X-Message-Number: 7644
From:  (Brian Wowk)
Newsgroups: sci.cryonics
Subject: Re: Freezing cells
Date: 4 Feb 97 05:53:36 GMT
Message-ID: <>
References: <>

In <> Jack JWA van Loon <> writes:

>Does any of you have any experience in freezing (and storing) single
>cells at -20 dgr.C for a prolonged period? I am aware that the best
>storage temp. is liquid Nitrogen but I only have -20 degr.C for this 
>particular experiment. I have to store the cells for at least a few 
>What happens if a cell suspension (with DMSO or any other preservative) 
>goes trough a temperature profile of -10 to -70 degr.C? Is there a change 
>in water crystal formation ?

	A 10% solution of glycerol or DMSO should limit ice formation
at -20'C to tolerable amounts (<80%) for single cells.  Add solution
slowly to avoid osmotic injury, and cool at about 1'C per minute.
Faster cooling may result in fatal intracelluar freezing.  These
suggestions are theoretical and not based on experience.  Consult
backissues of the journal Cryobiology for more detailed protocols
for freezing cell suspensions.

	Holding at -20'C for long periods of time may be difficult.
The cryoprotectant will become highly concentrated in the unfrozen
solution, and exposure to such concentrations at -20'C for days 
will likely be fatal.

	There definitely will be a change in water crystal formation
between -10'C and -70'C.  With slow cooling, an increasing percentage
of the solution will convert to ice.  A greater concentration of
cryoprotectant will be required to limit ice to tolerable amounts
at ultralow temperatures.

 Brian Wowk          CryoCare Foundation               1-800-TOP-CARE
 President           Human Cryopreservation Services   

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