title: Effect of different culture media and deswelling agents on survival of human corneal endothelial and epithelial cells in vitro.
abstract: OBJECTIVE
  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.
authors.affiliation: Institute of Anatomy, Faculty of Medicine Carl Gustav Carus of the TU Dresden, Fetscherstr. 74, 01307, Dresden, Germany. monika.valtink@tu-dresden.de.
authors.forename: Monika
authors.identifier: http://orcid.org/0000-0003-3205-1876
authors.initials: M
authors.lastname: Valtink
authors.affiliation: Institute of Anatomy, Faculty of Medicine Carl Gustav Carus of the TU Dresden, Fetscherstr. 74, 01307, Dresden, Germany.
authors.forename: Patricia
authors.initials: P
authors.lastname: Donath
authors.affiliation: Department of Ophthalmology, Klinikum Chemnitz gGmbH, Flemmingstr. 2, 09116, Chemnitz, Germany.
authors.forename: Katrin
authors.initials: K
authors.lastname: Engelmann
authors.affiliation: Institute of Anatomy, Faculty of Medicine Carl Gustav Carus of the TU Dresden, Fetscherstr. 74, 01307, Dresden, Germany.
authors.forename: Lilla
authors.initials: L
authors.lastname: Knels
mesh_terms: D017209:Apoptosis; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003470:Culture Media / Q000494:pharmacology*; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D003911:Dextrans / Q000494:pharmacology*; D004728:Endothelium, Corneal / Q000473:pathology*; D004791:Enzyme Inhibitors / Q000633:toxicity; D019573:Epithelium, Corneal / Q000473:pathology*; D005434:Flow Cytometry; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D006895:Hydroxyethyl Starch Derivatives / Q000494:pharmacology*; D008858:Microscopy, Phase-Contrast; D009336:Necrosis; D009924:Organ Culture Techniques; D016877:Oxidants / Q000633:toxicity; D010952:Plasma Substitutes / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D019311:Staurosporine / Q000633:toxicity
chemical_list: D003470:Culture Media; D016895:Culture Media, Serum-Free; D003911:Dextrans; D004791:Enzyme Inhibitors; D006895:Hydroxyethyl Starch Derivatives; D016877:Oxidants; D010952:Plasma Substitutes; D017382:Reactive Oxygen Species; D006861:Hydrogen Peroxide; D019311:Staurosporine
keywords: Cell survival; Corneal organ cultivation; Deswelling substance; Human corneal endothelium; Human corneal epithelium; Serum-free medium