Getting Started with Tissue Culture

Summary

This instructional video offers essential guidance for scientists new to mammalian tissue culture. It emphasizes the importance of a sterile environment, using a CO2 incubator, inverted microscope, and biosafety cabinet to prevent contamination. The video advises on proper lab attire, handling techniques, and regular screening of cell lines to maintain genetic integrity. It also highlights the significance of gentle cell treatment and even distribution for healthy cell growth, aiming to ensure reliable experimental outcomes and data reproducibility.

Takeaways

• 🔬 Tissue culture is a critical tool for studying biological processes at the cellular level and producing lab tools like antibodies and viruses.
• 🛠️ Essential equipment for tissue culture includes a carbon dioxide incubator, an inverted microscope, and a class 2 biosafety cabinet.
• 🚫 Preventing contamination is of utmost importance in tissue culture, starting with maintaining a sterile work environment.
• 🔍 Proper operation of the biosafety cabinet is crucial, including setting the sash to the correct height and allowing the unit to warm up before use.
• 🧴 All pipettes and surfaces should be wiped with 70% alcohol, and reagent bottles and plasticware should be decontaminated before use.
• 📦 Organize materials needed for the experiment before starting to minimize the risk of contamination.
• 🧤 Wear a clean lab coat and gloves, and ensure full coverage to prevent body fluids from contaminating the culture.
• 🔬 Regularly examine cultures for signs of contamination, both macroscopically and microscopically, including changes in odor or media color.
• 🛑 Change serological pipettes or tips after contact with culture to avoid cross-contamination.
• 🔄 Use different reagent bottles for different cell lines and thoroughly clean equipment when switching cell lines.
• 🧊 Create a cell bank of early passage cells to maintain cell line integrity and reproducibility over time.
• 🌱 Handle cells gently during procedures like aspirating and trypsinizing to ensure even distribution and culture health.

Q & A

What is the primary purpose of mammalian cell culture in a laboratory setting?

Mammalian cell culture is a critical tool for studying biological processes at the cellular level and for producing vital lab tools such as antibodies or viruses.

Why is it important for scientists to learn tissue culture techniques?

Learning tissue culture techniques is important because it allows scientists to work with cells directly, avoiding the need to work with whole organisms, and it is essential for producing and studying various biological tools.

What are some key instruments required for tissue culture experiments?

Key instruments include a carbon dioxide incubator, an inverted microscope, and a class 2 biosafety cabinet.

Why is preventing contamination so critical in tissue culture?

Preventing contamination is critical because it ensures the accuracy and reliability of experimental results and maintains the integrity of the cultured cells.

What is the proper operating height for a biosafety cabinet sash and why is it important?

The proper operating height for a biosafety cabinet sash is usually about 8 to 10 inches. This height is important because it ensures the correct airflow within the cabinet, preventing contamination of the cells.

How long should a biosafety cabinet be allowed to warm up before use?

The biosafety cabinet should be allowed to warm up for at least 5 minutes before use to purge unwanted particulates.

What should be done to pipettes and surfaces before beginning an experiment in the biosafety cabinet?

All pipettes and surfaces should be wiped with 70% alcohol and allowed to evaporate to ensure a sterile environment.

Why is it recommended to use filtered serological pipettes and tips in tissue culture?

Using filtered serological pipettes and tips reduces the risk of contaminating common equipment, such as pipettes and pipettemen, thus minimizing the chance of introducing contaminants into the culture.

What are some measures to prevent body floor contamination in tissue culture?

To prevent body floor contamination, wear a clean lab coat and gloves when working in a hood, ensure wrists and arms are covered, and avoid exposure by wearing disposable sleeves if necessary.

How can one check for signs of contamination in tissue culture?

Signs of contamination can be checked both macroscopically, such as bad odors or changes in media color, and microscopically, using the high power objective of an inverted microscope to observe low levels of contamination.

Why is it necessary to change serological pipette or pipette tips after they contact the culture?

Changing serological pipette or pipette tips after contact with the culture ensures that you are not contaminating your media or other reagents with cells, thus preventing cross-contamination.

What is the recommended procedure for switching to a new cell line in tissue culture?

When switching to a new cell line, dispose of all trash, rinse the aspirator line with bleach, wipe down all equipment and surfaces with alcohol, and change your gloves before handling the new line.

Why is it important to create a cell bank of early passage cells?

Creating a cell bank of early passage cells helps maintain the original genotype and phenotype of the cells, ensuring the reproducibility of experimental data over time.

What should be done to treat cells gently during tissue culture procedures?

To treat cells gently, aspirate carefully to not disrupt the cells, avoid adding enzymes directly to the culture medium, and gently rotate the flask to evenly disperse the liquid when detaching cells.

How can one ensure even distribution of cells when seeding a new culture vessel?

Using a serological pipet to thoroughly mix the cell suspension ensures even distribution. Additionally, preparing a master mix containing all cells and media for all dishes can result in better uniformity and more reliable data.

What is the recommended source for additional protocols and information on tissue culture?

For additional protocols, videos, and useful blog posts on tissue culture, one can visit Addgene's website.