precision cut tissue slice | Visikol

Why Use a Precision Cut Tissue Slice Model?

Carol Tomaszewski — Thu, 20 Apr 2023 13:18:03 +0000

Maximum intensity projection of a whole mount human precision cut liver slice (PCLS) labeled with antibodies against pan-collagen, α-smooth muscle actin and vimentin.

Over the last five years, Visikol has executed many precision cut tissue slice (PCTS) projects for clients despite the fact that we have a multitude of 2D and 3D (e.g., spheroid, organoid) cell culture models that we can use instead. The reason for this popularity is complicated, but boils down to the fact that even the best in vitro models are imperfect and tissue slice models provide a few unique features which simply are not possible in these systems.

When designing an in vitro assay with clients, the Visikol team puts a lot of focus on the specific research question at hand and work backwards towards determining which assay and model are best for that specific question. In the case of in vitro assays, we tend to do a lot of spheroid work as companies are very familiar with our various high content imaging approaches which uniquely allow our team to image these models in their entirety and to leverage them to answer novel research questions (e.g., antibody penetration, T-cell infiltration). However, a few years ago we launched a suite of precision cut tissue slice models which now include both animal and human kidney and liver tissue slice models. The reason for this addition was that we wanted to provide our clients with the broadest suite of models possible and these models meet a distinct customer need.

That need is that while we are making great strides in the advanced cell culture space, these models simply do not develop the complex physiology and structure that we see in vivo. For many research questions, this structure and innate functionality is critical and cannot be effectively replicated in current in vitro models. However, these same researchers are not able to develop effective animal models for the question at hand or want to see a different human model. It is for these cases where precision cut tissue slice models are particularly useful as they harness fresh patient or animal tissue that can be kept in culture for days-to-weeks depending on the type of tissue that is being used. These tissues are sliced into ultra-thin sections and kept functional on permeable supports (PermaCell) where various therapeutics can be tested. With human tissues, this provides a unique opportunity to test the efficacy and safety of a therapeutic in human tissues without a clinical trial in a medium throughput approach.

Precision Cut Tissue Slice Models

Though there are great benefits of these models, there are also some significant downsides which need to be taken into account when leveraging these models for assays. The first of which is that while these tissues are the innate tissue and the innate structure is generally preserved, the tissues after being removed from the body and sectioned tend to lose functionality and viability precipitously. This means that A) your desired functionality might not be present, B) the assay window for you research question could be too short at only a few days for some tissues (e.g., disease state liver tissue) and C) the single donor you evaluate might not be indicative of the whole patient population you are trying to model. Together, all of these advantages and disadvantages need to be assessed to determine which model is best for your research question. Currently, we have the following precision cut tissue slice models that our clients can leverage:

If you are interested in one of these models or discussing an in vitro assay with our team, please reach out.

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Retaining Phase 1 Metabolic Activity After Cryopreservation in Mouse Kidney Slices

Carol Tomaszewski — Mon, 23 May 2022 17:34:24 +0000

In this recent blog post from Dr. Ramesh Dasari and Dr. Amir Wahba from Visikol, they evaluate the ability to cryopreserve mouse kidney slices. As a company, Visikol is highly focused on providing clients with advanced cell culture assays and one of the many models that Visikol offers its clients are tissue slice assays. While these assays leverage highly relevant in vivo tissues in an in vitro format, the availability of tissues makes the assays operationally challenging to execute, and thus having a cryopreservation approach allows for easy scheduling and execution. Within this work, Dr. Dasari and Dr. Wahba look at multiple functional parameters of kidneys before and after cryopreservation to evaluate the approach for routine use.

Cryopreservation has become a key enabling technology in regenerative and translational medicine to provide a stable and secure extended cell storage for primary tissue isolates and cell preparations. Cryopreservation can bring about a number of cellular injuries, potentially leading to deleterious changes in cell morphology, characteristics, metabolic activity, function, and cell death. Temperature decrease can be responsible for triggering specific stress response pathways and can activate apoptotic and necrotic pathways after thawing. However, with appropriate precautions adopted during the methodology, cryopreservation would overcome the shortage of human tissues during emergency conditions.

