|
| Status |
Public on Sep 02, 2009 |
| Title |
3D-cultured chondrocytes from OA donor 2 |
| Sample type |
RNA |
| |
|
| Source name |
human subcultured chondrocytes (passage 2) from OA donors maintained for 14 days in scaffold culture
|
| Organism |
Homo sapiens |
| Characteristics |
disease group: osteoarthritis
cell type: chondrocytes
cell culture: 3-D
|
| Treatment protocol |
Human chondrocytes were isolated by enzymatic digestion. Minced cartilage was digested for 16 hours in a spinner bottle in 10 ml of culture medium containing clostridial collagenase (1 mg per milliliter [150 U per liter]) and deoxyribonuclease I (0.1 mg per milliliter [25,000 U per liter]). . For scaffold culture, 2x106 chondrocytes of passage 2 were per cm2 were seeded in Hyaff-11 and were maintained under serum-free chondotions in presence of TGFbeta 1.
|
| Growth protocol |
Human chondrocytes obtained from non-weight bearing area of 3 donors undergoing ACT treatment (normal donor) and from biopsies of 3 donors undergoing total knee replacement (OA donors) were expanded by cultivation in DMEM/F12 supplemented with L-ascorbic acid (0.025 mg/ml, gentamicin sulphate (50 mg/l), amphotericin B (250 µg/ml) and L-glutamine (2 mM; Gibco) and 10% human serum for 2 passages, 3D cultured in Hyaff-11 scaffolds for 14 days in DMEM high glucose supplemented with 5.0 µg/ml linoleic acid, insulin-transferrin-selenium-G, 1.0 mg/ml human serum albumin, 10 ng/ml transforming growth factor beta 1 (TGF-b1), 10-7 M dexamethasone , 14 µg/ml L-ascorbic acid and 1% penicillin-streptomycin
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from chondrocytes cultured in monolayer (passage 2) was isolated applying protocols for animal tissues of the RNeasy Mini Kit (Qiagen, Hilden, Germany). For scaffold cultures, an 8 mm punch was prepared, snap-frozen in liquid nitrogen, and stored at -80°C until further use. Frozen samples were transferred to 1 ml TriReagent (Sigma-Aldrich) and mechanically homogenized. Subsequently, 133 µl 1-Bromo-3-chloro-propane (Sigma-Aldrich) was admixed followed by centrifugation for 45 min at 13000xg. The aqueous phase was collected and nucleic acids were precipitated by addition of an equal volume ice-cold isopropanol. After 30 min incubation the precipitated nucleic acids were pelletized and resolved in 350 µl RLT buffer (Qiagen). Further purification was performed according to protocols for animal tissues of the RNeasy Mini Kit (Qiagen). RNA from each donor was separately handled; no pooling was done at any stage.
|
| Label |
biotin
|
| Label protocol |
2 µg of total RNA was amplified and labeled using the GeneChip® one-cycle target labeling and control reagents (Affymetrix)
|
| |
|
| Hybridization protocol |
50µg/ml of labeled cRNA was hybridized at 45°C for 16h using the Affymetrix GeneChip hybridization oven. Washing and staining was performed on the Affymetrix Fluidics 450 using the EukGE-WS2v5 protocol.
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GeneChip Scanner 3000
|
| Description |
OA_2_Chon_CHyaff_D14_133P
human chondrocytes cultured in DMEM/F12 supplemented with L-ascorbic acid (0.025 mg/ml, gentamicin sulphate (50 mg/l), amphotericin B (250 µg/ml) and L-glutamine (2 mM; Gibco) and 10% human serum for 2 passages, 3D culture in Hyaff-11 scaffolds for 14 days maintained in DMEM high glucose supplemented with 5.0 µg/ml linoleic acid, insulin-transferrin-selenium-G, 1.0 mg/ml human serum albumin, 10 ng/ml transforming growth factor beta 1 (TGF-b1), 10-7 M dexamethasone , 14 µg/ml L-ascorbic acid and 1% penicillin-streptomycin; OA donor group (60-64 years, symptoms of severe OA, undergoing total knee replacement, radiological evidence of OA, OA grade 2-3 according to Ahlbäck, and exhibiting a Mankin score above 3)
human chondrocytes isolated from non-weight bearing area of the knee cartilage of patients undergoing ACT treatment, subcultured up to passage 2 (primary cells = passage 0), seeded onto hyaff-11 scaffold, maintained for 14 days in the presence of TGFbeta 1, chondrocytes obtained from OA donor 2
|
| Data processing |
Data were processed with GCOS 1.4 (TGT = 150) and subsequently imported and further analyzed in the BioRetis online database (www.bioretis.de).
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| |
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| Submission date |
Jun 05, 2009 |
| Last update date |
Sep 02, 2009 |
| Contact name |
Tilo Dehne |
| E-mail(s) |
[email protected]
|
| URL |
http://www.charite.de/te
|
| Organization name |
Charité - University Medicine Berlin
|
| Department |
Rheumatology and Clinical Immunology
|
| Lab |
Tissue Engineering Laboratory
|
| Street address |
Tucholskystraße 2
|
| City |
Berlin |
| State/province |
Berlin |
| ZIP/Postal code |
10117 |
| Country |
Germany |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE16464 |
Chondrogenic differentiation potential of OA chondrocytes and their use in autologous chondrocyte transplantation |
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