Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

Brian Czaya; Saurav Singh; Christopher Yanucil; Karla Schramm; Christian Faul; Alexander Grabner

doi:10.3791/55319

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

JoVE Journal
Immunology and Infection

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JoVE Journal Immunology and Infection

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

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08:32 min

March 10, 2017

DOI: 10.3791/55319-v

Brian Czaya*^1,2, Saurav Singh*^1,2, Christopher Yanucil^1,2, Karla Schramm^1,2, Christian Faul^1,2, Alexander Grabner¹

¹Katz Family Drug Discovery Center and Division of Nephrology and Hypertension, Department of Medicine,University of Miami Leonard M. Miller School of Medicine, ²Department of Cell Biology and Anatomy,University of Miami Leonard M. Miller School of Medicine

Overview

This article presents an enzymatic method for isolating primary hepatocytes from adult mice. It also details the quantification of inflammatory responses using ELISA and real-time PCR.

Key Study Components

Area of Science

Hepatology
Inflammatory response
Cell isolation techniques

Background

Understanding hepatocyte function is crucial for studying liver diseases.
Inflammatory cytokines play a significant role in various liver conditions.
Current methods for isolating hepatocytes can be complex and time-consuming.
This study aims to provide a more efficient approach.

Purpose of Study

To isolate functional murine hepatocytes for research.
To analyze the expression and secretion of inflammatory cytokines.
To investigate the hepatic acute phase response in chronic kidney disease.

Methods Used

Enzymatic isolation of hepatocytes from mouse liver.
Use of ELISA for quantifying cytokines.
Real-time PCR for gene expression analysis.
Preparation of liver perfusion and digest media.

Main Results

Successful isolation of viable hepatocytes.
Quantification of cytokines such as CRP and IL6.
Demonstrated reproducibility of the method.
Insights into the role of hepatic cytokines in inflammation.

Conclusions

The method provides a robust tool for hepatic research.
It facilitates the study of liver inflammation and drug responses.
This approach can enhance understanding of liver-related diseases.

Frequently Asked Questions

What is the main advantage of this method?

It offers a simple, fast, and reproducible analysis of hepatic metabolomics and inflammation.

How does this study contribute to chronic kidney disease research?

It helps elucidate the role of hepatic cytokines in a prolonged inflammatory environment.

What cytokines were measured in this study?

Cytokines such as CRP and IL6 were quantified using ELISA.

What techniques were used for gene expression analysis?

Real-time PCR was employed to analyze gene expression.

Is the isolation method applicable to other studies?

Yes, it can be used for various studies involving liver function and inflammation.

What temperature is required for the liver perfusion medium?

The liver perfusion medium should be preheated to 37 degrees Celsius.

Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR.

The overall goal of this procedure is to successfully isolate functional murine hepatocytes for studying the expression and secretion of inflammatory cytokines such as CRP and IL6 in response to mediators of the hepatic acute phase response. This method could help answer key questions of hepatocellular function and the contribution of hepatic cytokines to a prolonged inflammatory environment as found in chronic kidney disease. The main advantage of this technique is that it provides a robust tool for a simple, fast, and reproducible analysis of hepatic metabolomics and inflammation as well as a response to drugs.

To begin, preheat the liver perfusion medium and liver digest medium in a water bath at 37 degrees Celsius. Set up a perfusion pump as shown here. Then use liver perfusion medium to carefully pre-fill the tubing system of the pump.

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Hepatocytes Inflammatory Response Cytokines CRP IL6 Hepatic Acute Phase Response Hepatocellular Function Hepatic Cytokines Chronic Kidney Disease Hepatic Metabolomics Inflammation Liver Perfusion Liver Digest IVC Cannulation Liver Dissection