Surface-adhering Human Leucocytes: An In Vitro Model for Cytotoxicity Testing of Fluids

Abstract

A rapid screening method was developed for assessment of the toxic effects of fluid materials on the respiratory burst response of polymorphonuclear neutrophils (PMNLs). The method was used to detect adverse effects of peritoneal dialysis (PD) fluids. Intoxication of the respiratory burst response attenuates the bacterial killing capacity of PMNLs, and increases the sensitivity of patients to peritoneal infection. Capillary blood was taken from healthy donors, placed in drops on commercially available titanium pieces, and incubated in a humidified chamber at 37°C for up to 1 hour. The blood was rinsed off with saline, and the adhering cells were characterised by immunofluorescence by using antibodies directed against specific cell-differentiation antigens. A majority (> 95%) of the adhering leucocytes were PMNLs. The surface expression of selectins was down-regulated after 30 minutes, and the expression of integrins was down-regulated after blood exposure for 1 hour. NADPH-oxidase activity of the adhering cells was stimulated by f-MLP peptide and by opsonised zymosan. The zymosan-induced activation showed a lag-phase after 1 hour, consistent with the down-regulated expression of integrin. The zymosan-stimulated enzyme activity was used as an indicator of the cytotoxicity of PD fluids. NADPH-oxidase activity was inhibited by PD fluids with a pH of 5.7 and by heat-sterilised PD fluids. The results were compared with data obtained by using isolated circulating cells and cells from peritoneal dwell fluid.

Cytotoxicity Testing of Wound-Dressing Materials

“…The differences in results between the microtitre-plate assay and the titaniumsurface assay are not assigned to the different materials used for PMN adhesion. Earlier work shows that the methods are equivalent (8). The differences are due to different responses to the stimuli used.…”

“…The plate was incubated for 4 hours at 37°C, in order to obtain a homogeneous clot. After rinsing with saline, the adhering leucocytes (>95% PMNs [8]) were incubated with wound gel (A-H), dissolved 1:10 in Dulbecco's phosphate-buffered saline. After incubation for 1 hour at 37°C in a humidified chamber, the wells were rinsed with saline.…”