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Optimized Protocol for Regeneration of the Conjunctival Epithelium Using the Cell Suspension Technique

Bertolin, Marina, MSc*; Breda, Claudia, BSc*; Ferrari, Stefano, PhD*; Van Acker, Sara Ilse, MSc; Zakaria, Nadia, MD†,‡; Di Iorio, Enzo, PhD*,§; Migliorati, Angelo, PhD§; Ponzin, Diego, MD*; Ferrari, Barbara, MSc*; Lužnik, Zala, MD; Barbaro, Vanessa, PhD*

doi: 10.1097/ICO.0000000000001670
Basic Investigation

Purpose: To develop autologous tissue-engineered conjunctival epithelial sheets to be used as advanced therapy medicinal products for severe ocular surface disorders involving the conjunctiva.

Methods: Methods used aimed at 1) mapping the conjunctiva for identification of the stem cell location, 2) establishing proper cell culturing conditions, 3) identifying the proper scaffold, and 4) characterizing the conjunctival grafts better. For these purposes, immunostaining and PAS staining, serial cultivation of cells, and quantitative polymerase chain reaction ([INCREMENT]Np63α and MUC5AC) were performed.

Results: The inferior fornix represents the ideal area where to take the conjunctival biopsies from, with at least +3.58% of clonogenic colonies and higher percentages of stem cells compared with other areas, as confirmed by [INCREMENT]Np63α expression levels (6.79% ± 1.18%). The standard culture conditions are necessary when cells are cultured on bare plastic, while animal-free media can be used for conjunctival cell culture on the scaffold. Fibrin glue represents the ideal scaffold for production of epithelial conjunctival grafts because it allows physiological expression of the main conjunctival cell markers, with K19 as the ideal one (98.5% ± 0.5% positive cells). The presence of goblet cells (6.3% ± 1.3%) and expression of the stem cell marker [INCREMENT]Np63α (1.65% ± 0.35% positive cells) were also assessed.

Conclusions: Our findings pave the way for ex vivo cultivation of conjunctival epithelial cells onto a scaffold using the cell suspension technique by means of animal-free media. This would allow us to obtain conjunctival grafts for clinical purposes, thus giving a therapeutic option to patients with conjunctival diseases refractory to current therapies.

*Fondazione Banca degli Occhi del Veneto, Venice, Italy;

Department of Ophthalmology, Visual Optics and Visual Rehabilitation, University of Antwerp, Wilrijk, Belgium;

Department of Ophthalmology, Antwerp University Hospital, Edegem, Belgium;

§Department of Molecular Medicine, University of Padua, Padua, Italy; and

Eye Hospital, University Medical Centre, Ljubljana, Slovenia.

Correspondence: Marina Bertolin, MSc, Fondazione Banca degli Occhi del Veneto, c/o Padiglione G. Rama—Via Paccagnella 11, 30174 Zelarino (Venice), Italy (e-mail:

This study was partly supported by the 5 x 1000 funds (2015) from the Italian Ministry of Health and the Italian Ministry of University and Research (MIUR). S.I.V.A. holds a PhD fellowship from the Research Foundation Flanders (FWO, grant number 1196418N) and received an EU-COST scholarship to perform a Short-Term Scientific Mission to the Veneto Eye Bank Foundation in Italy.

M. Bertolin and C. Breda contributed equally to this study.

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Received March 14, 2018

Received in revised form May 09, 2018

Accepted May 10, 2018

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