The objective of this study was to evaluate the effects of cold storage (4°C) on osteoblast viability and interleukin-6 (IL-6) production. Osteoblasts were harvested from murine calvaria utilizing sequential collagenase digestion and the Ficoll-Paque technique. Twelve-well plates were then inoculated with 2 X 104 cells/ml and placed in cold storage for up to 14 days. The cells were then incubated at 37°C for up to 20 days. During the incubation periods supernatants were collected daily and the cells were evaluated for alkaline phosphatase activity. We developed an enzyme immunoassay (EIA) for IL-6 which was sensitive to 50 pg/ml (standard curve r~ .996) with no cross reactivity with other recombinant murine cytokines. The EIA was th~n employed to assay the supernatants for IL-6 production. Storage at 4 oc for up to 48 hours resulted in a pre9ipitous drop in IL-6 production. After 48 hours in cold storage there was no bone cell via~ility.

Abstract

The objective of this study was to evaluate the effects of cold storage (4°C) on osteoblast viability and interleukin-6 (IL-6) production. Osteoblasts were harvested from murine calvaria utilizing sequential collagenase digestion and the Ficoll-Paque technique. Twelve-well plates were then inoculated with 2 X 104 cells/ml and placed in cold storage for up to 14 days. The cells were then incubated at 37°C for up to 20 days. During the incubation periods supernatants were collected daily and the cells were evaluated for alkaline phosphatase activity. We developed an enzyme immunoassay (EIA) for IL-6 which was sensitive to 50 pg/ml (standard curve r~ .996) with no cross reactivity with other recombinant murine cytokines. The EIA was th~n employed to assay the supernatants for IL-6 production. Storage at 4 oc for up to 48 hours resulted in a pre9ipitous drop in IL-6 production. After 48 hours in cold storage there was no bone cell via~ility.