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Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

* These authors contributed equally
Medicine

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Summary

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

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Pearen, M. A., Lim, H. K., Gratte, F. D., Fernandez-Rojo, M. A., Nawaratna, S. K., Gobert, G. N., Olynyk, J. K., Tirnitz-Parker, J. E. E., Ramm, G. A. Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology. J. Vis. Exp. (157), e60992, doi:10.3791/60992 (2020).

Abstract

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

Introduction

The pathogenesis of most chronic liver diseases (i.e., viral hepatitis, nonalcoholic steatohepatitis, cholestatic liver injury and liver cancer) involves complex interactions between multiple different liver cell types that drive inflammation, fibrogenesis, and cancer development1,2. To understand the molecular mechanisms underlying these chronic liver-based diseases, the interactions between multiple liver cell types must be investigated. While multiple hepatic cell lines (and more recently, organoids) can be cultured in vitro, these models do not accurately emulate the complex structure, function, and cellular diversity of the hepatic microenvironment3. Furthermore, cultured liver cells (in particular, transformed cell lines) may deviate from their original source biology. Animal models are used experimentally to investigate the interactions between multiple liver cell types. However, they may become significantly reduced in scope for experimental manipulation, due to significant off-target effects in extrahepatic organs (e.g., when testing potential therapeutics).

The use of precision-cut liver slices (PCLS) in tissue culture is an experimental technique first used in drug metabolism and toxicity studies, and it involves the cutting of viable, ultrathin (around 100−250 µm thick) liver slices. This permits the direct experimental manipulation of liver tissue ex vivo4. The technique bridges an experimental gap between in vivo animal studies and in vitro cell culture methods, overcoming many drawbacks of both methods (i.e., practical limits on the range of experiments that can be performed in whole animals as well as loss of structure/function and cellular diversity with in vitro cell culture methods).

Furthermore, PCLS vastly increases experimental capacity compared to whole animal studies. As one mouse can produce more than 48 liver slices, this also facilitates the use of both control and treatment groups from the same liver. In addition, the technique physically separates the liver tissue from other organ systems; therefore, it removes potential off-target effects that can occur in whole animals when testing the effects of exogenous stimuli.

In this protocol, PCLS are generated using a vibratome with a laterally vibrating blade. Other studies have successfully used a Krumdieck tissue slicer, as described in Olinga and Schuppan5. In the vibratome, lateral vibration of the blade prevents tearing of the ultrathin tissue caused by shear stress, as the blade is pushed into the tissue. Both the vibratome and Krumdieck tissue slicer work effectively without structural embedding of liver tissue, which streamlines the slicing procedure. This technique can also be used to create PCLS from diseased livers, including those from mouse models of fibrosis/cirrhosis6 and hepatic steatosis7.

In addition to demonstrating the techniques required for preparation and tissue culture of PCLS, this report also examines the viability of these ex vivo tissues by measuring adenosine triphosphate (ATP) levels and examining tissue histology to assess necrosis and fibrosis. As a representative experimental procedure, PCLS are treated with pathophysiological concentrations of three different bile acids (glycocholic, taurocholic, and cholic acids) to simulate cholestatic liver injury. In the context of cholestatic liver injury, taurocholic acid in particular has been shown to be significantly increased in both the serum and bile of children with cystic fibrosis-associated liver disease8.

Liver progenitor cells have also been treated in vitro with taurocholic acid to simulate the elevated taurocholic acid levels observed in patients, and this treatment caused increased proliferation and differentiation of liver progenitor cells towards a biliary (cholangiocyte) phenotype9. Subsequently, PCLS were treated ex vivo with elevated levels of taurocholic acid, and increased cholangiocyte markers were observed. This supports the in vitro observation that taurocholic acid drives biliary proliferation and/or differentiation in the context of pediatric cystic fibrosis-associated liver disease9.

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Protocol

All animal experiments were performed in accordance with the Australian code for the care and use of animals for scientific purposes at QIMR Berghofer Medical Research Institute with approval from the institute animal ethics committee. Male C57BL/6 mice (15−20 weeks old) were obtained from the Animal Resources Centre, WA, Australia.

