Cryopreservation of keratinocytes without a -80oC freezer - (Jan/17/2012 )

I currently use a Mr Frosty to control the rate of freezing of keratinocyte cells down to -80oC and then transfer to liquid nitrogen and I've been helping out a lab in India and they don't have access to a -80oC freezer. Does anyone know the best way of cryopreserving cells slowly without one?

Cardice (solid CO2) has a temperature of -78 degrees centigrade. You could try putting the Mr. Frosty in a polystyrene box (big enough to take the Mr. Frosty) AND large enough for there to be a full 24 hrs worth of cardice.
Leave the Mr. Frosty overnight and I cannot think of a reason why this should not work.

I have never used this method as I have always had either a controlled rate cell freezer OR a -80 freezer available to me.

Hope this is a good possible plan

Kindest regards

Uncle Rhombus

rhombus,

I have primary keratinocytes from ATCC and when I contacted them about how I can make a cryopreserved stock, they told me it is not possible,
From ur experience, Can I make a stock by ordinary method for cryopreservation.
Or is there any especially method to freez a stock of primary cells such as human epidermal keratinocytes.
Best Regards

madelingirly on Thu Jan 19 09:09:47 2012 said:

Dear madelingirly,

The primaries have come from the ATCC......did they come as a growing cells (in a flask) or frozen?

If they came growing then they maybe correct.

However if they came frozen then you should easily be able to freeze them down.

Just a word of warning......primary cells can be frozen......BUT the viabilties you get back when initiating them back from liquid nitrogen will be a lot lower than immortalised cell lines.

You will need to optimise freezing conditions. Normally I use the standard method 90% Full growth media( containing 10% FCS) + 10% DMSO. You may need to increase the FCS concentration to 20% in order to achieve greater viability.

Hope this is useful

Kindest regards

Uncle Rhombus

Our keratinocytes are isolated from skin biopsies, we've never had a problem cryopreserving them and get good viabilities resurrecting them (used to cryopreserve in 90% serum, 10% DMSO, now use an animal free defined cryopreservative).

I'm not sure the lab in India will have access to solid CO2 (unless they buy it in which will be costly if it's a regular occurence). I'll check with them though....

rhombus.

Thanks for your helpful advice
they come as frozen, however they are cultured in Serum free condition, so When I tried to make cryopreserved stock, I used my cultured medium + 6%-10% DMSO.

or I should added Serum to my cryopreserved medium ??? despite the fact, Keratinocyte could be differentiated into fibroblast in presence of serum (According to my ATCC protocol).
Thanks again
Best Refards