Improved cryopreservation media formulation reduces costs of maintenance while preserving function of genetically modified insect cells.

ABSTRACT

Insect cell lines are an invaluable resource that facilitate various fundamental and applied research programs. Genetically engineered insect cell lines, in particular, serve as a platform through which the function of heterologously expressed proteins can be studied. However, a barrier to more widespread utilization and distribution of insect cell lines, genetically modified or not, is the technical and operational challenge associated with traditional cryopreservation methods, including their dependence on the use of liquid nitrogen facilities, animal or human serum products, and relatively high concentrations of permeating cryoprotectants (e.g., DMSO). Recent innovations in cryopreservation technologies have produced reagents with improved abilities to effectively preserve mammalian cell lines for long periods in regular laboratory deep freezers without using serum products, but their effectiveness in preserving genetically engineered insect cell lines has not yet been evaluated. In this study, we engineered Sf9 cells to express a dopamine receptor and used them as a model for evaluating the efficacy of a novel cryopreservation medium product, C80EZ®-INSECT, in not only preserving cell viability and proliferation efficiency but also maintaining the insect cell line's "functionality" after storage at -80°C. We found that the engineered Sf9 cells frozen using C80EZ®-INSECT with 5% DMSO alone and stored at -80°C for 6 mo displayed higher viability and growth rates than cells frozen using traditional fetal bovine serum (FBS)-based cryopreservation media with 10% DMSO that were stored at -80°C or in liquid nitrogen for the same period of time. We also found that after 6 mo of storage at -80°C or in liquid nitrogen the cells retained a responsiveness to dopamine comparable to that of the initial cell line, regardless of the cryopreservation reagent used. These results suggest that, due to the unique characteristics of C80EZ®-INSECT in preventing ice recrystallization and reducing ice crystal size and cellular apoptosis during cryostorage procedures, it is an effective cryopreservation reagent for genetically engineered Sf9 cells, and it practically eliminates the need for liquid nitrogen-based storage facilities and FBS-based cryopreservation formulations, as well as reduces the use of permeating cryoprotectants.