NOTE: All solutions, media, instruments, hardware, and tubes that contact the samples must be sterilized or thoroughly disinfected with a 70% ethanol solution and handled using sterile techniques to minimize the risk of culture contamination.

1. Setup of the vibratome

  1. Prepare sterile Krebs-Henseleit buffer solution with 2 g/L glucose (Table of Materials). Check that the pH of the buffer is 7.4. If the pH is higher, saturate the buffer with carbogen (95% O2 + 5% CO2) or incubate within a 5% CO2 tissue culture incubator to correct the bicarbonate buffering system. Refrigerate the sealed sterile buffer solution at 4 °C.
  2. Insert the vibratome blade into the cutting arm. Make sure the blade is tightly fixed to the cutting arm, as vibrations can cause the blade to come loose.
  3. Disinfect the blade and cutting arm with a 70% ethanol solution.
    CAUTION: Avoid contact with the blade.
  4. Disinfect the buffer tray with a 70% ethanol solution and wipe it with a sterile tissue.
  5. Insert the buffer tray into the vibratome and tighten the mounting mechanism.
  6. Set the cutting arm to the maximum available height.
  7. Set the blade angle to 10° below horizontal, sloping downwards to the sample. Make sure that the blade angle is tightly fixed.
    NOTE: The optimal blade angle may differ depending on the vibratome model and tissue characteristics.
  8. Disinfect the specimen holder with a 70% ethanol solution.
  9. Connect the cooling water for the Peltier thermoelectric cooler located under the buffer tray.
    NOTE: Some vibratome models use an ice-bath for cooling instead.
  10. Fix the water-out tube to a drain and turn on the water. Set the cooler to 4 °C. Run cooling water at a rate of >400 mL/min.
  11. Fill the buffer tray almost to the top with sterile ice-cold Krebs-Henseleit buffer (with 2 g/L glucose). Leave additional Krebs-Henseleit buffer solution on ice.

2. Liver removal and preparation

  1. Sterilize or disinfect all surgical instruments including curved forceps, flat square-tip forceps, tweezers, surgical clamps, and scissors using autoclaving.
  2. Prior to liver removal, deeply anesthetize mice using an intraperitoneal injection of 100 mg/kg ketamine and 12.5 mg/kg xylazine.
    NOTE: Other anesthetics may be used, or mice can be euthanized by CO2 asphyxiation/cervical dislocation if the liver is quickly removed to prevent hypoxia-induced damage.
  3. Disinfect skin surfaces on mice by wetting with a 70% ethanol solution. Secure mice on their backs with all extremities pinned to a dissecting board.
  4. Make a vertical midline incision into the skin from the base of the abdominal cavity to just above the diaphragm.
    NOTE: Incisions towards the extremities are made to facilitate the retraction of the skin.
  5. Pull the skin back while cutting any connective tissue between the skin and abdominal cavity. Prevent hair from being transferred to the abdominal cavity as much as possible. Pin or clamp the skin away from the abdominal cavity.
  6. Using clean forceps and scissors, open the abdominal cavity and lower thoracic cavity.
  7. Removing the liver quickly without damaging the lobes
    1. Using blunt tools (e.g., flat square-tip forceps), guide the upper liver lobes downwards towards the abdomen. Cut the connective tissue surrounding the upper liver lobes using scissors.
    2. Guide the liver lobes upwards towards the diaphragm. Hold the central vascular bundle of the liver with forceps.
      NOTE: It will not affect the procedure if the central vascular bundle is damaged.
    3. Pull the central vascular bundle away from the mouse and cut the remaining connective tissue, blood vessels, etc. to remove the liver. Take care not to 1) damage the liver lobes and 2) cut into the gastrointestinal tract, as this may contaminate the liver with microorganisms.
  8. Place the removed liver into a sterile 10 cm tissue culture dish half-filled with ice-cold Krebs-Henseleit buffer solution. Using a blunt instrument to guide the lobes, cut the center of the liver to divide it into separate lobes.
  9. Select one liver lobe (use the largest first), and place it flat side down onto a new sterile 10 cm dish. Keep all other liver lobes in the dish of Krebs-Henseleit buffer solution stored on ice for subsequent use.
  10. Trim around 10% of the material from the tallest edge of the liver lobe while lying flat.
    NOTE: Trimming is important, as this tissue edge will contact the cutting blade first; therefore, a tissue edge that is relatively perpendicular to the cutting blade will prevent tissue compression that can occur because of shallow angles in the uncut liver lobes.
  11. Trim the other three tissue edges.
    NOTE: This removes some of the fibrous Glisson's capsule and will make separation of the tissue slices easier.
  12. Mounting the liver lobe on the specimen holder
    1. Place a thin layer of cyanoacrylate glue (superglue or medical/veterinary grade cyanoacrylate glue) on the specimen holder towards the front, sized slightly larger than the trimmed liver lobe.
      NOTE: Check that the cyanoacrylate glue does not contain non-cyanoacrylate additives.
    2. Gently pick up the liver lobe with sterile forceps using a corner away from the cutting edge, then pat dry any residual buffer from the flat side of the liver lobe using sterile absorbent material.
    3. Place the large flat side of the liver lobe on the cyanoacrylate glue patch with the largest edge facing towards the front. Allow it to cure in the air for 1−2 min.
      NOTE: Cyanoacrylate adhesives cure quickly (~2 min) in response to residual moisture and tissue proteins, and they will firmly attach the tissue to the specimen holder.

