Primary Cell Culture: A Synopsis

Cell Applications, Inc. YouTube

26 Feb 201504:39

Summary

TLDRCell Applications' video script outlines a step-by-step guide for optimal cell storage and handling. It instructs users to promptly transfer cryovials to liquid nitrogen upon arrival and to thaw cells in a 37°C water bath, ensuring partial ice remains. The script details cell plating, subculturing, and tips for healthy cell growth, emphasizing the importance of using the correct growth medium and sterile techniques for reliable cell culture data. For more information, visit www.cellapplications.com.

Takeaways

❄️ Upon receiving cryovials, immediately remove them from dry ice and minimize exposure to room temperature.
🌡️ Set a water bath to 37 degrees Celsius for cell thawing and confirm the temperature with a thermometer.
🧪 Prepare a T75 flask with 15 ml of growth medium, ensuring the flask is cell culture treated and the media is appropriate for the cells.
🧊 Keep the cryovial on dry ice while loosening and retightening the cap to release pressure before thawing.
🚿 Thaw the cells partially in the water bath, ensuring a small amount of ice remains inside the vial.
🔍 Decontaminate the tube with alcohol before removing the cryovial cap, being careful not to touch the threads or rim.
🌱 Gently resuspend the cells with a pipette to avoid foaming, and transfer them into the prepared flask with growth medium.
🔄 Loosen the flask cap for gas exchange and incubate the cells undisturbed for at least four hours before changing the medium.
📈 Replace the growth medium every other day until the cells reach 60% confluence, then double the volume of growth medium.
🔄 Subculture cells when confluence reaches 80% by aspirating the medium, washing with HBSS, and using trypsin-EDTA to detach the cells.
🔬 After trypsinization, neutralize the trypsin with a trypsin neutralizing solution and centrifuge the cells before resuspending and counting them.

Q & A

What is the first step to take when you receive cryovials with primary cells?
-Upon arrival, remove the cryovial from dry ice packaging and minimize its exposure to room temperature.
Where should cryovials be stored after removing them from dry ice packaging?
-Cryovials should be transferred to liquid nitrogen for storage.
What is the recommended temperature for a water bath to thaw cells?
-The water bath should be set to 37 degrees Celsius for thawing cells.
How much growth medium should be pipetted into a T75 flask before thawing cells?
-15 ml of growth medium should be pipetted into a T75 flask.
What should be done to release pressure from the cryovial before thawing?
-Loosen and then re-tighten the vial cap to release pressure before submerging the cryovial in the water bath.
How should cells be resuspended after thawing?
-Cells should be resuspended three times with 1 ml pipette, pipetting gently to avoid foam.
What is the purpose of loosening the cap of the T75 flask after transferring cells?
-The cap should be loosened for gas exchange and then incubated, leaving the cells undisturbed for at least four hours.
When should fresh growth medium be changed after thawing the cells?
-Fresh growth medium should be changed after four hours of initial incubation.
At what confluence level should the cells be subcultured?
-Cells should be subcultured once the confluence reaches 80 percent.
What is the procedure to detach cells from the flask during subculturing?
-After adding trypsin-EDTA, cells should be observed under a microscope. When they are rounded but still attached, the flask can be hit against the palm to detach the cells. If necessary, check and detach cells again.
How should cells be counted after subculturing?
-Cells should be counted with a hemocytometer or cell counter after pelleting and resuspending them in growth medium.

Outlines

00:00

🧪 Cell Cryovial Storage and Thawing Process

This paragraph outlines the procedure for handling and thawing primary cells stored in cryovials. Upon arrival, the cryovial should be swiftly moved from dry ice to liquid nitrogen to minimize exposure to room temperature. For cell thawing, a water bath set at 37 degrees Celsius is used. A T75 flask with 15 ml of growth medium is prepared, ensuring the flask is cell culture treated. The cryovial is partially submerged in the water bath, thawing until a small amount of ice remains. The tube is decontaminated with alcohol, and the cap is removed without touching the threads or rim. Cells are then resuspended gently to avoid foaming and transferred to the flask, which is then capped and rocked gently. The flask's cap is loosened for gas exchange, and the cells are left undisturbed for at least four hours. After this period, the growth medium is replaced, and the medium is changed every other day until the cells reach 60% confluence, at which point the volume of growth medium is doubled.

Mindmap

Keywords

💡Cryovial

A cryovial is a small, usually plastic, vial designed to hold biological samples at extremely low temperatures, typically in liquid nitrogen. In the context of the video, cryovials are used for the storage of primary cells, ensuring their preservation until they are ready for use. The script emphasizes the importance of minimizing exposure to room temperature upon arrival to maintain cell viability.

