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In vitro establishment, validation and characterisation of conjunctival epithelium outgrowth using tissue fragments and amniotic membrane
  1. Marina Bertolin1,
  2. Vanessa Barbaro1,
  3. Claudia Breda1,
  4. Stefano Ferrari1,
  5. Giorgio Marchini2,
  6. Emilio Pedrotti2,
  7. Barbara Ferrari1,
  8. Ponzin Diego1,
  9. Adriano Fasolo1,2
  1. 1Research Unit, Fondazione Banca degli Occhi del Veneto, Mestre, Veneto, Italy
  2. 2Eye Clinic, Department of Neurological and Movement Sciences, Università degli Studi di Verona, Verona, Veneto, Italy
  1. Correspondence to Dr Marina Bertolin, Fondazione Banca degli Occhi del Veneto, Mestre, Veneto, Italy; marina.bertolin{at}


Background/Aims To set up the in vitro conditions for renewal of the conjunctival epithelium using healthy fragments of conjunctival tissue glued over an amniotic membrane.

Methods We evaluated the capability of conjunctival tissue fragments to generate conjunctival cell outgrowth after seeding them onto amniotic membrane and culture plates; we then assessed conjunctival molecular marker expression by immunofluorescence. We also evaluated the efficiency of glueing the fragments over the amniotic membrane to determine the best setting and the feasibility of shipping preloaded amniotic membranes.

Results Epithelial outgrowth was detected in 65%–80% of conjunctival fragments starting 48–72 hours after glueing, without major differences between type of membrane preparation and fragment size. Within 6–13 days, a full epithelium covered the surface of the amniotic membrane. Specific marker expression (conjunctival epithelium, Muc1, K19, K13; stemness, p63; tight junctions, ZO-1) was detected. Results of the shipping test showed that only 31% of the fragments were still glued over the epithelial side of the membrane within 24 hours compared to more than 90% of fragments stayed attached in the remaining conditions.

Conclusion The in vitro regeneration of conjunctival epithelium following outgrowth from conjunctival tissue fragments glued over an amniotic membrane may offer a viable strategy to renew the epithelium in vivo once applied over the ocular surface at the recipient site.

  • conjunctiva
  • ocular surface
  • vision

Data availability statement

All data relevant to the study are included in the article.

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Data availability statement

All data relevant to the study are included in the article.

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  • Correction notice The paper has been updated since it was published online. The results section in the abstract has been updated.

  • Contributors MB, CB and BF performed the experiments and collected the data. AF, SF, GM, EP, PD and VB contributed to the revision and expert opinion.

  • Funding The Italian Ministry of Health and the Italian Ministry of University and Research 5×1000 funds partially supported this study.

  • Competing interests None declared.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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