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Effect of different culture media and deswelling agents on survival of human corneal endothelial and epithelial cells in vitro

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An Erratum to this article was published on 15 February 2016

Abstract

Purpose

To examine the effects of media and deswelling agents on human corneal endothelial and epithelial cell viability using a previously developed screening system.

Methods

The human corneal endothelial cell line HCEC-12 and the human corneal epithelial cell line HCE-T were cultured in four different corneal organ culture media (serum-supplemented: MEM +2 % FCS, CorneaMax®/CorneaJet®, serum-free: Human Endothelial-SFM, Stemalpha-2 and -3) with and without 6 % dextran T500 or 7 % HES 130/0.4. Standard growth media F99HCEC and DMEM/F12HCE-T served as controls. In additional controls, the stress inducers staurosporine or hydrogen peroxide were added. After 5 days in the test media, cell viability was assessed by flow cytometrically quantifying apoptotic and necrotic cells (sub-G1 DNA content, vital staining with YO-PRO-1® and propidium iodide) and intracellular reactive oxygen species (ROS).

Results

The MEM-based media were unable to support HCEC-12 and HCE-T survival under stress conditions, resulting in significantly increased numbers of apoptotic and necrotic cells. HCEC-12 survival was markedly improved in SFM-based media even under staurosporine or hydrogen peroxide. Likewise, HCE-T survival was improved in SFM with or without dextran. The media CorneaMax®, CorneaJet®, and CorneaMax® with HES supported HCEC-12 survival better than MEM-based media, but less well than SFM-based media. HCE-T viability was also supported by CorneaJet®, but not by CorneaMax® with or without HES. Stemalpha-based media were not suitable for maintaining viability of HCEC-12 or HCE-T in the applied cell culture system.

Conclusions

The use of serum-supplemented MEM-based media for corneal organ culture should be discontinued in favour of serum-free media like SFM.

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Acknowledgments

We thank Cornelia Schroeder for critically reading the manuscript.

Contributors

MV designed the study and was involved in data evaluation and manuscript writing. PD did all experiments. KE supervised the project and was involved in manuscript writing. LK supervised the laboratory work and was involved in data evaluation and manuscript writing.

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Authors and Affiliations

  1. Institute of Anatomy, Faculty of Medicine Carl Gustav Carus of the TU Dresden, Fetscherstr. 74, 01307, Dresden, Germany

    Monika Valtink, Patricia Donath & Lilla Knels

  2. Department of Ophthalmology, Klinikum Chemnitz gGmbH, Flemmingstr. 2, 09116, Chemnitz, Germany

    Katrin Engelmann

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  1. Monika Valtink
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  2. Patricia Donath
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  3. Katrin Engelmann
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  4. Lilla Knels
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Corresponding author

Correspondence to Monika Valtink.

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This article does not contain any studies with human participants or animals performed by any of the authors.

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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.

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Valtink, M., Donath, P., Engelmann, K. et al. Effect of different culture media and deswelling agents on survival of human corneal endothelial and epithelial cells in vitro. Graefes Arch Clin Exp Ophthalmol 254, 285–295 (2016). https://doi.org/10.1007/s00417-015-3235-4

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