels, how they've been used with liver cultures in the past. Now I'm going to have a couple of questions. I'd really like to know from the audience if you could please vote. So here's the first question, are you working in 3D or with animal derived hydrogels and are you looking to migrate over to animal free 3D culture? So the first options you've got a yes I'm looking to, I'm working with 2D and I'd like to move over to three DB. I'm working with animal Dr. Hydrogels and I'd like to move, move, move over to animal free CI use a non matrix that would be for example, you bought a Mueller and I would like to use a matrix and then D is. I am not interested in changing at the moment to a 3D culture, so I'll give a few moments if you could vote for those. If you could submit your votes. We've got 10 so far. Give that a little bit of time. Hopefully most people have had AB and C If you vote D, it'll be really interesting to to understand why. So do you also put that in the Q&A? If you don't want to change over, I'd love to know why. OK, so we've got just over 50% attendees voted, so I'm going to click over next and have a look at the results. So for those who have joined live so far we've got 22% have said that they are currently working in 2D and would like to move over to 3D 41%, just over 41% have said that they are using animals arrived and would like to know to move over to 3D animal free and 22% are working with non matrix. And 14% who don't really want to move over at the moment. So yeah, send in your send in your comments in the Q&A please, will be really interesting to know. OK, I have just one more question then. And by any chance are you working in high throughput applications? Yes, A, yes, you're working in 2D high throughput. B. Yes, I'm working in 3D high put high throughput or C, no, I do not work in high throughput. So it'll just be really interesting to know if you are or if you're not and if you are is a 2D or 3D that helps me to to understand the audience. Also if you wanted a follow up call, we can actually help to tailor how we can talk about the application of the Hydrogels. As you saw from that previous example where they were using 1536 well format, this is a low 96 well format. OK. So again just over 50% attendees. So now we can see from the results that most people are not working with high throughput currently with the live station and a few are working in 2D and then so that's 13.5% and just under 30% are working in 3D. So that's great. Thanks very much for your attention. I'm now going to hand over to Darren and he's going to take over from here. Thank you, Jonathan. So my name is Darren Haywood and I'm one of the Product Managers at Premier UK. And today I will be presenting on our Multiplex 3D optimized cell based assays to get more biologically relevant data from your 3D cultures. So to fully understand the jigsaw puzzle on the right hand side, you need to have all the pieces of the jigsaw puzzle in place. So this can be a great analogy for understanding the effects of compounds on your cells for example. So to fully understand and to get a complete picture of the effects of your compounds on your cells, you need to measure more than one parameter at a time, as shown here. Now if any of those pieces are missing, the results of tank can be quite misleading. So you may see a decrease in viability for example, but does that translate into an increase in cycle in insights? Toxicity for example? So measuring multiple parameters in your cells provides a more complete picture of what's going on with your cells. So multiplexing can be described as running two or more assays to gather more than one set of data from the same sample. So when multiplexing cell based assays and to get the most from your cells, certain criteria have to be followed. So, for example, the assays that you Multiplex, they must be biologically and chemically compatible, and to ensure that the assays do not negatively impact each other's cellular measurements. Also the signals from these assays, they must be spectrally different, or if they're not, they the signals must be measured separately. And quite importantly as well, the multiplexing assays they must fit in the available volume of the well. If not, then they must be be separable. So how can multiplexing cell based assays help you? So multiplexing cell based assays, They obviously give you a more complete picture of what's happening to the cell and when you're multiplexing the data it gives you, sorry, multiplexing the assays it gives you more data from that same sample. So in the long term that will obviously save you time, but it will also allow you to conserve your samples and by multiplexing assays, it means that you can reduce the 40 interpretation or any ambiguity that you have in your data. And also multiplexing assays, you can use one assay as an internal control, we can normalize against that. So in my presentation today, I in the first part I will address the first three points that I've highlighted and in the next part of my presentation I'll highlight the last two points. But before I do that, I'd like to introduce you to our portfolio of our 3D cell based assets. So here are 3D optimized cell based assays. So we have assays for monitoring cell health and we have assays for for monitoring metabolism. Now with these assays they can fall into three modes if you like. So we have these assays can either be lytic endpoint