Characterization of a Unique Spontaneous Calcifying Cell Line (CJ): a Novel Tool for the Study of Ectopic Calcification

PurposeDue to the lack of an appropriate in vitro evaluation system, there is no effective prevention or treatment for ectopic calcification diseases (ECD). We obtained canine renal adenocarcinoma cells (CJ cells) that spontaneously form large amounts of calcified precipitates (CaP-ppt) and examined whether they could serve as a model for the early stages of ectopic calcification (EC). MethodsDispersed cells obtained by collagenase-DNase digestion were cultured in 10% FBS, antibiotic-containing DMEM-high glucose medium (standard medium). CaP-ppt was stained with Alizarin Red (AR) and colorimetrically quantified after acid extraction. Cell volume was determined by Crystal violet (CV) staining followed by extraction and colorimetric quantification. Calcium (Ca) and phosphate (PO4) were determined with a commercial kit to obtain the Ca/P ratio. Screening of anti-CaP-ppt compounds was performed in the same 96-well plate in the order of cell culture {Rightarrow} CaP-ppt quantification {Rightarrow} cell quantification and evaluated by IC50 value. ResultsCJ cells produced large amounts of CaP-ppt on standard medium alone without external phosphate addition; CaP-ppt formation was not accompanied by cell death, but on the contrary, CaP-ppt increased at physiological pH values around pH 7.4 due to active cell metabolism. The Ca2+ and PO43- partitioning kinetics to CaP-ppt were found, and the Ca/P ratio of CaP-ppt was stable at 1.35. The anti-Cap-ppt effects of bisphosphonates and all-trans retinoic acid (ATRA) were also confirmed in this CJ cell CaP-ppt system. DiscussionThere was doubt about the vascular calcifying cell model with the addition of high concentrations of phosphoric acid, but there were no suitable alternative cells. Spontaneously calcifying CJ cells provide a fundamental solution to this problem. Anti-CaP-ppt screening also eliminates the need for medium exchange, thus saving labor and cost. The Ca/P ratio of CaP-ppt in CJ cells is 1.35, the same as that of amorphous Ca phosphate (ACP), corresponding to the early (reversible) stage of EC. Therefore, it has favorable conditions as an evaluation system for drug discovery. ConclusionCJ cells, which calcify at physiological phosphate concentrations (0.9 mM) in standard media, are useful and novel research material for basic and preventive studies of ECD and for drug development studies.