Precision cut tissue slices (PCTS) represent a suitable and convenient tool for pharmacological, toxicological and morphological studies. We investigated the potential of cryopreservation of mouse kidney slices as a morphological tool by snap freezing the slices (without any medium) in liquid nitrogen and freezing them at -80^oC. The application of a rapid freezing method (direct immersion in liquid nitrogen) allows for maintaining urea synthesis, sulfo-conjugation, and CYP-dependent oxidation of ethoxycoumarin, testosterone hydroxylation, and N-de-ethylation of lidocaine at the same level as a non-cryopreserved rat or human PCLS after 2 or 3 hours of incubation.

Mouse kidney tissues were procured from Melior Discovery. The tissues were packaged in a temperature-controlled container and maintained on ice until transported to Visikol. Processing began immediately by obtaining 5 mm biopsy punch cores, followed by cutting using the Compresstome automatic tissue slicers. The cores were bonded to sample holders one at a time, embedded in agarose, mounted, and sectioned (section thickness: 250μm). The slices were collected in a Petri dish containing ice-cold UW solution and immediately transferred to cryovials which were snap-frozen in liquid nitrogen and stored at -80^oC. Cryopreserved kidney slices were thawed by transferring the freeze vials directly from liquid nitrogen into a water bath at 37^oC. After thawing, slices were washed with slice washing medium (DPBS supplemented with 1x B27 minus insulin, trypsin inhibitor, aprotinin, chymostatin and D-glucose) at room temperature. Then the slices were cultured in 24-well transwell inserts up to 72 hours in culture medium ( William’s E medium supplemented with 1x B27 minus insulin, trypsin inhibitor, aprotinin, chymostatin, 1% FBS, 1x insulin transferrin-selenium, dexamethasone, GlutaMAX and D-glucose). Before the end of the incubation period, the slices were cultured with substrates (midazolam, tolbutamide, dextromethorphan, and phenacetin) for one hour and the conditioned medium was collected. The CYP metabolites were separated on an Agilent PoroShell 120 EC-C18 column with an isocratic solvent system composed of 90% H₂O (0.1% (v/v) acetic acid) and 10% ACN (0.1% (v/v) acetic acid) (Figure 1).

Figure 1. (A) LC-MS/MS chromatogram of the conditioned medium of kidney PCTS following one-hour incubation with CYP enzyme substrates: midazolam, tolbutamide, dextromethorphan, and phenacetin. (B) Selected base peak of acetaminophen, the metabolic product of CYP1A2, at m/z 152.1/110.1, which was the only enzyme that showed activity. The metabolites were separated on an Agilent PoroShell 120 EC-C18 column with an isocratic solvent system composed of 90% H2O (0.1% (v/v) acetic acid) and 10% ACN (0.1% (v/v) acetic acid).

Our results indicate that phase I metabolic enzymes were well maintained after cryopreservation in kidney slices. While this does not guarantee that this cryopreservation approach will maintain all functionality within the slices, it does demonstrate a viable path towards preserving tissue slices for routine use over long periods of time from the same donor.

Therefore, if you are looking to adopt PCTS assays into your work please reach out to Visikol, we have a lot of flexibility in designing assays and can work to develop a solution that best meets your research needs.

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Precision cut liver slices (PCLS) as models for the study of liver disease

Mike Johnson — Wed, 29 Jul 2020 14:11:36 +0000

Precision-cut tissue slices (PCTS) are thin tissue slices with precise thickness generated from fresh, viable tissues through the use of a vibrating microtome (e.g. vibratome, compresstome). These slices can be cultured ex vivo and used to assess various metrics of interest to drug discovery, including pharmacological, toxicological, and physiological studies. The use of precision-cut tissue slices as an experimental model has become popular because of several major advantages over conventional 2D or 3D cell culture models. Unlike 2D or 3D cell culture models—which may lack tissue architecture, intracellular matrix, or cell heterogeneity—precision-cut tissue slices can recapitulate in vivo tissue environment because PCTS retain intact tissue architecture, extracellular matrix (ECM), cell-cell or cell-ECM interactions and heterogenous cell populations of the tissue of interest. Several studies (discussed below) have suggested that using precision-cut human tissue slices can bridge the translational gap between animal studies and human observations. Because precision-cut tissue slices are an ideal platform for pharmacology and toxicology studies, helping to overcome limitations offered by the traditional cell culture models, Visikol has established and validated service offerings using PCTS ex vivo culture model using healthy and/or diseased human or rodent tissues.

