cryopreservation

[fery]

Dear Friends

I would like to know what is the simplest way for storage tissue samples(polyps and biopsies)   for a period ot time ( up to 3 months)  for  flow cytometry analysis. I know that the best way is using cryomedia (RPMI + DMSO ) and freezing overnight in -70 and then transfer to liquies nitrogen but what happen if I put my samples immediately in liquid nitrogen or store them for long time in -20 freezer.
any comment or suggestion would be very appreciated.

Thanks in advance

Yours

Fery

[excalibur]

After the samples are frozen, they should be stored in a -70 freezer. A normal refrigerator freezer at -20 goes through a defrost cycle every night and will not be good for your specimens. Cryostats also go through this defrost cycle.

[fery]

Dear Excalibur

Thank you very much for you comment, but please let me know if there is any method that I can use it instead of -70 freezer. I have heard that if i put my samples in isopropanol and then transrer it to liquied nitrogen, it protect sample from sudden freezing.

Thanks alot

Yours

Fery

[excalibur]

You probably mean isopentane. You cool a container of isopentane in a container of liquid nitrogen and then freeze your specimen in the isopentane.
Here is the protocol on IHC World: http://www.ihcworld.com/_protocols/histology/frozen_section.htm

If you do not have access to a -70 freezer, your sample integrity may degrade in the -20.

Why will it be 3 months before you can perform the flow cytometry?

[fery]

Dear Excalibur

Thank you again. you are right, it is isopentane but the protocol which you reffer me is about cryostat.
I   take samples from different centers in different cities and tI have to store them for a while because I don't have access to flow cytometer in all centers.
it seems that I have to ship my samples immediately to central lab where there is a -70 freezer, so I appreciate you if you tell me the best way for short term storage of tissue samples (up to 48 hours) during shipping time

Truly yours

Fery

[excalibur]

McCarey-Kaufman modified tissue culture media is what donor corneas are stored in for up to 48 hours prior to transplantation. Contact a local ophthalmologist that performs corneal transplants or the eye bank for their supplier.

Michel's transport medium is used for immunofluoresents, but is not recommended for flow.

You can use the cryostat protocol, you only need the freezing part.