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Graefes Arch Clin Exp Ophthalmol. 2016 Feb;254(2):285-95. doi: 10.1007/s00417-015-3235-4. Epub 2015 Dec 19.

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

Author information

1
Institute of Anatomy, Faculty of Medicine Carl Gustav Carus of the TU Dresden, Fetscherstr. 74, 01307, Dresden, Germany. monika.valtink@tu-dresden.de.
2
Institute of Anatomy, Faculty of Medicine Carl Gustav Carus of the TU Dresden, Fetscherstr. 74, 01307, Dresden, Germany.
3
Department of Ophthalmology, Klinikum Chemnitz gGmbH, Flemmingstr. 2, 09116, Chemnitz, Germany.

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.

KEYWORDS:

Cell survival; Corneal organ cultivation; Deswelling substance; Human corneal endothelium; Human corneal epithelium; Serum-free medium

PMID:
26686232
DOI:
10.1007/s00417-015-3235-4
[Indexed for MEDLINE]

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