Name: murine precision-cut liver slices as an ex vivo model of liver biology
Uploaded: 2020-03-14T13:00:00.000+00:00
Duration: 12 min 36 s
Description: Watch this Scientific Journal Video about Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology at JoVE.com

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).

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&#8722;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
<ol>
	<li>Prepare sterile Krebs-Henseleit buffer solution with 2 g/L glucose (<a href="https://www-jove-com.remotexs.ntu.edu.sg/pdf-materials/60992?refresh=1" target="_blank">Table of Materials</a>). 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 &#176;C.</li>
	<li>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.</li>
	<li>Disinfect the blade and cutting arm with a 70% ethanol solution. 
	CAUTION: Avoid contact with the blade.</li>
	<li>Disinfect the buffer tray with a 70% ethanol solution and wipe it with a sterile tissue.</li>
	<li>Insert the buffer tray into the vibratome and tighten the mounting mechanism.</li>
	<li>Set the cutting arm to the maximum available height.</li>
	<li>Set the blade angle to 10&#176; 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.</li>
	<li>Disinfect the specimen holder with a 70% ethanol solution.</li>
	<li>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.</li>
	<li>Fix the water-out tube to a drain and turn on the water. Set the cooler to 4 &#176;C. Run cooling water at a rate of &#62;400 mL/min.</li>
	<li>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.</li>
</ol>
2. Liver removal and preparation
<ol>
	<li>Sterilize or disinfect all surgical instruments including curved forceps, flat square-tip forceps, tweezers, surgical clamps, and scissors using autoclaving.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Using clean forceps and scissors, open the abdominal cavity and lower thoracic cavity.</li>
	<li>Removing the liver quickly without damaging the lobes
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Trim the other three tissue edges. 
	NOTE: This removes some of the fibrous Glisson&#39;s capsule and will make separation of the tissue slices easier.</li>
	<li>Mounting the liver lobe on the specimen holder
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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&#8722;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.</li>
	</ol>
	</li>
</ol>
3. Production of liver slices
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#181;m.</li>
	<li>Repeat the cutting process until the top of the tissue is removed. Discard the first one or two slices, as these will contain Glisson&#39;s capsule and therefore do not contain much functional liver tissue compared to other slices.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#181;m liver slices are produced from each lobe.</li>
	<li>Cut the tissue slices until near the cyanoacrylate glue. Do not cut into the glue.</li>
	<li>Repeat the whole process with other liver lobes until the required number of tissue slices is obtained.</li>
	<li>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.</li>
</ol>
4. Tissue culture
NOTE: All tissue culture work must be performed in a sterile laminar flow hood.
<ol>
	<li>In a tissue culture laminar flow hood, pipette 1 mL of William&#39;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 &#181;g/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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Incubate the liver slices at 37 &#176;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).</li>
	<li>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.</li>
</ol>
5. Example application of PCLS
<ol>
	<li>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 &#181;M) for 2 days.</li>
	<li>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 (<a href="https://www-jove-com.remotexs.ntu.edu.sg/pdf-materials/60992?refresh=1" target="_blank">Table of Materials</a>).</li>
	<li>Purify the RNA using an RNA isolation kit (<a href="https://www-jove-com.remotexs.ntu.edu.sg/pdf-materials/60992?refresh=1" target="_blank">Table of Materials</a>) and create cDNA from the purified RNA.</li>
	<li>Perform quantitative polymerase chain reaction (<a href="https://www-jove-com.remotexs.ntu.edu.sg/pdf-materials/60992?refresh=1" target="_blank">Table of Materials</a>) using specific primers (Table 1) on the cDNA to examine gene expression.</li>
</ol>

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The authors have nothing to disclose.

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&#38;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-&#946;]). In this context, a recent article by Bigaeva et al.24 suggests that spontaneous fibrosis in PCLS is in part mediated by TGF-&#946; signaling, as incubation of PCLS with TGF-&#946; 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.

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&#8722;250 &#181;m 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.

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

murine precision-cut liver slices as an ex vivo model of liver biology

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.