assays or they can be non lytic media sampling assays or they can be non lytic lifestyle assays. Now with some of these assays they can either be lytic or media sampling depending what assay that is and what protocol that you follow if it's a lytic or media sampling protocol. Now in addition to that, we have manual and automated systems for analyzing the genome, DNA, and also we have automated and manual systems for monitoring changes in gene expression, all in 3D. So promigas, sorry, what in this slide, actually, I'd like to go through the those 3 modes in a little more detail. So it gives you an understanding of how the assays work. So we have our lytic assays which are endpoint assays. So they measure an enzyme or biomarker inside the cell. So here you have your 3D cells in culture, you add in the assay reagent, it lies as the cells and it produces A luminescence signal. And that luminescence signal is then proportional to the enzyme or biomarker inside the cell. So with these assays, you can generate a data point per assay. So moving on to the nominated assay, so we have the media sampling assays. So they measure an enzyme or a biomarker that's been released from the cell into the cell culture media. So here you have your cells and culture. Then what you do is you take a small volume of that media and you add it into a separate well. You then add in the assay reagent that results in the luminescence signal. And then that luminescence signal is then proportional to that enzyme or the biomarker that's been released from the cell into the cell culture media. Now because you're only taking a small volume of media from that initial well, that means that you can do multiple sampling from that that well over time. So with these assays you can generate a single data point per sample. Now moving on to the non lytic assays. So these are our lifestyle real time assays and they continuously measure an enzyme or bio marker in the Sam in the same sample well over time. So here you have your 3D cells in culture and you add in the assay reagent that results in a luminescent or fluorescent signal. Now again because this is a non lytic assay, it means that you can continuously measure that that generation of signal over time. So with these assays you can generate a single data point per read. So with Pramiga cell based assays, yes they're validated for use in 3D cell cultures. They're really simple to use. So you can they follow this ad mix and measure as a format. They are scalable. So the majority of our assays can be used in 96 and 384 well plates, but we do have some assays that can be miniaturized down and used in 1536 well plates. The majority of our assays used by luminescence. We do have some that uses fluorescent signals, but they're incredibly sensitive and just they're multiplexing friendly and with these assays to read the signals you don't need a specialized piece of equipment to do so. You just need a standard plate reader that has luminescence and fluorescence capabilities. So in this example I'd like to show you how our non lytic lifestyle assays can be multiplexed, allowing you to generate more data from the same sample, saving time and conserving cells. So in this example we've used the real time glow empty cell viability assay and we've used the cell top screen cytotoxicsteine assay and the cell top screen cytosoxicity assay is one of the assays that Johanna is used in her liver model. So looking at the real time glow empty cell viability assay. So this assay determines the number of viable cells in culture and it does that by measuring the reducing potential of the cell. It's a very similar mechanism to an MTT assay. So here you have your 3D cell in culture. This assay has two components to it, so it has a pro substrate, which is the grain molecule on the left hand side and it has nano luciferase, which is the luciferase on the right hand side. So the pro substrate is cell permeable, so it gets into the cell where it's reduced down and then it's exported out of the cell as the substrate for non elusive rays. So that binds to the luciferase generating a luminescence signal and that luminescence signal is then proportional to the amount of viable cells that you have in culture. Now with this assay you continuously monitor that pro substrate substrate conversion to luciferase luminescence signal over time moving on to the cell top screen assay. So this is a cell in permeable asymmetric cyanine dye that you add that determines the number of dead cells in culture. So with this assay you add it to your cell culture medium and when it comes across a dead cell that has a damaged membrane, it gets into the cell and binds to the DNA. Then when you take those cells and interrogate them with your plate reader, you can read the fluorescence and the amount of fluorescence that you have is proportional to the amount of dead cells that you have in culture. Now both of these assays, because the the signals are different, they can be multiplexed and put together in the same sample well in the same plate. So with these assays you can continuously monitor viability and such toxicity in that same plate in real time for up to 72 hours. And with these assays you can either add them when plating the cells or dosing the cells or actually you can use these. You can add these at any time point during your experiment and use them as non lytic endpoint assays. So here's some data that we