Christian Wertheimer1, Kirsten H Eibl-Lindner2, Denise Compera3, Alexander Kueres2, Armin Wolf2,3, Denitsa Docheva4,5, Siegfried G Priglinger2,3, Claudia Priglinger2, Ricarda G Schumann3. 1. Laboratory for Cell- and Molecular Biology, Department of Ophthalmology, Ludwig-Maximilians-University Munich, Mathildenstrasse 8, 80336, Munich, Germany. Christian.Wertheimer@med.uni-muenchen.de. 2. Laboratory for Cell- and Molecular Biology, Department of Ophthalmology, Ludwig-Maximilians-University Munich, Mathildenstrasse 8, 80336, Munich, Germany. 3. Laboratory for Vitreoretinal Pathology, Department of Ophthalmology, Ludwig-Maximilians-University Munich, Munich, Germany. 4. Laboratory for Experimental Trauma Surgery, Department of Trauma Surgery, University Regensburg Medical Centre, Regensburg, Germany. 5. Laboratory for Experimental Surgery and Regenerative Medicine, Department of Surgery, Ludwig-Maximilians-University Munich, Munich, Germany.
Abstract
PURPOSE: To introduce a human cell culture technique for investigating in-vitro behavior of primary epiretinal cells and membrane contraction of fibrocellular tissue surgically removed from eyes with idiopathic macular pucker. METHODS: Human epiretinal membranes were harvested from ten eyes with idiopathic macular pucker during standard vitrectomy. Specimens were fixed on cell culture plastic using small entomological pins to apply horizontal stress to the tissue, and then transferred to standard cell culture conditions. Cell behavior of 400 epiretinal cells from 10 epiretinal membranes was observed in time-lapse microscopy and analyzed in terms of cell migration, cell velocity, and membrane contraction. Immunocytochemistry was performed for cell type-specific antigens. RESULTS: Cell specific differences in migration behavior were observed comprising two phenotypes: (PT1) epiretinal cells moving fast, less directly, with small round phenotype and (PT2) epiretinal cells moving slowly, directly, with elongated large phenotype. No mitosis, no outgrowth and no migration onto the plastic were seen. Horizontal contraction measurements showed variation between specimens. Masses of epiretinal cells with a myofibroblast-like phenotype expressed cytoplasmatic α-SMA stress fibers and correlated with cell behavior characteristics (PT2). Fast moving epiretinal cells (PT1) were identified as microglia by immunostaining. CONCLUSIONS: This in-vitro technique using traction application allows for culturing surgically removed epiretinal membranes from eyes with idiopathic macular pucker, demonstrating cell behavior and membrane contraction of primary human epiretinal cells. Our findings emphasize the abundance of myofibroblasts, the presence of microglia and specific differences of cell behavior in these membranes. This technique has the potential to improve the understanding of pathologies at the vitreomacular interface and might be helpful in establishing anti-fibrotic treatment strategies.
PURPOSE: To introduce a human cell culture technique for investigating in-vitro behavior of primary epiretinal cells and membrane contraction of fibrocellular tissue surgically removed from eyes with idiopathic macular pucker. METHODS:Human epiretinal membranes were harvested from ten eyes with idiopathic macular pucker during standard vitrectomy. Specimens were fixed on cell culture plastic using small entomological pins to apply horizontal stress to the tissue, and then transferred to standard cell culture conditions. Cell behavior of 400 epiretinal cells from 10 epiretinal membranes was observed in time-lapse microscopy and analyzed in terms of cell migration, cell velocity, and membrane contraction. Immunocytochemistry was performed for cell type-specific antigens. RESULTS: Cell specific differences in migration behavior were observed comprising two phenotypes: (PT1) epiretinal cells moving fast, less directly, with small round phenotype and (PT2) epiretinal cells moving slowly, directly, with elongated large phenotype. No mitosis, no outgrowth and no migration onto the plastic were seen. Horizontal contraction measurements showed variation between specimens. Masses of epiretinal cells with a myofibroblast-like phenotype expressed cytoplasmatic α-SMA stress fibers and correlated with cell behavior characteristics (PT2). Fast moving epiretinal cells (PT1) were identified as microglia by immunostaining. CONCLUSIONS: This in-vitro technique using traction application allows for culturing surgically removed epiretinal membranes from eyes with idiopathic macular pucker, demonstrating cell behavior and membrane contraction of primary human epiretinal cells. Our findings emphasize the abundance of myofibroblasts, the presence of microglia and specific differences of cell behavior in these membranes. This technique has the potential to improve the understanding of pathologies at the vitreomacular interface and might be helpful in establishing anti-fibrotic treatment strategies.
Entities:
Keywords:
Cell culture; Epiretinal membrane; Idiopathic macular pucker; Time lapse microscopy
Authors: Franco Klingberg; Melissa L Chow; Anne Koehler; Stellar Boo; Lara Buscemi; Thomas M Quinn; Mercedes Costell; Benjamin A Alman; Elisabeth Genot; Boris Hinz Journal: J Cell Biol Date: 2014-10-20 Impact factor: 10.539