In VitroandIn VivoValidation of Human and Goat Chondrocyte Labeling by Green Fluorescent Protein Lentivirus Transduction

Abstract

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, immunostaining 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 indicates the potential of this stable labeling technique for tracking animal chondrocytes for in cartilage repair studies.

Influence of in vitro maturation of engineered cartilage on the outcome of osteochondral repair in a goat model

“…In order to determine if the repair fi brocartilaginous tissue was derived from the implanted graft or from the host, implanted chondrocytes could have been labelled using fl uorescent dye such as PKH26 (Dell'Accio et al, 2003) or transduced by a lentivirus allowing long term expression of a green fl uorescent protein (Miot et al, 2009). …”

Validation of an Automated Procedure to Isolate Human Adipose Tissue–Derived Cells by Using the Sepax®Technology

“…17 To quantify the sulfated glycosaminoglycan (GAG) content, other pellets were digested with a proteinase K solution, incubated with a dimethylmethylene blue dye, and read spectrophotometrically, with chondroitin sulfate as a standard, as previously described. 18 The GAG content was normalized to the deoxyribonucleic acid (DNA) amount that was measured using a CyQUANT cell proliferation assay kit (Molecular Probes), with calf thymus DNA as a standard.…”

Viruses: Friends and Foes

“…Toward this, many different animal cells have been used, including human, rat, rabbit, goat, and cattle [92][93][94][95]. One group showed that transduction of chondrocytes with GFP was associated with an approximate 60% success rate [92].…”