3. Production of liver slices

  1. Place the specimen holder with the attached liver lobe into the vibratome buffer tray. Make sure the liver lobe is fully covered by buffer.
    NOTE: If needed, top up the level of ice-cold Krebs-Henseleit buffer solution. If the buffer level obscures the cutting process, increase or decrease the level.
  2. Set the cutting speed.
    NOTE: This may need to be empirically determined depending on the vibratome model and blade characteristics. As a starting guide, use a speed of 57 Hz/3,420 rpm. The vibratome speed can be measured using an audio spectrum analyzer application on a smartphone.
  3. Lower the cutting blade until it is located just above the liver lobe using the height dial. Run the vibrating cutting arm over the sample.
  4. Return the blade back using the crank handle in reverse. Activate the vibrations while returning the blade. After the blade has returned and is not above the sample, lower the blade height by 250 µm.
  5. Repeat the cutting process until the top of the tissue is removed. Discard the first one or two slices, as these will contain Glisson's capsule and therefore do not contain much functional liver tissue compared to other slices.
  6. To cut liver slices, slowly advance the vibrating blade into the tissue by turning the handle. Use a small paintbrush (disinfected with a 70% ethanol solution) to gently guide the tissue during the cutting process.
  7. Use the paintbrush to lift the cut tissue slices and place the tissue slice into a sterile tube of ice-cold Krebs-Henseleit buffer. When not in use, keep this tube on ice between slices.
    NOTE: Sometimes, the tissue does tear during the cutting process, but for most purposes the tissue is still usable.
  8. Repeat the cutting process until the base of the liver lobe is reached. Discard any liver slices that have visible cured glue.
    NOTE: The cured glue is visible as white areas on the tissue slices. A typical liver lobe will only have a useable thickness of around 2 mm. Therefore, only around eight 250 µm liver slices are produced from each lobe.
  9. Cut the tissue slices until near the cyanoacrylate glue. Do not cut into the glue.
  10. Repeat the whole process with other liver lobes until the required number of tissue slices is obtained.
  11. To clean the cured cyanoacrylate glue and tissue from specimen holder, either use a blade to scrape the cured glue/tissue mixture off, or soften and clean the mixture with acetone or dimethyl sulfoxide.

4. Tissue culture

NOTE: All tissue culture work must be performed in a sterile laminar flow hood.