generated using both of those assays. So here we took MCF 7 cells and plated them in media containing the real time glow assay and the cell tox green assay and these these cells were treated with the topside and the signal from viability in the side. Toxicity assays were measured from the same sample well every hour for 72 hours. So looking at the results from the real time glow assay, looking at luminescence, as you can see the topside resulted in a dose dependent decrease in the amount of viable cells in culture over time. And then looking at the cell top screen toxicity assay read out for looking at fluorescence, we can show that a topside resulted in a dose dependent increase in amount of dead cells in culture over time. Now these have been used in MCF 7 cells which were two D cell. But because both of these assays, the real time glow and the cell top screen have been optimized for use in 3D cells then you can use those in 3D and generate similar readouts and graphs. So with our non lytic lifestyle assays these can be multiplexed together in the same sample plate to generate multiple data points. So to generate the same amount of data using lytic endpoint assays, you would need multiple plates, meaning you need more cells. And obviously this will take a lot of time to read these. So multiplexing live cell assays minimize cell culture use, it saves on time and it saves on your pressure cell culture samples. So in this next example, in addition to generating more data from the Sam sample, saving time and conserving samples by multiplexing assays together, I'd like to show you how multiplexing can be used to reduce false positives leading to more reliable results and how multiplexing can be used as an internal control. So in this paper published by MacKinnon, he reviewed or they reviewed some of the latest developments in pancreatic cancer organoid research and they obviously reviewed some of the latest developments in novel treatment design as well. So in addition to this they also summarized their own experiences and their own work with pancreatic cancer, organoid drug sensitivity and resistant testing. So with their work they took these pancreatic cancer organoids from 11 patients and profiled them for viability and cell death responses to various anti cancer agents. So for viability they use the cell Type 2 glow cell viability assay, and for cell death they used to sell top screen cytotoxicity assay. Before we look at those results though, I'd just like to take you through the essays that they used in a little more detail. So the Cell Type 2 glow in lesson cell viability assay. So again, this is one of the assays that Johanna will be using in her liver or has used in her liver model. So this assay, it determines a number of viable cells in culture. So here you have your 3D cell culture, your 3D cells which are viable. So you add the cell type to glow reagent, which is a lytic reagent, so it lies the cells. It releases that ATP. That ATP is then used in the luciferin luciferase reaction which generates A luminescence signal, and that signal is then proportional to the number of viable cells you have in culture. So moving on to the cell top screen assay that determines the number of death cells in culture as we've already discussed earlier. So looking at their results, so they took these human pancreatic cancer organoids and they treated them with paclitaxel. And as you can see paclitaxel resulted in a dose dependent decrease in viability as seen in the blue trace and it also resulted in the dose dependent increase in the amount of dead cells that shown by the bread trace. So they conclude that the concurrent loss in viability and there was an increase in cell death. Now interestingly when they're treated with gemcitabine, they showed that there was a decrease in cell viability, but there was no increase or no official increase in cell death. So they showed that there was an apparent loss of viability without any significant change in cytotoxicity. So the effect that gemcitabine was having was more of a a such a static effect rather than a such a toxic effect. So they conclude that it would be important to include an actual cell death readout as organoid growth inhibition as a reduction in viability without cell death can potentially be a source of false positive predictions when looking at the cytotoxic agents. So in this next example I'd like to show you how one of our non lytic media sampling assays can be multiplexed with a non lytic lifestyle viability assay. So in this example we'll take a closer look at our metabolite assays and the real time glow empty cell viability assay and how that assay can be used as an internal control. So our metabolite detection assays. So we have assays for looking at glucose and lactate. So we have glucose glow and lactate glow. And those assays can be used to monitor changes in glycolysis or gluconeogenesis. And we also have glutamine and acids. Look at glutamine and glutamate. So we have glutamine glow and glutamate glow. And both these assays can monitor changes in glutam analysis. So how do these assays work? So each metabolite has a specific dehydrogenase, and that dehydrogenase oxidizes that specific metabolite with a concurrent reduction in NAD to NADH. And in the presence of the NADH, the reductase, it catalyzes luciferin from proluciferin. And that luciferin is then used in the luciferin luciferase reaction to generate