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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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.

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 cellul....

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 solutio.......

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 (

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 expression

Acknowledgements

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

NameCompanyCatalog NumberComments
10 cm Petri DishGREINER664160Sterile Dish
12 Well Tissue Culture Plate Flat BottomGreiner Bio-one665180
70% Ethanol Solution (made with AR Grade)Chem-Supply Pty LtdEA043-20L-PDisinfection solution
AcetoneChem-Supply Pty LtdAA008-2.5L
Cholic acidSigma-AldrichC1129-100G
Cyanoacrylate Super GlueParfix, DuluxGroup (Australia)Other brands should work
Disposable Single Edge Safety Razor BladesMixed
Dissection BoardMade in-houseSterile material over polystyrene
Fetal Bovine SerumGE Healthcare Australia Pty LtdSH30084.02
Forceps sharp point 130 mm longThermoFisher ScientificMET2115-130
Forma Steri-Cycle CO2 IncubatorThermoFisher Scientific371
GlutamineLife Technologies Australia Pty Ltd25030081
Glycocholic acid hydrateSigma-AldrichG2878-100G
ISOLATE II RNA Mini KitBioline (Aust) Pty LtdBIO-52073
Ketamine 50 mlProvetKETAI1
Krebs-Henseleit Buffer with Added Glucose 2000 mg/LSigma-AldrichK3753Can also be made in house
Laminar Flow HoodHepa air filtration
NanoDrop 2000/2000c SpectrophotometersThermoFisher Scientific
Penicillin-Streptomycin, Liq 100 mlLife Technologies Australia Pty Ltd15140-122
Picro Sirius RedABCAM Australia Pty Ltdab246832
Pipette Tips Abt 1000 µl Filter InterpathInterpath24800
Pipette Tips Abt 10 µl Filter InterpathInterpath24300
Pipette Tips Abt 200 µl Filter InterpathInterpath24700
Pipette Tips Abt 20 µl Filter InterpathInterpath24500
Precellys HomogeniserBertin InstrumentsP000669-PR240-A
ProtractorGenericTo measure blade angle
Quantstudio 5 QPCR Fixed 384 BlockApplied Biosystems/ ThermoFisher Scientific
Scalpel BladeMixed
Scalpel Blade HolderMixed
SensiFAST cDNA Synthesis KitBioline (Aust) PTY LTD
Small Paintbrush with Plastic HandleMixedPlastic handle resists ethanol
Square-Head ForecepsMixed
Sterile 50 ml Plastic TubesCorning Falcon352098
Surgical ClampsMixed
Surgical ForcepsMixed
Surgical PinsMixed
Surgical ScissorsMixed
Taurochoic acidSigma-AldrichT-4009-5G
Vibratome SYS-NVSLM1 Motorized VibrosliceWorld Precision InstrumentsSYS-NVSLM1With thermoelectric cooling
Williams Medium ELife Technologies Australia Pty Ltd125510322.0 g/l glucose
Xylazine 100 mg/mL 50 mLProvetXYLAZ4

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.

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Precision cut Liver SlicesEx Vivo ModelLiver BiologyVibrating BladeTissue CultureBile AcidsCholestatic Liver InjuryHepatic FibrogenesisKrebs Henseleit BufferPeltier Thermoelectric Cooler

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