JBiSE  Vol.1 No.3 , November 2008
In vitro and in vivo cell tracking of chondrocytes of different origin by fluorescent PKH 26 and CMFDA
Tissue engineering techniques for cartilage re-pair 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 de-fect. 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 ap-plied 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 and or quickly lose their tracking ability. New fluorescent probes are easier to handle and do not to interfere with cells. PKH 26劌 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-chloromethylfluorescein diacetate - CMFDA (¨cell tracker green〔) is an established fluores-cent 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 chondro-cytes. The first aim of the study was to evaluate qualitative differences in fluorescence pattern after labeling of articular, auricular and costal chondrocytes. Secondly, we evaluated the influ-ence of labeling with CMFDA on cellular adhe-sion properties. The third aim was to compare the duration of cell labeling of chondrocytes of different origin with established CMFDA as stan-dard and PKH 26潴 for 3 cell generations in vitro and 12 weeks in vivo. We show that chondro-cytes from different origin can be labeled effec-tively with both PKH 26潴 and CMFDA. The PKH 26潴 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.

Cite this paper
nullChristian, W. , TS, J. and TJ, G. (2008) In vitro and in vivo cell tracking of chondrocytes of different origin by fluorescent PKH 26 and CMFDA. Journal of Biomedical Science and Engineering, 1, 163-169. doi: 10.4236/jbise.2008.13027.
[1]   Albertine KH, Gee MH. (1996) In vivo labeling of neutrophils using a fluorescent cell linker. J Leukoc Biol 59, 631 – 638.

[2]   Andsberg G, Kokaia Z, Bj鰎klund A, Lindvall O, Martinez ?Serrano A. (1998) Amelioration of ischemia - induced neural death in the rat striatum by NGF ?secreting neural stem cells. Eur J Neurosci 10, 2026 ?2036.

[3]   Batard P, Monier M-N, Fortunel N, Ducos K, Sansilvestri – Morel P, Phan T-H, Hatzfeld A, Hatzfeld J. (2000) TGF-beta 1 maintains the hematopoitic immaturity by a reversible negative control of cell cycle and induces CD 34 antigen up-modulation. J Cell Sci 113, 383 – 390.

[4]   Butler PEM, Lee WP, Sims DC, Randolph MA, Vacanti CA, Yaremchuk MJ. (1998) Cell transplantation from limb allografts. Plast Reconst Surg 102, 161 – 168.

[5]   Deutsch M, Kaufman M, Shapiro H, Zurgil N. (2000) Analysis of enzyme kinetics in individual living cells utilizing fluorescence intensity and polarization measurements Cytometry 39, 36 – 44.

[6]   Evans CH, Ghivizzani SC, Oligino TA, Robbins PD. (2001) Fu-ture of adenoviruses in the gene therapy of arthritis. Arthritis Res 3, 142-6.

[7]   Fricker RA, Carpenter MK, Winkler C, Greco C, Gates M., Bj鰎klund A. (1999) Site - specific migration and neural differen-tiation of human neural progenitor cells after transplantation into the adult rat brain. J Neurosci 19, 5990 ?6005.

[8]   Givan AL, Fisher JL, Waugh M, Ernstoff MS, Wallace PK. (1999) A flow cytometric method to estimate the precursor frequencies of cells proliferating in response to specific antigens. J Immunol Methods 230, 99 – 112.

[9]   Haas SJP, Bauer P, Rolfs A, Wree A. (2000) Immunocytochemical characterization of in vitro PKH 26 labeled and intracerebrally transplanted neonatal cells. Acta Histochem 102, 273 – 280

[10]   Horan PK, Slezak SE. (1989) Stable cell membrane labeling. Nature 340, 167 – 168.

[11]   Johnson I. (1998) Fluorescent probes for living cells. Histochem J 30, 123 – 140.

[12]   Lai CC, Gouras P, Doi K, Lu F, Kjedlbye H, Goff SP, Pawliuk R, Leboulch P, Tsang SH. (1999) Tracking RPE transplants labeled by retroviral gene transfer with green fluorescent protein. Invest Ophtalmol Vis Sci 40, 2141 – 2160.

