In vitro evaluation of chitosan cytotoxic properties

Introduction. Chitosan immunomodulatory and mucoadhesive properties render it a promising vaccine adjuvant. Safety – particularly the absence of cytotoxicity – is a key requirement for adjuvant candidates. In vitro biocompatibility assessments enable evaluation of chitosan preparations prior to animal testing. Objective. To evaluate low molecular weight chitosan solution at a concentration of 10 mg/mL for its cytotoxic effect on chicken embryo fibroblast (CEF) cultures and calf coronary artery epithelial-like cells (CCEC) to justify its further use as a vaccine adjuvant. Materials and methods. Low molecular weight (LMW) chitosan (degree of deacetylation: 90%) prepared with a 1% glutamic acid solution (pH 6.9) was used. Cytotoxicity was comprehensively assessed using three methods: trypan blue vital staining (for cell viability), live-cell microscopy (for morphological evaluation), and calculation of the proliferation index after 72 hours of incubation at 37 °C in a 5% CO2 atmosphere. Results and discussion. Following 2-hour incubation with chitosan, viable CEF and CCEC were 97.4 and 98.7%, respectively, with no significant differences from controls (97.6 and 96.4%). Microscopy at 72 hours showed dense, homogeneous monolayers in test groups, free of cytopathic effects, vacuolization, or morphological changes – indistinguishable from controls. Proliferation indices aligned closely (CEF: 3.9 and 3.6; CCEC: 3.7 and 3.8), evidencing no cytostatic effect of the chitosan preparation. Conclusion. Low-molecular-weight chitosan (10 mg/mL) exhibited no in vitro cytotoxic or cytostatic effects on the tested cell lines. The findings confirm its biocompatibility and justify advancement to in vivo studies for developing safe, effective vaccines for veterinary use.