Webinar | Visikol https://visikol.com Advanced Drug Discovery and Bioimaging Services Thu, 28 Sep 2023 13:47:58 +0000 en-US hourly 1 https://wordpress.org/?v=6.8.5 Join Us for a Free Webinar Presented by CrestOptics https://visikol.com/blog/2023/09/28/webinar-crestoptics/ Thu, 28 Sep 2023 13:47:12 +0000 https://visikol.com/?p=19994

CrestOptics ‘Best practices from clearing methods to high content microscopy: exploring the 3D cellular complexity in depth’ webinar

Sessions: 8:00 AM | 2:00 PM | 8:00 PM (UTC/GMT +2)

Date: October 11, 2023 

Abstract

Studying the three-dimensional (3D) cellular structure is crucial for gaining a more meaningful understanding of biological systems as it remarkably preserves the physiological characteristics of the cellular architecture. The study of 3D cellular structures has gained increasing significance in recent years due to advances in techniques such as clearing and microscopy. Together, these techniques provide researchers with the tools to deeply visualize complex 3D cellular architectures at high resolution, enabling detailed analysis of cellular structures and functions.

Performing volumetric imaging at a considerable Z-depth is often challenging due to the light-scattering properties and opacity limits of native tissue. To address this problem, Visikol Inc. developed the Visikol HISTO-M which is an optical clearing technique (i.e., the process of making opaque biological structures transparent) designed specifically for 3D cell culture models and plate-based high-throughput processing. During the webinar, the Visikol HISTO-M technique will be described in combination with fluorescent labeling and high-content imaging allowing for more accurate 3D cell culture model characterization and drug screening.

The webinar will also feature highly relevant speakers who have made significant contributions to the clearing and organoid fields: Prof. Nicolas Baeyens,as well as Prof. Silvia Di Angelantonio and her collaborator Dr. Erica Debbi. Prof. Baeyens, a leading scientist in the field of mechanobiology, has developed an advanced clearing protocol that enables the volumetric visualization of the intricate 3D architecture of various organs, such as bones and human teeth. In the webinar, he will present how this clearing method can be applied to study tissues effectively through the detailed analysis of 3D structures. Prof. Di Angelantonio, an expert in developing innovative techniques for generating 3D brain organoids from hiPSCs, together with Dr. Debbi will share ground-breaking research on complex brain organoid structures and their potential applications taking advantage of high-resolution imaging.

In addition to that, you will learn how CrestOptics X-Light V3 Spinning Disk Confocal technology allows the collection and analysis of 3D images of whole cleared samples at a higher resolution than a wide-field microscope and at a faster scan rate than a point-scanning confocal microscope.

Altogether, our joint webinar promises to provide valuable insights into the cutting-edge techniques and applications of clearing methods, and high-content 3D imaging.

The post Join Us for a Free Webinar Presented by CrestOptics first appeared on Visikol.]]> Cytometric Analysis of Immune Cell Populations in Archived FFPE Biopsies https://visikol.com/blog/2022/03/01/cytometric-analysis-of-immune-cell-populations-in-archived-ffpe-biopsies/ Tue, 01 Mar 2022 14:28:59 +0000 https://visikol.com/?p=16542

Video Transcript

Slide 1

Hi Everybody, and thanks so much Maria for the invite to give this presentation today. My name is Tom Villani and I’m going to be giving an overview of Cytometric Analysis of Immune Cell Populations in Archived clinical FFPE Biopsies for Immuno-oncology Research.

Slide 2

So, before we dive in, I wanted to give a little bit of an introduction about the company that I work for, Visikol. We are an industry leader in advanced models and tissue imaging, we’re focused on improving and accelerating drug discovery and development through contract research services.

We are an innovative and profitable company based in the US and offer research services for drug discovery, development, and clinical research. We have a worldwide customer base of startups, small, medium, and large pharma, and a number of prestigious research institutions.

Our team is composed of a number of experts in drug discovery, assay design, oncology, immunology, inflammation, liver disease, and many other therapeutic areas. We leverage that expertise to do several hundred drug-screening, preclinical and clinical campaigns per year.

We have dozens of unique technologies and patents related to histology, tissue imaging, 3D cell culture, and other kinds of advanced techniques for biological research and our 2 main pillars of our business are our advanced tissue imaging and our advanced cell culture and tissue model assay services.

Slide 3

So, a brief overview of the presentation today. So, this presentation is going to be focused on how we extract cytometric data from FFPE tissue and towards that end I wanted to start with a brief overview of immuno-oncology, which is an emerging, one of the leading paradigms in cancer research these days. Touch base with a brief overview of flow cytometry and its applications to cancer research. We’ll talk about some of the limitations of flow cytometry applied to clinical specimens, especially archived specimens. We’ll dive into some applications of our FFPE technic for doing cytometry on tissue sections and conclude with an overview of some of the limitations and the future direction we’re going wit this technique.

