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A cell culture technique for human epiretinal membranes to describe cell behavior and membrane contraction in vitro

  • Retinal Disorders
  • Published:
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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.

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Author information

Authors and Affiliations

  1. Laboratory for Cell- and Molecular Biology, Department of Ophthalmology, Ludwig-Maximilians-University Munich, Mathildenstrasse 8, 80336, Munich, Germany

    Christian Wertheimer, Kirsten H. Eibl-Lindner, Alexander Kueres, Armin Wolf, Siegfried G. Priglinger & Claudia Priglinger

  2. Laboratory for Vitreoretinal Pathology, Department of Ophthalmology, Ludwig-Maximilians-University Munich, Munich, Germany

    Denise Compera, Armin Wolf, Siegfried G. Priglinger & Ricarda G. Schumann

  3. Laboratory for Experimental Trauma Surgery, Department of Trauma Surgery, University Regensburg Medical Centre, Regensburg, Germany

    Denitsa Docheva

  4. Laboratory for Experimental Surgery and Regenerative Medicine, Department of Surgery, Ludwig-Maximilians-University Munich, Munich, Germany

    Denitsa Docheva

Authors
  1. Christian Wertheimer
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  2. Kirsten H. Eibl-Lindner
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  3. Denise Compera
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  4. Alexander Kueres
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  5. Armin Wolf
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  6. Denitsa Docheva
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  7. Siegfried G. Priglinger
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  8. Claudia Priglinger
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  9. Ricarda G. Schumann
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Corresponding author

Correspondence to Christian Wertheimer.

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Conflict of interest

All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript.

Funding

No funding was received for this research.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Financial interest

The authors have no proprietary interest in this study.

Electronic supplementary material

Movie 1

Time-lapse microscopy of idiopathic epiretinal membrane pinned on cell culture plastic with tangential traction application. Differences in cell migration behavior were observed: (1) epiretinal cells moving fast, less directly, with small round phenotype, and (2) epiretinal cells moving slowly, directly, with elongated large phenotype. (MOV 1662 kb)

Movie 2

Time-lapse microscopy of idiopathic epiretinal membrane pinned on cell culture plastic demonstrating membrane contraction. The membrane edge was marked with a reference marker. Based on 10 measurements per specimen, mean drift of the membrane edges towards the centre was 3.1 ± 0.96 μm within 15 h. (MOV 1430 kb)

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Wertheimer, C., Eibl-Lindner, K.H., Compera, D. et al. A cell culture technique for human epiretinal membranes to describe cell behavior and membrane contraction in vitro. Graefes Arch Clin Exp Ophthalmol 255, 2147–2155 (2017). https://doi.org/10.1007/s00417-017-3767-x

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  • DOI: https://doi.org/10.1007/s00417-017-3767-x

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