Novel renal replacement therapies, such as a bioartificial kidney (BAK), are needed to improve current hemodialysis treatment of end-stage renal disease (ESRD) patients. As BAK applications may reveal safety concerns, we assessed the alloimmunization and related safety aspects of readily available conditionally immortalized human proximal tubule epithelial cell (ciPTEC) lines to be used in BAK. Two ciPTEC lines, originally derived from urine and kidney tissue, were characterized for the expression and secretion of relevant molecules involved in alloimmunization and inflammatory responses, such as HLA class-I, HLA-DR, CD40, CD80, CD86, as wells as soluble HLA class I and proinflammatory cytokines (IL-6, IL-8 and TNF-α). A lack of direct immunogenic effect of ciPTEC was shown in co-culture experiments with peripheral blood mononuclear cells (PBMC), after appropriate stimulation of ciPTEC. Tight epithelial cell monolayer formation on polyethersulfone flat membranes was confirmed by zonula occludens-1 (ZO-1) expression in the ciPTEC tight junctions, and by restricted inulin-FITC diffusion. Co-culture with (activated) PBMC did not jeopardize the transepithelial barrier function of ciPTEC. In conclusion, the absence of allostimulatory effects and the stability of ciPTEC monolayers show that these unique cells could represent a safe option for BAK engineering application.