Slide 4

To start off, Immuno-oncology has been a rapidly emerging paradigm in cancer research over the last decade or so. And this is of course potentially unlocking the power of the immune system to fight cancer. Over the past 10 years there’s been an explosion of interest in understanding the immune system and how it responds when a tumor is growing. And there’s been a huge amount of development towards pharmaceutical strategy to leverage to immune system to help out in fighting cancer. A lot of this started in the 70s and early 80s, as we were learning more about the immune system to try to combat AIDS, it was understood that these cells were able to attack cancer cells in some clinical experiments. By the late 80s it was shown that we could genetically engineer T-cells to recognize and attack tumor cells directly. Into the 90s there was a lot of research done looking at adoptive cell therapy and understanding immune checkpoint inhibition. And by the early 2000s there was already the first trial to test the immune checkpoint inhibiting drugs. The first one was tested in 2000 and it was approved in 2011, Ipilumab also known as Yervoy. And by 2012 the first child with leukemia had been treated with CAR-T cell therapy. Over the last 5-10 years there’s been a huge amount of development-Keytruda was approved in 2016. The FDA granted accelerated approval to avelumab, a PD-L1 checkpoint inhibitor for Merkel cell carcinoma. With all the success in a very short period of time there’s been an explosion of interest in research in this space and there’s a huge number of companies right now seeking to modulate the immune response to try to treat cancer.

Slide 5

So, let’s step back for a second and talk a little bit about Flow Cytometry. Flow Cytometry is a critical tool for understanding immunology and really for diving into the population of cells. Flow Cytometry is used to quantify cells, but they need to be suspended so that you can run them through the flow cytometer.  In a flow cytometer we measure the intensity of the fluorescent signal of the cell, we typically get them to be fluorescent by immunolabeling, we also measure Diameter and the refraction/reflection. And this allows us to analyze the population of cells by selective parameters, such as intensity ranges, size and shape. And this gating allows us to do multiparametric analysis on complex cell populations to really quickly count cells, understand changes in phenotype, protein expression. To understand the composition of the immune cells that are located inside a tissue, understand the difference in cell cycle, apoptosis, activation of immune cells-there’s obviously a wide variety of applications that flow cytometry is critical for and it’s routinely used in research, obviously, and in clinical practice such as hematology.

Slide 6

So, flow cytometry has been a critical tool for a long time. It allows us to take extremely complex population of cells and essentially come up with a systematic way to evaluate differences in those populations and how they may change or shift with treatment or the progression of a disease. In immuno-oncology it’s incredibly important to understand the immune cell population within the tumor, so this is typically done in research by homogenization of a culture tumor or a fresh biopsy sample. They essentially disincorporate the cell, de-cellularize the tissue and then label it and put it into the flow cytometer and then can do this multi-parametric analysis to better understand the different immune cell populations in the specimens they’re looking at. Determining the relative receptor expression in the immune cell subpopulations is critical to understanding the mechanisms of oncogenesis, resistance, and drug effects and so at the end of the day the more data you can get about these cell populations the more information you can then uncover about the different factors that lead to the progression of the tumor itself.

Slide 7

So, a brief overview of flow cytometry and cancer research. Obviously, there’s a lot of pros. It’s very high throughput and highly automatable. You can do high-plex labeling, so it’s easy to do 7, 10, 15 labels on a single population cell. It’s extremely quantitative and you get very high statistical power due to the sheer number of cells you are measuring. It’s got widespread adoption and familiarity; most immunologists are familiar with how to read these scatter plots. And of course, the major advantage is that you can recover viable, sorted cells for further experimentation. Now, there are some limitations to flow cytometry. Flow cytometry can only measure a total of signal, it can’t really distinguish between the specificity of staining of internal structures, and sometimes this is important for different phenotypes. Another major con is that you lose all the spatial information in the tissue. Since you have to dis-incorporate the tissue you end up with just a mixture of cells and you don’t know which ones were near which others, and that can be a difficulty when you’re trying to quantify the changes in the immune cell population in terms of how close the immune cells are to interacting with one another or with the tumor cell.  Furthermore, only suspended cells can be put through a flow cytometer, and as I mentioned you have to disaggregate these tissues using enzymes and so because of this you can’t use flow cytometry on archived Formalin Fixed Paraffin Embedded tissue specimens, it’s just not an option. Now, of course, a huge amount of the tissue specimens stored in the world from former clinical trials in research are stored as FFPE tissue blocks, and so enabling a technique that would allow us to do this type of immune cell population research on FFPE tissues will be very valuable to cancer research and to research in general.

