Abstract
PurposeSimple limbal epithelial transplantation (SLET) and cultivated limbal epithelial transplantation (CLET) are proven clinical techniques for treating limbal stem cell deficiency (LSCD). However, the ideal size and number of the limbal explants required for transplantation has not been clearly elucidated. This in vitro study aimed to determine the optimal limbal explant size required for complete corneal epithelialization by characterizing the cell expansion.MethodsLimbal explants obtained from both live and cadaveric biopsies were cultured on the denuded amniotic membrane. Explant size and the explant cell outgrowth (expansion) were measured using ImageJ software with respect to days. Cultures were characterized by assessing the rate of proliferation of cells with 5-bromo-2’-deoxyuridine (BrdU) assay along with the expression of different stem cell markers (ABCG2, p63α), corneal epithelial (CK3+12) and adherens junction molecules (E-Cadherin) by immunofluorescence.ResultsExplants from live biopsies had 80% growth potential in vitro whereas 40% of the cadaveric tissue failed to grow. Minimum explant sizes of 0.3 mm2 for live and ≥0.5 mm2 for cadaveric tissue had a mean expansion areas of 182.39±17.06 mm2 and 217.59±16.91 mm2 respectively suggesting adequate growth potential of the explants. Mean total percentage of proliferative cells was 31.80±3.81 in live and 33.49±4.25 in cadaveric tissue expansion. The expression was noted to be similar in cells cultured from cadaveric compared to cells cultured from live limbal tissue with respect to ABCG2, p63α, CK(3+12) and E-cadherin.ConclusionOur findings show that a minimal amount of 0.3 mm2 live tissue would be sufficient for ample limbal cell expansion in vitro. Cadaveric explants <0.5 mm2 had poor growth potential. However, larger explants (≥ 0.5 mm2) had growth rate and proliferative potential similar to the live tissue. These findings could prove to be critical for clinical success especially while attempting cadaveric limbal transplantation. This study provides a novel clinical strategy for enhancing efficacy of the limbal transplantation surgery and opens the probability of even using the cadaveric tissue by considering the size of explant.
Highlights
Epithelium of the cornea undergoes continuous regeneration and renewal through cell proliferation and migration [1, 2]
Our findings show that a minimal amount of 0.3 mm2 live tissue would be sufficient for ample limbal cell expansion in vitro
Larger explants (! 0.5 mm2) had growth rate and proliferative potential similar to the live tissue. These findings could prove to be critical for clinical success especially while attempting cadaveric limbal transplantation
Summary
Epithelium of the cornea undergoes continuous regeneration and renewal through cell proliferation and migration [1, 2] The source of these cells is usually from the periphery of the cornea [3] where a specialized region known as ‘limbus’ harbors stem cells [4, 5] and their niche [6]. Limbal transplantation has historically been successful in most of the LSCD cases [8, 9] but, it had encountered failures in cases of patients who underwent penetrating keratoplasty previously or suffering from persistent dry eye disease [10] Cell based therapies such as cultivated limbal epithelial transplantation (CLET) [11, 12] and simple limbal epithelial transplantation (SLET)) [13, 14] utilizes limbal biopsy from the healthy eye of the donor from which the progenitor cells originate, facilitating wound healing. Reconstruction of the healthy limbal niche requires a good source of limbal cells and the sufficient size of an explant that supports efficient growth in optimal time after the surgery for better successful outcomes
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