Primary Cell Culture: A Synopsis

Summary

Cell 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.