luminescence signal, and that signal is then proportional to the lactate. Sorry to the particular metabolite in your medium and the real time gloam T cell viability as we've already discussed determines the number of viable cells in culture. So in this example we are measuring the metabolic activity of HCT 116 spheroids. So here we plated the the spheroids out and 1005 thousand cells and as you can see in a no drug control the cells are quite happily checking out lactate as monitored or measured by the lactate glow assay. But when we dose into DG we can see that there's a reduction in that lactate secretion. Now similarly with the looking at glutamate secretion, these cells as measured by the glutamate glow assay. So in the no drug controls we can see that these cells are quite happy checking out or secreting glutamate. Now when we add in the BPTES, we can see that there's a reduction in glutamate secretion. So in this particular time point, we don't necessarily know if that reduction in that metabolite is a compound specific change or if it's a change in cell number. So what we did is we added in the real time glow empty cell viability assay and as you can see across these we see no differences in the in the amount of viable cells. So from this that we conclude that the reduction in these metabolites are definitely compound specific changes rather than a change in cell number as in some sort of cytotoxic effect of these these agents. Now interestingly with with that experiment because you still have viable cells there, you can actually take, we could have actually taken it further and done more with the live cells. So after running that real time glow empty cell viability assay, we could then purified the RNA from the cells, then quantified that RNA then then done an RT qPCR step to check for any changes in the transcriptome. So in this last example I'd like to show you how our non lytic cell based Cytochrome P-450 assays can be multiplexed with a lytic viability assay which is used as an internal control in this case and can be used to normalize data against. So Johanna in her liver model also uses one of these Cytochrome P-450 assays. So the SIP three O 4 assay. So the SIP, sorry the cell based P-450 SIP assays. So they measure a specific Cytochrome P-450 activity in cells. So here you have your your 3D cell culture and you have a Luciferin IPA. So this is this is cell permeable. So that red arrow indicates the site of modification by that particular cipassa. So you add this into your media. It then gets into the cell, then the active SIP enzymes, and these can be basal SIP enzymes or they can be induced SIP enzymes. Then they modify where that red arrow is producing luciferin, then that luciferin then diffuses out of the cell is then used in the luciferin luciferase reaction to generate a luminescence signal which are then proportional to the activity of that particular SIP assay, that enzyme, Sorry. And in this example we use the cell tricolonescent cell viability assay and as already discussed we know that that assay determines the number of viable cells in culture, so in this example to human liver micro tissues and they were treated with rifampicin for 48 hours. After that point we added in the luciferase IPA substrate and that was added for an hour that media that was then taken and the signal was then red in the remaining cells. We then added in cell 5 to glow 3D cell viability assay and as you can see rifampicin resulted in a dose dependent increase in the Sip 3A4 activity and we can see you using the cell 5 to glow viability assay that there was no decrease or there's no effect on cell viability. Now when it comes to normalization, the Sip 450 results can be normalized to cell number by dividing the P-450 values by the cell. Try to glow 3D values then. This would obviously compensate for any variability in cell number or any proliferative effects that some compounds might have. So in summary, so Promiga offers a range of 3D optimized cell based assays that can be multiplexed giving you more biologically relevant data and a better insight into cell health, allowing you to get the most from yourselves and culture. Now before I hand over to you Hannah, I would just like to ask you a quick polling question. So are you hoping to Multiplex more of your assays? So for A is yes for 2D cultures, What answer B for yes for 3D cultures? Or C is no. I don't want to Multiplex my assays, so if you wouldn't mind answering those questions, that would be most appreciated. So we'll give it a few more seconds. See some of you are starting to to answer those questions, which is great. I can see that the numbers of people answering those are increasing. So I'll give another couple of seconds for you to answer those questions. So we're at just over 50% now, so which is great. Thank you. So looking at those results that's that's that's great to know that they're very interesting results. So thank you ever so much. So without further ado, I will hand over to Johanna. Thank you, Darren. Hello, my name is Johanna Lampa. I'm a scientist in the product development team at UPM Biomedicals and working with our life science products. And we've now learned a lot from Jonathan about the UPM hydrogels and from there and about the Promega SA kits and how these can be multiplexed. And I would like to show you how we brought all of this together in a liver model. You might wonder why did we do this? Why