Gibco’s Recovery Cell Culture Freezing Medium is used to cyropreserve mammalian cells, either primary or immortalized, for adherent or suspension cells. This optimized version is a ready-to-use formula which provides an improved cell viability and cell recovery after thawing. The product sheet shows that this freezing medium has a very good performance on a broad spectrum of mammalian cell lines (AE-1, CHO, HeLa, etc.) when compared to other leading brands (Sigma, Cambrex, Biolife Solutions). From my experience, this medium works very well on difficult primary cell lines, such as mouse melanocytes, when compared with other classical combinations such as 10% DMSO in fetal bovine serum (FBS), 7.5% DMSO in culture medium or even with this medium’s previous version (prepared in Dulbecco’s modified Eagle’s medium, high-glucose and containing fetal bovine serum, calf serum with iron and 10% DMSO). The media provides better results if aliquoted in Eppendorf tubes containing the volume needed to freeze the cells. Recovery™ Cell Culture Freezing Medium is based on the same formulation as Cell Culture Freezing Medium (Cat. no. 11101-011) but is manufactured with an optimized ratio of fetal bovine serum to bovine serum. According to its manual, the new version does not seem to be prepared with calf serum with iron.
No protocol change is necessary upon adoption of the new, improved Recovery™ Cell Culture Freezing Medium. The protocol I have used for freezing and thawing primary melanocytes was as follows: cells were washed with phosphate buffered saline (PBS), detached using trypsin-EDTA, and centrifuged at 1000g for 3-5min at 4°C. Centrifugation speed and duration as well as the temperature may vary depending on cell type. Cells ~90–95% confluent were then resuspended in 1 ml of freezing medium and frozen in cryovials using a cryo freezing container with isopropanol (Nalge Nunc Int., Naperville, IL) to allow gradual freezing. They were kept overnight at -70°C and then moved to the liquid nitrogen tank for long-term storage.
The thawing procedure of melanocytes started with the incubation of the frozen vial in a warm water bath (37°C) for a maximum of 3-5 min followed by the resuspension of the cells very slowly in 5-6 ml of the appropriate medium and then briefly centrifuged at 1000g at 4°C to avoid toxic effects of DMSO in the freezing medium. Each pellet was seeded as necessary for each type of experiments. If the cells were thawed early in the morning, the media was changed at the end of the day (and again in the following day) or only the next day if the thawing occurred late during the day and the cells did not attach to allow the media to be refreshed.
The above protocols worked very well for my primary or immortalized mouse melanocyte lines which can be very challenging, especially if they carry particular mutations that make them very sensitive to the freeze-thaw cycles. In general terms, the protocols suggested by the manual for Gibco’s Recovery™ - Cell Culture Freezing Medium are useful and can be easily adapted to each cell line’s requirements. However, the manual does not provide the catalog numbers of its components, especially the FBS and calf serum in case anyone would like to prepare in house formulations for a particular cell line and compare those with this product in terms of recovery efficiency.