Assessment of Rose Bengal Photodynamic Therapy on Viability and Proliferation of Human Keratolimbal Epithelial and Stromal Cells In Vitro

Abstract

Purpose To investigate the effect of Rose Bengal photodynamic therapy (RB-PDT) on viability and proliferation of human limbal epithelial stem cells (T-LSCs), human corneal epithelial cells (HCE-T), human limbal fibroblasts (LFCs), and human normal and keratoconus fibroblasts (HCFs and KC-HCFs) in vitro.

Methods T-LSCs and HCE-T cell lines were used in this research. LFCs were isolated from healthy donor corneal limbi (n = 5), HCFs from healthy human donor corneas (n = 5), and KC-HCFs from penetrating keratoplasties of keratoconus patients (n = 5). After cell culture, RB-PDT was performed using 0.001% RB concentration and 565 nm wavelength illumination with 0.14 to 0.7 J/cm² fluence. The XTT and the BrdU assays were used to assess cell viability and proliferation 24 h after RB-PDT.

Results RB or illumination alone did not change cell viability or proliferation in any of the cell types (p ≥ 0.1). However, following RB-PDT, viability decreased significantly from 0.17 J/cm² fluence in HCFs (p < 0.001) and KC-HCFs (p < 0.0001), and from 0.35 J/cm² fluence in T-LSCs (p < 0.001), HCE-T (p < 0.05), and LFCs ((p < 0.0001). Cell proliferation decreased significantly from 0.14 J/cm² fluence in T-LSCs (p < 0.0001), HCE-T (p < 0.05), and KC-HCFs (p < 0.001) and from 0.17 J/cm² fluence in HCFs (p < 0.05). Regarding LFCs proliferation, no values could be determined by the BrdU assay.

Conclusions Though RB-PDT seems to be a safe and effective treatment method in vivo, its dose-dependent phototoxicity on corneal epithelial and stromal cells has to be respected. The data and experimental parameters applied in this study may provide a reliable reference for future investigations.

Zusammenfassung

Ziel Die photodynamische Therapie kann eine alternative Behandlungsmethode bei antibiotikaresistenten Keratitiden darstellen. In dieser Studie wurde die Wirkung der photodynamischen Therapie mit Bengalrosa (RB-PDT) auf die Viabilität und Proliferation von humanen limbalen Epithelstammzellen (T-LSCs), humanen Hornhautepithelzellen (HCE-T), limbalen Fibroblasten (LFCs), normalen und Keratokonus-Fibroblasten (HCFs und KC-HCFs) in vitro untersucht.

Material und Methoden Für die Experimente an den limbalen Epithelstammzellen wurden 2 verschiedene Zelllinien (T-LSCs und HCE-T) verwendet. Die primären LFCs und HCFs wurden aus den Korneoskleralringen von Hornhautspendern, die KC-HCFs aus perforierenden Keratoplastiken von Keratokonus-Patienten isoliert und kultiviert (jeweils n = 5). Die RB-PDT wurde mit einer 0,001%igen RB-Konzentration bei einer Wellenlänge von 565 nm und einer Energiedosis von 0,14 bis 0,7 J/cm² durchgeführt. Zur Bestimmung der Viabilität wurde 24 h nach der Bestrahlung der XTT-, zur Bestimmung der Proliferation der BrdU-Assay verwendet.

Ergebnisse Die ausschließliche Verwendung von RB oder Bestrahlung hatten keinen messbaren Einfluss auf die Viabilität oder Proliferation der unterschiedlichen Zelltypen (p ≥ 0,1). Unter Verwendung der RB-PDT mit einer Dosis von 0,17 J/cm² sank die Viabilität jedoch in den HCFs (p < 0,001) und KC-HCFs (p < 0,0001), und bei einer Dosis von 0,35 J/cm² in den T-LSCs (p < 0,001), HCE -T (p < 0,05) und LFCs (p < 0,0001). Die Zellproliferation verringerte sich signifikant ab einer Dosis von 0,14 J/cm² in T-LSCs (p < 0,0001), HCE-T (p < 0,05) und KC- HCFs (p < 0,001) und ab einer Dosis von 0,17 J/cm² Fluenz in HCFs (p < 0,05). Für die Proliferationsbestimmung der LFCs konnten mit dem BrdU-Assay keine Werte ermittelt werden.

Schlussfolgerung Die RB-PDT zeigt eine dosisabhängige Phototoxizität auf Hornhautepithel- und Stromazellen. Die in dieser Studie ermittelten Daten und experimentellen Parameter bieten eine verlässliche Referenz für zukünftige Untersuchungen für die photodynamische Therapie mit Bengalrosa.

Conclusions

Already known:

• RB-PDT may be a potential treatment method of infectious keratitis and an effective corneal stiffening procedure.

• In animal models, corneal RB-PDT was a safe treatment procedure. However, there is no previous report focusing on the cytotoxicity of RB-PDT on human corneal cells in vitro.

• The effect of RB-PDT on viability and proliferation of corneal epithelial and stromal cells should be evaluated in vitro to provide a practical experimental model for future analysis.

Newly described:

• The fluence-dependent phototoxicity of RB-PDT on human corneal epithelial and stromal cells should be kept in mind.

• The data and the experimental parameters applied in this study provide a reliable reference for future investigations.

Key words

photodynamic therapy - Rose Bengal - human cornea fibroblasts - keratocytes

Schlüsselwörter

photodynamische Therapie - Bengalrosa - humane Hornhautfibroblasten - Keratozyten

Publikationsverlauf

Eingereicht: 19. Dezember 2022

Angenommen: 16. Februar 2023

Accepted Manuscript online:
20. Februar 2023

Artikel online veröffentlicht:
19. Juni 2023

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