did we want to develop a liver model? It is known that drug induced liver injury is a major reason for the failure of clinical trials and also for post approval withdrawal of drugs from the market. So we need better models to detect the drug induced livery liver injury early on in the process of drug development. There are some animal models but these have limited predictive power due to the species differences and also we don't want to use more animal models, we want to stop using them. So we need to develop more in vitro models and of course this starts with the cell types you're using. Primary human hepatocytes have some great features but they also have some disadvantages like their limited availability. There are some hepatomas cell lines. Many of these do not retain the liver specific functionalities but some do. For example, they have RRG cell line that we have been using on the project I will present. And then you also have to think about the the culture model, the culture method And as we learned from Jonathan, the UPM growth X hydrogels are great for automation and this is very important in the process of drug development when you're dealing with a with a high number of novel compounds. Of course, when deciding on the the features of your model, it's also important to think about the applications and the questions that you're asking. And if you are interested in in basic liver biology or modeling liver disease, maybe the high throughput isn't the most important, but as I mentioned the Hepato toxicity and the drug drug interactions there, it is really important. And also the multiplexing can help here to reduce the consumption of time and material. So like I said, we used the Heparogy cell line in Grodex Hydrogels and to develop this model we plated the cells in 3D and Grodex. And we did this in 96 well plates preparing for identical plates. And we also dispensed products only without any cells in it to some of these wells to be able to use those for blank measurements. And then once weekly at 714-2128 days, we took one of these plates, treated the cells with Refampine to induce Sip 3 for activity or with itraconazole to inhibit the activity, incubated for two more days and then started the multiplexing readout. This is how the cells look like in Grotex. They started clustering very soon after plating and within one week these steroids had formed to which were around 50 to 60 micrometer in diameter. And this size remained stable throughout the whole experiment because the separate GSL line does not divide. So there's no cell growth observed in this model. And then as I mentioned, we started the drug treatment always after 1234 weeks after plating and then ran the Multiplex essay. The first step in the Multiplex essay was the Saltox Green to quantify the cell death. And this was very easy because we had the Saltox Green die in the culture all the time. So whenever we wanted to read the cell death, we could just put the plate into the plate reader and read the fluorescence. After this we collected the supernatant and just stored it at -20. And then later we quantified the albumin secretion from these supernatant samples and there's a lot of leftover. So we would be able to to add other supernatant based readouts here without interfering with the whole protocol. Then in the next step, we quantified the Cytochrome P-450 activity with AP450 Glow SA and we've optimized this for two of the Cytochrome P-450 SIP 2C9 and Sip 3A four. And so we added the the substrate to the cells in the first step and then after the incubation transferred the supernatant to a new plate and added the detection reagent and then quantified the SIP activity by rating the luminescence. And because we still had the cells in the original plate in the grow decks, we were able to quantify the viability with a cell Titaglow 3 DSA. OK, let's look at some of the results now. As I mentioned, we used the compound treatment to induce and inhibit SIP activity and it is very important that these compounds do not kill the cells so that we are still able to measure the Cytochrome P-450 activity in otherwise healthy cells. This shows the cell death, so basically the cytotoxicity of the compounds at the four time points 9/16/23 and 30 days. In Gray we can see the control treatment and green the rifampin, so the SIP 3A4 inducer and below the SIP 3A4 inhibitor etroconazole and this color code will remain the same throughout the whole presentation. The controls are set to 1 here for each of the days and you can see the effect on or the induction of cyto cell death in by the compounds and in general they didn't induce much cell death. There's a little bit here at day 23 by itraconazole, but in general we can say that the cells were happily growing or surviving there in the Grotex even after the drug treatments. And to confirm this we can move on to the results from the cell viability. I say the cell Titaglow 3D this, if you only look at the Gray bars, the viability decreased over time. And this was expected because as I mentioned, the cell line does not divide, so there's no growth observed. But we can really nicely see here that the drug treatment did not reduce cell viability. So the cells were healthy and we can move on and look at the liver cell specific functionality. We're first taking a look at the results from the P-450 GLOW essays. So the Sip 3-4 activity in the left panel you can see the basal