Abstract

DO.02.07

A comparative in vitro toxicity analysis of preservatives in primary and immortalized keratocytes

Kagan L., Rieck P.
Augenklinik, Campus Virchow Klinikum, Charité - Universitätsmedizin Berlin

Objective: Immortalized cell lines become more important in research in the course of increasing standardization. In this study, human primary (HCKp) and immortalized keratocytes (HCKi) were tested to evaluate their behaviour with regard to proliferation and toxicity, in order to make a statement about the use of immortalized keratocytes to replace primary cells.
Methods: HCKp and HCKi were cultivated, mixed with cetrimide or benzalkonium chloride (BAC) in various concentrations (100-0.01microg/ml or 40-0.1 microg/ml respectively) and incubated for 72 hours. A qualitative and quantitative evaluation of the cells were made under the microscope or with a cellcounter, respectively, also considering the doubling time. The rate of apoptotic keratocytes was determined by a TUNEL-Assay and an Annexin-V-Propidiumiodide-assay.
Results: After treatment with BAC no vital cells remained detectable in concentrations of 40-4 microg/ml, neither after treatment with cetrimide in concentrations of 100-10 microg/ml. At less than 0.4 microg/ml a significant increase in the number of cells could be shown in HCKi as opposed to HCKp treated with BAC in comparison with the control. Under cetrimide treatment HCKi showed a highly significant decrease in the number of cells, whereas for HCKp, concentration levels below 0.1 microg/ml already proved no statistically detectable difference in comparison to control cultures. Evaluation of the doubling time showed clearly shorter times for HCKi. Furthermore, BAC appears to have a proliferative effect in small doses, as doubling time was reduced tendentially in HCKp, and significantly in HCKi. Cetrimide, on the other hand, led to a significant prolongation of doubling time in HCKp up to concentration levels of 0.1microg/ml, and up to 1microg/ml in HCKi.
Conclusions: Immortalized keratocytes are especially recommendable for preliminary experiments towards defining toxic concentration levels in further studies. Still, there were clearly detectable differences between the two cell lines with regard to proliferation as well as sensitivity to toxic agents. In contrast to HCKp, HCKi were more resistant and had a higher regenerative capacity.

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