Chawla Kanika, Klein Travis J, Schumacher Barbara L, Schmidt Tannin A, Voegtline Michael S, Thonar Eugene J-M A, Masuda Koichi, Sah Robert L
Department of Bioengineering, University of California-San Diego, 9500 Gilman Dr., MC 0412, La Jolla, California 92093-0412, USA.
J Orthop Res. 2006 Jul;24(7):1499-508. doi: 10.1002/jor.20116.
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.
区分植入细胞和宿主来源的细胞，以及不同的细胞表型，对于评估基于细胞的软骨修复机制将是有用的。荧光追踪染料PKH26先前已应用于多种细胞类型，以评估体外增殖并在体内追踪细胞。本研究的目的是评估PKH26用于追踪表层和中层区域软骨细胞及其增殖的效用，并确定PKH26对软骨细胞功能的影响，特别是对蛋白聚糖4（PRG4）的增殖和分泌的影响。将PKH26标记和未标记的表层和中层区域软骨细胞接种在低密度或高密度单层培养中，并在第0天和第7天通过流式细胞术和荧光显微镜分析PKH26的保留情况。分别分析细胞悬液和条件培养基中的DNA和PRG4的分泌情况。对流式细胞术直方图进行反卷积分析，以便估计每个倍增代中对最终细胞群体有贡献的细胞数量。软骨细胞在7个分裂周期中始终被PKH26强烈标记。在培养的第7天，低密度或高密度接种的表层区域细胞中>97%可被鉴定为荧光细胞，高密度接种的中层区域细胞也是如此。PKH26标记后细胞荧光的保留以及对细胞增殖和PRG4合成没有不利影响，表明PKH26可用于确定体内植入软骨细胞的命运和功能，以及监测体外增殖。
