PhD Scientific Days 2025

Molecular Medicine III.

Establishing an In Vitro Protocol for the Investigation of Fluvoxamine in the Treatment of Corneal Fibrosis

Name of the presenter

Buzogany Zsuzsanna

Institute/workplace of the presenter

Semmelweis University Pediatric Center Bokay Street

Authors

Zsuzsanna Buzogany^1,2, Judit Hodrea^1,2, Illes Kovacs³, Anna Takacsi-Nagy⁴, Attila J. Szabo², Andrea Fekete^1,2

1: MTA-SE Lendület “Momentum” Diabetes Research Group, Semmelweis University, Budapest
2: Pediatric Center, MTA Center of Excellence, Semmelweis University, Budapest
3: Department of Ophthalmology, Semmelweis University, Budapest
4: PannonPharma Pharmaceutical Ltd, Pécsvárad

Text of the abstract

Introduction:
Primary human corneal fibroblasts are critical for studying corneal fibrosis mechanisms and testing therapeutic interventions. Fibrotic eye diseases may result from diabetes mellitus (DM), injuries, chemical burns, and traumas, often as primary conditions or secondary complications. Our previous work showed that Sigma-1 Receptor (S1R) agonist Fluvoxamine (FLU) reduces fibrotic damage in the anterior segment of the eye, prompting us to hypothesize that FLU may also benefit corneal fibrosis.
Aims:
We aimed to optimize the isolation of primary corneal fibroblasts from human, wild-type, and S1R-/- mice by comparing explant and enzymatic methods, with or without growth supplements. We also investigated FLU’s molecular effects on PDGF-BB and TGFβ-1-activated fibroblasts and explored pathway differences between WT and S1R-/- mouse cells to establish a reliable protocol and validate FLU’s anti-fibrotic and anti-inflammatory impact.
Methods:
Primary fibroblasts were isolated from human, WT, and S1R-/- mouse corneas using both explant and enzymatic digestion. The cells were cultured in DMEM:F12 medium with 10-20% FBS, with or without 2 µg/ml FGF. MTT and LDH assays assessed viability and proliferation. Immunocytochemistry evaluated morphology and markers of fibrosis and proliferation, including FN, αSMA and Ki67.
Results:
Effective isolation protocols were established for all models. MTT and LDH assays demonstrated that FLU (0.5- 40 µM) and PDGF-BB (5-70 µg/ml) were non-toxic. Experimental durations were optimized for reproducibility. FGF slightly increased FN and αSMA expression in human fibroblasts suggesting a more activated state. While both methods yielded viable cultures, enzymatic digestion proved more efficient and time-effective, and was chosen as the standard protocol.
Conclusion:
We established efficient, reproducible protocols for isolating primary corneal fibroblasts from human and mouse tissues, enzymatic digestion is preferred due to reduced culture time. FLU and PDGF-BB were non-toxic within tested ranges, PDGF significantly promoted proliferation. Our future perspective is to investigate the effect of this novel treatment with sigma-1 receptor agonist on primary corneal fibrosis cells.
Funding:
OTKA-K135398, LP2021-3/2021, TKP2021-EGA-24, 2023-2.1.2-KDP-2023-00016

zsuzsi.buzogany@gmail.com
Semmelweis University
Prof. Andrea Fekete