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Klin Monbl Augenheilkd 2024; 241(08): 972-981
DOI: 10.1055/a-2038-8899
Experimentelle Studie

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

Untersuchung der photodynamischen Bengalrosa-Therapie auf die Viabilität und Proliferation humaner keratolimbaler Epithel- und Stromazellen in vitro
Ning Chai 
1   Dr. Rolf M. Schwiete Center for Limbal Stem Cell and Aniridia Research, Saarland University, Homburg/Saar, Germany
,
Tanja Stachon
1   Dr. Rolf M. Schwiete Center for Limbal Stem Cell and Aniridia Research, Saarland University, Homburg/Saar, Germany
,
Mahsa Nastaranpour
1   Dr. Rolf M. Schwiete Center for Limbal Stem Cell and Aniridia Research, Saarland University, Homburg/Saar, Germany
,
Zhen Li 
1   Dr. Rolf M. Schwiete Center for Limbal Stem Cell and Aniridia Research, Saarland University, Homburg/Saar, Germany
,
Berthold Seitz 
2   Department of Ophthalmology, Saarland University Hospital and Saarland University, Faculty of Medicine, Homburg/Saar, Germany
,
Myriam Ulrich
1   Dr. Rolf M. Schwiete Center for Limbal Stem Cell and Aniridia Research, Saarland University, Homburg/Saar, Germany
,
Achim Langenbucher
3   Institute of Experimental Ophthalmology, Saarland University, Homburg/Saar, Germany
,
Nóra Szentmáry
1   Dr. Rolf M. Schwiete Center for Limbal Stem Cell and Aniridia Research, Saarland University, Homburg/Saar, Germany
4   Department of Ophthalmology, Semmelweis University, Budapest, Hungary
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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

Supporting Information



Publication History

Received: 19 December 2022

Accepted: 16 February 2023

Accepted Manuscript online:
20 February 2023

Article published online:
19 June 2023

© 2023. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

 

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