β-FGF, TGF-β, and PDGF are sufficient to support MSC growth in serum-free medium. (A) MSCs grown under serum-free conditions without growth factors displayed a flattened, fibroblastic morhpology, while cells grown in the presence of growth factors displayed a distinct spindle-shaped morphology, which was closer to the morphology displayed by MSCs. Micrographs were acquired with a Nikon Diaphot TMD microscope (Nikon, Melville, NY) fitted with a 20×/0.4 NA objective and a Moticam 2000 camera (Motic Instruments, Richmond, BC). (B) Human MSCs (passage 5) grown under serum-free conditions displayed robust expansion in the presence of the 3 growth factors, which was comparable with MSCs grown in DMEM containing 10% FBS. The initial seed number for MSCs in serum-containing cultures was 28 800, whereas in serum-free cultures the seed number was 96 600. This difference in initial seeding density was necessary, since the optimal cell density for serum-containing cultures was 3000 cells/cm², whereas cells in serum-free medium grew best at a density of 10 000 cells/cm². The total number of cells in each passage was calculated as a ratio of total number of cells harvested to total number of cells seeded multiplied by the total number of cells from the previous passage. Although 28 800 cells and 96 600 cells were seeded, respectively, in serum-containing and serum-free cultures at every passage, the total cell number was calculated as described above. This enabled us to compute the total numbers of cells we would have harvested if we had cultured all cells from each passage between passages 5 and 9. It is important to note that since the cultures started with different seed numbers, the final number of cells at each passage were proportionately different. (C) MSCs grown in serum-free medium retain their ability to differentiate into all 3 lineages. To induce differentiation, hMSC grown for 5 passages in serum-free medium were seeded under respective differentiation conditions, cultured for 14 days, and stained with Oil Red O (Adipocytes), Alcian Blue (Chondrocytes), and Alkaline Phosphatase (Osteoblasts). Adipocytes and chondrocytes at 10× magnification; osteoblasts at 4× magnification. Micrographs were acquired with a Nikon Diaphot TMD microscope fitted with a 10×/0.25 NA (Adipocyte & Chondrocyte) and a 4×/0.13 NA objective (Osteoblast) and a Moticam 2000 camera. Brightness and contrast of all images were adjusted using Adobe Photoshop Elements 5.0 (Adobe Systems, San Jose, CA).