Hello Caique Figueiredo,

Freezing of monocytes is usually not recommended because monocytes are sensitive to freezing. Cryopreservation may not affect cell survival, but they are likely to lose some of the functions which you may be interested in, although expression of surface markers may not be affected on cryopreservation.

Protocol for cryopreservaton of monocytes.

Monocytes may be frozen for future use by suspending 5 - 10 × 10^6 cells/ml in ice cold freezing medium. The freezing medium may be prepared by combining 10% DMSO and 90% heat-inactivated FBS on ice. One-millilitre aliquots may be dispensed to the cryovials. The cells may be transferred to a cryofreezing container (like Mr. Frosty) and placed in a −80°C freezer. On the following day, the vials may be transferred to the gas phase of liquid nitrogen and stored there for a duration of 2 days up to 4 weeks before use. The cells may be thawed quickly in a 37°C water bath and transferred into ten volumes of prewarmed RPMI medium supplemented with 10 % FBS.

Best.

We have used CryoStor successfully.

Malcolm Nobre Thank you for your answer!

James E Talmadge

What kind of CryoStor are you using to frozen monocytes? I have seen that Sigma has different types (CS2, CS5...)? Could you provide the number of cells that you have frozen, the storage term and viability?

Thank you very much!

Here are two papers on such processes. The one we did address the use of cryostor and was used to validate the process for GMP manufacturing. It is on a different scale than you are doing but will work well for you. The paper from Mcmannis addresses a more traditional approach. Again validated for GMP manufacturing. I addresses your questions. The approach works for monocytes, DCs and PBMCs/apheresis cells.

James E Talmadge Thank you very much!