The RT-PCR analysis of hAECs.

<p>The RT-PCR analysis of hAECs after de-epithelialization and each passage (P0-P5). The iPS cells were used as a positive (iPS) and corneal fibroblasts as negative control (CF). Sample without cDNA (NTC) was used as non-template control. One representative experiment of 3 (with identical results) is shown.</p

Primer sequences used for real-time PCR.

<p>Primer sequences used for real-time PCR.</p

Comparison of the DNA concentrations.

<p>Comparison of the DNA concentration in the tissues form the intact (Control) and denuded HAMs with TrypLE Express (TrypLE), trypsin/EDTA and thermolysin treatment directly after de-epithelialization. Each bar represents mean ± SD from 3 determinations (***<i>P</i> < 0.001).</p

The viability of hAECs.

<p>Comparison of the hAECs viability after TrypLE Express, trypsin/EDTA and thermolysin treatment. Cells were stained with trypan blue and counted via hemocytometer. Each bar represents mean ± SD from 15 determinations (***<i>P</i> < 0.001).</p

Immunostaining of BM.

<p>Distribution of BM collagen type IV α2 chain (green; A) or laminin α5 (green; B) in intact (Control) and denuded HAM: TrypLE Express, trypsin/EDTA, thermolysin treatment. Intact HAM (primary antibody omitted), was used as negative control. Cell nuclei were stained with the propidium iodide (red). Scale bar represents 100 μm.</p

The morphology of hAECs.

<p>The comparison of morphology of cultured hAECs after trypsin/EDTA treatment in complete DMEM medium. The cells for the light microscopy were photographed before each passage (after de-epithelialization, before 1^st, 2^nd, 3^rd, 4^th and 5^th passage). Results of one out of 3 identical experiment is shown. Scale bars represent 100 μm.</p

The metabolic activity of hAECs.

<p>Comparison of metabolic activity of the epithelial cells unstimulated (Uns) and stimulated with EGF (EGF) after each passage. WST-1 reagent was added to the cell cultures for 4 h to form formazan. The absorbance was measured at a wave-length of 450 nm. Each bar represents mean ± SD from 3 determinations.</p

Comparison of the intact and denuded HAMs and HAM cryosections.

<p>Comparison of the intact (Control) and denuded HAMs (A) and HAM cryosections (B) after TrypLE Express, trypsin/EDTA and thermolysin treatment stained with H/E for light microscopy. Scale bar represents 100 μm.</p