Cytotoxicity associated with prolonged room temperature storage of serum and proposed methods for reduction of cytotoxicity

Canine serum preserved at room temperature (25 °C) for longer than 24 h is known to exhibit significant cytotoxicity. This phenomenon is one of the major reasons for the failure of virus neutralization tests. In this study, a method for reducing this cytotoxicity was investigated by applying several treatments to dog, cat and human serum prior to room temperature storage. Additionally, the identity of the cytotoxic factor generated during room temperature storage was investigated. Heat-inactivation at 56 °C or 65 °C and the addition of protease inhibitor prior to storage were found to be effective for reducing cytotoxicity in the serum. Furthermore, heat-inactivation at 65 °C reduced the cytotoxicity that was induced under room temperature storage. Several protein factors in serum were suspected to play a role in the observed cytotoxicity. According to this study, the membrane-attack-complex in serum was not involved in the cytotoxicity. This study provides useful information for development and improvement of cell culture and virus neutralization tests.