Measuring the area of expansion using Image J.

<p>Scale is set initially using the micron bar on the collaged picture. Here, 1μm = 0.415 pixels. White dotted line indicates the area of the explant and black dotted line indicates the outgrowth of cells from explant. AM—Amniotic membrane as scaffold, Ex—Expansion of cells, E—Explant of the limbus. Cell expansion from limbal explant without dotted lines is shown in the inset image. Scale—40X.</p

Immunofluorescence.

<p>Respective live and cadaveric cultures expressing corneal epithelial cell marker cytokeratin 3+12 <b>(A&B)</b>, limbal stem cell markers ABCG2 <b>(C&D)</b>, p63α <b>(E&F)</b> and epithelial tight junction marker E-Cadherin <b>(G&H)</b>. White arrow heads represents the edge of expansion. Ex- Explant; White dotted lines in panel (A) represents the explant border.</p

Optimizing the role of limbal explant size and source in determining the outcomes of limbal transplantation: An <i>in vitro</i> study

<div><p>Purpose</p><p>Simple 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 <i>in vitro</i> study aimed to determine the optimal limbal explant size required for complete corneal epithelialization by characterizing the cell expansion.</p><p>Methods</p><p>Limbal 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.</p><p>Results</p><p>Explants from live biopsies had 80% growth potential <i>in vitro</i> 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.</p><p>Conclusion</p><p>Our findings show that a minimal amount of 0.3 mm² live tissue would be sufficient for ample limbal cell expansion <i>in vitro</i>. 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.</p></div

Rate of cell proliferation.

<p><b>A)</b> Comparative analysis of mean cell proliferation for live and cadaveric cultures both at center and peripheral positions at different time points. <b>B)</b> Total mean percentage of proliferative cells in live and cadaveric cultures. Cadaveric cell proliferation is significantly greater on day 5 at the culture periphery (p = 0.013) in addition to the total proliferation rate (p = 0.018). Statistical test used is ‘Two sample <i>t</i>-test’ with variability of data represented by standard error (SE).</p

Growth potential of limbal explants.

<p>Mean area of live limbal tissue cell expansion with time for live <b>(A)</b> and cadaveric <b>(B)</b> tissues. Exponential cell expansion can be seen in explants of different size ranges. <b>C)</b> Mean area of cell expansion obtained from a single explant of different sizes in live and cadaveric cases. Data shows that a single explant placed on the amniotic membrane has the ability to expand its cells to an area equal to that of anterior corneal surface area (i.e., 132 mm²) in a period of 6.5 to 7.5 days. <b>D)</b> Expansion potential of cadaveric limbal explants is depicted to be equal to that of live limbal explants. Statistical test used is ‘Linear mixed-effects model fit by maximum likelihood’ with variability of data represented by standard error (SE).</p

Day wise cell expansion (outgrowth) of a single limbal explant cultured using amniotic membrane as scaffold.

<p>Increase in the area of the expansion can be observed with respect to days.</p

Mean area of expansions with respect to the explant sizes.

<p>Mean area of expansions with respect to the explant sizes.</p

Limbal cell expansion from explant <i>in vitro</i>.

<p><b>A)</b> Limbal explant culture at day 2 showing round clusters of cells around explant edge. Inset with white arrows showing the same at higher magnification; <b>B)</b> Culture at day 3—Disappearance of round cell clusters and expansion of polygonal shaped cells as a sheet. <b>C & D)</b> Streak like appearance at the periphery of the outgrowth and the presence of fibroblast shaped stromal cells indicated by black arrows.</p

BrdU cell proliferation assay for limbal culture from cadaveric tissue.

<p>Cells of the explant expansion showing BrdU (Red) staining denoting cell proliferation compared to the total number of cells represented by nuclear counterstain DAPI (Blue). Yellow dotted lines indicate edge of the explant and white solid lines indicate the periphery of the cell expansion. Explants in left hand pane appear as red patches due to staining of BrdU indicating cell proliferation inside the tissue.</p

Percentage of proliferating cells in limbal cultures.

<p>Percentage of proliferating cells in limbal cultures.</p