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&#39;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 (<a href="https://www-jove-com.remotexs.ntu.edu.sg/pdf-materials/60992?refresh=1" target="_blank">Table of Materials</a>) 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&#38;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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60992/60992fig1.jpg" /> 
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&#8722;5 days post-isolation. (B) To examine cell morphology, PCLS were fixed, paraffin-embedded, sectioned, and stained with H&#38;E immediately (day 0) and at 1, 2, and 5 days post-isolation. A Kruskal-Wallis test with Dunn&#39;s multiple comparisons test was performed relative to T + 0. All data are represented as mean &#177; SEM (n = 2&#8722;3 mice) with *p &#60; 0.05. The scale bars represent 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60992/60992fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60992/60992fig2.jpg" /> 
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 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60992/60992fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60992/60992fig3.jpg" /> 
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 &#956;M 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 &#947;-glutamyl transpeptidase 1 (Ggt1; C) was examined relative to the geometric mean of Gapdh and Hprt1. Dunnett&#39;s multiple comparisons test was performed relative to untreated control tissue slices (n = 15). All data are represented as mean &#177; SEM (n = 4 mice, triplicate slices; *p &#60; 0.05, **p &#60; 0.01). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60992/60992fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/60992/60992table1.jpg" /> 
Table 1: qPCR primers.

Watch this Scientific Journal Video about Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology at JoVE.com

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

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.

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

murine-precision-cut-liver-slices-as-an-ex-vivo-model-of-liver-biology

Nanyang Technological University

Research

JoVE Journal

Medicine

15.5K Views.  QIMR Berghofer Medical Research Institute. Deze video toont de productie en toepassing van precisie-gesneden lever plakjes als een experimenteel ex vivo model van leverweefsel biologie. Precisiegesneden leversegmenten bezetten een experimentele niche die bestaat tussen in vivo dierstudies en in vitro celkweekmethoden. Deze techniek heeft verschillende belangrijke voordelen ten opzichte van in vivo dierstudies, waaronder de mogelijkheid om controle- en behandelingsmonsters uit dezelfde lever te gebruiken, de isolatie van leverweefsel o...

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

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

Cancer Research

Ex Vivo Hepatic Perfusion Through the Portal Vein in Mouse

Metabolic diseases such as diabetes, pre-diabetes, non-alcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH) are becoming increasingly common. Ex vivo liver perfusions allow for a comprehensive analysis of liver metabolism using nuclear magnetic resonance (NMR), in nutritional conditions that can be rigorously controlled. As in silico simulations remain a primarily theoretical means of assessing hormone actions and the effects of pharmaceutical intervention, the perfused liver remains one of the most valuable test beds for understanding hepatic metabolism. As these studies guide basic insights into hepatic physiology, results must be accurate and reproducible. The greatest factor in the reproducibility of ex vivo hepatic perfusion is the quality of surgery. Therefore, we have introduced an organized and streamlined method to perform ex vivo mouse liver perfusions in the context of in situ NMR experiments. We also describe a unique application and discuss common issues encountered in these studies. The overall purpose is to provide an uncomplicated guide to a technique we have refined over several years that we deem the golden standard for obtaining reproducible results in hepatic resections and perfusions in the context of in situ NMR experiments. The distance to the center of the field for the magnet as well as the inaccessibility of the tissue to intervention during the NMR experiment makes our methods novel.

Ex Vivo Hepatic Perfusion Through the Portal Vein in Mouse

The protocol describes a straightforward method of resectioning an intact mouse liver for metabolic studies through portal vein perfusion.

Metabolic diseases such as diabetes, pre-diabetes, non-alcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH) are becoming ...

Biochemistry

Normothermic Ex Vivo Liver Machine Perfusion in Mouse

This protocol presents an optimized erythrocytes-free NEVLP system using mouse livers. Ex vivo preservation of mouse livers was achieved by employing modified cannulas and techniques adapted from conventional commercial ex vivo perfusion equipment. The system was utilized to evaluate the preservation outcomes following 12 h of perfusion. C57BL/6J mice served as liver donors, and the livers were explanted by cannulating the portal vein (PV) and bile duct (BD), and subsequently flushing the organ with warm (37 &#176;C) heparinized saline. Then, the explanted livers were transferred to the perfusion chamber and subjected to normothermic oxygenated machine perfusion (NEVLP). Inlet and outlet perfusate samples were collected at 3 h intervals for perfusate analysis. Upon completion of the perfusion, liver samples were obtained for histological analysis, with morphological integrity assessed using modified Suzuki-Score through Hematoxylin-Eosin (HE) staining. The optimization experiments yielded the following findings: (1) mice weighing over 30 g were deemed more suitable for the experiment due to the larger size of their bile duct (BD). (2) a 2 Fr (outer diameter = 0.66 mm) polyurethane cannula was better suited for cannulating the portal vein (PV) when compared to a polypropylene cannula. This was attributed to the polyurethane material&#39;s enhanced grip, resulting in reduced catheter slippage during the transfer from the body to the organ chamber. (3) for cannulation of the bile duct (BD), a 1 Fr (outer diameter = 0.33 mm) polyurethane cannula was found to be more effective compared to the polypropylene UT - 03 (outer diameter = 0.30 mm) cannula. With this optimized protocol, mouse livers were successfully preserved for a duration of 12 h without significant impact on the histological structure. Hematoxylin-Eosin (HE) staining revealed a well-preserved morphological architecture of the liver, characterized by predominantly viable hepatocytes with clearly visible nuclei and mild dilation of hepatic sinusoids.

