Fig. 3: Passaging in Beefy-9.

a Growth analysis via live-cell imaging of BSCs passaged in B8/Beefy-9 media. Results showed that cells needed to be passaged in the absence of supplemental rAlbumin (Delayed rAlbumin), and that a coating (e.g., iMatrix-511 laminin) was required for adhesion and growth. Specifically, cells without any coating (No coating) were unable to grow, as were cells with iMatrix-511 (Lmn) coating that were passaged in the presence of rAlbumin (Lmn; Passage w/ rAlbumin). In contrast, cells that were passaged onto Lmn coated flasks and allowed to adhere overnight before the addition of rAlbumin (“Lmn Delayed rAlbumin”) showed exponential growth. n = 9 image fields of view; statistical significance calculated by two-way ANOVA with multiple comparisons between conditions, and significant difference between “Lmn Delayed rAlbumin” and all other conditions are indicated by asterisks, in which p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****). A 95% confidence interval calculated via nonlinear regression (least squares regression; exponential (Malthusian) growth) is given. b Schematic of B8/Beefy-9 passaging system developed. BSCs were plated in identical conditions (BSC-GM) on day 0 to ensure that an equal number of cells adhered to the plate initially. On day 1, cells were rinsed with PBS and media was changed to Beefy-9. At 70% confluency (day 3), cells were passaged using TrypLE and plated in B8 (no rAlbumin) along with adhesive peptides. One day after passaging, media was changed to Beefy-9, and cells were proliferated and analyzed for adhesion, growth, and myogenicity. c PrestoBlue adhesion and growth analysis of BSCs plated with various animal-free coatings. Truncated vitronectin (Vtn) at 1.5 μg/cm² showed superior cell attachment and growth compared to iMatrix-511 laminin (Lmn) or Poly-D-Lysine (PDL). n = 6 (two reads for three biological replicates); statistical significance was calculated by one-way ANOVA performed separately for day 1 or day 4 with multiple comparisons between Vtn-N 1.5 μg/cm² and all other samples, and is indicated by asterisks (day 1) or hashes (day 4), in which p < 0.05 (*, #), p < 0.01 (**, ##), p < 0.001 (***, ###), and p < 0.0001 (****, ####). d Immunofluorescence staining for nuclei (DAPI), actin (Phalloidin), and Myosin Heavy Chain (MF20) in BSCs passaged in Beefy-9 media with 1.5 μg/cm² Vtn-N and delayed rAlbumin. Cells were proliferated to confluency and differentiated for 6 days in a previously described serum-free differentiation medium. Scale bars are 50 μm.