💡Liquid Nitrogen

Liquid nitrogen is a cryogenic liquid with a temperature of -196°C, commonly used in laboratories for the long-term storage of biological samples. It is crucial for maintaining the integrity of cells in cryovials, as mentioned in the script. The video instructs transferring the cryovial from dry ice to liquid nitrogen to ensure optimal cell preservation.

💡Thawing

Thawing refers to the process of raising the temperature of frozen biological samples to room temperature or body temperature to restore their original state. In the script, the process of thawing cells involves setting a water bath to 37 degrees Celsius and submerging the cryovial partially to avoid complete thawing, which could harm the cells.

💡Growth Medium

Growth medium is a nutrient-rich substance, often a liquid, used to support the growth and maintenance of cells in vitro. The script specifies pipetting 15 ml of growth medium into a T75 flask, which is a type of cell culture flask. The correct growth medium is essential for cell survival and proliferation.

💡Cell Culture Treated Flask

A cell culture treated flask is a specialized type of flask that has been treated to promote cell adhesion and growth. The script mentions ensuring the flask is cell culture treated before adding the growth medium and cells, which is vital for successful cell attachment and growth.

💡Trypsin-EDTA

Trypsin-EDTA is a solution used in cell culture to detach cells from the surface of a culture vessel. In the script, trypsin-EDTA is added to the flask to facilitate the detachment of cells that have reached confluence. It is a critical step in subculturing cells for further growth and experimentation.

💡Subculturing

Subculturing is the process of transferring a portion of a confluent cell culture to a new culture vessel to allow for continued growth. The script describes the conditions under which subculturing should occur, such as when confluence reaches 80 percent, and the steps involved, including aspirating medium, washing with Hanks Balanced Salt Solution (HBSS), and using trypsin-EDTA.

💡Cell Pellet

A cell pellet refers to a concentrated mass of cells that forms after centrifugation. In the script, cells are centrifuged at 220 times g for five minutes to form a pellet, which allows for the removal of the supernatant and resuspension of the cells in growth medium. This step is essential for preparing cells for counting and further manipulation.

💡Hemocytometer

A hemocytometer is a laboratory instrument used to count cells in a sample, typically used in conjunction with a microscope. The script mentions using a hemocytometer or cell counter to determine the cell count, which is necessary for accurately plating cells at the appropriate density in a new culture vessel.

💡Confluence

Confluence in cell culture refers to the state when a monolayer of cells has grown to cover the entire surface of the culture vessel. The script discusses monitoring confluence levels, with subculturing recommended once it reaches 80 percent, indicating that the cells are ready to be transferred to a new vessel for expansion.

💡Incubator

An incubator is a controlled-environment chamber used to maintain optimal temperature, humidity, and sometimes gas conditions for cell growth. The script instructs placing the flask in an incubator after cell plating and subculturing, which is essential for providing a stable environment that supports cell growth and maintenance.

Highlights

Upon arrival, remove cryovial from dry ice packaging to minimize exposure to room temperature.

Transfer cryovial to liquid nitrogen for optimal cell storage.

Set water bath to 37 degrees Celsius for cell thawing and confirm temperature with a thermometer.

Prepare a T75 flask with 15 ml of growth medium for cell transfer.

Ensure the flask is cell culture treated for optimal cell growth.

Loosen and re-tighten the vial cap to release pressure before thawing.

Submerge the lower half of the vial in a water bath without thawing completely.

Decontaminate the tube with alcohol before removing the cryovial cap.

Resuspend cells gently to avoid foaming and transfer to the prepared flask.

Loosen the cap for gas exchange and incubate the cells undisturbed for at least four hours.

Change to fresh growth medium after four hours and replace every other day until 60% confluence.

Double the volume of growth medium when confluence reaches 60%.

Subculture cells once confluence reaches 80% by aspirating the medium and washing with HBSS.

Add trypsin-EDTA to detach cells and observe under a microscope for readiness.

Detach cells by hitting the flask against the palm and check under the microscope.

Add trypsin neutralizing solution to inactivate trypsin and transfer cells to a centrifuge tube.

Pellet cells at 220 x g for five minutes and resuspend in growth medium.

Count cells with a hemocytometer or cell counter for accurate seeding.

Seed cells at 5000 cells per square centimeter in a T175 flask for further growth.

Maintain proper cell culture conditions for healthy cells and reliable data.

Visit www.cellapplications.com for more information on cell culture techniques.