In Vitro and In Vivo Validation of Human and Goat Chondrocyte Labeling by Green Fluorescent Protein Lentivirus Transduction Sylvie Miot, Ph.D., 1 Roberto Gianni-Barrera, Ph.D., 1 Karoliina Pelttari, Ph.D., 1 Chitrangada Acharya, Ph.D., 1 Pierre Mainil-Varlet, M.D., 2 Henriette Juelke, M.D., 2 Claude Jaquiery, M.D., 1 Christian Candrian, M.D., 1,3 Andrea Barbero, Ph.D., 1 and Ivan Martin, Ph.D. 1 We investigated whether human articular chondrocytes can be labeled efficiently and for long-term with a green fluorescent protein (GFP) lentivirus and whether the viral transduction would influence cell proliferation and tissue-forming capacity. The method was then applied to track goat articular chondrocytes after autologous implantation in cartilage defects. Expression of GFP in transduced chondrocytes was detected cytofluorimetrically and immunohistochemically. Chondrogenic capacity of chondrocytes was assessed by Safranin-O staining, im- munostaining for type II collagen, and glycosaminoglycan content. Human articular chondrocytes were efficiently transduced with GFP lentivirus (73.4  0.5% at passage 1) and maintained the expression of GFP up to 22 weeks of in vitro culture after transduction. Upon implantation in nude mice, 12 weeks after transduction, the percentage of labeled cells (73.6  3.3%) was similar to the initial one. Importantly, viral transduction of chondrocytes did not affect the cell proliferation rate, chondrogenic differentiation, or tissue-forming capacity, either in vitro or in vivo. Goat articular chondrocytes were also efficiently transduced with GFP lentivirus (78.3  3.2%) and maintained the expression of GFP in the reparative tissue after orthotopic implantation. This study demonstrates the feasibility of efficient and relatively long-term labeling of human chondrocytes for co-culture on integration studies, and indi- cates the potential of this stable labeling technique for tracking animal chondrocytes for in cartilage repair studies. Introduction C artilage tissue engineering can provide functional cartilaginous constructs that can be used for controlled in vitro studies of chondrogenesis and potentially for in vivo articular cartilage repair. 1 For example, tissue-engineered cartilage has been introduced as a model to investigate in- teractions between different zonal populations of articular chondrocytes, 2 between primary and passaged chon- drocytes, 3 or between chondrocytes and other cell types, including osteoblasts, 2,4 human embryonic stem cells, 5 and synoviocytes. 6 Engineered cartilaginous tissues have also been used as in vitro model systems to study the integration process of a cartilage graft with native cartilage 7–9 or bone tissue. 10 In most of these coculture or integration studies, however, some of the key mechanistic questions related to the functional contribution of specific cell populations could not be addressed, since chondrocytes were either unlabeled or labeled with a membrane fluorescent dye, 2 which is sub- jected to natural fading with time. Due to limited scientific evidence of the role of implanted chondrocytes in a cartilage defect and of their extent of participation in the repair process, chondrocyte labeling would also be extremely important before cell grafting in animal models. So far, only Dell’Accio et al. 11 used a fluo- rescent dye, PKH26, to label goat articular chondrocytes and showed that implanted cells persisted for at least 14 weeks in the defects and contributed to structural cartilage repair in a goat model of autologous chondrocyte implantation. This study represents an important milestone in the field, but the method used for labeling was not ideal for long-term studies because of the natural fading of PKH26 and the decrease in intensity of labeling with cell divisions. 11,12 With the perspective of performing controlled in vitro or in vivo studies on the interaction between defined chon- drocyte populations and other cell systems, a reliable tech- nique for sustained chondrocyte labeling should be used. Besides membrane fluorochromes, 11,13 other methods for cell tracking include the use of radioactive isotopes, 14 Y chromo- some–specific probes, 15 or delivery of reporter genes. Among 1 Departments of Surgery and of Biomedicine, University Hospital Basel, Basel, Switzerland. 2 Institute of Pathology, University of Bern, Bern, Switzerland. 3 Ospedale Regionale di Lugano, FMH Chirurgie, Orthopa ¨die und Traumatologie des Bewegungsapparates, Lugano, Switzerland. TISSUE ENGINEERING: Part C Volume 16, Number 1, 2010 ª Mary Ann Liebert, Inc. DOI: 10.1089=ten.tec.2008.0698 11