  1. In a tissue culture laminar flow hood, pipette 1 mL of William's E medium containing 2.0 g/L glucose, 10% fetal bovine serum (FBS), 2 mM L-glutamine supplement, 100 U/mL penicillin, and 100 µg/mL streptomycin into 12 well tissue culture plates.
    NOTE: Other antibiotics and antifungal agents may also be added. Some studies have used additives in the tissue incubation medium (i.e., dexamethasone and insulin10), but these can interfere with cell signaling.
  2. Place the tube containing liver slices from ice into the tissue culture hood. To remove the tissue slices, gently swirl the mixture and gently tip into a sterile 10 cm dish while swirling.
  3. Using sterile sharp-point forceps and a scalpel, cut the tissue slices into roughly uniform sizes (e.g., 15 mm2).
    NOTE: This step is performed to ensure a consistent surface area. Because mouse liver lobes are significantly different in size and some tissue slices will tear, this will produce a range of PCLS with varying surface areas. The final surface area sizes of the PCLS depend on how much material is needed in subsequent analysis applications and how many replicates are needed. Cutting large PCLS into smaller multiple pieces of the same approximate size will innately allow an increased number of experimental replicates.
  4. Using sterile forceps or a small paintbrush, transfer the cut tissue slices to the wells of a 12 well plate containing 1 mL of medium.
    NOTE: Take care to confirm the consistency of thickness and shape of tissue slices. Sections that are darker than average suggest the tissue thickness is larger and need to be discarded. Lighter slices suggest the presence of cured cyanoacrylate glue and need to be discarded.
  5. Incubate the liver slices at 37 °C and 5% CO2 under 95% humidity.
    NOTE: Other groups have used methods to enhance oxygen delivery to PCLS, which appear to prolong tissue survival time6,10,11,12,13 (see discussion section).
  6. On the following day, change the culture medium using a manual pipette (rather than suction) to prevent the loss of tissue through suction errors. Subsequently, change the medium on the PCLS daily. The PCLS are now ready for experimental use.

5. Example application of PCLS

  1. At 16 h post-generation, treat PCLS with three different bile acids: glycocholic acid (GCA), taurocholic acid (TCA), and cholic acid (CA) (as sodium salts, all at a concentration of 150 µM) for 2 days.
  2. After 2 days of bile acid treatment, extract mRNA by placing tissue slices in ice-cold RNA lysis buffer and quickly homogenize using beads with a bead homogenizer (Table of Materials).
  3. Purify the RNA using an RNA isolation kit (Table of Materials) and create cDNA from the purified RNA.
  4. Perform quantitative polymerase chain reaction (Table of Materials) using specific primers (Table 1) on the cDNA to examine gene expression.

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Representative Results

To determine the cell viability of PCLS over time, tissue ATP levels were measured. ATP levels are typically proportional to viability. PCLS (around 15 mm2 in area) were cultured in normal William's E medium with 10% FBS, then at specific timepoints, liver slices were removed from tissue culture and homogenized with both ATP and protein (for normalization) concentrations (Table of Materials) being measured (Figure 1A). For biochemical assays like this, normalization is important, as the cut liver slices are not necessarily identical in dimensions. ATP levels (relative to protein) were suppressed immediately post-isolation and after 1 h (Figure 1A), suggesting short-term metabolic stress from the cooling and cutting procedures. However, ATP levels recovered by 3 h. ATP levels remained elevated at up to 5 days of tissue culture, indicating no significant decrease in viability. Hematoxylin and eosin (H&E) staining of liver slices suggested that limited tissue necrosis (characterized by nuclear pleomorphism) occurred in culture from around days 2 and 3. Tissue necrosis levels progressed to severe by day 5 (Figure 1B). Considering these morphological data, taken together with the ATP results, it is recommended to use this experimental tissue model for up to 3 days.

PCLS also displayed increasing collagen accumulation at later culture timepoints, as shown by thickening of Picro-sirius red-stained collagen fibers at day 5 (Figure 2). This thickening of collagen fibers suggests that spontaneous fibrogenic processes are active in PCLS obtained using this method. This process appears independent of PCLS isolation methodology with the thickening of collagen fibers6 and profibrogenic gene expression14 occurring from both vibratome and Krumdieck tissue slicers, respectively, over time. The development of these spontaneous fibrogenic processes needs be taken into account when interpreting PCLS biology, particularly with experiments associated with fibrotic processes.

