Getting Started with Tissue Culture

Addgene

30 Nov 201806:25

Summary

TLDRThis 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.

Outlines

00:00

🔬 Introduction to Mammalian Tissue Culture Techniques

This paragraph introduces the fundamental aspects of mammalian tissue culture, a critical tool for scientific research. It emphasizes the importance of studying cellular processes in a controlled environment and the use of mammalian cells for producing essential lab tools like antibodies and viruses. The script highlights the challenges faced by both novice and experienced scientists in learning tissue culture techniques and offers guidance on setting up a sterile lab environment. Key instruments such as a carbon dioxide incubator, inverted microscope, and biosafety cabinet are mentioned, along with best practices for preventing contamination, including proper sash height, airflow, and the use of alcohol for decontamination. The importance of maintaining sterile conditions, such as wearing lab coats and gloves, is also stressed.

05:01

🛠 Best Practices for Tissue Culture and Cell Handling

This paragraph delves into the best practices for handling and maintaining mammalian cell cultures. It provides detailed instructions on how to prepare for an experiment, including the setup of materials and the use of filtered serological pipettes to reduce contamination risks. The script advises on the importance of body floor control, proper attire, and handling techniques to prevent contamination. It also discusses the signs of contamination, both macroscopic and microscopic, and the steps to take when switching between cell lines to avoid cross-contamination. The paragraph further covers the importance of cell line screening and maintaining early passage cells for consistency in research. Additionally, it offers tips on cell detachment and seeding, emphasizing the need for gentle handling and even distribution of cells for optimal culture health. The video concludes with a resource recommendation for further learning on tissue culture.

Mindmap

Keywords

💡Mammalian Cells

Mammalian cells are cells derived from animals and are used in laboratory settings for various scientific studies. They are central to the video's theme as they are the primary subjects of tissue culture. The script mentions that these cells allow scientists to study biological processes at the cellular level without needing to work with whole organisms, highlighting their importance in research.

💡Tissue Culture

Tissue culture is a technique used to maintain or grow biological tissues, such as mammalian cells, outside their natural environment. It is a critical tool for scientists as explained in the script, allowing them to produce vital lab tools like antibodies or viruses. The video is essentially an instructional guide on best practices for those new to tissue culture.

💡Sterile Work Environment

A sterile work environment is crucial for preventing contamination in tissue culture. The script emphasizes the importance of maintaining sterility by using a class 2 biosafety cabinet, wiping down surfaces with 70% alcohol, and ensuring proper airflow. This concept is integral to the success of any tissue culture experiment.

💡Contamination

Contamination refers to the unwanted presence of microorganisms or other substances that can compromise the integrity of a tissue culture. The script discusses various measures to prevent contamination, such as wearing gloves and lab coats, using filtered pipettes, and checking for signs of contamination like changes in media color or turbidity.

💡Carbon Dioxide Incubator

A carbon dioxide incubator is a piece of laboratory equipment that provides a controlled environment for cell growth, maintaining specific conditions like temperature and CO2 levels. The script lists it as a key instrument for tissue culture, indicating its role in simulating the natural environment for the cells.

💡Inverted Microscope

An inverted microscope is used to observe cells that are growing on surfaces, such as in tissue culture dishes. The script mentions its use for examining cultures both macroscopically and microscopically to detect signs of contamination, making it a vital tool for monitoring cell health.

💡Class 2 Biosafety Cabinet

A class 2 biosafety cabinet is a type of enclosed workspace that provides personnel, product, and environmental protection in laboratory settings. The script describes its importance in establishing a sterile work environment by maintaining proper sash height and airflow, which is essential for tissue culture.

💡Serological Pipettes and Tips

Serological pipettes and tips are used for transferring liquids in a sterile manner. The script recommends using filtered versions to reduce the risk of contamination, illustrating their importance in maintaining sterility during tissue culture procedures.

💡Cell Bank

A cell bank refers to a collection of cells that are stored for future use, typically in a cryogenic freezer. The script advises creating a cell bank of early passage cells to ensure the availability of genetically consistent cells, which is crucial for maintaining the reproducibility of research data.

💡Passaging

Passaging is the process of transferring cells from one culture vessel to another to maintain or expand the cell population. The script discusses the importance of keeping track of passage numbers and replacing cells with those from an early passage cell bank after a certain number of passages to prevent genotypic variations.

💡Master Mix

A master mix is a solution containing all the necessary components for a particular procedure, such as seeding multiple culture vessels. The script suggests preparing a master mix for better uniformity and more reliable data, highlighting its use in streamlining tissue culture processes.

Highlights

Tissue culture is a critical tool for studying biological processes on the cellular level and producing vital lab tools like antibodies and viruses.

Learning basic tissue culture technique can be daunting for both new and experienced scientists.

Ensure the lab is equipped with necessary materials like a CO2 incubator, inverted microscope, and biosafety cabinet before starting tissue culture.

Preventing contamination is critical for tissue culture experiments, starting with establishing a sterile work environment.

Set biosafety cabinet sash to proper operating height (8-10 inches) to avoid disrupting airflow and compromising cells.

Allow biosafety cabinet to warm up for 5 minutes before use to purge unwanted particulates.

Wipe all pipettes and surfaces with 70% alcohol and allow to evaporate before starting an experiment.

Decontaminate reagent bottles and plasticware with alcohol before transferring to the biosafety cabinet.

Position all materials needed for the experiment in the biosafety cabinet to minimize opening and closing the sash.

Use filtered serological pipettes and tips to reduce risk of contaminating common equipment.

Wear a clean lab coat and gloves to prevent body fluids from contaminating tissue culture.

Check lab coat to ensure wrists and arms are covered, and wear disposable sleeves if needed.

Examine cultures macroscopically and microscopically for signs of contamination like bad odors, color changes, or turbidity.

Change serological pipette or tips anytime they contact the culture to prevent cross-contamination.

Use different reagent bottles for different cell lines to limit the chance of cross-contamination.

Screen all cell lines 2-3 weeks after thawing and rescreen every 2-3 months to detect genotypic variations.

Create a cell bank of early passage cells to maintain reproducibility and discard flasks after 30-40 passages.

Treat cells gently during growth procedures, like aspirating and trypsinizing, to prevent disrupting cell layers.

Prepare a master mix for seeding multiple dishes to ensure even distribution and uniformity.