Table 1.
Primer sequences used for real-time PCR.
Fig 1.
Comparison of the intact and denuded HAMs and HAM cryosections.
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.
Fig 2.
Comparison of the DNA concentrations.
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 (***P < 0.001).
Fig 3.
Topography of intact and denuded HAM.
Scanning electron micrographs (A, D, G, J) and stereo anaglyphs (B, C, E, F, H, I, K, L) of the intact (A, B, C) and denuded HAM by TrypLE Express (D, E, F), trypsin/EDTA (G, H, I) and thermolysin (J, K, L). Areas of damaged BM are marked by arrows, ruptured gaps by *, the residues of ECM by Δ. Red-green or red-cyan glasses required for proper view of stereo anaglyphs.
Fig 4.
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.
Fig 5.
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 (***P < 0.001).
Fig 6.
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 1st, 2nd, 3rd, 4th and 5th passage). Results of one out of 3 identical experiment is shown. Scale bars represent 100 μm.
Fig 7.
The metabolic activity of hAECs.
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.
Fig 8.
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.