Gene Expression Profile during Chondrogenesis in Human Bone Marrow derived Mesenchymal Stem Cells using a cDNA Microarray

Yoo, Hyun Jung; Yoon, Sung Soo; Park, Seon Yang; Lee, Eun Young; Lee, Eun Bong; Kim, Ju Han; Song, Yeong Wook

Mononuclear cells from BM aspirates were isolated by density Ficoll-Paque gradient separation. BM was placed in a 50 mL syringe containing 5,000 units of preservative-free heparin, diluted 1:1 with phosphate buffered saline (PBS), resuspended in PBS to a final volume of 10 mL, and layered over an equal volume of Histopaque-1,077 (Sigma Chemical Co., St. Louis, MO, USA). After centrifugation at 2,000 rpm for 30 min, mononuclear cells were recovered from the gradient interface, rinsed twice in PBS, adjusted to a concentration of 1.5 × 10 7 cells/10 mL, and seeded onto 100-mm culture plates in Dulbecco's Modified Eagle's Medium-Low Glucose (DMEM-LG; 1 g/L glucose, JBI, Seoul, Korea) containing 1% penicillin-streptomycin (P/S; 10,000 units/mL, Gibco/BRL, New York, NY, USA) and 10% (v/v) heat-inactivated fetal bovine serum (FBS; Hyclone, Logan, UT, USA). Total numbers of nucleated and viable cells were determined using a hemocytometer and trypan blue (Gibco/BRL, Gaithersburg, MD, USA) staining. Cells were incubated at 37°C in a humidified 5% CO2 atmosphere and allowed to adhere for 72 hr. Non-adherent cells were then removed. The medium was changed twice a week. When cells were 80%-90% confluent, adherent cells were trypsinized (0.05% trypsin, Gibco/BRL) at 37°C for 5 min and replated in 100-mm culture plates. After passage 3, a morphologically homogenous population of adherent cells was obtained. During this expansion, medium was changed every 4-5 days.