Abstract
Purpose :
Corneal scar formation has been investigated extensively in animal models. To reduce animal experiments, we developed 2D and 3D tissue-engineered cornea models based on human cells, and optimized assays to assess scar-modulating substances.
Methods :
Human corneal stroma from donor corneas was digested with collagenase I for 1 h to grow primary corneal stromal keratocytes (CSK) in DMEM medium containing 10% FCS. For 2D analysis, 15.000 CSK per coverslip were seeded and cultured in DMEM with 2% FCS.
The 3D cornea model consists of epithelium and stromal equivalent. For the stroma equivalent, 45.000 CSK were suspended in 700 µL type I collagen (10.5 mg/mL) and seeded onto cell culture inserts (snapwell 0.4 µm). The collagen was compressed for 30 minutes leaving behind stromal equivalent of approx. 500 µm thickness followed by 24 h culture. On apical side, 500.000 immortalized human epithelial cells (hTCEpi, EverCyte) were seeded in serum-free EpiLife® basal medium. Culture at the air-liquid interface induces the formation of a multilayered, non-keratinizing squamous epithelium.
Transformation of CSK to myofibroblasts was induced by TGF-β2 (10 ng/ul), visualized by α-SMA immunofluorescence. Contraction of the 3D models was assessed by H&E histology and optical coherence tomography (Leica Bioptigen Envisu) and quantified with ImageJ.
Results :
In the 2D model, CSK showed expression of actin stress fibers and α-SMA after 2 days of incubation with TGF-β2. Pre-incubation with lovastatin, an inhibitor of Rho-GTPase signaling, partially inhibited α-SMA expression.
In the 3D model, a model contraction of 6% ±2.3% was observed under the influence of TGF-β2 from day 5 onwards. This contraction increased steadily and significantly over time i.e. 12.4% ±3.8% and 16.6% ±1.9% on day 7 and day 9, respectively. Like 2D analysis, no significant contraction was observed under simultaneous incubation with lovastatin. Controls without TGF-β2 expressed significantly lower contraction i.e. 2.8% ±1.1% on day 5; 0.6% ±2.4 on day 7; 3.6% ±0.6% on day 9. Histologically, CSK under the influence of TGF-β2 showed a loss of their elongated collagen arrangement.
Conclusions :
CSK myofibroblast transdifferentiation can be induced in the 3D cornea model and can serve as a surrogate for fibrosis and corneal scarring. This represents a new alternative method to animal experiments to pre-screen and evaluate fibrosis-modulating substances in vitro.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.