Slide 8

So, to that end, we thought to develop a technique to do exactly that. We wanted to characterize the immune cell population in these archival tissues to take advantage of the millions of archived tissue specimens that pharma clients, researcher repositories, universities, research hospitals, etc. have at their disposal. The most critical aspect is that these stored clinical archived samples have a huge amount of information about the progression of the tissue, disease, etc. so this represents an extremely large repository of information about the needs that we can get access to. So how are we going to do this? So the way that we accomplish this is to adapt multiplex immunofluorescent techniques that we had developed for doing high plex immune-histochemistry using digital slide scanning and we developed some open-source data mining and visualization tools, that allowed us to develop what we call Tissue Cytometry. This is essentially doing the same kind of analysis, where you’re measuring the intensity and count of the different cells in your sample, but we are doing it in tissue. So, directly from the FFPE slide.

Slide 9

So how does this work? The approach is essentially sequential, multi-round immuno-labeling. So, the way that this works is by taking a tissue section, labeling with 3-5 antibodies, then we do digital fluorescent slide scanning. After that we strip the antibodies off the tissue using a proprietary reagent that we developed here. We repeat this process over and over until we’ve covered all the targets on that same tissue. Then we use a computational co-registration algorithm to assemble this into one, big mega stack of an image that contains all of different channels and that allows us to conduct quantitative analysis, where we conduct cell counting , colocalization, intensity-gating, etc.

Slide 10

This is a brief overview of how our approach works, and some of the preliminary information to demonstrate why we can use it this way. So the approach that we developed involves using a chemical technique to essentially denature the antibodies from the tissue, and this is a very gentle technique that we developed. It’s very rapid, and this allows us to do multi-round immuno-labeling. It’s essentially like an antibody eraser. And so we can take a tissue, you can see on the far left panel we have a tissue that is before staining, this is obviously nothing. Then we stain it with our selected antibodies. We can strip the tissue, which will get us back to the tissue as it was with no signal, and now to demonstrate that we have fully removed all the primary, and secondary, antibodies from that tissue we add just the secondary antibody here, and this will demonstrate that indeed all the primary has been removed as we get no background signal. Then we can repeat with new markers. In this case we just demonstrated that the antigens is preserved, we can stain the same markers and we get an almost indistinguishable image. One of the coolest advantages of this technique in the eyes of many pathologists is that after you’re done collecting all this fluorescent data you can still go back and get an H&E slide or cross-validate with traditional immunohistochemistry techniques, and so it makes it very easy to cross-validate this technique with existing pipeline.

Slide 11

So, the critical instrument that allows us to do this, in our lab, is the Aperio VERSA. And so this allows us to do multiple channel slide scanning at 40x on the samples extremely rapidly, it usually takes between 15 minutes to an hour for these samples depending on the size. We get extremely high image quality, they’re extremely well suited for qualitative image analysis, and this allows us to move directly into the next part of our pipeline.

 

Slide 12

The most important part of this process, at least from a data science standpoint, is assembling these images together into one continuous image. If you’re going to do colocalization of these different biomarkers in the cell population that is required for tissue cytometry, then we have to make sure that the different rounds of imaging have perfectly aligning cells. The way that we accomplish this is by using a technique that’s known as computational elastic co-registration. This is a technique that was originally developed to essentially stitch satellite images together way back, several decades ago, but has been re-adapted for use in bioimaging. And so, we actually used, for this particular project, an ImageJ plugin that’s publicly available-it’s free, which essentially takes the DAPI channels of the different rounds of imaging, uses those as a guide, and manages to align these images by slightly deforming the space between the cells to account for the slight deformation of cells that happen when you remove the cover slip and repeat the process of staining. This allows us to get one continuous image set out of our multiplex image run, and that way we can get a single image with 10,12,15 channels which represent the different markers that we’re trying to look at in the panel.

Slide 13

So how does this all fit together? In the next couple of slides we’ll show you an example of how we have applied this technique to take a look at a couple of patients, and understand the differences between the immune cell population within these patients. What we thought to do was develop a panel of 12 targets, you can see these targets listed on this slide here. They’re mostly for T-cells, cytotoxic T-cells, we have memory T-Cell markers in here, we’re looking at proliferating markers, we have Pan-CK in there to distinguish between the tumor regions and the non-tumor stroma regions, as well as some checkpoint markers such as PD-1 and PD-L1.

Slide 14

And so we put together this panel and labeled the 3 different patients who are all suffering from the same type of breast cancer, and you can see the representative images here displayed with a zoomed in cut-out down below. And as you can see from a quick glance, there’s not a huge amount of difference between these patients here, and so moving along here.

Slide 15

For each of these patients we get a 13 plex image stack, where we have 12 antibody targets- a channel for each one, as well as the nuclear space. Here is just a representation of that. The composite image is shown on the left, and in the middle you’re seeing a sequential, essentially a video, of the different channels going one after the other on the same image. And also on the right you can see just a montage.

