TissueCulture:Thawing cells

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Current revision (02:14, 25 May 2012) (view source)
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*10-50 ml centrifuge tube
*10-50 ml centrifuge tube
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*cryo tubes
 
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*DMSO, Tissue culture (TC)-tested (e.g. [http://www.sigmaaldrich.com Sigma], D2650)
 
*Cell culture medium (depends on cell line/cells)
*Cell culture medium (depends on cell line/cells)
*Trypsin-EDTA (e.g. from [http://www.paa.com PAA], L11-004) - for adeherent cells
*Trypsin-EDTA (e.g. from [http://www.paa.com PAA], L11-004) - for adeherent cells
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*'''Freeze-down medium'''
 
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**6-10% DMSO
 
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**Cell culture medium (I recommend to use at least 20% FCS and you can use 100% fetal calf serum (FCS) instead of cell culture medium)
 
*Basic TC centriguge (0 - 2500 rpm)
*Basic TC centriguge (0 - 2500 rpm)
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*Freeze-down box (this could be simply made from a small styropore box and cotton or from two styropore racks from 15-ml centrifuge tubes )
 
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==Freeze-down (suspension cells)==
 
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#Centrifuge at 80-100 x g for 4-5 min at RT.
 
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#Suck out the medium.
 
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#Resuspend 1-5 x 10*6 cells in appropriate volume of freezing medium (e.g. 1.5 ml for 1.8ml Cryo-tube).
 
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#Transfer cryo-vials with cells into freeze-down box and store at -80 gr.C.
 
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*Leave over-night at -80 gr.C.
 
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*You can store cells at -80 gr.C for up to 6 months.
 
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*Transfer cells into liquid nitrogen a day later for longer storage (1-20 years or so).
 
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==Freeze-down (adherent cells)==
 
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#Wash cells with PBS.
 
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#Add thin layer of Trypsin-EDTA on cells (approx. 1ml for 90 mm Petri dish)
 
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#Observe cells under microscope until these are rounded.
 
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#Add serum-containing medium and resuspend cells. Transfer into centrifuge tubes.
 
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#Centrifuge at 1000-1200 rpm for 4-5 min at RT.
 
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#Suck out the medium.
 
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#Resuspend cells in appropriate volume of freezing medium (e.g. 1.5 ml for 1.8ml Cryo-tube).
 
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#Transfer cells into freeze-down box and place at -80 gr.C. (Comments as for suspension cells).
 
==Thawing==
==Thawing==
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#Keep frozen cells on dry ice until ready for thawing.
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# Setup two vials of cold (4 C) media (10 ml) in a 15 ml tube
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#Swirl a vial with frozen cells in 37gr.C water bath and remove just while a sliver of ice remains.
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# Warm remaining media (~5-8 ml) in a 15 ml tube to 37C
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#Spray tube with 70% ethanol for sterilization.
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# When ready to thaw, remove vial of cells from liquid nitrogen
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#Aspirate cells from cryo-tube into pre-half-filled pipette with warm culture medium.
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#:Keep frozen cells on dry ice until ready for thawing.
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#Fill centrifugation tube with culture medium: at least 10 ml total volume.
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# Swirl the vial in the water bath (37C) until a small ice chip is left in the tube (~1 min)
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#Centrifuge for 5 min at 80-100 x g (RT).
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# Place the vial in the hood and clean it with 70% ethanol
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#Resuspend pelleted cells in appropriate volume of culture medium and culture o/n in CO2 incubator.
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# Immediately remove the contents of the vial and place into the cold media
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#Optional: Exchange medium next day.
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# Rinse the tube down with some of the cold media from the second vial
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# Spin down cells at 2000 rpm (80-100 x g) for 5 min
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# Aspirate off the cold media and resuspend the cells in the warm media
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# Transfer the cells and the media into a petri dish
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# Count cells
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# Place in incubator
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# Change the media once the cells have attached to the plate
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#: Removes remaining DMSO
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# Allow cells to grow to 80-90% confluency
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# Split cells (1:3) into petri dishes
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# Continue to split cells and freeze down as needed
==Notes==
==Notes==
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#You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
#You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
#Anecdotal observations that might be of use to others can also be posted here.   
#Anecdotal observations that might be of use to others can also be posted here.   
 +
 +
'''Alternative Thawing'''
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* From step 5 above, using a 1 ml pipet add 1 ml of the cold media from the 15 ml tube drop by drop to the vial (about 1 drop every 10 seconds).
 +
* Plate immediately with the cold media and change the media once cells have attached (12 - 24 hours later) to remove DMSO that was in the freezing media. Usually a 1:3 ratio is good for MC3T3s and a 1:4 ratio for MLOY4s.
Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
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==
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See also
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:[[Jacobs:Protocol_Freezing_and_Thawing_Cells]]
 +
:[[WangLab:Thawing_Cells]]
 +
:[[Marek:_Freeze-down/Thaw]]
 +
:[[Wittrup:_Thawing_Protocol]]
==Contact==
==Contact==

Current revision

Contents

Overview

This short protocol describes how to freeze down and thaw (or bring up) mammalian cells (e.g. HeLa, KEK293, CHO, Jurkat T cells).

Materials

List reagents, supplies and equipment necessary to perform the protocol here.

  • 10-50 ml centrifuge tube
  • Cell culture medium (depends on cell line/cells)
  • Trypsin-EDTA (e.g. from PAA, L11-004) - for adeherent cells
  • Basic TC centriguge (0 - 2500 rpm)

Thawing

  1. Setup two vials of cold (4 C) media (10 ml) in a 15 ml tube
  2. Warm remaining media (~5-8 ml) in a 15 ml tube to 37C
  3. When ready to thaw, remove vial of cells from liquid nitrogen
    Keep frozen cells on dry ice until ready for thawing.
  4. Swirl the vial in the water bath (37C) until a small ice chip is left in the tube (~1 min)
  5. Place the vial in the hood and clean it with 70% ethanol
  6. Immediately remove the contents of the vial and place into the cold media
  7. Rinse the tube down with some of the cold media from the second vial
  8. Spin down cells at 2000 rpm (80-100 x g) for 5 min
  9. Aspirate off the cold media and resuspend the cells in the warm media
  10. Transfer the cells and the media into a petri dish
  11. Count cells
  12. Place in incubator
  13. Change the media once the cells have attached to the plate
    Removes remaining DMSO
  14. Allow cells to grow to 80-90% confluency
  15. Split cells (1:3) into petri dishes
  16. Continue to split cells and freeze down as needed

Notes

  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. Anecdotal observations that might be of use to others can also be posted here.

Alternative Thawing

  • From step 5 above, using a 1 ml pipet add 1 ml of the cold media from the 15 ml tube drop by drop to the vial (about 1 drop every 10 seconds).
  • Plate immediately with the cold media and change the media once cells have attached (12 - 24 hours later) to remove DMSO that was in the freezing media. Usually a 1:3 ratio is good for MC3T3s and a 1:4 ratio for MLOY4s.

Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

== See also

Jacobs:Protocol_Freezing_and_Thawing_Cells
WangLab:Thawing_Cells
Marek:_Freeze-down/Thaw
Wittrup:_Thawing_Protocol

Contact

  • Who has experience with this protocol?

or instead, discuss this protocol.

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