59-05-032 Proceeding

198 Proceedings of the Princess Maha Chakri Sirindhorn Congress nMdexamethasone, 10mM β -glycerophosphate, and 50 µg/mL ascorbate-2 phosphate. Adipogenic medium consisted of culture medium supplemented with 100 nM dexamethasone, 50 µg/mL indomethacin, 10 µg/mL insulin and 0.45 mM 3-isobutyl-1-methylxanthine. For chondrogenic differentiation approximately 1.0 x 106 cells were grown in a pellet culture. Chondrogenicmedium consisted of culture medium supplemented with 10 nM dexamethasone, 10 ng/mL transforming growth factor- β 3, and 6.25mg/ml insulin–transferrin–selenium(ITS supplement). For histological staining, osteogenic cultures were stainedwithAlizarin Red, adipogenic cultures were stainedwith Oil Red O, and chondrogenic pellets were embedded in paraffin, cut into 4 µM thin sections and stained with Toluidine blue. Gene expression the osteogenic, adipogenic and chondrogenic genes was analyzed using reverse transcription-polymerase chain reaction. III. Results High-proliferating, spindle-shaped, plastic adherent cells emerged from the planted tissue samples after 1-2weeks of incubation. CFU-F assay confirmed that all cell lineswere clonogenicwith approximately 40% colony-forming efficiency for all cell lines. Cells exhibited high-proliferation and cell proliferation assay evinced that cells could be grown for maximum 5 passages when seeded at low density and reaching up to 36.39 population doublings (Figure 1). Flow cytometry revealed cultures to be partly or fully positive for common MSCs markers CD29, CD44, CD90 and CD105, whilst being fully negative for hematopoietic markers CD34 and CD45 (Figure 2). All cell lines were able to undergo tri-linage differentiation. Calciumaccumulation in osteogenetic Figure 1 Cell proliferation assay. MSCs from facet joints (FJ) and interspinous ligaments (IL) and ligamentum flavum (LF) proliferated at similar rates.