We have previously shown the significant induction of cholangiocyte-specific gene connexin 43 (Cx43) and secretion of glutamyl transpeptidase (Ggt1) protein by TCA in PCLS9. The expression of cholangiocyte-specific genes relative to housekeeping controls (glyceraldehyde 3-phosphate dehydrogenase [Gapdh] and hypoxanthine phosphoribosyltransferase 1 [Hprt1]) in PCLS treated with TCA, GCA, or CA were examined by qPCR using specific primers. Consistent with a previous report, a significant induction in the expression of cholangiocyte-specific genes cytokeratin 19 (CK19; Figure 3A) and connexin 43 (Cx43; Figure 3B) were observed by both GCA and TCA. Ggt1 expression was increased by both bile acids; although, this did not reach statistical significance, possibly due to experimental variation (Figure 3C). The expression of CA was unaffected by any bile acid. The induction of cholangiocyte-specific genes suggests that GCA may also be involved in cholestatic liver injury, as previously reported for TCA8,9.

Figure 1
Figure 1: Tissue viability of precision-cut liver slices (PCLS). (A) ATP and protein levels in PCLS were measured immediately (T + 0) and at 1 h, 3 h, 6 h, and 1−5 days post-isolation. (B) To examine cell morphology, PCLS were fixed, paraffin-embedded, sectioned, and stained with H&E immediately (day 0) and at 1, 2, and 5 days post-isolation. A Kruskal-Wallis test with Dunn's multiple comparisons test was performed relative to T + 0. All data are represented as mean ± SEM (n = 2−3 mice) with *p < 0.05. The scale bars represent 100 µm. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Assessment of collagen deposition in PCLS. PCLS were fixed, paraffin-embedded, sectioned, and stained with Picro-Sirius Red to visualize collagen fibers at days 0, 1, 2, and 5 post-isolation. The scale bars represent 200 µm. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Bile acids induce cholangiocyte-specific gene expression. At 16 h post-isolation, medium on PCLS was changed, and new medium was added along with 150 μM glycocholic (GCA), taurocholic (TCA), or cholic (CA) acids (sodium salts). PCLS were harvested after 2 days, and the expression of cytokeratin 19 (CK19; A), connexin 43 (Cx43; B), and γ-glutamyl transpeptidase 1 (Ggt1; C) was examined relative to the geometric mean of Gapdh and Hprt1. Dunnett's multiple comparisons test was performed relative to untreated control tissue slices (n = 15). All data are represented as mean ± SEM (n = 4 mice, triplicate slices; *p < 0.05, **p < 0.01). Please click here to view a larger version of this figure.

Table 1
Table 1: qPCR primers.

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Discussion

The protocol demonstrates the application of murine PCLS isolation and tissue culture, and the procedures are designed to assess both viability and utility as well as examine impacts of exogenous mediators of liver pathobiology using biochemical assays, histology, and qPCR. The experimental utility of PCLS tissue culture in rodents and humans has been demonstrated in a wide range of applications, including experimental investigations in microRNA15/RNA9/protein expression16, metabolism17, viral infection dynamics10,18, infection signaling19, tumor invasion12, toxicity studies4,13,15,20, DNA damage studies21, cell biology14, and secretion studies9.

While ATP levels indicate cellular viability in PCLS up to 5 days in culture, H&E staining suggest that severe necrosis was occurring by 5 days in culture. The main factor that appears to limit the viability of PCLS is oxygen availability6,11. Several studies have included enhanced oxygen availability and consequently increased the viability time of PCLS in tissue culture. Methods used to increase oxygen availability include the incubation of tissue in oxygen-rich atmospheres10,11, use of the oxygen carrier perfusion perfluorodecalin12, shaking/rolling the culture during incubation6,10,13, and tissue culture plates designed to maximize oxygenation6.

Recently, an innovative air-liquid interface tissue culture system has been described with functional utility stated at longer than 7 days in culture14. This protocol uses ambient atmospheric oxygen in a normal tissue culture incubator. The method allows PCLS to be accessible to laboratories that do not have highly specialized, custom equipment to safely enhance oxygen delivery. However, if such methods to enhance oxygen delivery are available, they may improve long-term PCLS viability in culture.

