Supplemented media used in the culture of human corneal endothelial cells.

<p>Supplemented media used in the culture of human corneal endothelial cells.</p

Expression of cultured P3 hCECs.

<p>Representative sets of photomicrographs showing expression of Na⁺K⁺/ATPase and ZO-1 by immunocytochemistry: Immunostaining of Na⁺K⁺/ATPase in <b>A</b>: M2 and <b>B</b>: M4. Immunostaining of ZO-1 in <b>C</b>: M2 and <b>D</b>: M4. Control staining <b>E</b>: Isotype matched IgG₁ negative control. (<i>n</i> = 6; Scale bars = 50 µm).</p

Schematic diagram depicting processes involved in the isolation and propagation of hCECs.

<p><b>A</b>: All research-grade corneas used in this study were procured from Lions Eye Institute for Transplant and Research Inc. (Tampa, FL). Research corneas were preserved and transported in Optisol-GS, and were used within 10 days from preservation. <b>B</b>: Once received, corneas were washed thrice in an antibiotic, antimycotic wash solution. The DM-CE was peeled and the hCECs were isolated and plated into passage 0 cultures within a day. <b>C</b>: Isolated hCECs were seeded and propagated in the 4 culture conditions for up to 4 weeks. <b>D</b> and <b>E</b>: Confluent cells at each time point were trypsinized using TrypLE Express and seeded at a matched density of 5,000 cells/cm².</p

Donor information.

<p>COD: cause of death. Donor age ranged from 10 year-old to 42 year old with a median age of 26 year old. Days taken from death of donor to the initiation of corneal endothelial cell culture ranged from 3 days to 13 days with a median of 8 days. Experiment A: morphological assessment/growth profile - P0 to P1; Experiment B: morphological assessment/growth profile - P1 onwards; Experiment C: Cell adherence analysis – xCelligence; Experiment D: Cell proliferation – Click-iT EdU; Experiment E: Immunofluorescence staining.</p

Morphology of cultured hCECs P3 to P5.

<p>Representative sets of photomicrographs showing morphology of hCECs at passage 3, passage 4 and passage 5 cultured in M2 and M4. (<i>n</i> = 6; Scale bars = 100 µm).</p

Morphology of cultured hCECs P0 to P1.

<p>Representative sets of photomicrographs showing morphology of hCECs at passage 0 and passage 1 cultured in the 4 culture conditions over various time points. <b>A</b> to <b>D</b>: S/N07 passage 0 day 1 after attachment (adaptive phase). <b>E</b> to <b>H</b>: S/N10 passage 0 week 4 at the end of the proliferative phase before passaging. <b>I</b> to <b>L</b>: S/N09, <b>M</b> to <b>P</b>: S/N01, and <b>Q</b> to <b>T</b>: S/N09 are passage 1 week 2 cultures derived from three pairs of donor corneas.</p

Expansion profile of hCECs in the four culture media.

<p>For passage 0–1 (<i>n</i> = 6), significance for non-parametric Kruskal-Wallis test with pair wise comparisons corrected for multiple comparison using Mann-Whitney U test was achieved between M1 & M2 (^*<i>p</i><0.01); M1 & M4 (^†<i>p</i><0.01); M2 & M3 (^‡<i>p</i><0.01); M3 & M4 (^§<i>p</i><0.01); For passage 1–2 (<i>n</i> = 4), significance was achieved between M1 & M2 (^*<i>p</i><0.05); M1 & M4 (^†<i>p</i><0.05); M2 & M3 (^‡<i>p</i><0.05); M3 & M4 (^§<i>p</i><0.05); For passage 2–3 (<i>n</i> = 4), significance was achieved between M2 & M3 (^*<i>p</i><0.05); and M3 & M4 (^†<i>p</i><0.05).</p

Expression ofα-smooth muscle actin (α-SMA) in the post-operative central corneas and peripheral flaps.

<p>(A–D) α-SMA (green), a marker of myofibroblasts, was not present in the central corneas on week 8 and 16 after both ReLEx and refractive lenticule re-implantation. (E–H) α-SMA (green) was expressed at the flap periphery and co-localized with F-actin (red) subepithelially on week 8 post-ReLEx and lenticule re-implantation, but was absent 16 weeks after both surgical procedures. In pane A–H, α-SMA (green) was double immunostained with F-actin marker (red), phalloidin. Nuclei were counterstained using DAPI (blue). Arrowheads indicate the location of the laser incision site or lenticular interface. PR: post-ReLEx, PLR: post-lenticule re-implantation. Scale bar: 50 µm.</p

Mean corneal curvature measured by keratometer and mean spherical error measured by refractometer (n = 7).

a<p>PR = post-ReLEx.</p>b<p>PLR = post-lenticule re-implantation.</p>c<p>D = diopter.</p>d<p><i>p</i> values relative to the keratometry before ReLEx.</p>e<p><i>p</i> values relative to the spherical error before ReLEx.</p

Atomic force microscope (AFM) images showing surface topography of native DM (DM; A–D) and DM with fibrin glue (FG) facing up (E–H).

<p>A and E: Height data; B and F: Amplitude data; C and G: Phase data. D and H represent a 3D presentation of topographical map of native DM and DM+FG, respectively. Image scale = 10 µm×10 µm.</p