Fig. 4
Effect of long-term AA supplementation in MSC manufacturing. (A) Schematic diagram of the experiment setup. MSCs frozen at P1 were thawed and recovered for 1 passage in standard expansion media. From passage 2 to 9, AA was supplemented with standard expansion media in the experimental group (AA), whereas the Untreated control was cultured in standard expansion media without AA. MSCs were harvested at passage 9 for subsequent analysis. (B) Chondrogenic differentiation is indicated by the ratio of total sGAG to total DNA in chondrogenic pellets; (C) osteogenic differentiation is represented by the amount of Alizarin red stain for calcium deposits; and (D) adipogenic differentiation is represented by the amount of oil red stain for oil droplets. (E) Comparison between chondrogenic, osteogenic and adipogenic differentiation efficiency of AA-treated MSCs in relative to Untreated. (F) Metabolic profile of Untreated and AA treated MSCs, presented as OCR: ECAR at passage 9 (P9) (G) µMRR T2 relaxation time of Untreated and AA treated MSCs at P9. (H) Suspended cell diameter of Untreated and AA-treated MSCs from P3 to P9. Measurements were calculated from 400–500 cells. Data are presented in violin plots with the first dotted line as the 75th percentile, the second dotted line as the mean and the last dotted line as the 25th percentile. (I) Population doubling time (PDT) and (J) cumulative population doubling level (CPDL) of Untreated and AA-treated MSCs from P3 to P9. With an initial cell seeding density of 2.0 × 103 cells/cm2, expansion to 7 passages yielded 9.24 ± 0.27 × 103 cells/cm2 and 2.76 ± 0.49 × 103 cells/cm2 from AA treated and Untreated groups at P9, respectively. Experiments were performed in 3 technical replicates. Data are presented as mean ± standard deviation. * P < 0.05 and ** P < 0.01 compared to Untreated