Primary Cell Culture: A Synopsis
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
🧪 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
💡Liquid Nitrogen
💡Thawing
💡Growth Medium
💡Cell Culture Treated Flask
💡Trypsin-EDTA
💡Subculturing
💡Cell Pellet
💡Hemocytometer
💡Confluence
💡Incubator
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.
Transcripts
cell applications recommends the user
follow these steps for optimal success
with their primary cells
cryovial storage
upon arrival remove cryovial from dry
ice packaging
minimize vial exposure to room
temperature
transfer to liquid nitrogen
cell thawing
to thaw set water bath to 37 degrees
celsius
confirm temperature with thermometer
pipette 15
ml growth medium into t75 flask then cap
use correct growth media ensure flask is
cell culture treated
retrieve cryovial from liquid nitrogen
and keep on dry
ice loosen re-tighten
vial cap to release pressure submerge
lower half a vial and bath
do not thaw completely small amount of
ice should remain
decontaminate tube with alcohol
remove cryovial cap
do not touch threads or rim
cell plating
resuspend cells three times with one ml
pipette
pipette gently to avoid foam
transfer cells to t75 flask containing
15
ml growth medium cap flask and rock
gently
loosen cap for gas exchange
and incubate leave cells undisturbed at
least four
hours change to fresh growth medium
after four hours
replace growth medium every other day at
sixty percent confluence
double the volume of growth medium
subculturing the cells
subculture once confluence reaches 80
percent
aspirate medium from flask
wash attached cells with hbss and remove
hbss do not warm
hbss or other subculture reagents
add 5 ml trypsin edta
gently rock to cover cells
immediately remove 4.5 ml of trypsin
edta
recap tightly observe cells under
microscope
cells are ready when rounded but still
attached
hit flask against palm to detach cells
check and detach cells again if needed
add 5 ml trypsin neutralizing solution
to flask
transfer cell suspension to sterile 50
ml centrifuge tube
add another 5 ml trypsin neutralizing
solution to flask
rock gently and transfer remaining cells
to same tube
examine cells under microscope
re-trypsinize if over 20 percent remain
pellet cells at 220 times g for five
minutes
remove tube from centrifuge aspirate
supernatant without disturbing pellet
flick tube to loosen pellet add 2 ml
growth medium
and pipette gently to resuspend
count cells with hemocytometer or cell
counter
pipet 30 ml growth medium into t175
flask
transfer to flask at 5000 cells per
square centimeter
attach cap and rock gently
loosen cap and move to incubator
and that's it a few tips mean healthy
cells and solid data
learn more at www.cellapplications.com
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