Slide 16

So, this enables us to get really beautiful multi-channel composite images that look really cool and they can go up on the wall, but now what, right? This doesn’t really tell us a lot. Here we’re looking at 12 different channels and at the end of the day there are so many different colors your eyes can see. These images are pretty, but they don’t really tell you much on their own.

Slide 17

The next step of this process involves computational analysis and so we utilize image analysis software to do cell segmentation and marker colocalization. So, we utilize one of several pieces of software. We have several pieces of software that we have developed internally, we also use ImageJ as well as commercially available software HALO to process these image sets, depending on the need. From that image analysis we get huge data, we get usually more than a million rows per sample, because there are so many cells that we are detecting in these patients’ tumors. We take that huge amount of data and we feed it through the multi-source data analysis pipeline that allows us to generate the conventional scatter plots that are typically produced when doing flow cytometry analysis. Just to point out, the kind of data that we get for this analysis is also compatible with FlowJo and other commercially available flow cytometry software, for those who are more comfortable with it. We just wanted to emphasize that you don’t need commercial software to do this analysis this is very straightforward and can be reproduced with a basic understanding of Python coding and some of these open-source libraries.

Slide 18

So, what is this overall process of doing the data analysis for tissue cytometry? So, first off with the raw data we filter by size and shape and colocalize by signals. Then, we do processing, just background subtraction, normalization, log-scaling. Finally we will apply our actual analysis, this is where we are doing the gating and counting, the statistical analyses, and one of the advantages that we have of doing tissue cytometry is that we can do spatial analysis. So after we are done looking at cell population’s direct colocalizations we can dive into which cell populations were located near one another in tissue, and that is critically important for understanding the roles the multiple immune cell phenotypes play in the progression of cancer.

Slide 19

Here is just a representative of example of some selected tissue cytometry scatter plots here. Just for a point of emphasizing how much data we are really dealing with, we’ve got 12 markers, we have 132 one-to-one primary marker comparisons, and if we want to compare the coincidence of every marker to every other marker, that’s 12 factorial possible phenotypes. That’s about five hundred million possible combinations to look at if you want to be complete. And so obviously that’s a lot of data. And so the best way to do this is obviously systematically, and drive by hypothesis.

Slide 20

And so we were particularly interested in understanding the differences in T cell exhaustion between these patients. And so you can see here on the top we have the scatter plots representing the PD-1 intensity is the Y-axis and the CD3 is the X-axis. And you can see here that Patient 2 displays a particular shift in its phenotype, compared to Patient 1, and we see that same trend with Patient 3. Patient 1 has a significantly higher relative percentage of CD3 and PD-1 positive cells, this would be exhausted T cells, compared to Patient 2 and Patient 3. Patient 2 and Patient 3 we see a much smaller percentage of the total T cell population, especially in the PD-1 marker. This goes hand in hand with what we see when we look at the PD-L1 tumor, and so the figure on the bottom we see PD-L1 intensity in the Y-axis and PanCK intensity in the X-axis, and here we see 2 distinct populations of cells in our samples. We have a number of PD-L1 PanCK positive cells, and we also have a number of PD-L1 that are not PanCK expressing. And so you can see when we look at the bar graph that is out on the right, patient 1 had a significant decrease, I guess significantly fewer, PD-L1/PanCK negative cells compared to Patient 2 or Patient 3.

Slide 21

Moving along here, we were interested in understanding the tumor associated macrophages, and so this is where we had started to look at the advantages of spatial analysis, and so in our study we can correlate the CD68 positive cells with the nearby PanCK positive cells and do a scatter plot here to look at the relative expression of the CD68 in the region where we see PanCK positive cells. So you can see on the figure at the top we have a kind of bi-modal distribution of between our 3 patients. We have in Patient 1 a significantly higher concentration of our macrophages contained within the tumor comparison to the stroma, whereas Patient 2 and 3 show very similar relative percentage of macrophages in the tumor region compared to the stroma. And so to summarize in Patient 1 we are seeing a significant decrease in the tumor associated macrophages in the stroma compared to Patients 2 and 3.

Slide 22

Now finally, the spatial analysis is really what brings this tissue cytometry to the forefront interest to immuno-oncology because of the way that we do this analysis from the actual immunohisto fluorescence images. We can take the data analysis and start to bucket these cells into the distance from cell types of interest. And so we were particularly interested here in understanding the engagement between T cells and tumor associated macrophages in Patient 1, we saw such distinct difference in some of the other t cell exhaustion and some of the other factors that we measured here in Patient 1, and so we generated a histogram of the distribution of the distance between CD3 cells and the nearest CD68 positive cell. We see these figures that are shown in the middle here. And so look at the top one, we are looking at the distance between CD3+ cells and CD68+ cells you can see that CD68 has considerably higher percentage of its t cell population in close proximity to CD68+ cells. And on the graph at the bottom we are looking at a the histogram of distances between CD3/PD-1+ cells and the nearest CD68+ cell. And you can see that trend continues here-we have a much higher percentage of the total population of exhausted T cells in close proximity to a CD68 cell, withing the tumor in Patient 1 compared to Patient 2 and Patient 3.

