Matrix-Dependent Spreading and Metabolic Activity of Bovine Satellite Cell Myospheres in Three-Dimensional Culture

Abstract Three-dimensional culture provides a useful platform for examining how extracellular matrix (ECM) environments influence bovine satellite cell behavior. In this study, OPO-MOO-M17 bovine satellite cells were used to establish myosphere cultures and evaluate responses to Matrigel, collagen I, and LunaGel under conditions spanning nominal stiffness values from the pascal to kilopascal range. Myogenic differentiation capacity was first confirmed, and initial seeding density was evaluated to establish reproducible myosphere formation. Myosphere spreading and relative alamarBlue fluorescence were then quantified across the three matrix systems, while selected cell-state-associated markers were further examined in Matrigel cultures by immunofluorescence and quantitative reverse transcription polymerase chain reaction. Matrix encapsulation promoted peripheral outgrowth compared with non-encapsulated myospheres. In Matrigel, increasing matrix concentration was associated with greater spreading, whereas relative alamarBlue fluorescence increased at low and intermediate concentrations but decreased under the highest-concentration condition. Collagen I and LunaGel also produced matrix-dependent differences in spreading and relative metabolic activity, with the two readouts showing more similar directional responses within the tested conditions. In Matrigel, selected comparisons showed lower PAX7 and CDKN2A/p16INK4a transcript levels and higher MKI67 transcript levels relative to non-encapsulated controls. Overall, morphological spreading and relative metabolic activity did not consistently change in parallel across matrix conditions. These findings demonstrate that bovine satellite cell responses depend on the specific three-dimensional matrix environment and highlight the value of integrating morphological, metabolic, and cell-state-associated readouts when evaluating culture conditions.