Functional assessment of gap junctions in monolayer and three-dimensional cultures of human tendon cells using fluorescence recovery after photobleaching

• Published in: Journal of biomedical optics Vol. 19; no. 1; p. 015001
• Main Authors: Kuzma-Kuzniarska, Maria, Yapp, Clarence, Pearson-Jones, Thomas W, Jones, Andrew K, Hulley, Philippa A
• Format: Journal Article
• Language: English
• Published: United States Society of Photo-Optical Instrumentation Engineers 01-01-2014
• Subjects: Adult; Carbenoxolone - chemistry; Cell Communication; Cell Culture Techniques; Cells, Cultured; Collagen - chemistry; Culture; Diffusion; Female; Fluorescence; Fluorescence Recovery After Photobleaching - methods; Gap Junctions - pathology; Glycyrrhetinic Acid - analogs & derivatives; Glycyrrhetinic Acid - chemistry; HeLa Cells; Human; Humans; Imaging, Three-Dimensional; Immunohistochemistry; Inhibitors; Male; Middle Aged; Monolayers; Recovery; Research Papers: General; Tendons; Tendons - cytology; Tendons - pathology; Three dimensional; gap junctions; three dimensional; fluorescence recovery after photobleaching; human tenocytes
• DOI: 10.1117/1.JBO.19.1.015001
• EISSN: 1560-2281
• ISSN: 1083-3668

Gap junction-mediated intercellular communication influences a variety of cellular activities. In tendons, gap junctions modulate collagen production, are involved in strain-induced cell death, and are involved in the response to mechanical stimulation. The aim of the present study was to investigate gap junction-mediated intercellular communication in healthy human tendon-derived cells using fluorescence recovery after photobleaching (FRAP). The FRAP is a noninvasive technique that allows quantitative measurement of gap junction function in living cells. It is based on diffusion-dependent redistribution of a gap junction-permeable fluorescent dye. Using FRAP, we showed that human tenocytes form functional gap junctions in monolayer and three-dimensional (3-D) collagen I culture. Fluorescently labeled tenocytes following photobleaching rapidly reacquired the fluorescent dye from neighboring cells, while HeLa cells, which do not communicate by gap junctions, remained bleached. Furthermore, both 18 β-glycyrrhetinic acid and carbenoxolone, standard inhibitors of gap junction activity, impaired fluorescence recovery in tendon cells. In both monolayer and 3-D cultures, intercellular communication in isolated cells was significantly decreased when compared with cells forming many cell-to-cell contacts. In this study, we used FRAP as a tool to quantify and experimentally manipulate the function of gap junctions in human tenocytes in both two-dimensional (2-D) and 3-D cultures.