Slide 23

So, to summarize, Tissue Cytometry is possible by combining the multiplex immunohistochemistry, digital slide scanning, and open-source data processing packages. The sequential multi-round immunohistochemistry technique that we use allowed for 10-20 targets to be labeled in a single sample, and this allows rapid multi-parametric data analysis so that we can explore the cell populations within archived FFPE tissue specimens. The differential analysis of cell populations in archived patient tumor biopsies is rapid, it’s quite inexpensive, and you get a huge matrix of quantitative data for analysis. We end up with an extremely high statistical power with this type of analysis, since we usually end up measuring millions of cells in every single sample and it’s at relatively high throughput. With our technique we can do 50-100 slides per day per scanner that we have, we have 2 VERSAs pretty much running around the clock here. But it’s a fairly high throughput technique for histopathology. Most importantly, in my opinion, is the ability to then do the spatial analysis and understand the network of immune cells and how they’re interacting in contributing to the progression of the disease.

So, there are a few limitations to this technique. So, compared to flow cytometry we have a much lower dynamic range, the imagers typically take 8-bit images so we’re dealing with between 0-255 as our possible intensity values. That can make it a little bit trickier to get separation between your cell populations, unless you’re careful in how you do your data processing. Because a lot of tissues have Autofluorescence this requires either background correction or some kind of chemical processing ahead of time to reduce the background. Of course since these tissues are fixed you cannot isolate the cells easily after this analysis, and because these tissues are often archived for decades they all require very careful antigen retrieval and it’s well known that some antigens are destroyed by FFPE targeting, and so not every target will remain available in archived specimens.

The next steps, at least on the Visikol side, we are currently expanding this for the analysis of tissue microarrays so that we can offer more complex services for patient stratification. We’re next incorporating a number of panels to measure aspects about the extra cellular matrix and tumor micro environment. We’re currently in the process of validating a wide library of standard panels for immune markers and other disease markers, and we are continuing to improve our techniques by incorporating AI where it fits best.

So, with that I will draw our presentation to a close.

The post Cytometric Analysis of Immune Cell Populations in Archived FFPE Biopsies first appeared on Visikol.]]> Modern Approaches to Drug Discovery Panel https://visikol.com/blog/2022/01/19/modern-approaches-to-drug-discovery-panel/ https://visikol.com/blog/2022/01/19/modern-approaches-to-drug-discovery-panel/#comments Wed, 19 Jan 2022 13:00:19 +0000 https://visikol.com/?p=16306
Modern Approaches to Drug Discovery Panel

Visikol’s Dr. Tom Villani, Ph.D. and Dr. Michael Johnson, Ph.D. participated in the ‘Modern Approaches to Drug Discovery Panel’ at the 2021 BICO Partnership Conference.

Joining them in this discussion is Dr. Pankaj Karande, Ph.D., Assistant Professor of Chemical and Biological Engineering at Rensselaer Polytechnic Institute and Dr. Peter Yeow, Ph.D., Postdoctoral Research Associate at Johns Hopkins University.

The post Modern Approaches to Drug Discovery Panel first appeared on Visikol.]]> https://visikol.com/blog/2022/01/19/modern-approaches-to-drug-discovery-panel/feed/ 1 HUREL Micro Liver Overview Webinar https://visikol.com/blog/2021/11/09/hurel-micro-liver-webinar/ Tue, 09 Nov 2021 13:00:15 +0000 https://visikol.com/?p=16044

Visikol’s CEO Michael Johnson, Ph.D. gives an overview of Visikol’s HUREL Micro Liver products and services.

Video Transcript:

Hurel Slide 1

Hello! My name is Michael Johnson and I am the CEO and Co-Founder of Visikol. Today, I will be talking about the HUREL portfolio of micro liver products and services.

HUREL slide 2

Now, before I dive into talking more about the HUREL products and services I wanted to give you a little bit of background about Visikol as a company. Visikol is focused on accelerating the drug discovery and development process by providing pharmaceutical and biotech companies with advanced cell culture models and assay services, as well as imaging services. These are the two pillars of our business, and we’ve worked with 18 of the top 20 pharmaceutical companies in helping them accelerate their drug discovery and development processes, as well as dozens of other small and mid-sized pharmaceutical and biotech companies.

