Tracking chondrocytes and assessing their proliferation with PKH26: Effects on secretion of proteoglycan 4 (PRG4)

Abstract

Distinguishing between implanted and host-derived cells, as well as between distinct cell phenotypes, would be useful in assessing the mechanisms of cell-based repair of cartilage. The fluorescent tracker dye, PKH26, was previously applied to several cell types to assess proliferation in vitro and to track cells in vivo. The objectives of this study were to assess the utility of PKH26 for tracking chondrocytes from superficial and middle zones and their proliferation, and determine the effects of PKH26 on chondrocyte functions, in particular, proliferation and secretion of Proteoglycan 4 (PRG4). PKH26-labeled and unlabeled superficial and middle zone chondrocytes were plated in either low- or high-density monolayer culture and analyzed for retention of PKH26 by flow cytometry and fluorescence microscopy at days 0 and 7. Cell suspensions and conditioned media were analyzed for DNA and secretion of PRG4, respectively. Flow cytometric histograms were deconvolved so that the number of cells in each doubling generation contributing to the final cell population could be estimated. Chondrocytes were consistently and intensely labeled with PKH26 through 7 cycles of division. At day 7 of culture, >97% of superficial zone cells seeded at low or high density could be distinguished as fluorescent, as could middle zone cells seeded at high density. Retention of cell fluorescence after PKH26 labeling and lack of adverse effects on cell proliferation and synthesis of PRG4 suggest that PKH26 can be useful in determining the fate and function of implanted chondrocytes in vivo, as well as monitoring proliferation in vitro. © 2006 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.