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Cultivation and Characterization of Limbal Epithelial Stem Cells on Contact Lenses With a Feeder Layer: Toward the Treatment of Limbal Stem Cell Deficiency

Gore, Ariel PhD; Horwitz, Vered PhD; Gutman, Hila MSc; Tveria, Liat MSc; Cohen, Liat BSc Pharm; Cohen-Jacob, Orit DVM; Turetz, Joseph MD; McNutt, Patrick Michael PhD; Dachir, Shlomit PhD; Kadar, Tamar PhD

doi: 10.1097/ICO.0000000000000002
Basic Investigation

Purpose: Limbal epithelial sheets are used to promote corneal surface reconstruction after the detection of limbal epithelial stem cell deficiency. The aim of this study was to evaluate a novel combination of limbal stem cells (LSCs) maintained on contact lenses (CLs) in the presence of a 3T3 feeder cell layer regarding preservation of stem cell phenotype and the potential use for future in vivo transplantation.

Methods: Limbal epithelial cells were isolated from rabbit cornea and cultured with 3T3 cells on CLs. The preservation of LSC phenotype was determined using p63α and ABCG2 immunostaining, whereas epithelial differentiation was evaluated using CK3 and CK19. The colony-forming assay was used to determine the percentage of LSCs in cultures. Finally, CLs seeded with PKH26-labeled LSCs were transferred to rabbit eyes after performing a surgical keratectomy, and the transition and phenotype of labeled cells on the corneal surface were evaluated in whole-mount corneas.

Results: Proliferation of individual limbal cells was observed on CLs with a 3T3 feeder cell layer, showing holoclone formation and retention of viable stem or progenitor cell phenotype. Finally, a higher transition of cultivated cells after a dual sequential CL transplantation to the ocular surface was observed, showing the preservation of the LSC phenotype in the corneal surface.

Conclusions: Limbal cells cultivated on a CL carrier overlaying a 3T3 feeder layer are mitotically active and retain the LSC phenotype. This novel technique of using CLs as a carrier offers an easily manipulable and nonimmunogenic method for transferring LSCs for ocular surface reconstruction in patients with limbal epithelial stem cell deficiency.

This study characterizes the potential use of contact lenses, with a 3T3 feeder layer, as a carrier of limbal cells for future in vivo transplantation in treating limbal epithelial stem cell deficiency.

*Department of Pharmacology, Israel Institute for Biological Research, Ness Ziona, Israel; and

Cell and Molecular Biology, USAMRICD, Aberdeen Proving Ground, MD.

Reprints: Ariel Gore, Department of Pharmacology, Israel Institute for Biological Research, Ness Ziona 74100, Israel (e-mail:

Part of this work was supported by the Defense Threat Reduction Agency and US Army Medical Research and Material Command under Contract # W81XWH-08-2-0129.

The authors have no funding or conflicts of interest to disclose.

Received May 12, 2013

Accepted September 04, 2013

Copyright © 2014 Wolters Kluwer Health, Inc. All rights reserved.