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

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

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

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

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

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

Immunochemical analysis of primary hCECs on RAFT.

<p>Primary hCECs seeded on glass slides and fixed after 4 days, stained with (A) ZO-1 or (B) Na⁺/K⁺-ATPase (green) counterstained with DAPI (blue). Primary hCECs seeded onto RAFT stained with either (C and E) ZO-1, (D and F) Na⁺/K⁺-ATPase (green) counterstained with DAPI (blue). (C and D) fixed after 4 days in culture or (E and F) after 14 days. Scale bars 50 µm.</p

Immunochemical analysis of hCECL cells on RAFT.

<p>hCECL seeded on RAFT (A) at 3000 cells/mm² with C/L coating or (B) 3000 cells/mm² on FNC coating stained with ZO-1 (green) and counterstained with propidium iodide (red). hCECL seeded on RAFT (C) at 2000 cells/mm² with C/L coating or (D) seeded at 4000 cells/mm² on FNC coating stained with Na⁺/K⁺-ATPase (green) and counterstained with propidium iodide. (E) Negative isotype control. (F) hCECL on permanox slides with FNC coating stained with Na⁺/K⁺-ATPase (green) and counterstained with propidium iodide. Scale bars 50 µm.</p

Scanning electron microscope characterisation of RAFT with hCECs.

<p>(A) Representative micrograph of the surface of RAFT constructs. (B) Representative low magnification SEM image showing confluent monolayer of hCECs on RAFT. (C) Higher magnification image showing cell borders between cells and (D) at high magnification over-lapping finger-like projections onto juxtaposed cells. Scale bars A 5 µm, B 50 µm, C 10 µm, D 2 µm.</p