Development of a novel serum-free freezing medium for mammalian cells using the silk protein sericin

Abstract

Cryopreservation is a pivotal process in cellular engineering for creating a continuous source of generated functional cell lines and for the convenience of various medical treatments that involve cell culture. FBS (fetal bovine serum) supplemented with 10% (v/v) DMSO is extensively used as a freezing medium for mammalian cells using conventional methods. However, FBS should ideally be avoided, owing to serious concerns regarding bovine spongiform encephalopathy and other infections such as viruses, and an alternative to FBS is eagerly awaited. Furthermore, bio-medicines and cells for transplantation should not be infectious. The present study aimed to develop a novel serum-free freezing medium. For this purpose, we focused on using the silk protein sericin as a cryoprotectant for storage and developed a novel serum-free freezing medium consisting of PBS, 1% (v/w) sericin, 0.5% (v/w) maltose, 0.3% (v/w) proline, 0.3% (v/w) glutamine and 10% DMSO. This novel freezing medium was compared with the conventional FBS supplemented with DMSO and also with three purchased freezing media with respect to cryopreservation of the P 3 U1 myeloma cell line and Chinese-hamster ovary cells. As a result, the constructed medium containing sericin successfully cryopreserved both cell types as efficiently as the conventional medium of FBS containing 10% DMSO and was superior to all three of the purchased media. The constructed medium containing sericin also cryopreserved normal human dermal fibroblasts, human epidermal keratinocytes, the rat phaeochromocytoma cell line PC12 and insect (Spodoptera frugiperda) cell line S f 9 as effectively as the conventional medium of FBS and DMSO.