ObjectiveTo evaluate protocols of root canal irrigation and dentin pretreatment in a cell culture model simulating immature teeth. Cytotoxic, migration, and angiogenic effects of Sodium hypochlorite associated with EDTA (NaOCl/EDTA), NaOCl associated with Smear Clear (NaOCl/SC), and QMix were compared. DesignThree roots of mandibular first premolars had their length and root canal diameter standardized. Root canals were irrigated, and the resulting solutions were diluted in culture medium. Sulforhodamine B (SRB) assay was performed with apical papilla cells and with endothelial cells (HUVECs) to assess cytotoxicity. Polarity index and migration assays of apical papilla cells and sprouting of HUVECs were evaluated. Data were analyzed by ANOVA and Tukey post-hoc tests (p < .05). ResultsIn apical papilla cells, NaOCl/SC and QMix promoted higher cytotoxicity, decreased fraction of elongated cells, and had lower migration speed and shorter migration distance of cells compared to NaOCl/EDTA. Also, HUVECs treated with NaOCl/SC and QMix showed decreased tubule formation in comparison with NaOCl/EDTA. ConclusionsNaOCl/SC and QMix showed unfavorable biological responses of cells involved in revascularization in comparison to NaOCl/EDTA. Further studies with other intracanal irrigants should be performed to improve the balance of root canal disinfection with biological responses.