Enhanced Exosome Production in Mesenchymal Stem Cells via Extracellular Matrix-Incorporated 3D Spheroid Printing

Mesenchymal stem cell (MSC)-derived exosomes (MSC-exosomes) are emerging as promising cell-free therapeutic agents that address many challenges associated with traditional cell-based therapies. However, conventional methods for isolating MSC-exosomes using 2D culture systems are often limited in their efficiency, posing challenges to large-scale production. This study introduces a novel approach to boost MSC-exosome production by promoting cell–cell and cell–extracellular matrix (ECM) interactions. Specifically, ECM-integrated MSC spheroid bioprinting technology is employed to optimize exosome secretion, analyzing the effects of spheroid size and ECM composition on exosome production. It is demonstrated that smaller spheroids constructed using MSCs exhibit an enhanced production of exosomes. Additionally, incorporating ECM components, such as fibrin, Matrigel, and collagen, particularly at higher concentrations, further boosts exosome production. Among these, MSC spheroids with a 150 μm diameter and 0.6% w/v collagen integration demonstrate the highest exosome secretion, achieving an 18.4-fold increase compared to traditional 2D culture systems. Furthermore, exosomes derived from ECM-enhanced MSC spheroids exhibit strong efficacy in an in vitro scratch wound assay, underscoring their therapeutic potential. Thus, the newly developed ECM-incorporated spheroid bioprinting technology offers a highly effective strategy for scaling up MSC-exosome production, paving the way for exosome-based therapeutic applications.