Cryopreservation of differentiated HL-60 cells for pyrogen testing.
All-trans retinoic-acid (ATRA) differentiated HL-60 cells can be used to detect pyrogens such as bacteria, bacterial components, yeasts and fungi. Differentiated HL-60 cells obtain neutrophil like characteristics and if stimulated the differentiated HL-60 cells produce reactive oxygen species in a dose dependent manner. Culturing and differentiation of cell lines are time consuming activities and require suitable facilities; cryopreservation of pre-differentiated cells could provide the basis for an easily distributable pyrogen testing kit. Cryopreservation of granulocytes has proven to be very complicated and neutrophils are especially difficult to cryopreserve, most likely due to their large degree of granulation. Here we present evidence that HL-60 cells can be differentiated with ATRA and subsequently cryopreserved. Upon thawing the cells retain their ROS producing capabilities and reactivity towards pyrogens. Further, the cells retain their ability to react dose dependently towards lipopolysaccharide (LPS), lipoteichoic acid (LTA) and zymosan. At pathophysiologically relevant concentrations of LPS, LTA and zymosan the cells retain full reactivity for at least two months when stored in liquid nitrogen. In conclusion, ATRA differentiated HL-60 cells are cryopreservable and retain reactivity upon thawing. It is therefore possible to produce an in-vitro in-house pyrogen test kit for medicines and related products.
Department of Pharmacology and Pharmacotherapy, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark. mt@farma.ku.dk
Michael
M
Timm
Stine
S
Bartelt
Lise
L
Moesby
Erik Wind
EW
Hansen
D000970:Antineoplastic Agents / Q000494:pharmacology; D001681:Biological Assay / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects; D015925:Cryopreservation; D004305:Dose-Response Relationship, Drug; D018922:HL-60 Cells; D006801:Humans; D009504:Neutrophils / Q000166:cytology / Q000276:immunology*; D011749:Pyrogens / Q000032:analysis* / Q000276:immunology / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000032:analysis / Q000276:immunology; D012680:Sensitivity and Specificity; D014212:Tretinoin / Q000494:pharmacology
D000970:Antineoplastic Agents; D011749:Pyrogens; D017382:Reactive Oxygen Species; D014212:Tretinoin