HUREL slide 3

Now the focus of today’s presentation is on advanced cell culture, and advanced cell culture, the objective, is to try and bridge the gap between in vitro studies and ex vivo studies. Ideally, what we would like to do is have in vitro studies mimic what happens within the body at an inexpensive and high throughput so that we can predict what’s going to happen very early on the drug discovery process. But unfortunately, this isn’t always the case, so we are constantly trying to better mimic the body in a dish and develop complex models that can accomplish that task. We, as a company, are focused on liver cell culture models and assays, as well as oncology models and assays. This tends to be about 80-90 percent of the work that we do. In the liver space we have several models that I will talk about that have been developed and validated for various applications, and in the oncology space we also have several models that tend to be confused on immuno-oncology applications, such as the T-Cell infiltration assay that I am showing here. We’re quantifying how T-Cells are infiltrating into a spheroid model of cancer. And then lastly, while we focus a lot on oncology and liver cell culture, we as a company are model agnostic. We’ve developed all sorts of models for custom application and disease states and as a company that works everything from trans wells to ex vivo tissue slices and really can focus on developing custom assays and models for projects based upon a customer specific requirement and are very open to doing that and working hand in hand with our clients.

HUREL Slide 4

But, the focus today will be on liver culture assays and models. A really important thing to consider is that there is not a ‘one size fits all’ in vitro model. 3D cell culture, bio-print models, tissue slice models, they’re not going to replace all the other type of models. Every in vitro model has its place because when you look at a model it’s a balance between through put, price, and relevancy. A model can be extremely relevant, such as a tissue slice model, but the price can be quite high and the throughput can be quite low, so depending on your research question there is a specific model that meets those specific criteria. So, depending on what question you’re specifically answering, what your budget is, what your throughput requirement is, how many compounds you’re looking at, and also what types of in vivo recapitulation do you need to actually answer your research question? If you can get by with a very simple model that’s at low cost, why not use that model? So, we are always focusing on this as a company and trying to provide models that meet our client’s specific criteria for their assays.

HUREL slide 5

As a company we have broad spectrum of models, spanning from 2D cell culture, all the way to ex vivo tissue slices of human, rodent, feline, and also canine for use in the liver space. Prior to acquiring HUREL in May of 2021, we were very focused on spheroids, organoids, and tissue slice models. All 3D or tissue slice models that are quite complex, difficult to characterize, and useful for various applications but not all applications. These models tend to have metabolic stability for only a few days to a few weeks, and the phenotypic stability is not as strong as it is in some other models. And this is one of the main reasons that we wanted to acquire HUREL. HUREL, as a company, their services and products were based upon 10 different species of primary hepatocyte 2D models that are extremely stable phenotypically, and also metabolically, for long periods of time. And thus, by adding this portfolio of models to our portfolio, we are now able to provide our clients with the broadest breadth of liver cell culture models in the entire marketplace, from 2D to spheroids to organoids to tissue slices, we have all these different models within our portfolio that we can pull into DMPK studies, looking at non-alcoholic steatohepatitis, looking at fibrosis, looking at immune cell infiltration into liver tissues, and also traditional drug-induced liver injury So we have a very broad portfolio of liver models as a company, and the focus on today’s presentation is on the HUREL micro liver models, which I will get to in a second, but what I am showing here is not every model answers every research question, and it really depends on what that research question is that we are going after before we are going to pick which model we are going to use because it’s not a one-size fits all solution.

HUREL Slide 6

The HUREL micro livers which are now part of the Visikol portfolio of models and assays are a 2D liver co-culture system. The process by which we culture these systems is patented and proprietary, but it allows us to keep primary hepatocytes in culture and metabolically and phenotypically stable for over 4 weeks, up to 2 months of time. Which just isn’t something we see with more complex spheroid models and something we don’t see in traditional 2D cell culture. So, it’s a very unique model, but it’s also very well validated. We have over 100 publications using this model for various applications. We sell it as both a product and leverage it as a service, which I’ll get to in a moment. But, as a product it is shipped live and it can be shipped in 12, 24, 48, 96, and 384 well plate formats, ready for use in our assays. And because it’s a standard plate format it is compatible with standard automation and analytical equipment-any imager, any other type of equipment that you use in your lab for plates is compatible with this system. So we don’t have any special fluidic devices, we don’t need any special equipment, this is a standard plate format, and it’s used by most major pharmaceutical companies for various applications.

HUREL Slide 7

And as I mentioned, this has been in various publications for lots of different types of applications, we have looked at over 10 species. And you can see here just from a sub-sampling of papers that are out there that they cover a wide range of topics, but the models have been extensively validated by both academia and GO’s and of course the pharmaceutical and biotech industry. It’s also been extensively compared, which I’ll get to in a moment, with other types of 2D and spheroid systems that are in the marketplace today for the applications that I have described here.