The accumulation of collagen in PCLS after day 3 in culture suggests that spontaneous fibrogenic processes are active within this model. Spontaneous fibrosis has also been observed previously in PCLS6,14,22,23,24 and is possibly mediated by damage-associated molecular patterns (DAMPs) released by the cutting process or tissue necrosis. DAMPs act as signaling molecules that subsequently activate pro-fibrotic signaling pathways25,26. Furthermore, spontaneous fibrosis in PCLS could be mediated by chemokines released from the activation of stellate cells and/or Kupffer cells (i.e., transforming growth factor beta [TGF-β]). In this context, a recent article by Bigaeva et al.24 suggests that spontaneous fibrosis in PCLS is in part mediated by TGF-β signaling, as incubation of PCLS with TGF-β inhibitor Galunisertib inhibited gene expression changes associated with spontaneous hepatic fibrosis. Another possible mechanism is that the slicing procedure initially induces cell proliferation signaling pathways and entry of hepatocytes into the cell cycle; however, this mechanism fails and results in cell cycle arrest in mid-G1 phase27. Cell cycle arrest is associated with hepatic fibrosis rather than hepatic regeneration28.

In the representative experimental procedure, PCLS are treated with three different bile acids (GCA, TCA, and CA) for 2 days to simulate cholestatic liver injury. Two of the bile acids (GCA and TCA) induce significant expression of CK19 and Cx43, which are genes associated with cholangiocyte function. This suggests the expansion or differentiation of cells towards a cholangiocyte lineage in PCLS treated with these bile acids. This is consistent with our previous work showing a similar effect using TCA9 on PCLS. Furthermore, it is also consistent with in vivo observations that show feeding taurocholate to rats increases cholangiocyte numbers29. The treatment of liver tissue slices with bile acids simulates hepatic cholestasis, and it is speculated that the increase in the expression of genes associated with cholangiocyte function is an attempt by the liver tissue to create additional bile ductules to increase bile secretion. Given the specific induction of these genes is produced by the conjugated bile acids (GCA and TCA) but not the unconjugated CA, it is suspected that these effects are mediated by the sphingosine-1-phosphate receptor 2, a cell surface receptor with preferential activation by conjugated bile acids30.

One key limitation of the application of PCLS is individual replicate variability. Since the liver is not homogenous and there is variability in production, liver slices tend to have larger biological variation than cell culture experimentation. In experimental studies with few variables, such as the representative procedure demonstrated above, this is overcome by using an increased number of replicates. However, this may become an issue for larger screening studies. In summary, the described protocol is a straightforward and reliable method to study aspects of liver pathobiology ex vivo, requiring little specialized equipment except for the vibratome.

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Disclosures

The authors have nothing to disclose.

Acknowledgments

This work was supported by research grants from the National Health and Medical Research Council (NHMRC) of Australia (Grant No. APP1048740 and APP1142394 to G.A.R.; APP1160323 to J.E.E.T., J.K.O., G.A.R.). Grant A. Ramm is supported by a Senior Research Fellowship from the NHMRC of Australia (Grant No. APP1061332). Manuel Fernandez-Rojo was supported by the TALENTO program of Madrid, Spain (T1-BIO-1854).