Normothermic Ex Vivo Liver Machine Perfusion in Mouse

A normothermic ex vivo liver perfusion (NEVLP) system was created for mouse livers. This system requires experience in microsurgery but allows for reproducible perfusion results. The ability to utilize mouse livers facilitates the investigation of molecular pathways to identify novel perfusate additives and enables the execution of experiments focused on organ repair.

This protocol presents an optimized erythrocytes-free NEVLP system using mouse livers. Ex vivo preservation of mouse livers was achieved by employing ...

Engineering

A High-Throughput In Situ Method for Estimation of Hepatocyte Nuclear Ploidy in Mice

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as cholangiocytes, myofibroblasts, progenitors and inflammatory cells also indicate chronic liver damage, tissue remodeling and disease progression. In this protocol, we describe a simple high-throughput approach for calculating changes in the cellular composition of the liver that are associated with injury, chronic disease and cancer. We show how information extracted from two-dimensional (2D) tissue sections can be used to quantify and calibrate hepatocyte nuclear ploidy within a sample and enable the user to locate specific ploidy subsets within the liver in situ. Our method requires access to fixed/frozen liver material, basic immunocytochemistry reagents and any standard high-content imaging platform. It serves as a powerful alternative to standard flow cytometry techniques, which require disruption of freshly collected tissue, loss of spatial information and potential disaggregation bias.

We present a robust, cost-effective, and flexible method for measuring changes in hepatocyte number and nuclear ploidy within fixed/cryopreserved tissue samples that does not require flow cytometry. Our approach provides a powerful sample-wide signature of liver cytology ideal for tracking the progression of liver injury and disease.

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as ...

Precision Cut Lung Slices as an Efficient Tool for Ex vivo Pulmonary Vessel Structure and Contractility Studies

The visualization of murine lung tissue provides valuable structural and cellular information regarding the underlying airway and vasculature. However, the preservation of pulmonary vessels that truly represents physiological conditions still presents challenges. In addition, the delicate configuration of murine lungs result in technical challenges preparing samples for high-quality images that preserve both cellular composition and architecture. Similarly, cellular contractility assays can be performed to study the potential of cells to respond to vasoconstrictors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these technical issues, the precision-cut lung slice (PCLS) method can be applied as an efficient alternative to visualize lung tissue in three dimensions without regional bias and serve as a live surrogate contractility model for up to 10 days. Tissue prepared using PCLS has preserved structure and spatial orientation, making it ideal to study disease processes ex vivo. The location of endogenous tdTomato-labeled cells in PCLS harvested from an inducible tdTomato reporter murine model can be successfully visualized by confocal microscopy. After exposure to vasoconstrictors, PCLS demonstrates the preservation of both vessel contractility and lung structure, which can be captured by a time-lapse module. In combination with the other procedures, such as western blot and RNA analysis, PCLS can contribute to the comprehensive understanding of signaling cascades that underlie a wide variety of disorders and lead to a better understanding of the pathophysiology in pulmonary vascular diseases.

Precision Cut Lung Slices as an Efficient Tool for Ex vivo Pulmonary Vessel Structure and Contractility Studies

Presented here is a protocol for preserving the vascular contractility of PCLS murine lung tissue, resulting in a sophisticated three-dimensional image of the pulmonary vasculature and airway, which can be preserved for up to 10 days that is susceptible to numerous procedures.

The visualization of murine lung tissue provides valuable structural and cellular information regarding the underlying airway and vasculature. However, ...

