CellFreezing

Reagents/Solutions

• Cells!(atleast5x10⁶).
• Foetalcalfserum(FCS),heatinactivatedat65癈for1hr.
• Dimethylsulfoxide(DMSO).
• Freezingvials.
• Hi-TechCellfreezer(polystyreneboxwaddedwithcottonwool[!]).
• Liquidnitrogenbank.

Protocol

1. Spindowncells,removeanddiscardthesupernatant.
2. ResUSPendthecellsat5x10⁶permlinFCS.
3. AddDMSOto10%(byvolume)dropwise,agitatinggentlyaftereachdrop.Dothisslowly.
4. Place1mlaliquotsintothevials.Remembertolabelthevialsprecisely.
5. Putvialsintocentralpositionsofapolystyrenerack(frome.g.BoehringerEnzymeStoragebox),andplacethatrackintoanexpandedpolystyrenebox(e.g."Eprack"assuppliedbyScotlab)whichiswaddedwithcottonwool.
6. Placelidonboxandsealinplacewithtape.
7. Placeboxinthevapourphaseoftheliquidnitrogenbank.Itmustnotbeplacedintheliquidphase.Leaveformorethan4hrstofreeze.
8. Removevialsfromboxandplaceinliquidphaseinthecellbank.
9. Testtheefficiencyofthefreezingprocedurebythawing(method)andgrowingononevial.

Ifthecellswerehealthy(>95%viable)atthestartofthisprocedure,thenweobtainaculture80-90%viable24hoursafterthawingandgrowingon(method)thetestvialfrompoint9.

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