Supplementary Figure 1 Expansion of MSCs in 20% oxygen vs 5% oxygen
Supplementary Figure 2 Expansion of Kaneka-filtered cells in Flasks (P4) and the Quantum (P2).
Supplementary Figure 3 Adipogenesis Osteogenesis Chondrogenesis 2F3504 MSC from Quantum 2F3504 MSC from Flasks
Supplementary Figure 4a Gated in Negative Panel
Supplementary Figure Legends S1. The same lot of cells were thawed and 2x107 cells were placed in one Quantum supplied with a gas mixture of 20% oxygen or 5% oxygen. When the lactate exceeded 3 mM the Quantums were harvested and the cells were counted. S2. 50 mL of bone marrow was filtered per Kaneka’s recommendation and split into either flasks or the Quantum. Cells were cultured as described in the materials and methods and then harvested after 2 passages (Quantum) or 4 passages (Flasks). S3. Trilineage potential of MSCs. Shown are MSCs after treatment with lineage-specific stimuli (see materials and methods). Shown is staining for adipogenesis (top), osteogenesis(middle), and chondrogenesis(bottom). S4. Phenotyping of a representative example of MSCs. (A) Positive markers for MSCs (CD73, CD90, CD105) as well as the negative marker HLA-DR. (B) Lineage markers(FITC) and individual positive markers for CD73, CD90, and CD105. (C) Phenotypic comparison of cells expanded at P2 in the Quantum (black bars) and flasks (grey bars).
Supplemental Materials and Methods S3. Tri-lineage differentiation was performed as described by Pittenger et al. 6 well plates were seeded with 2X104 cells in growth medium (Alpha-modified Minimum Essential Medium containing ribo and deoxyribonucleotides supplemented with 10% Fetal Bovine Serum) at 37°C, 5% CO2. One day later, the plates prepared for osteogenesis had the growth medium replaced by inducing medium containing dexamethasone (10-8 M), ascorbic acid (50 µg/ml), and -glycerophosphate (10 mM). Cultures were maintained in osteogenic medium, which was changed twice a week, for two weeks. Osteogenesis was evaluated by histochemistry methods including von Kossa staining of Calcium deposits and detection of lysosomal Alkaline Phosphatase using the VECTASTAIN ABC-AP KIT. Adipogenesis was initiated when cell confluence was about 50 to 60%. Growth medium was replaced with adipogenic differentiation medium containing Insulin (10 µg/ml), methyl-iso-butylxanthine (0.5 mM), indomethacin (200 µM) and dexamethasone (1 µM). 48 to 72 hours later, cultures were placed in adipogenic maintenance (growth medium plus 10 µg/ml Insulin) for 24 hours. Adipogenic cultures were fed as described for 14 days. Adipocytes were detected by staining intracellular lipid droplets with Oil Red. Chondrogenic differentiation was induced in cell pellets. 0.2X106 cells were washed twice in PBS. After the final wash, PBS was removed carefully without disturbing the pellet and then chondrogenic medium was added to the pellet. Cultures were maintained in Alpha-modified MEM supplemented with with 1% Insulin Transferrin Selenium (ITS) Universal Culture Supplement Premix (BD Biosciences,Franklin Lakes, NJ), 50µg/ml ascorbate-2-phosphate (Sigma, St. Louis, MO), 40 µg/ml L-proline (Sigma), 100 nM dexamethasone (Sigma), and 10 ng/ml recombinant human TGFβ-3 (R&D Systems, Minneapolis, MN). Alcian Blue staining was used to demonstrate differentiated chondrocytes.