Precision-cut tissue slices from diseased human or mouse tissues can be utilized to predict toxicology, drug efficacy and therapeutic effects^1-3. For example, precision-cut liver slices (PCLS) can be generated from diseased human liver with late stage liver fibrosis (e.g. F3 or F4 liver). Our data shows that precision-cut liver slices from F4 human liver can be cultured and retain viability up to at least 72 hours, and hepatocytes , immune cells, and vesicles indicative of steatosis can be maintained throughout the culture period (Fig. 1). Immunostaining of whole mount precision cut liver slices shows an extensive collagen network surrounding many vimentin-expressing hepatic stellate cells (HSC), which also very frequently express high levels of alpha smooth muscle actin indicative of HSC activation (Fig. 2).

Figure 1. H&E staining showed the presence of lymphocytes and macrovesicular steatosis in human F4 precision-cut liver slices. Lymphocytes and macrovesicular can be detected at 0 hr and maintained after 72 hrs under culture conditions.

Figure 2. Structure integrity of F4 human precision-cut liver slices after 72 hours of ex vivo culture. Precision-cut liver slices of 250 µm thickness were cultured ex vivo for 72 hours. Intact precision-cut liver slices were immunostained for vimentin (red), pan-collagen (green) and alpha smooth muscle actin (ACTA2, grey).

Treatment with a specific ALK5 inhibitor in F4 human precision-cut liver slices (PCLS) reduced the expression of fibrotic genes such as metallopeptidase Inhibitor 1 (TIMP) and collagen type 1 alpha 1 (COL1A1) compared to vehicle treated PCLS (Fig. 3A). In collaboration with Melior Discovery, Visikol has also established precision-cut liver slice screening platform using carbon tetrachloride (CCl4)-induced liver fibrosis mouse model. Blocking ALK5 function in CCl4-treated mouse precision cut liver slices significantly reduced the mRNA levels of platelet derived growth factor beta (PDGFRβ) and collagen 1A1 (COL1A1; Fig. 3B). These data indicate the usefulness of precision-cut liver slices as a drug screening platform to assess compounds intended to mitigate fibrosis.

Figure 3. Gene expression analysis of precision-cut liver slices generated from F4 human liver and CCl4-induced liver fibrosis mouse model. A) Alk5 inhibition reduced the expression of COL1A1 and TIMP1 in F4 human precision cut liver slices; B) The expression of COL1A1 and PDGFRβ were significantly suppressed after Alk5 inhibition in precision-cut liver slices from CCl₄-treated mice.

In addition, several studies have shown that precision-cut liver slices are an excellent ex vivo model to study viral infection and replication^{4, 5}. Alternatively, liver fibrosis can be modeled using precision-cut liver slices ex vivo using chemicals or inflammatory cytokine cocktails⁶. Treatment of transforming growth factor β1 (TGFβ-1) and platelet growth factor, PDGF-BB caused fibrogenesis in precision-cut liver slices derived from both humans and rats⁶. Paish et al also demonstrated that anti-fibrotic compound treatment successfully attenuated ECM deposition and reduced the expression of fibrotic genes in these PCLS.

The utilization of precision-cut tissue slices is a powerful tool for drug discovery research which can yield great insights to those in pursuit of an IND for a new drug compound, or for those engaged in basic research to better understand fundamental pathways for disease. Expert utilization of precision-cut tissue slices requires the accumulation of many disciplines: 3D cell culture, pharmacology, histopathology, tissue-clearing and confocal imaging, genetics, biochemistry, and logistics. As such, rest assured that when you work with Visikol, our experts will work diligently to oversee all the details, work with you to plan an excellent experiment to your exact specifications, and give you access to unique insights for pre-clinical drug discovery. Reach out today to discuss our precision-cut liver slice and precision-cut tissue slice service offerings.