HUREL Slide 8

The products themselves, there are 10 different species and they’re shipped in a flexible format. So if we want to ship that with multiple species on a single plate, we refer to that as a Zooplate, and that’s something that we are able to easily do. And then when the plates arrive, after about a 4 hour exchange period (a media exchange) we are able to use them right away for our assays. But, the process is very flexible, we ship live right to your lab and you can use them in your assays right away. And like I mentioned before, there is a 4 week, greater than 4 weeks, stability of these, which is really just fantastic compared to most of the other models out there and of course fantastic compared to traditional 2D cell culture models that have been used for years, specifically primary human hepatocytes, which have been the go-to. We are able to get longer metabolic and phenotypic stability with the patented HUREL culturing process.

HUREL Slide 9

If we look at the landscape and compare the HUREL models to micropatterned models, spheroids, what we see is that with the HUREL models we have a far higher number of hepatocytes per cell, what this means is that we have much better phenotypic stability and we are much more easily able to see low clearance compounds and what happens to them; how they metabolize, compared to other models where you have 1,000 or 5,000 hepatocytes. This system can go anywhere from 12 to 384 wells and one of the unique things about the HUREL micro livers, which I will get to later, is that we can actually look at hepatitis infection, it sustains it for a long period of time. It is also compatible with seahorse, and for high content imaging it’s compatible with traditional high content imaging approaches. So, it’s very extensible to a lot of different applications, of course DMPK and DILI. But overall, the greater number of hepatocytes in the process allows this to be the most phenotypically and metabolically stable type of liver model in the market.

Hurel Slide 10

Like I mentioned previously, the HUREL micro liver models have a very high degree of metabolic competency. And we see here the ability to generate reactive metabolites as exemplified by HUREL’s viability after exposure to hepatotoxicant cyclophosphamide incubated in the absence and presence, respectively, of broad cytochrome p450 inhibitor aminobenzotriazole. And you see that on the right here in this graph, which allows for a high degree of sensitivity and also predictive capability of the model itself.

HUREL Slide 11

And I mentioned this as well, previously, by the phenotypic stability of the model has been peer reviewed and we see this for over 30 days. It’s immutable compared to other models that are out there that we have the highest degree of phenotypic stability in the HUREL model platform and this is especially compared to hepatocytes in suspension, in monoculture, in micropatterned arrays, and also in spheroids.

HUREL Slide 12

One of the unique things about the HUREL micro liver platform is that we exposed, for example, the human micro liver model to midazolin in the presence of CYP 3A4 rifampin and CYP 3A4 potent inhibitor ketoconazole, which we administer every 24 hours for 72 hours and we remove these, what we see is the micro livers demonstrated a return to basal enzyme activity in a time-dependent manner, which illustrates the dynamic responsiveness and resilience of HUREL micro liver model metabolic competency, which is just incredible. It returns to basal state after we remove these, which really demonstrates the metabolic competency of these models and also their resistance.

HUREL Slide 13

And, when we look at this model for DILI prediction and we look at sensitive and specificity as compared to 3D spheroid models and micropatterned co-cultured models, all primary human hepatocytes, what we see is the accuracy and also the specificity and sensitivity of the HUREL micro liver models is significantly higher, as you see here, based on the publications and literature out there in the research space, which is really incredible to see and shows another application of the model.

HUREL Slide 14

And then we look at metabolite generation, what we see is based upon a study from a major US pharma company, we show that the predictive capability of the HUREL micro liver models is improved compared to suspension, 2D cell culture, and other approaches that have been traditionally used. Which demonstrates, again, the ability for this model to replicate what’s happening in vitro compared to known in vivo outcomes.

HUREL Slide 15

After going through the features of the product itself and some applications and data, I want to touch upon the services that we offer as a company. Visikol operates primarily as a contract research services company, in addition to selling the HUREL products as well as other reagents and kits.

HUREL Slide 16

What researchers are able to do is send compounds along to Visikol that we can test in HUREL micro liver plates. We have LCMS capabilities for metabolite generation and ID studies and the HUREL micro liver coculture system is robust and reliable for human in vivo clearance studies, especially slowly metabolized drugs where we can’t see the clearance in other types of models especially suspension primary human hepatocyte models.

HUREL Slide 17

The next application that we routinely do is HUREL TOX, this is a simple TOX assay that we do leveraging the system and various types of readouts-we can do imaging based readouts, we can do ATP assays, we can look at albumin, there are various endpoints we can do as a company but again, what’s happening here is clients are sending along to us compounds and we are running those through simple and traditional TOX assays within the HUREL system. We can go from as simple as ATP assays all the way up to looking at high content confocal imaging if needed at high resolution, and everything in between-qPCR, RNA-Seq, ELISAs, we really have a wide array of different endpoints we can look at, but it really depends on what the research question is. We can also add a mechanistic read out to it as well to look at reactive metabolites and cholestatic liability as well. This is a very flexible platform, but essentially we are taking in compounds and testing them in the HUREL plates.