activities are only the control treated cells. There is a decrease over time, but if you look at the right panel, we can really nicely induce the Sip 3-4 activity at all of these time points and also at almost all of them we can inhibit it. So the the this essay works also if you're for example studying drug drug interactions because sometimes one drug can inhibit step three or four activity, but this enzyme would be needed for metabolism of another drug and this is then prevented and this can lead of course to to an overdose then in vivo. So it's really important to be able to measure SIP activities in such a model and we can do this in our model with the multiplexing approach. And then we also looked at another liver cell specific functionality, the secretion of albumin. You can see that here the the basal secretions or the Gray bars increased a bit from 9 to 16 days and then there was a slight decrease. But in general it remained roughly in the same level and there was no effect of the compounds on the album in secretion. Which is great because the those are were supposed to specifically interfere with ZIP activity, but not generally with functionalities of the cells. And in this 96 well format we collected 100 microlita supernatant from the from each well. And we used only one microlita here for the ELISA, so there's plenty of material left for other readouts which can be added to the multiplexing approach without interfering with this protocol in general. So in summary, I hope I have convinced you that we can culture Heparogy Cells Syndrome X for at least 30 days and we can analyze this model by the multiplexing of assays to quantify cell death and cell viability and to to measure the liver cell specific functionality. By with AP450 GLOW SA and an ELISA for albumin secretion. More supernatant based assays can easily be added and in this model the liver specific functionality is retained and the CIPRIAN 3A4 activity can be modified with drug treatments. Because of the automation friendly characteristics of Grotex and of the essays, this model has the potential to be automated for drug screens and there are many different possible applications, for example in liver toxicity assays or quality controls of primary human hepaticide batches. Now I've been talking a lot about 3D essays and of course I would be interested to know if you are performing liver toxicity essays in 2D and if you would like to move on and perform these in 3D. So there are two options. A yes I currently use 2D and want to transition to 3D or B know I want to keep using 2D and of course we hope that everyone wants to transition to 3D. But if you wanna keep using 2D then we would of course also be interested why this is the case. I can see that there's still some modes coming in, so I'll give you a little bit more time. OK, let's move on so that we may be still also have some time for question and answers. So well, this is great. We have 90.92 point, 3% of attendees who use 2D and want to transition to 3D and then 7.7% who want to keep using 2D. So now I will hand over to Simon and hope that we still have time for one or two questions. Oh, sorry. I didn't realize I was telling mute. My apologies. So we'll start with the live Q&A. So these are all questions that were submitted during the talks. So great job everyone. The first question is, are the Bioinks FDA approved or is there a plan to get a proper certification for clinical use? Yeah, That's a that's a great question. Thanks Simon and thank you for asking that question. So basically the the hydrogel which is going to be released this year for clinical use is a more viscous version than the Gradex hydrogels and therefore it is compatible for bioprinting. But what the way that we are releasing it is that it would be an excipient or it would be a part of a particular product or workflow that you might be producing yourself. So if you'd like to talk more about that, really great question, great application, do connect with us in the Technical Support team and we can put you in contact with the right people to talk about that. So thanks. All right. And then the next question someone asked what plate do you recommend to use for Multiplex viability assays, knowing that white is used for luminescence and black for fluorescence. I can say that that, so that's that's a great question. So when using a luminescent assay, yes, we recommend to use a white plate as you said when using a fluorescent assay to use a a black plate. But when you're multiplexing these assays where you've got a luminescence and a fluorescent signal, that we recommend to use a black plate. All right. And then for time, we'll probably just have one final question. So this one is, hi, Johanna. How long can you incubate the cells with cell Tox Green without affecting their biology? Well we had the the cell Tox green in the culture all the time and this is what I learned from Darren that it's non-toxic to the cells. So we didn't observe any effect on the cells by the dye. Great. All right. So there were a bunch of other questions that were submitted. Since the webinar is coming to a close, we'll do our best to follow up with everyone that submitted them. We really appreciate everyone showing up, spending time listening to us talk about really great science. And yeah, we hope to invite you to a webinar soon in the future. So thank you very much. Thank you very much. Thanks, Simon. _1732296320327