References:

de Graaf et al., Preparation and incubation of precision-cut liver and intestinal slices for application in drug metabolism and toxicity studies. (2010) Nature Protocols (5) p1540–1551
de Bovenkamp et al., Precision-Cut Liver Slices as a New Model to Study Toxicity-Induced Hepatic Stellate Cell Activation in a Physiologic Milieu. (2005) Toxicological Sciences (85) Issue 1, p632–638
Westra et al., The Effect of Antifibrotic Drugs in Rat Precision-Cut Fibrotic Liver Slices. (2014) PLoS ONE 9(4): e95462
Lagaye et al., Efficient replication of primary or culture hepatitis C virus isolates in human liver slices: a relevant ex vivo model of liver infection. (2012) Hepatology 56(3) p861–872
Palma et al., Precision-cut liver slices: a versatile tool to advance liver research. (2019) Hepatology International (13) p51–57
Paish et al, A Bioreactor Technology for Modeling Fibrosis in Human and Rodent Precision‐Cut Liver Slices (2019) Hepatology 70(4) p.1377-1391

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Visikol Now Offering Precision Cut Tissue Slice Models for Use in Services

Mike Johnson — Wed, 15 Apr 2020 11:29:32 +0000

Many advanced in vitro cell culture models such as 3D co-culture systems or organ-on-a-chip have been developed in order to recreate the cellular complexity and architecture seen in vivo. At Visikol, we use many different types of 3D cell culture models to evaluate the efficacy, toxicity and PK/PD of potential therapeutics. However, a short-coming of these models is that they still cannot fully recapitulate the complex cellular structure and interactions of in vivo tissues. Furthermore, generating iPSC derived models de novo to increase complexity and improve in vivo functionality greatly increases challenges associated with validating the model and the reproducibility of any assays. Therefore, in our efforts to continually improve our advanced cell culture service portfolio, we have begun to offer precision cut tissue slices (PCTS) as an ex vivo culture model wherein ultrathin slices are taken from living tissues from animals and humans and subsequently cultured. Because PCTS are taken from living animals, they are able to better recapitulate in vivo tissue architecture, cellular heterogeneity and surrounding extracellular matrix (ECM). In addition, cell-cell and cell-ECM interactions are fully preserved in PCTS.

Like all cell culture models, PCTS have their respective advantages and disadvantages and we see them fitting into our drug discovery services to address specific types of research questions where the most physiologically relevant model is required. For example, human PCTS can be used to bridge the translational gap between animal studies to human whereas the use of mouse PCTS to predict drug efficacy prior to preclinical studies can reduce the number of animal experiments. While PCTS better mimic in vivo systems compared to traditional spheroid and organoid 3D cell culture models, they are more expensive and lower throughput due to the sourcing requirements as well as the processing required to obtain and generate them and are thus not applicable to all applications.

PCTS can be generated from explanted human tissues, diseased human tissues or animal models of diseases using a specialized vibrating microtome that can reproduce tissue slices with precise thickness. These tissue slices (~250um) retain the tissue structure and cellular components within and can be cultured ex vivo up to 5 to 15 days under optimized conditions. Fibrosis models can be generated ex vivo by treating PCTS with pro-fibrotic cytokines such as transforming growth factor beta (TGFb), platelet-derived growth factors (PDGFs), a cocktail of free fatty acids or organic compounds such as carbon tetrachloride (CCl4). For example, non-alcoholic fatty liver disease (NAFLD) can be induced by treating liver slices with a cocktail of linoleic and oleic acids. Fat accumulation can be detected in human PCLS after 24 hours, suggesting the usefulness of PCLS as a fatty liver model.

In addition to fibrosis induction models, PCTS from diseased tissues can also be used as a drug screening platform to test the efficacy of anti-fibrotic drugs or as a tool to study drug metabolism. One of the major benefits of PCTS is that patient-derived biopsy tissue can be used to predict treatment efficacy for personalized drug testing. Since PCTS can be an invaluable tool in drug discovery research, Visikol has thoroughly built out this biological platform as an additional tool for use in its drug discovery research services and has used it with Clients for a wide range of research applications such as using human liver PCTS to screen for anti-fibrotic compounds. This ex vivo model when coupled with high content imaging and biochemical analyses is a robust platform for translational drug discovery research and provides Clients a great level of in vivo recapitulation while also providing moderate throughput.

Interested in discussing your project with our scientific team?

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