Recirculating glass pipettes constitute a high risk when working with freshly isolated immune cells - the presence of bacterial pyrogenic material

Srinivas AkulaSandra LaraAnna-Karin OlssonLars Torkel Hellman^*

• Uppsala University, Uppsala, Sweden

Abstract: A number of immune cells are highly sensitive to pyrogenic substances such as bacterial lipopolysaccharides (LPS). It is therefore crucial to ensure that all material coming in contact with these cells are pyrogen free. We here present a comparative analysis of the induction of inflammatory cytokines by freshly isolated human peripheral blood monocytes when handling the cells with recirculating glass pipettes or disposable plastic pipettes. The glass pipettes were sterilized but previously used for multiple projects including work with bacteria. Using these glass pipettes resulted in the same induction of a set of inflammatory cytokines and chemokines as when adding 1 ug of LPS/ml to the culture medium, an exceptionally high level of LPS. The most extreme upregulation was seen for IL-6, which increased in expression by a factor of more than 75 000-fold already by four hours of in vitro culture indicating that great care should be taken when using glass pipettes that are recycled in the lab for culturing of eukaryotic cells. IL-8 became the most highly expressed gene by four hours incubation exceeding the previous top transcript, which was lysozyme, by 50%. This finding clearly shows that glass pipettes, despite the careful washing procedures and sterilizing during recirculation, is not advisable to use when working with cells, in particular freshly isolated immune cells. Based on these data it seems as pyrogen free plastic pipettes, in spite of the fact that this involves an increase in plastic disposal, is the only acceptable solution to obtain biologically relevant results. A possible alternative could be to use glass pipettes that are used only for work with eukaryotic cells and that these pipettes are kept in containers with anti-microbial substances to avoid any microbial growth during storage in the lab between washings. However, such procedures need to be carefully tested and monitored during long term use.