Cellular and Molecular Biology


In vivo confocal microscopy assessment of the corneoscleral limbal stem cell niche before and after biopsy for cultivated limbal epithelial transplantation to restore corneal epithelium

Beatriz E. Ramírez1, Darío A. Victoria1, Giovana M. Murillo1, José M. Herreras1,2 and Margarita Calonge1,2

1Cell Therapy Unit, IOBA (Institute of Applied Ophthalmobiology), University of Valladolid, Valladolid, Spain and 2CIBER-BBN (Networking Research Center on Bioengineering, Biomaterials and Nanomedicine), Carlos III National Institute of Health, Spain

Offprint requests to: Margarita Calonge, MD, IOBA, Campus Miguel Delibes, University of Valladolid, Paseo Belen 17, E-47011 Valladolid, Spain. e-mail: calonge@ioba.med.uva.es or beatrize_oft@hotmail.com

Summary. Autologous cultivated limbal epithelial transplantation (CLET) is a successful therapy to restore corneal epithelium when limbal epithelial stem cells are damaged unilaterally, which can result in corneal blindness. We used in vivo confocal microscopy (IVCM) to identify the best location in the corneoscleral limbal niche and to harvest autologous epithelial stem cells for CLET. We also ascertained the completeness of limbal structure removal after biopsy and followed the healing process for any evidence of limbal structure reappearance. The 4 meridians of the corneoescleral limbus of 5 healthy donor eyes were scanned clinically and by IVCM before biopsy and 1 week, 1, 3, and 6 months after. IVCM detected palisades of Vogt, the limbal niche hallmark, more efficiently (100%) than clinically (60%), and were consistently better defined at the 12 o’clock meridian, and so this was the site selected for biopsy. The depth of palisades was 80.4±19.8 µm, and of the limbal biopsies was 136.8±19.1 µm, thus assuring that the limbal niche was completely harvested in all cases. Re-epithelialization of the donor site was complete at 1 week. The limbal wound was refilled with fibrovascular tissue, and no limbal-like structures reappeared. The study shows that clinical absence of palisades of Vogt is not necessarily an exclusionary criterion for autologous CLET. IVCM was useful to select the best place for limbal biopsy and identified features not visible clinically. IVCM also confirmed complete removal of limbal tissue by the biopsy. Limbal niche structures did not reappear by 6 months after surgery. Histol Histopathol 30, 183-192 (2015)

Key words: Palisades of Vogt, Limbal biopsy, In vivo confocal microscopy

DOI: 10.14670/HH-30.183