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

Authors: Corcoran, Jacob; HAN, XU - University Of Missouri

Submitted to: In Vitro Cellular and Developmental Biology - Animal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/16/2022
Publication Date: 12/14/2022
Citation: Corcoran, J., Han, X. 2022. Improved cryopreservation media formulation reduces costs of maintenance while preserving function of genetically modified insect cells. In Vitro Cellular and Developmental Biology - Animal. 58:867-876. https://doi.org/10.1007/s11626-022-00741-3.
DOI: https://doi.org/10.1007/s11626-022-00741-3

Interpretive Summary: Tissues can be dissected from insects and used to establish replicating cell lines that are used by scientists to conduct various types of research. Certain insect cell lines are commonly genetically modified to express proteins not normally present in the cells, providing a system that allow scientists to study the function of the proteins in a cellular environment. However, one requirement for using insect cell lines is to have liquid nitrogen-based storage facilities for their long-term preservation. Liquid nitrogen storage facilities are dangerous and costly to set-up and maintain. These costs can be so high that they preclude smaller labs from working with insect cell lines entirely. New technologies are being developed that allow for the preservation of cell lines in standard “ultra-low” freezers, thereby eliminating the need for liquid nitrogen-based storage, however these reagents have not yet been tested on genetically modified insect cell lines. In this study we produced a genetically modified insect cell line and evaluated the ability of a novel cell freezing medium, C80EZ, to maintain cell viability, growth rates, and functionality after storing the cell line in ultra low freezers or in liquid nitrogen for various lengths of time. We found that engineered cells frozen using C80EZ and stored in ultra low freezers for six months displayed higher viability and growth rates than cells frozen using traditional cryopreservation media that were stored in ultra low freezers or in liquid nitrogen for the same period. These results suggest that C80EZ is an effective cryopreservation reagent that may eliminate the need for liquid nitrogen-based storage facilities to preserve genetically engineered insect cells.

Technical Abstract: Insect cell lines are an invaluable resource that facilitate various fundamental and applied research programs. Moreover, genetically engineered insect cell lines serve as a platform through which the function of heterologously expressed proteins can be studied. However, a barrier to more widespread use of insect cell lines, genetically modified or not, is the requirement for liquid nitrogen (LN2)-based storage facilities for their long term preservation. Recent innovations in cryopreservation technologies have produced reagents that show improved abilities to preserve cell lines for long periods in “ultra-low” freezers, however 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 to evaluate not only cell viability and growth rates, but also variation in the cell line’s “functionality” after storage in various conditions using the novel cryopreservation reagent, C80EZ. We found that engineered Sf9 cells frozen using C80EZ and stored at -80 'C for 6 months displayed higher viability and growth rates than cells frozen using traditional, FBS-based cryopreservation media that were stored at -80 'C or in LN2 for the same period. We also found that after 6 months of storage at -80 'C or in LN2 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 C80EZ is an effective cryopreservation reagent for genetically engineered Sf9 cells and may eliminate the need for FBS-based cryopreservation formulations and LN2-based storage facilities.