Functionality of lyophilized osteoinductive EVs: a mechanistic study.

INTRODUCTION

Mesenchymal stem cell-derived extracellular vesicles (MSC EVs) hold significant promise for regenerative medicine. Lyophilization of EVs significantly enhances their translational potential. While, lyophilized EVs have been studied from a morphological perspective, the functional stability of these EVs and their cargo following lyophilization need to be mechanistically investigated.

METHODS

In this study, we investigated the functional and mechanistic bioactivity of fresh versus lyophilized MSC EVs, specifically focusing on functionally engineered osteoinductive EVs developed in our laboratory. We utilized dimethyl sulfoxide (DMSO) as a cryoprotectant and conducted pathway-specific and experiments to assess the stability and functionality of the EVs.

RESULTS

Our findings show that using DMSO as a cryoprotectant before lyophilization preserves the functional stability of engineered MSC EVs. experiments demonstrated that the endocytosis, cargo integrity, and pathway-specific activity of lyophilized EVs were maintained when DMSO was used as the cryoprotectant. Additionally, bone regeneration studies revealed that the functionality of cryoprotected lyophilized EVs was comparable to that of freshly isolated EVs.

DISCUSSION

These results provide a foundation for evaluating the functionality of lyophilized EVs and exploring the use of DMSO and other cryoprotectants in EV-based therapies. Understanding the functionality of lyophilized naïve and engineered EVs from a mechanistic perspective may enhance validation approaches for tissue regeneration strategies.