Orbital adipose/connective tissue explants were obtained from seven GO individuals undergoing surgical decompression for severe proptosis associated with increased orbital fat volume, and tissue from seven control individuals with no history of GO or autoimmune thyroid disease was obtained in the course of orbital surgery for other noninflammatory problems (Table 1 ). The GO patients were not on steroid medication for at least 3 months before surgery and were euthyroid at the time of surgery. The orbital adipose tissue volumes were seriously enlarged in all GO patients. However, the clinical activity score at the time of harvest was below four in all patients (i.e., all the GO patients were not in an active inflammatory disease state). Orbital decompression surgery is usually not performed in the active disease, as surgery itself can aggravate inflammation and proptosis can recur postoperatively. None of the patients had been previously treated with orbital radiotherapy. The protocol for obtaining orbital adipose/connective tissue was approved by the Institutional Review Board of Severance Hospital, and written informed consent was obtained from all patients.
Tissue explants were minced and placed directly in plastic culture dishes in DMEM containing 20% FBS, penicillin (100 U/mL), and gentamycin (20 µg/mL), allowing preadipocyte fibroblasts to proliferate. After fibroblasts had grown out from the explants, monolayers were passaged serially by gently treating with trypsin/EDTA, and cultures were maintained in 80-mm flasks containing DMEM with 10% FBS and antibiotics. Cell cultures were grown in a humidified 5% CO2 incubator at 37°C. The strains were stored in liquid N2 until needed, and they were used between the third and seventh passage.
After cells reached confluence in 6-well plates, differentiation of adipocytes was initiated by the following protocol. The culture medium were changed to serum-free DMEM supplemented with 33 µM biotin, 17 µM pantothenic acid, 10 µg/ml transferrin, 0.2 nM T3, 1 µM insulin (Boehringer-Mannheim, Mannheim, Germany), and 0.2 µM carbaprostaglandin (cPGI2; Calbiochem, La Jolla, CA, USA). For the first 4 days, 1 µM insulin, 1 µM dexamethasone, and 0.1 mM isobutylmethylxanthine were included in the media. The differentiation was continued for 10 days, during which the media was replaced every 3 days. A PPARγ agonist, rosiglitazone (10 µM, Cayman, Ann Arbor, MI, USA), was added from day 1 for further stimulation of adipogenesis.
Tissue explants were minced and placed directly in plastic culture dishes in DMEM containing 20% FBS, penicillin (100 U/mL), and gentamycin (20 µg/mL), allowing preadipocyte fibroblasts to proliferate. After fibroblasts had grown out from the explants, monolayers were passaged serially by gently treating with trypsin/EDTA, and cultures were maintained in 80-mm flasks containing DMEM with 10% FBS and antibiotics. Cell cultures were grown in a humidified 5% CO2 incubator at 37°C. The strains were stored in liquid N2 until needed, and they were used between the third and seventh passage.
After cells reached confluence in 6-well plates, differentiation of adipocytes was initiated by the following protocol. The culture medium were changed to serum-free DMEM supplemented with 33 µM biotin, 17 µM pantothenic acid, 10 µg/ml transferrin, 0.2 nM T3, 1 µM insulin (Boehringer-Mannheim, Mannheim, Germany), and 0.2 µM carbaprostaglandin (cPGI2; Calbiochem, La Jolla, CA, USA). For the first 4 days, 1 µM insulin, 1 µM dexamethasone, and 0.1 mM isobutylmethylxanthine were included in the media. The differentiation was continued for 10 days, during which the media was replaced every 3 days. A PPARγ agonist, rosiglitazone (10 µM, Cayman, Ann Arbor, MI, USA), was added from day 1 for further stimulation of adipogenesis.
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