3D Imaging of the Liver Extracellular Matrix in a Mouse Model of Non-Alcoholic Steatohepatitis

Non-alcoholic steatohepatitis (NASH) is the most common chronic liver disease in the United States, affecting more than 70 million Americans. NASH can progress to fibrosis and eventually to cirrhosis, a significant risk factor for hepatocellular carcinoma. The extracellular matrix (ECM) provides structural support and maintains liver homeostasis via matricellular signals. Liver fibrosis results from an imbalance in the dynamic ECM remodeling process and is characterized by excessive accumulation of structural elements and associated changes in glycosaminoglycans. The typical fibrosis pattern of NASH is called &#34;chicken wire,&#34; which usually consists of zone 3 perisinusoidal/pericellular fibrosis, based on features observed by Masson&#39;s trichrome stain and Picrosirius Red stains. However, these traditional thin two-dimensional (2D) tissue slide-based imaging techniques cannot demonstrate the detailed three-dimensional (3D) ECM structural changes, limiting the understanding of the dynamic ECM remodeling in liver fibrosis.
The current work optimized a fast and efficient protocol to image the native ECM structure in the liver via decellularization to address the above challenges. Mice were fed either with chow or fast-food diet for 14 weeks. Decellularization was performed after in situ portal vein perfusion, and the two-photon microscopy techniques were applied to image and analyze changes in the native ECM. The 3D images of the normal and NASH livers were reconstituted and analyzed. Performing in situ perfusion decellularization and analyzing the scaffold by two-photon microscopy provided a practical and reliable platform to visualize the dynamic ECM remodeling in the liver.

The present protocol optimizes the liver in situ perfusion/decellularization and two-photon microscopy methods to establish a reliable platform to visualize the dynamics of extracellular matrix (ECM) remodeling during non-alcoholic steatohepatitis (NASH).

Non-alcoholic steatohepatitis (NASH) is the most common chronic liver disease in the United States, affecting more than 70 million Americans. NASH can ...

The Murine Choline-Deficient, Ethionine-Supplemented (CDE) Diet Model of Chronic Liver Injury

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual inflammation, progressive destruction and regeneration of the hepatic parenchyma, liver progenitor cell proliferation, and fibrosis. The end-stage of every chronic liver disease is cirrhosis, a major risk factor for the development of hepatocellular carcinoma. To study processes regulating disease initiation, establishment, and progression, several animal models are used in laboratories. Here we describe a six-week time course of the choline-deficient and ethionine-supplemented (CDE) mouse model, which involves feeding six-week old male C57BL/6J mice with choline-deficient chow and 0.15% DL-ethionine-supplemented drinking water. Monitoring of animal health and a typical body weight loss curve are explained. The protocol demonstrates the gross examination of a CDE-treated liver and blood collection by cardiac puncture for subsequent serum analyses. Next, the liver perfusion technique and collection of different hepatic lobes for standard evaluations are shown, including liver histology assessments by hematoxylin and eosin or Sirius Red stainings, immunofluorescent detection of hepatic cell populations as well as transcriptome profiling of the liver microenvironment. This mouse model is suitable for studying inflammatory, fibrogenic, and liver progenitor cell dynamics induced through chronic liver disease and can be used to test potential therapeutic agents that may modulate these processes.

The Murine Choline-Deficient, Ethionine-Supplemented CDE Diet Model of Chronic Liver Injury

Here we describe a common method to induce chronic liver injury in mice by feeding of a choline-deficient and ethionine-supplemented (CDE) diet. We demonstrate health monitoring, liver perfusion, isolation, and preservation. A time course of six weeks can inform about liver injury, pathohistology, fibrosis, inflammatory, and liver progenitor cell responses.

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual ...

Biology

In Situ Mouse Liver Perfusion Model to Study Metabolic Responses to Substrates

Hepatic Glucose Production, Ureagenesis, and Lipolysis Quantified using the Perfused Mouse Liver Model

The liver has numerous functions, including nutrient metabolism. In contrast to other in vitro and in vivo models of liver research, the isolated perfused liver allows the study of liver biology and metabolism in the whole liver with an intact hepatic architecture, separated from the influence of extra-hepatic factors. Liver perfusions were originally developed for rats, but the method has been adapted to mice as well. Here we describe a protocol for in situ perfusion of the mouse liver. The liver is perfused antegradely through the portal vein with oxygenated Krebs-Henseleit bicarbonate buffer, and the output is collected from the suprahepatic inferior vena cava with clamping of the infrahepatic inferior vena cava to close the circuit. Using this method, the direct hepatic effects of a test compound can be evaluated with a detailed time resolution. Liver function and viability are stable for at least 3 h, allowing the inclusion of internal controls in the same experiment. The experimental possibilities using this model are numerous and may infer insight into liver physiology and liver diseases.

