Isolation of Small Extracellular Vesicles Derived from Mesenchymal Stem Cells from Large-Volume Samples with GMP Compliance for Clinical Trials

Yixiao Zhou; Chunge Ren; Qiongfang Zhang; Pan Xie; Min Chen; Hongjia Zhang; Yanxia Wang; Qin Niu; Bianbaduojie; Yong Liu; Baishijiao Bian

doi:10.3791/67644

Method Article

Isolation of Small Extracellular Vesicles Derived from Mesenchymal Stem Cells from Large-Volume Samples with GMP Compliance for Clinical Trials

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DOI:

10.3791/67644

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March 28th, 2025

Yixiao Zhou^*¹^,² , Chunge Ren^*¹^,² , Qiongfang Zhang³ , Pan Xie³ , Min Chen¹^,² , Hongjia Zhang¹^,² , Yanxia Wang⁴ , Qin Niu⁴ , Bianbaduojie⁵ , Yong Liu¹^,²^,⁶ , Baishijiao Bian¹^,²^,⁵^,⁶^,⁷

¹Southwest Hospital/Southwest Eye Hospital, Third Military Medical University (Army Medical University), ²Key Lab of Visual Damage and Regeneration & Restoration of Chongqing, ³Department of Nephrology, First Affiliated Hospital, Third Military Medical University (Army Medical University), ⁴Institute of Pathology and Southwest Cancer Center, Southwest Hospital, Third Military Medical University (Army Medical University) and Key Laboratory of Tumor Immunopathology, ⁵Army 953 Hospital, Shigatse Branch of Xinqiao Hospital, Third Military Medical University (Army Medical University), ⁶Jinfeng Laboratory, ⁷Department of Trauma Medical Center, Daping Hospital, State Key Laboratory of Trauma and Chemical Poisoning, Army Medical University

^* These authors contributed equally

Summary

Small extracellular vesicles derived from mesenchymal stem cells (MSC-sEVs) have been underscored as a cell-free treatment modality with minimal adverse effects. This study provides a protocol combining hemodialysis with ultracentrifugation, significantly reducing the time spent on the entire process and ensuring compliance with good manufacturing practice (GMP) standards.

Abstract

Small extracellular vesicles (sEV) derived from mesenchymal stem cells (MSC-sEVs) have been underscored as a cell-free treatment modality with minimal adverse effects. In contrast, traditional extraction methods such as ultracentrifugation and size-exclusion chromatography are limited by their time intensity, cost, and scalability. To overcome these limitations, we propose a method integrating a hemodialyzer and ultracentrifugation. This approach utilizes a hemodialysis device with a 100 kDa molecular weight cut-off (MWCO) membrane, which selectively concentrates sEVs while filtering out a plethora of proteins, thereby enhancing the yield and purity of sEVs. This initial purification step is followed by ultracentrifugation to further refine the sEV preparation. The integration of these two technologies not only significantly reduced the time spent on the entire process but also ensured compliance with good manufacturing practice (GMP) standards. The method here demonstrates high efficiency in isolating sEVs from a large volume of samples, offering a significant advancement over traditional methods. This protocol holds promise for accelerating the translation of EV-based therapies into clinical practice by providing a scalable, cost-effective, and GMP-compliant solution.

Introduction

Small extracellular vesicles derived from mesenchymal stem cells (MSC-sEVs) are heterogeneous vesicles enriched with multiple components such as mRNA, micro-RNA, cytokines, lipids, and metabolites¹. In recent years, many studies have underscored the immense therapeutic potential of MSC-sEVs as a cell-free treatment modality with minimal adverse effects², showing promise in addressing a spectrum of conditions, including aging, tissue degeneration, cancer, and inflammatory disorder³^,⁴^,⁵^,⁶. Nevert....