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Daniel Kampik, Ioana-Sandra Tarau, Hong Han, Heike Walles, Florian Kai Groeber-Becker, Jost Hillenkamp, Christian Lotz; A 3D-hemi-cornea wound healing model for pre-clinical testing. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4117.
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© ARVO (1962-2015); The Authors (2016-present)
To test a novel bioengineered 3D hemi-cornea model for its suitability to emulate corneal irritation and wound healing.
Hemi-cornea models comprised epithelium and stroma. For stroma, human primary corneal keratocytes were added to type 1 rat tail collagen (11 mg/ml) to form a hydrogel which was compressed at 6.5 * 10-6 m/s to a final thickness of 500 µm. For the epithelium, immortalized human corneal epithelial cells were seeded on top of the stroma substitute and left in submerse culture for 1 day, followed by air-lift culture for 12 days. Cornea models were then wounded with an excimer laser at 150 µm ablation depth, 2 mm ablation diameter. Two different media, epidermal keratinocyte medium (EpiLife™ supplemented with human keratinocyte growth supplement and 1.5 mM CaCl2, “E3”) and DMEM-F12 medium containing 5% FCS and 1.5 mM CaCl2 (“F12”), were tested. Models were characterized by H&E staining, immunohistochemistry, trans-epithelial electrical resistance (TEER) and Calcein live cell staining, and compared to native human corneas.
After 12 days in culture, hemi-cornea models showed non-keratinized, stratified, squamous epithelium expressing cornea-specific markers Keratin-12, Keratin-14, α-Enolase, and basal membrane marker Col-4 at the basal side. Stromal marker Col-1 could be observed in the stromal equivalent. E3-cultured models showed a marker expression pattern more similar to human cornea than F12-cultured models. In all experiments (n=12), TEER at 100 Hz was consistent at 275 ±25 Ω*cm2 independent of the culture medium.Re-epithelialization after laser wounding was dependant on culture medium: Epithelium in F12-cultured models regenerated faster (10-14 days) than E3-cultured models (14-18 days). In F12, TEER decreased to 100 ± 30 Ω*cm2, and recovered to 300 ±15 Ω*cm2 after 7 days. In E3, TEER did not recover to levels above 200 ±25 Ω*cm2. Calcein live staining confirmed this trend.
Re-epithelialisation after wounding was reproducible with low variability. Re-epithelialisation speed and tightness of the cell layer can be modified by the type of culture medium. This model can be used for pre-clinical tests of new therapies for corneal wound healing and thereby reduce animal tests.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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