Master the Thawing and Plating of Primary Hepatocytes

Table of Contents:

1. Introduction
2. Life Science Research Tools
3. Lonza Bioscience Solutions
4. Hepatic Cells: A Key Tool for Researchers
   • Basic Research and Therapeutics Development
   • Importance of Hepatic Cells in Research
5. Thawing and Plating Protocol for Primary Hepatocytes
   • Preparing for Thawing and Plating
   • Thawing Process
   • Preparation of Thawing Medium
   • Cell Counting
   • Plating Hepatocytes
6. Incubation and Maintenance of Cells
   • Incubation Conditions
   • Maintenance Medium and Overlay
7. Tips and Troubleshooting
8. Contacting our Scientific Support Team
9. Conclusion

Hepatic Cells: A Key Tool for Life Science Research

Hepatic cells play a pivotal role in advancing life science research, particularly in the field of therapeutics development. Lonza Bioscience Solutions, a part of Lonza's Pharma and Biotech segment, is dedicated to providing researchers with the highest quality hepatic cells and the necessary tools to support their work. In this article, we will explore the importance of hepatic cells in research and provide a detailed protocol for thawing and plating primary hepatocytes.

Introduction

The field of life science research is constantly evolving, with scientists tirelessly working to develop new and effective therapeutics. The journey from basic research to the final product release involves numerous stages, and hepatic cells serve as a crucial tool throughout this process. Lonza Bioscience Solutions recognizes the significance of high-quality hepatic cells and aims to support scientists by providing them with the necessary tools and expert technical instructions.

Life Science Research Tools

To conduct life science research effectively, scientists rely on a wide range of tools and techniques. These tools help them study various biological processes, understand the underlying mechanisms, and eventually develop innovative therapies. Hepatic cells, in particular, are a vital resource for researchers working in drug discovery, toxicity testing, and disease modeling, among other areas.

Lonza Bioscience Solutions

As a leader in the field of life science research, Lonza Bioscience Solutions is committed to delivering superior hepatic cells and support to scientists worldwide. Our comprehensive portfolio includes cryopreserved primary hepatocytes, which are characterized by their high viability and functionality. These cells are vital for accurately studying drug metabolism, toxicity, drug-drug interactions, and other hepatic processes.

Hepatic Cells: A Key Tool for Researchers

Basic Research and Therapeutics Development

Hepatic cells serve as a foundational tool in basic research and therapeutics development. Researchers require accurate models to study the liver's response to various compounds and to assess how drugs are metabolized and potentially toxic. Hepatocytes, the primary cell type in the liver, offer valuable insights into these processes and enable scientists to make informed decisions during drug development.

Importance of Hepatic Cells in Research

The importance of hepatic cells in research cannot be overstated. These cells simulate the liver's functions and provide a reliable model for testing and evaluating potential drug candidates. Hepatocytes play a crucial role in predicting drug metabolism and assessing drug-induced liver toxicity, enabling researchers to identify potential risks and make necessary adjustments to drug candidates before reaching clinical trials.

Thawing and Plating Protocol for Primary Hepatocytes

Accurate and efficient handling of primary hepatocytes is essential to maintain their viability and functionality. The following protocol outlines the steps for thawing and plating cryopreserved primary hepatocytes, ensuring optimal results in downstream applications.

Preparing for Thawing and Plating

Before starting the thawing process, it is crucial to prepare a 37-degree Celsius water bath and warm the appropriate media for your application. Disinfect the conical tube containing the medium and transfer it to your biological safety cabinet. It is recommended to read through the primary hepatocytes thawing and plating protocol in its entirety before proceeding.

Thawing Process

Remove the cryopreserved hepatocytes from their storage location and promptly submerge as much of the vial as possible, up to the cap, in the water bath. Take care to keep the vial's cap above the water line to prevent contamination. Thaw the vial for 90 to 120 seconds, allowing the ice inside to thaw gradually. After thawing, disinfect the vial and transfer it to the biological safety cabinet.

Preparation of Thawing Medium

Prepare the thawing medium by ensuring there are no bubbles in the liquid. Pipette one milliliter of thawing medium back into the original vial and pour the remaining cells back into the 50-milliliter tube of thawing medium. Suspend the cells by carefully rocking the tube by hand for a few seconds.

Cell Counting

To determine the viability and yield of your hepatocytes, use the trypan blue exclusion method for cell counting. Manual counting is preferred, as it provides the most accurate results due to the dual nuclei found in many species of hepatocytes. Determine the volume of plating medium to add to achieve your desired cell density using the formulas provided in the protocol.

Plating Hepatocytes

Using a one-milliliter pipettor, transfer the hepatocytes to a collagen-coated multi-well plate. Refer to the protocol to determine the correct volume of cell stock to add to each well and for an estimate of cells per well. Once the cells are added, gently shake the plate in a southeast-west motion to ensure even dispersion. Repeat the shaking motion at regular intervals following the guidelines provided.

Incubation and Maintenance of Cells

Proper incubation conditions and subsequent maintenance of cells are essential for their growth and functionality. Incubate the cells in a 37-degree Celsius carbon dioxide incubator for a minimum of four to six hours. The specific procedure after incubation differs for plates with or without an overlay.

Plates without an Overlay

If you are not using an overlay, replace the medium with warm maintenance medium or application-specific medium as per the guidelines provided in the protocol.

Plates with an Overlay

For plates with an overlay, prepare the maintenance medium by cooling it on ice. Determine the volume of maintenance medium needed to feed your plate using the provided formula. Find the protein concentration of the overlay matrix on its specification sheet and calculate the amount to add to the maintenance medium based on the formula in the protocol. Aspirate plating media from each well, replace with cold overlay solution, and return the plate to the incubator.

Tips and Troubleshooting

Proper handling of the hepatocytes is crucial to obtaining reliable results. In case of any difficulties or unexpected outcomes during the protocol, refer to the tips and troubleshooting section of the primary hepatocytes thawing and plating protocol. This section provides valuable insights and solutions to common issues that may arise during the experimental process.

Contacting our Scientific Support Team

At Lonza Bioscience Solutions, we understand the significance of having a reliable support system during your research journey. If you require assistance or have any questions regarding our products or protocols, please do not hesitate to contact our dedicated scientific support team. We are here to provide guidance, troubleshoot any issues, and ensure your research progress smoothly.

Conclusion

In conclusion, hepatic cells are an indispensable tool for life science researchers. Lonza Bioscience Solutions aims to support scientists by providing them with high-quality hepatic cells and expert technical instructions. By following the thawing and plating protocol outlined in this article, researchers can ensure the optimal viability and functionality of primary hepatocytes, enabling accurate and reliable research outcomes.