[13]   Lee-MacAry AE, Ross EL, Davies D, Laylor R, Honeychurch J, Glennie MJ, Snary D., Wilkinson RW. (2001) Development of a novel flow cytometric cell-mediated cytotoxicity assey using the fluorophores PKH 26 and TO-PRO-3 iodide. J Immunol Methods 252, 83 – 92.

[14]   Lundberg C, Field PM, Ajayi YO, Raisman G, Bj鰎klund A. (1996) Conditionally immortalized neural progenitor cell lines integrate and differentiate after grafting to the adult rat striatum. A com-bined autoradiographic and electron microscopic study. Brain Res 737, 295 ?300.

[15]   Lundberg C, Martinez ?Serrano A, Cattaneo E, McKay RDG, Bj鰎klund A. (1997) Survival, integration and differentiation of neural stem cell lines after transplantation to the adult rat striatum. Exp Neurol 145, 342 ?360.

[16]   Madry H, Trippel SB. (2000) Efficient lipid-mediated gene trans-fer to articular chondrocytes. Gene Ther 7, 286-91.

[17]   Maus U, Herold S, Muth H, Maus R, Ermert L, Ermert M, Weiss-mann N, Rosseau S, Seeger W, Grimminger F, Lohmeyer J. (2001) Monocytes recruited into the alveolar air space of mice show a monocytic phenotype but upregulate CD 14. Am J Physiol Lung Cell Mol Physiol 280, L58 – L68.

[18]   Messina LM, Podrazik RM, Whitehill TA, Ekhterae D, Brothers TE, Wilson JM, Burkel WE, Stanley JC. (1992) Adhesion and in-corporation of lacZ-transduced endothelial cells into the intact capillary wall in the rat. Proc Natl Acad Sci U S A. 89, 12018-12022.

[19]   Mitchell EA, Bergmeister LA, Doyle C, Brookes R, Hussain LA, Wang Y, Lehner T. (1998) Homing of mononuclear cells from iliac lymph nodes to the genital and rectal mucosa in non-human pri-mates. Eur J Immunol 28, 3066 – 3074.

[20]   Parish CR. (1999) Fluorescent dyes for lymphocyte migration and proliferation studies. Immunol Cell Biol 77, 499-508.

[21]   Rousselle C, Paillasson S, Robert-Nicoud M, Ronot X. (1999) Chromatin texture analysis in living cells. Histochem J 31, 63-70.

[22]   Sabate O, Horellou P, Vigne E, Colin P, Perricaudet M, Buc – Caron MH, Mallet J. (1995) Transplantation to the rat brain of human neural progenitors that were genetically modified using adenoviruses. Nat Genet 9, 256 – 260.

[23]   Sheen VL, Macklis JD. (1995) Targeted neocortical cell death in adult mice guides migration and differentiation of transplanted embryonic neurons. J. Neurosci 15, 8378 – 8392.

[24]   Slezak SE, Horan PK. (1989) Fluorescent in vivo tracking of hematopoietic cells. Part I. Technical considerations. Blood 74, 2172 – 2177.

[25]   Song L, Hennink EJ, Young IT, Tanke HJ. (1995) Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy. Biophys J 68, 2588 – 2600.

[26]   Weinand C, Xu, JW, Peretti GM, Randolph MA, Bonassar LJ, Savvidis E., Gill TJ. Conditions affecting chondrocyte seeding onto three-dimensional scaffolds. Submitted for Cells Tissues Or-gans

[27]   Weinand C., Peretti G.M., Adams SB, Bonassar L.J., Randolph M.A., Gill T.J. (2006) An allogeneic cell-based implant for me-niscus lesions. Am J Sports Med 34, 1779 – 1789

[28]   Yang XB, Roach HI, Clarke NMP, Howdle SM, Quirk R, Shakesheff KM, Oreffo ROC. (2001) Human osteoprogenitor growth and differentiation on synthetic biodegradable structures after surface modification. Bone 29, 523 – 531.