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Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology
Summary March 14th, 2020
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.
Transcript
This video demonstrates the production and application of precision-cut liver slices as an experimental ex vivo model of liver tissue biology. Precision-cut liver slices occupy an experimental niche that exists between in vivo animal studies and in vitro cell culture methods. This technique has several key benefits over in vivo animal studies, including the ability to use control and treatment samples from the same liver, the isolation of liver tissue to remove off-target effects from other organ systems, and the ability to use reagents that might have a negative effect if applied to a whole living mouse, for example, toxic pathway inhibitors.
The technique involves using a laterally vibrating blade to cut ultra-thin, liver slices of viable liver tissue followed by laboratory tissue culture. The vibrations of the blade prevents tearing caused by shear stress. In this example, we'll show the techniques for preparations of viable ex vivo liver slices, and demonstrate the utility of liver slice culture in example experiments where this ex vivo tissue is treated with bile acids to stimulate cholestatic liver injury to assess the mechanisms of hepatic fibrogenesis.
Insert the blade into the cutting arm. Disinfect the blade and the cutting arm with a 70%ethanol solution. Always be careful to avoid contact with the blade.
Disinfect the buffer tray with a 70%ethanol solution, and wipe with a sterile tissue. Insert the buffer tray into the vibratome and tighten the mounting screw. Set the cutting arm to the maximum available height.
Check the blade angle. On our vibratome, we use a 10-degree angle from horizontal, with the blade sloping down towards the sample. Make sure the blade angle is tightly fixed.
Disinfect the specimen holder, again, with a 70%ethanol solution. Connect the cooling water for the Peltier thermoelectric cooler, located under the buffer tray. Some vibratome models use an ice bath for cooling instead.
Fix the water-out tube to a drain, and turn on the water. Set the cooler to four degrees Celsius. Add ice-cold Krebs-Henseleit buffer to the buffer tray.
All surgical instruments and materials should be sterile. Mice should be deeply anesthetized or euthanized. Skin surfaces on the mice were disinfected by wetting with 70%ethanol solution.
Make a midline incision into the skin from the base of the abdominal cavity to the diaphragm. Using clean forceps and scissors, open the abdominal cavity. Remove the liver quickly, without damaging the lobes.
Push the liver downwards, and cut the connective tissue at the top of the liver. Push the liver upwards. Hold the center vascular area of the liver with the forceps and pull upward.
Cut connective tissue blood vessels. Take care not to damage the liver lobes, and also, take care not to cut into the gastrointestinal tract. Place the removed liver into a sterile dish of ice-cold Krebs-Henseleit buffer.
Separate the liver into individual lobes. Select one liver lobe and place flat side down on a new sterile dish. Keep other lobes on ice in Krebs-Henseleit buffer.
Trim the edges of the liver lobes. Trimming is important, particularly at the edge that will contact the cutting blade first, as having a tissue edge relatively perpendicular to the cutting blade will prevent tissue compression that can occur at shallow angles. Cutting the tissue around the other three edges removes much of the fibrous capsules at the tissue edge, and typically allows easier removal of the tissue slices.
Place a thin layer of cyanoacrylate glue on the front edge of the specimen holder, slightly larger than the trimmed liver lobes. Remove any residue of buffer from the tissue using sterile absorbent material. Place liver lobe on cyanoacrylate glue, with the largest edge facing towards the front.
Allow to cure in air for one to two minutes. Place the tissue attached to the specimen holder into the vibratome. Set the vibratome speed.
Lower the cutting blade until it's located just above the liver lobe. Run the vibrating cutting arm over the sample. Vibration of the blade on this machine is activated by the foot pedal.
Return the blade backwards, and lower the blade height by 250 micrometers. Repeat this process until the top layer of tissue is removed. Discard the first one or two slices, as these will contain Glisson's capsule, and therefore, won't contain much functional liver tissue.
To cut liver slices, slowly advance the vibrating blade into the tissue by turning the handle. Use a small paintbrush to gently guide the tissue during the cutting process. Use the paintbrush to pick up the cut tissue section, and then place the tissue section into a sterile tube containing Krebs-Henseleit buffer for storage.
When not in use, keep this tube on ice. Some of the time, the tissue does tear during the cutting process, but for most purposes, the tissue is still usable. Cut the tissue slices until near the cyanoacrylate glue.
Don't cut into the glue. Repeat with the other liver lobes, which are sitting on ice, until the required number of tissue slices is obtained. To clean the specimen holder, either use a blade to scrape the glue-tissue mixture off, or the mixture can be softened using solvents such as acetone or dimethyl sulfoxide.
In a tissue culture hood, pipette one mL of Williams'E medium containing 10%fetal bovine serum and antimicrobials into a 12-well plate. Remove the tissue slices by gently swirling the mixture and tip into a sterile dish. Cut the tissue into a roughly uniform size.
In this example, we aimed for tissue slices with a surface area around 50 millimeters square. Take care to examine the tissue slices for consistency. Darker slices suggest the tissue thickness is increased, and should be discarded.
Lighter slices suggest the presence of cured cyanoacrylate glue, and should be discarded. Transfer the tissue slices to the 12-well plate containing one milliliter of media. Incubate the liver slices under normal tissue culture conditions.
If possible, use methods to enhance oxygen availability to the tissue slices. On the day after, change the media using a manual pipette to prevent loss of tissue by suction. The precision-cut liver slices are now ready for experimental use.
For our application, we have treated the liver slices with bile acids for two days, and homogenized in lysis buffer for messenger RNA extraction and qPCR analysis. To examine the viability of precision-cut liver slices over time, we used ATP levels as a proxy for cell viability. ATP levels were normalized to protein, and were measured at multiple time points post-isolation.
ATP levels were decreased in the immediate and one-hour samples, however this recovered by three hours. Following this, the ATP levels were maintained for five days, although they trended downwards over time. H&E staining was used to examine precision-cut liver slices that were maintained in culture for up to five days.
Tissue morphology was suggestive of some necrosis occurring at day two, with severe necrosis happening by day five. Because of the necrosis happening between days two and five, we'd recommend the experimental utility of this technique is limited to around three days. However, this could be enhanced by using better oxygen delivery techniques to the tissue slices.
Precision-cut liver slices also appear to have thickening of collagen fibers relative to time in culture. This is suggestive of spontaneous fibrogenic processes occurring. In the example experiment, the precision-cut liver slices were treated with three separate bile acids for two days, to mimic hepatic cholestasis.
Looking at the mRNA expression of three cholangiocyte-specific genes, the two conjugated bile acids, glycocholic and taurocholic acids, significantly increased the expression of both cytokeratin-19 and connexin-43. This is consistent with cholangiocyte development. After watching this video, you should have a good understanding of how to create precision-cut liver slices, and how to perform basic tissue culture.
Precision-cut liver slices can be used for a very wide range of applications, including experimental investigations in RNA expression, protein expression, epigenetics, DNA binding, metabolism, infection mechanisms, cancer invasion, pharmacology, cell biology, and secretion studies, just to name a few.
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