In vitro and in vivo cell tracking of chondrocytes of different origin by fluorescent PKH 26 and CMFDA

Abstract

Tissue engineering techniques for cartilage repair to heal defects in joint surfaces is a clinical practice. Harvested autologous chondrocytes are expanded in culture and delivered in a suitable carrier medium back into the patient's joint defect. The defect is then subsequently filled by new cartilage. Whether the cells in the repair tissue originate from the engineered tissue of the host or are derived from the surrounding original cartilage remains a relevant question for the applied therapy. To answer this several methods exist to track cells, such as transfection of cells with LacZ carrying viruses, radio labeling with 111 IN or 51 Cr or fluorescent labeling with FDA. However, these techniques have drawbacks such as they may influence cellular properties, are radioactive or quickly lose their tracking ability. New fluorescent probes are easier to handle and do not to interfere with cells. PKH 26 ® is a relatively new cell-labeling agent, but few data exist on the application of this dye in chondrocytes in vitro and in vivo. 5-chlorome-thylfluorescein diacetate-CMFDA ("cell tracker green") is an established fluorescent probe for imaging the dynamic processes of cell proliferation in vitro and in vivo. Likewise, several studies exist on different cell types. However, little data are available for chondrocytes. The first aim of this study was to evaluate qualitative differences in fluorescence pattern after labeling of articular, auricular and costal chondrocytes. Secondly, we evaluated the influence of labeling with CMFDA on cellular adhesion properties. The third aim was to compare the duration of cell labeling of chondrocytes of different origin with established CMFDA as standard and PKH 26 ® for 3 cell generations in vitro and 12 weeks in vivo. We show that chondro-cytes from different origin can be labeled effectively with both PKH 26 ® and CMFDA. The PKH 26 ® labeled articular chondrocytes maintained fluorescence longer than CMFDA in vitro and in vivo. A higher percentage of articular chondro-cytes remained stained at 63 days than auricular or costal chondrocytes.