HUREL slide 18

And then lastly for services applications, like I mentioned previously, the HUREL micro liver model is very stable with hepatitis infection. We can keep infection for a long period of time within the model and of course subsequently test therapeutics against hepatitis, which makes it a really robust in vitro platform for this, compared to other systems that are out there. And there are several publications, like the Nature Communications publication you see here, referenced, that we can pull from for looking at Hepatitis and the HUREL micro liver model for various mounted species being used.

HUREL Slide 19

And then lastly, for working with HUREL micro liver services and products, these are your main points of contact. The HUREL micro liver team has joined the Visikol team and will be part of the Visikol team going forward, so if you have any questions on the models themselves, ordering the models, setting up your next study, please reach out to the contacts here and if you have any questions don’t hesitate to reach out.

Thank you.

The post HUREL Micro Liver Overview Webinar first appeared on Visikol.]]> Visualizing the Benefits of 3D Cell Culture Webinar https://visikol.com/blog/2021/10/27/3d-cell-culture-webinar/ Wed, 27 Oct 2021 12:57:27 +0000 https://visikol.com/?p=15917

Visualizing the Benefits of 3D Cell Culture
Presented by
Genetic Engineering & Biotechnology
&
Corning

Broadcast Date: November 5, 2021
Time: 8:00 am PT, 11:00 am ET, 17:00 CET

Drug discovery screening is a complex endeavor that can often yield promising results toward new therapies. When combined with advanced 3D cell culture techniques like spheroids, the screening process becomes more physiologically relevant, but the complexity magnifies tremendously. However, many organizations have developed new techniques and strategies that can immensely simplify the process, allowing for more meaningful data.

Join us for this GEN webinar where our guest presenters will share their experiences with 3D cell culture—from the development of tissue specific spheroids to new imaging modalities. Additionally, we will hear about an assay-ready 3D liver spheroid kit for the development of 3D liver models that better recapitulates in vivo behavior. Finally, we will learn about some of the latest technology for high content image analysis of 3D cultures and how to overcome some of the current limitations that have prevented this methodology from achieving its full potential.

A live Q&A session will follow the presentations, offering you a chance to pose questions to our expert panelists.

Presenters

MichaelJohnson

Michael Johnson, PhD
CEO and Co-Founder
Visikol

LiFeng

Feng Li, PhD
Development Associate
Corning Life Sciences

The post Visualizing the Benefits of 3D Cell Culture Webinar first appeared on Visikol.]]> Making Precision Cut Tissue Slices Webinar https://visikol.com/blog/2021/10/11/precision-cut-tissue-slices-webinar/ Mon, 11 Oct 2021 13:00:17 +0000 https://visikol.com/?p=15486

‘Making Precision Cut Tissue Slices for Ex Vivo Assay Services’ webinar was given in partnership with Precisionary Instruments.

Get Started Today

Video Transcript:

Precision Cut Tissue Slices Slide 1

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.

Precision Cut Tissue Slices Slide 1

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.

Precision Cut Tissue Slices Slide 3

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.

Precision Cut Tissue Slices Slide 4

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

  1. 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.
  2. 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.
  3. 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.
Precision Cut Tissue Slices Slide 5

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.

Precision Cut Tissue Slices Slide 6

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.

Precision Cut Tissue Slices Slide 7

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.

Precision Cut Tissue Slices Slide 8

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.

Precision Cut Tissue Slices Slide 9

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.

Precision Cut Tissue Slices Slide 10

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.

Precision Cut Tissue Slices Slide 11

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.

Precision Cut Tissue Slices Slide 12

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.

Precision Cut Tissue Slices Slide 13

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.

For instance-

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.

Precision Cut Tissue Slices Slide 14

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.

Precision Cut Tissue Slices Slide 15

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.

Precision Cut Tissue Slices Slide 16

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.

Q&A

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.

The post Making Precision Cut Tissue Slices Webinar first appeared on Visikol.]]> Visikol CEO and Co-founder to Participate in Startup Webinar https://visikol.com/blog/2020/11/17/visikol-ceo-and-co-founder-to-participate-in-startup-webinar/ Tue, 17 Nov 2020 15:33:21 +0000 https://visikol.com/?p=13704

Join us on Wednesday, December 2nd for an online webinar hosted by the Foundation for Health Advancement.

Michael Johnson, CEO and co-founder of Visikol, will be joined alongside four other New Jersey entrepreneurs and business leaders.

The panel will discuss their experiences in making startups successful.

The panelists will also discuss:

  • How to bring a conceptual business plan to commercial launch
  • How to maximize company resources
  • Stories about common pitfalls and how to avoid them

The event will be held over zoom from 2:00 to 3:00pm EST on 12/2/20.

The post Visikol CEO and Co-founder to Participate in Startup Webinar first appeared on Visikol.]]>