Materials

Name Company Catalog Number Comments
10 cm Petri Dish GREINER 664160 Sterile Dish
12 Well Tissue Culture Plate Flat Bottom Greiner Bio-one 665180
70% Ethanol Solution (made with AR Grade) Chem-Supply Pty Ltd EA043-20L-P Disinfection solution
Acetone Chem-Supply Pty Ltd AA008-2.5L
Cholic acid Sigma-Aldrich C1129-100G
Cyanoacrylate Super Glue Parfix, DuluxGroup (Australia) Other brands should work
Disposable Single Edge Safety Razor Blades Mixed
Dissection Board Made in-house Sterile material over polystyrene
Fetal Bovine Serum GE Healthcare Australia Pty Ltd SH30084.02
Forceps sharp point 130 mm long ThermoFisher Scientific MET2115-130
Forma Steri-Cycle CO2 Incubator ThermoFisher Scientific 371
Glutamine Life Technologies Australia Pty Ltd 25030081
Glycocholic acid hydrate Sigma-Aldrich G2878-100G
ISOLATE II RNA Mini Kit Bioline (Aust) Pty Ltd BIO-52073
Ketamine 50 ml Provet KETAI1
Krebs-Henseleit Buffer with Added Glucose 2000 mg/L Sigma-Aldrich K3753 Can also be made in house
Laminar Flow Hood Hepa air filtration
NanoDrop 2000/2000c Spectrophotometers ThermoFisher Scientific
Penicillin-Streptomycin, Liq 100 ml Life Technologies Australia Pty Ltd 15140-122
Picro Sirius Red ABCAM Australia Pty Ltd ab246832
Pipette Tips Abt 1000 µl Filter Interpath Interpath 24800
Pipette Tips Abt 10 µl Filter Interpath Interpath 24300
Pipette Tips Abt 200 µl Filter Interpath Interpath 24700
Pipette Tips Abt 20 µl Filter Interpath Interpath 24500
Precellys Homogeniser Bertin Instruments P000669-PR240-A
Protractor Generic To measure blade angle
Quantstudio 5 QPCR Fixed 384 Block Applied Biosystems/ ThermoFisher Scientific
Scalpel Blade Mixed
Scalpel Blade Holder Mixed
SensiFAST cDNA Synthesis Kit Bioline (Aust) PTY LTD
Small Paintbrush with Plastic Handle Mixed Plastic handle resists ethanol
Square-Head Foreceps Mixed
Sterile 50 ml Plastic Tubes Corning Falcon 352098
Surgical Clamps Mixed
Surgical Forceps Mixed
Surgical Pins Mixed
Surgical Scissors Mixed
Taurochoic acid Sigma-Aldrich T-4009-5G
Vibratome SYS-NVSLM1 Motorized Vibroslice World Precision Instruments SYS-NVSLM1 With thermoelectric cooling
Williams Medium E Life Technologies Australia Pty Ltd 12551032 2.0 g/l glucose
Xylazine 100 mg/mL 50 mL Provet XYLAZ4

