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| Status |
Public on Oct 08, 2021 |
| Title |
EyeBank_cultured_hCECs_monoculture_interval_post-mortem_17days_008_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Primary human corneal epithelial cells
|
| Organism |
Homo sapiens |
| Characteristics |
post-mortem interval: 17 days
Sex: F
age: 67
cell type: Primary human corneal epithelial cells
|
| Treatment protocol |
The two-layers hTECs were produced following the self-assembly approach. Briefly, corneal fibroblasts were cultured in the presence of ascorbic acid (50 μg/ml) for 35 days to promote the production of their own extracellular matrix (ECM). Two fibroblasts sheets were then superimposed to form a reconstructed stroma, on which hCECs were seeded. Reconstructed tissues were cultured for 7 days under submerged conditions in complete DHc supplemented with ascorbate and then transferred to the air-liquid interface for 7 days in EGF-free DHc to induce epithelial differentiation.
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| Growth protocol |
hCECs were isolated from the limbal area of the post-mortem corneas either directly upon reception of the corneal tissue (PMI 0) or at different post-mortem intervals (PMI), ranging from 1 to 19 . For both transportation and storage, corneas were maintained in Optisol-GS in corneal viewing chambers at 4°C. Once isolated, the ten individual hCEC populations (PMI 0, 1, 2, 3, 4, 14, 15, 17, 18, and 19 ) were seeded in tissue culture flasks along with a lethally irradiated (6000 rad) human fibroblasts feeder layer and cultured in Dulbecco–Vogt modification of Eagle's medium with Ham's F12 in a 3:1 ratio (DME-HAM) supplemented with 5% Fetal Clone II serum, 5 μg/ml of insulin, 0.4 μg/ml of hydrocortisone, 10 ng/ml of epidermal growth factor, 0.212 mg/ml of isoproterenol hydrochloride, 100 IU/ml of penicillin and 25 μg/ml of gentamycin. Cells were maintained in 37°C and 8% CO2 incubators until they reached near-confluence and culture medium was changed after 2 days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using the RNeasy Mini Kit (QIAGEN, Cat#74104)
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 200 ng RNA (hCECs monoculture) or 100 ng RNA (hCECs from hTECs) using the One-Color LowInput QuickAmp Labeling Kit One-Color (Agilent) according to the manufacturer's instructions
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| |
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| Hybridization protocol |
600 ng of Cy3-labelled cRNA (specific activity >8.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole SurePrint G3 Human GE 8x60K (G4851A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately.
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| Scan protocol |
Slides were scanned immediately after washing on the Agilent SureScanner (G4900DA) using one color scan setting for 1x60k array slides (Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%).
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| Description |
Gene expression of passage 1 hCECs cultivated in monoculture until they reached near-confluence with post-mortem interval of 17days
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| Data processing |
The scanned images were analyzed with Feature Extraction Software 10,7 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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| |
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| Submission date |
Oct 06, 2021 |
| Last update date |
Oct 08, 2021 |
| Contact name |
Gaëtan Le-Bel |
| E-mail(s) |
gaetan.lebel17@gmail.com
|
| Organization name |
CUO-Recherche
|
| Lab |
Sylvain Guérin
|
| Street address |
Local H2-00, Hôpital du Saint-Sacrement,1050 Chemin Sainte-Foy
|
| City |
Québec |
| State/province |
Quebec |
| ZIP/Postal code |
G1S 4L8 |
| Country |
Canada |
| |
|
| Platform ID |
GPL13607 |
| Series (1) |
| GSE185459 |
Gene expression in human corneal epithelial cells (hCECs) with different post-mortem intervals and cultivated in monoculture or used for human tissue‐engineered corneas (hTECs) |
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