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PLoS One. 2019 Mar 12;14(3):e0212524. doi: 10.1371/journal.pone.0212524. eCollection 2019.

Effects of explant size on epithelial outgrowth, thickness, stratification, ultrastructure and phenotype of cultured limbal epithelial cells.

Utheim OA1,2,3, Pasovic L1, Raeder S3, Eidet JR1,4, Fostad IG1, Sehic A5,6, Roald B2,7, de la Paz MF8, Lyberg T1, Dartt DA9, Utheim TP1,3,4,5,6,10,11,12,13,14,15.

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Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway.
Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway.
Norwegian Dry Eye Clinic, Oslo, Norway.
Department of Ophthalmology, Oslo University Hospital, Oslo, Norway.
Department of Oral Biology, Faculty of Dentistry, University of Oslo, Oslo, Norway.
Department of Maxillofacial surgery, Oslo University Hospital, Oslo, Norway.
Department of Pathology, Oslo University Hospital, Oslo, Norway.
Institut Universitari Barraquer, Universitat Autonoma de Barcelona, Barcelona, Spain.
Schepens Eye Research Institute/Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, MA, United States of America.
Department of Plastic and Reconstructive Surgery, Oslo University Hospital, Oslo, Norway.
Department of Ophthalmology, Stavanger University Hospital, Stavanger, Norway.
Department of Clinical Medicine, Faculty of Medicine, University of Bergen, Bergen, Norway.
Department of Ophthalmology, Soerlandet Hospital Arendal, Arendal, Norway.
Department of Ophthalmology, Drammen Hospital, Vestre Viken Hospital Trust, Drammen, Norway.
National Centre for Optics, Vision and Eye Care, Faculty of Health and Social Sciences, University of Southeast Norway, Kongsberg, Norway.



Transplantation of limbal stem cells is a promising therapy for limbal stem cell deficiency. Limbal cells can be harvested from either a healthy part of the patient's eye or the eye of a donor. Small explants are less likely to inflict injury to the donor site. We investigated the effects of limbal explant size on multiple characteristics known to be important for transplant function.


Human limbal epithelial cells were expanded from large versus small explants (3 versus 1 mm of the corneal circumference) for 3 weeks and characterized by light microscopy, immunohistochemistry, and transmission electron microscopy. Epithelial thickness, stratification, outgrowth, ultrastructure and phenotype were assessed.


Epithelial thickness and stratification were similar between the groups. Outgrowth size correlated positively with explant size (r = 0.37; P = 0.01), whereas fold growth correlated negatively with explant size (r = -0.55; P < 0.0001). Percentage of cells expressing the limbal epithelial cell marker K19 was higher in cells derived from large explants (99.1±1.2%) compared to cells derived from small explants (93.2±13.6%, P = 0.024). The percentage of cells expressing ABCG2, integrin β1, p63, and p63α that are markers suggestive of an immature phenotype; Keratin 3, Connexin 43, and E-Cadherin that are markers of differentiation; and Ki67 and PCNA that indicate cell proliferation were equal in both groups. Desmosome and hemidesmosome densities were equal between the groups.


For donor- and culture conditions used in the present study, large explants are preferable to small in terms of outgrowth area. As regards limbal epithelial cell thickness, stratification, mechanical strength, and the attainment of a predominantly immature phenotype, both large and small explants are sufficient.

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