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References

  1. Sircana, A., Paschetta, E., Saba, F., Molinaro, F., Musso, G. Recent Insight into the Role of Fibrosis in Nonalcoholic Steatohepatitis-Related Hepatocellular Carcinoma. International Journal of Molecular Sciences. 20, (7), 1745 (2019).
  2. Kohn-Gaone, J., Gogoi-Tiwari, J., Ramm, G. A., Olynyk, J. K., Tirnitz-Parker, J. E. The role of liver progenitor cells during liver regeneration, fibrogenesis, and carcinogenesis. American Journal of Physiology-Gastrointestinal Liver Physiology. 310, (3), 143-154 (2016).
  3. Ouchi, R., et al. Modeling Steatohepatitis in Humans with Pluripotent Stem Cell-Derived Organoids. Cell Metabolism. 30, (2), 374-384 (2019).
  4. Vickers, A. E., Fisher, R. L. Organ slices for the evaluation of human drug toxicity. Chemico-Biological Interactions. 150, (1), 87-96 (2004).
  5. Olinga, P., Schuppan, D. Precision-cut liver slices: a tool to model the liver ex vivo. Journal of Hepatology. 58, (6), 1252-1253 (2013).
  6. Paish, H. L., et al. A Bioreactor Technology for Modeling Fibrosis in Human and Rodent Precision-Cut Liver Slices. Hepatology. 70, (4), 1377-1391 (2019).
  7. Prins, G. H., et al. A Pathophysiological Model of Non-Alcoholic Fatty Liver Disease Using Precision-Cut Liver Slices. Nutrients. 11, (3), 507 (2019).
  8. Ramm, G. A., et al. Fibrogenesis in pediatric cholestatic liver disease: role of taurocholate and hepatocyte-derived monocyte chemotaxis protein-1 in hepatic stellate cell recruitment. Hepatology. 49, (2), 533-544 (2009).
  9. Pozniak, K. N., et al. Taurocholate Induces Biliary Differentiation of Liver Progenitor Cells Causing Hepatic Stellate Cell Chemotaxis in the Ductular Reaction: Role in Pediatric Cystic Fibrosis Liver Disease. The American Journal of Pathology. 187, (12), 2744-2757 (2017).
  10. Clouzeau-Girard, H., et al. Effects of bile acids on biliary epithelial cell proliferation and portal fibroblast activation using rat liver slices. Lab Investigation. 86, (3), 275-285 (2006).
  11. Szalowska, E., et al. Effect of oxygen concentration and selected protocol factors on viability and gene expression of mouse liver slices. Toxicology in Vitro. 27, (5), 1513-1524 (2013).
  12. Koch, A., et al. Murine precision-cut liver slices (PCLS): a new tool for studying tumor microenvironments and cell signaling ex vivo. Cell Communication and Signaling. 12, 73 (2014).
  13. Granitzny, A., et al. Maintenance of high quality rat precision cut liver slices during culture to study hepatotoxic responses: Acetaminophen as a model compound. Toxicology in Vitro. 42, 200-213 (2017).
  14. Wu, X., et al. Precision-cut human liver slice cultures as an immunological platform. Journal of Immunological Methods. 455, 71-79 (2018).
  15. Zarybnicky, T., et al. Inter-Individual Variability in Acute Toxicity of R-Pulegone and R-Menthofuran in Human Liver Slices and Their Influence on miRNA Expression Changes in Comparison to Acetaminophen. International Journal of Molecular Sciences. 19, (6), 1805 (2018).
  16. van de Bovenkamp, M., et al. Precision-cut liver slices as a new model to study toxicity-induced hepatic stellate cell activation in a physiologic milieu. Toxicology Sciences. 85, (1), 632-638 (2005).
  17. Buettner, R., et al. Efficient analysis of hepatic glucose output and insulin action using a liver slice culture system. Hormone and Metabolic Research. 37, (3), 127-132 (2005).
  18. Lagaye, S., et al. Anti-hepatitis C virus potency of a new autophagy inhibitor using human liver slices model. World Journal of Hepatology. 8, (21), 902-914 (2016).
  19. Gobert, G. N., Nawaratna, S. K., Harvie, M., Ramm, G. A., McManus, D. P. An ex vivo model for studying hepatic schistosomiasis and the effect of released protein from dying eggs. PLoS Neglected Tropical Diseases. 9, (5), 0003760 (2015).
  20. Jaiswal, S. K., Gupta, V. K., Ansari, M. D., Siddiqi, N. J., Sharma, B. Vitamin C acts as a hepatoprotectant in carbofuran treated rat liver slices in vitro. Toxicology Reports. 4, 265-273 (2017).
  21. Plazar, J., Hreljac, I., Pirih, P., Filipic, M., Groothuis, G. M. Detection of xenobiotic-induced DNA damage by the comet assay applied to human and rat precision-cut liver slices. Toxicology in Vitro. 21, (6), 1134-1142 (2007).
  22. van de Bovenkamp, M., Groothuis, G. M., Meijer, D. K., Olinga, P. Precision-cut fibrotic rat liver slices as a new model to test the effects of anti-fibrotic drugs in vitro. Journal of Hepatology. 45, (5), 696-703 (2006).
  23. Guyot, C., et al. Fibrogenic cell phenotype modifications during remodelling of normal and pathological human liver in cultured slices. Liver International. 30, (10), 1529-1540 (2010).
  24. Bigaeva, E., et al. Exploring organ-specific features of fibrogenesis using murine precision-cut tissue slices. Biochim Biophys Acta - Molecular Basis Disease. 1866, (1), 165582 (2020).
  25. Kiziltas, S. Toll-like receptors in pathophysiology of liver diseases. World Journal of Hepatology. 8, (32), 1354-1369 (2016).
  26. Mencin, A., Kluwe, J., Schwabe, R. F. Toll-like receptors as targets in chronic liver diseases. Gut. 58, (5), 704-720 (2009).
  27. Finot, F., et al. Combined Stimulation with the Tumor Necrosis Factor alpha and the Epidermal Growth Factor Promotes the Proliferation of Hepatocytes in Rat Liver Cultured Slices. International Journal of Hepatology. 2012, 785786 (2012).
  28. Marshall, A., et al. Relation between hepatocyte G1 arrest, impaired hepatic regeneration, and fibrosis in chronic hepatitis C virus infection. Gastroenterology. 128, (1), 33-42 (2005).
  29. Alpini, G., et al. Bile acid feeding increased proliferative activity and apical bile acid transporter expression in both small and large rat cholangiocytes. Hepatology. 34, (5), 868-876 (2001).
  30. Studer, E., et al. Conjugated bile acids activate the sphingosine-1-phosphate receptor 2 in primary rodent hepatocytes. Hepatology. 55, (1), 267-276 (2012).

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