As a lab technician or operator in biotechnology and cell-based research, you play a pivotal role in the establishment and maintenance of a cell bank. Here are the steps, considerations, and guidelines you need to keep in mind to ensure proper cell banking. 

Steps in Proper Cell Banking

Because cell banking involves securing valuable biological resources and cell lines for future studies and applications, it’s important that you know and follow these essential steps and best practices.

Cell Line Authentication

Before you start creating a cell bank, verify the identity of the cell lines you intend to use. Cell line misidentification or contamination can lead to unreliable experimental results, wasting valuable time and resources. To authenticate your cell lines, consider these key steps:

  • Short Tandem Repeat (STR) Analysis: Perform STR analysis to compare the DNA profiles of your cell lines with a reference database. This method is highly reliable for confirming cell line identity.
  • Authentication Frequency: Regularly authenticate your cell lines to ensure their continued integrity. Many experts recommend authentication at the time of acquisition, during the establishment of the master cell bank, and periodically thereafter.
  • Cross-Contamination Prevention: Implement rigorous measures to prevent cross-contamination between cell lines in your laboratory. This strategy includes maintaining separate equipment, avoiding shared workspaces, and practicing strict aseptic techniques.

Master Cell Bank Creation

After authentication, the establishment of a master cell bank (MCB) is a pivotal step in the cell banking process. The MCB serves as the primary source of cells for all subsequent activities, and its creation involves several critical considerations:

  • Cell Line Expansion: Begin by expanding the cell line of interest to an adequate scale. This expansion typically involves growing cells in culture dishes, bioreactors, or other suitable containers.
  • Cryopreservation: Once you’ve achieved the desired cell density, cryopreserve the cells in multiple vials. Cryopreservation ensures long-term storage and provides a backup in case of unforeseen issues with the working cell bank (WCB).
  • Characterization: Thoroughly characterize the cells in the MCB, documenting their growth kinetics, morphology, and relevant genetic markers. This information is vital for maintaining consistency and traceability.
  • Stability Testing: Perform stability testing on the MCB to assess its ability to maintain cell viability, identity, and functionality over time. This testing involves periodic thawing and culturing of vials to evaluate the MCB’s stability.

Working Cell Bank Preparation

From the MCB, you will create the working cell bank (WCB), which is the primary source of cells for routine experiments. Preparing the WCB involves the following steps:

  1. Vial Selection: Choose a representative vial from the MCB for the WCB preparation. This vial should accurately reflect the characteristics of the cell line.
  2. Thawing and Expansion: Thaw the selected vial and expand the cells in the culture. Pay close attention to cell density and viability during this process to ensure a successful WCB.
  3. Quality Control: Assess the WCB for cell line identity, purity, and functionality. This step verifies that the WCB maintains the same characteristics as the MCB.
  4. Documentation: Maintain thorough records of the WCB preparation process, including dates, cell counts, and test results. This documentation is essential for traceability and regulatory compliance.

Adhering to Good Cell Culture Practice (GCCP) Guidelines

To ensure the quality and reliability of your cell banking process, it is crucial to adhere to GCCP guidelines. These guidelines provide a framework for maintaining high standards in cell culture practices, which includes these key GCCP principles.

Aseptic Techniques

Follow strict aseptic techniques to prevent contamination of cell cultures. This process includes working in a laminar flow hood, using sterile equipment, and practicing proper hand hygiene.

Record Keeping

Maintain detailed and organized records of all cell banking activities, including authentication, expansion, cryopreservation, and stability testing. These records are essential for regulatory compliance and troubleshooting.

Quality Control

Implement robust quality control measures to monitor cell line identity, purity, and functionality throughout the cell banking process. Additionally, regularly test for mycoplasma contamination.

Training and Education

Ensure that all personnel involved in cell banking are adequately trained in GCCP principles and procedures. Ongoing training and education are essential to maintain consistency and compliance.

Other Considerations To Keep in Mind

Here are other considerations to keep in mind when performing cell banking.

Cell Line Characterization

Comprehensive characterization of your cell lines is essential for understanding their behavior and ensuring reproducible results. Consider the following aspects of cell line characterization:

  • Growth Characteristics: Determine the optimal culture conditions, such as media composition, temperature, and CO2 levels, that support robust cell growth.
  • Morphology: Document the typical morphology of your cell line, including cell shape, size, and adherence properties. Changes in morphology can indicate issues with the cell line.
  • Genetic Markers: Identify and monitor relevant genetic markers or mutations specific to your cell line. This information helps confirm cell line identity and track any genetic changes over time.
  • Functional Assays: Perform functional assays to assess the biological activity of your cells. These assays may include cell proliferation, differentiation, or response to specific stimuli.

Stability Testing

Ensuring the long-term stability of your cell bank is crucial for maintaining the reliability of your research. Stability testing involves subjecting cells from the MCB and WCB to various conditions to assess their viability and identity over time. Consider these key aspects of stability testing:

  • Temperature Variations: Evaluate how cells respond to fluctuations in storage temperature. Cells should remain viable and maintain their characteristics under different temperature conditions.
  • Long-Term Storage: Assess the stability of cells during extended storage periods, typically over several years. This testing is critical for ensuring that your cell bank remains a valuable resource.
  • Freeze-Thaw Cycles: Subject cells to multiple freeze-thaw cycles to determine their resilience and ability to recover after cryopreservation.
  • Monitoring Genetic Stability: Continuously monitor genetic stability by comparing the genetic markers of cells from the MCB and WCB over time.

Storage Conditions

Finally, proper storage conditions are essential for preserving the integrity of your cell bank. Consider the following recommendations:

  • Cryopreservation: Store cell banks in liquid nitrogen (-196°C) or ultra-low temperature freezers (-80°C). These temperatures ensure long-term viability and stability.
  • Backup Systems: Implement redundancy by storing duplicate cell banks in separate locations or with trusted collaborators to mitigate the risk of catastrophic loss.

If you don’t have the equipment or the facilities to replicate these storage solutions, you can get cell banking and storage services from Cell Culture Company. 

Final Word

Establishing and maintaining a cell bank is a meticulous process that demands attention to detail and adherence to best practices. By following these steps and adhering to the GCCP guidelines, you can protect the quality and reliability of your cell bank.