Mechanoregulation of MSC spheroid immunomodulation

This study quantified how controlled uniaxial cyclic compression alters the immunomodulatory secretome of human mesenchymal stromal cell (MSC) spheroids cultured in RGD-modified alginate hydrogels. Spheroid-laden gels (8 mm) were exposed to defined loading regimes that varied compressive stress magnitude (5 vs 10 kPa) and hold duration (30 vs 250 s), generating L5H30, L10H30, and L10H250 cycles with matched overall cycle time. Mechanical loading increased spheroid diameter without reducing viability, metabolic activity, or total DNA. Cytokine/chemokine profiling showed regime-dependent shifts: higher stress generally produced larger changes, with L10H30 prominently increasing IL-6 and IL-8 and altering multiple inflammatory/recruitment factors, while IL-10 was broadly decreased under compression; VEGF was enhanced at lower stress (L5H30) but suppressed at high stress with long holds (L10H250). Mechanistically, loading-dependent actin organization correlated with mechanoresponsive gene regulation; L10H30 increased CTGF expression, and ROCK inhibition (Y-27632) disrupted actin architecture, reduced CTGF, and globally dampened secreted analytes, linking spheroid immunomodulation to cytoskeletal/YAP-associated mechanosignaling. Conditioned media experiments demonstrated that loading and cytoskeletal perturbation together modulate THP-1 macrophage polarization outcomes.