Mild inactivation and inhibition of phenoloxidase in silkworm serum for xeno-free culture of insect cells

In insect cell culture, a fetal bovine serum is often used to maintain the cellular physiological conditions, irre-spective of its relatively high cost and unstable supply. Several serum-free media for insects were developed, but it is challenging to adopt some insect cells to these serum-free media. Silkworm serum was used as a re-placement of fetal bovine serum for a long time but not used widely due to the melanization of insect serum catalyzed by phenoloxidase (PO). PO inhibitors reported for insect serum preparation have a significantly nega-tive effect on the long-term culture of healthy insect cells. In this study, we evaluated several kinds of PO inhibi-tors and found the method to prepare silkworm serum, which is suitable for the maintenance of insect cells and the production of the recombinant proteins by the baculovirus expression system. When L-Glutathione (reduced form) or L-Cysteine is used as a PO inhibitor at the time of recovery of silkworm serum, it is possible to sup-press the browning of the silkworm serum. However, in this state of silkworm serum, browning is observed in long-term cell culture use. Therefore, it is preferable for cell culture to add 10% of silkworm serum treated at 50°C for 30 minutes to the medium with 2.7 mM L-Glutathione. Our results suggest that silkworm serum can be an alternative of mammalian serum for cell culture and used for producing the proteins for clinical application without any risk of contamination of zoonotic infectious disease viruses.