pathology: Normal tissue: Amniotic membrane-expanded limbal epithelium cultured: yes
Treatment protocol
Samples were stored at −70°C/–80°C prior to RNA isolation
Growth protocol
AMLE cultures were initiated using donor cadaveric limbal tissue within ten days of biologic death. Limbal rings were dissected and limbal explants (2×2×0.25mm) cultured on fresh frozen human amniotic membrane (Transplant Service Foundation, Spain; Code-30102-619). To prepare the amniotic membrane for limbal explant culture; cryo-preserved amniotic membrane was thawed, freezing-medium aspirated, nitrocellulose paper removed and membranes washed in PBS and incubated with trypsin/EDTA (20 min., 37°C). Cell scrapers (Costar-Corning, Corning, NY) were used to remove amniotic epithelium and mucus from the membrane. The amniotic membrane was held taut by wrapping around a sterile glass slide and limbal explants placed on the de-epithelialised surface. Explants were cultured in supplemented epithelial growth medium; 3:1 DMEM/HAM F12 (Sigma Aldrich, St. Louis, MO), 10% FCS (Invitrogen-Gibco, Grand Island, NY), 5μg /ml insulin, 10ng/ml rhEGF, 100ng/ml cholera toxin, 0.4μg /ml hydrocortisone,1E-9M triiodothyronine (Sigma Aldrich, St. Louis, MO). All cultures were incubated at 37 °C, 5% CO2 with media aspiration every 2-3 days until confluent. CE samples were isolated from donor cadaveric corneal buttons within ten days of biologic death. Upon receipt, the blunt edge of a scalpel blade (Swann-Morton, Sheffield, UK) was used to separate corneal epithelial tissue. Separation of the corneal epithelial sheet was confirmed using a Nikon Eclipse TS100 microscope (Nikon, Melville, NY). LFC samples were initiated using donor cadaveric limbal tissue within ten days of biologic death. Limbal rings were dissected and limbal explants (0.5×0.5×0.25mm) cultured on tissue culture-treated plastic (Costar-Corning, Corning, NY) using DMEM (Sigma Aldrich, St. Louis, MO) supplemented with 10% FCS (Invitrogen-Gibco, Grand Island, NY). All cultures were incubated at 37 °C, 5% CO2 with media aspiration every 2-3 days until confluent.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from cell samples using Qiagen RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. RNA concentration was verified using a Nanodrop spectrophotometer (ND-1000, Labtech International, UK), and RNA integrity verified using a 2100 Bioanalyser (Agilent, CA, USA).
5.5 μg of fragmented ssDNA was processed according to the manufacturers' instructions using the GeneChip® WT Terminal Labeling and Hybridization kit (P/N 702808). 100µl of hybridisation coctail was added to the arrays and they were hybridised for 16 hours at 45°C and 60 rpm. Arrays were subsequently washed (Affymetrix Fluidics Station 450) and stained with streptavidin-phycoerythrin.
Scan protocol
Arrays were scanned on an Affymetrix GCS GeneChip GeneArray scanner. Resulting data was analysed using GCOS, dCHIP, and GeneSpring (Agilent Technologies).
Description
AMLE_2 cultures demonstrated stratified corneal epithelial cell morphology with small, tightly packed, cobble-shaped cells on the basal layer and larger squamous cells on apical layers. AMLE_2 tissues were 2-3 cell layers thick.
Data processing
All microarray data were called using the robust multichip average method (RMA)