Author Spotlight: Investigating Hepatic Adaptations and Prediabetic Progression in Liver Diseases 

Here, we present a robust method for in situ perfusion of the mouse liver to study the acute and direct regulation of liver metabolism without disturbing the hepatic architecture but in the absence of extra-hepatic factors.

The liver has numerous functions, including nutrient metabolism. In contrast to other in vitro and in vivo models of liver research, the isolated perfused ...

Studying Liver Regeneration of ALLPS in Animal Models for Staged Hepatectomy

A Mouse Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy Procedure Aided by Microscopy

Hepatectomy is widely regarded as the primary treatment for hepatic malignancies; yet, postoperative liver failure remains a major cause of perioperative mortality, severely impacting patient outcomes. In a robust hepatic environment, the future liver remnant (FLR) must exceed 25%, and in cases of cirrhosis, this requirement increases to over 40%. The inadequacy of FLR is currently a major obstacle in the progression of hepatic surgery.
Traditional methods to enhance FLR hypertrophy mainly focus on portal vein embolization (PVE), but its effectiveness is considerably limited. In recent years, there have been numerous reports on a novel biphasic hepatectomy method involving hepatic partitioning and portal vein ligation, known as associating liver partition and portal vein ligation for staged hepatectomy (ALPPS). ALPPS surpasses PVE in efficiently and considerably inducing FLR hypertrophy. However, the detailed mechanisms driving ALPPS-facilitated hepatic regeneration are not fully understood. Thus, replicating ALPPS in animal models is crucial to thoroughly investigate the molecular mechanisms of hepatic regeneration, offering valuable theoretical and practical insights.

Author Spotlight: Advancing Liver Regeneration Research through ALPPS Mouse Model

Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy (ALPPS) has been shown to result in remarkable liver regeneration after several weeks post first-stage surgery. Animal models that have undergone ALPPS have been developed to explore potential liver regeneration ability and achieve favorable clinical outcomes.

Hepatectomy is widely regarded as the primary treatment for hepatic malignancies; yet, postoperative liver failure remains a major cause of perioperative ...

Noninvasive Intraocular In Vivo Imaging of Engrafted Liver Spheroids in Mice

In Vivo Imaging of Liver Spheroids Engrafted in the Anterior Chamber of the Mouse Eye

Biomedical studies of the liver in mammals are hindered by the lack of methods for in vivo noninvasive longitudinal imaging at cellular resolution. Until now, optical imaging of the liver in situ is possible by intravital imaging, which offers high-resolution imaging at the cellular level but cannot be performed multiple times and, therefore, longitudinally in the same animal. Noninvasive imaging methods, such as bioluminescence, allow repeated imaging sessions on the same animal but do not achieve cell resolution. To address this methodology gap, we have developed a platform for noninvasive in vivo imaging of liver spheroids engrafted in the anterior chamber of the mouse eye. In the workflow described in this study, primary mouse liver spheroids are generated in vitro and transplanted into the anterior chamber of the eye of recipient mice, where they engraft on the iris. The cornea acts as a natural body window through which we can image the engrafted spheroids by conventional confocal microscopy. The spheroids survive for months in the eye, during which the cells can be studied in contexts of health and disease, as well as being monitored in response to different stimuli over repeated imaging sessions using appropriate fluorescent probes. In this protocol, we provide a breakdown of the necessary steps to implement this imaging system and explain how to best harness its potential.

Author Spotlight: Harnessing Mouse Eye Chambers for Noninvasive Liver Spheroid Studies

Here, we describe a platform that allows noninvasive in vivo imaging of liver spheroids engrafted in the anterior chamber of the mouse eye. The workflow spans from generating spheroids from primary liver cells to transplantation into the mouse eye and in vivo imaging at cellular resolution by confocal microscopy.

Biomedical studies of the liver in mammals are hindered by the lack of methods for in vivo noninvasive longitudinal imaging at cellular resolution. Until ...

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

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