Pyrogens are heterogeneous group of fever-inducing substances derived from Gram-positive and Gram-negative bacteria, fungi, and viruses. They incite immune response by producing endogenous pyrogens such as prostaglandins and other proinflammatory cytokines like IL-1β, IL-6, and TNF-α. The present study was to analyze the influence of cryopreservation in IL-1βrelease, a marker for inflammatory response from human lymphocytes, in response to exogenous pyrogenic stimulants. Lymphocytes isolated from pooled blood of multiple healthy individuals were cryopreserved in DMSO and glycerol for periods of 7, 14, 30, and 60 days and were challenged with LPS and LTAin vitro. The inflammatory cytokine, IL-1βrelease, was measured by ELISA method. It was observed that the release of IL-1βincreases instantaneously after the initiation of incubation and reaches a maximum at 3 to 5 hours and then gradually decreases and gets stabilized for both pyrogens. Moreover it was also observed that the effect of cryoprotectants, DMSO (10%) and glycerol (10%), showed almost similar results for short-term storage, but DMSO-preserved lymphocytes yielded a better viability for long-term storage. Thus, the isolated cryopreserved lymphocytes system can be a promising approach for the total replacement/alteration to animal experimentation for pyrogenicity evaluation.
Fixation and long-term storage of human lymphocytes for surface marker analysis by flow cytometry