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

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 disorder^3,4,5,6. Nevert....

Protocol

The protocol was approved and conducted in accordance with the Human Research Ethics Committee of the Southwest Hospital.

1. Removing cell debris from the culture medium

NOTE: The procedures below should be operated in a GMP-compliant environment, especially when the samples may be directly exposed to the environment.

1. Given the requirement of microbiota-free, ensure that the filter and rubber tube are autoclaved for effective sterilization. Ensure all other consumables that might encounter the culture medium are sterile and in sealed packaging. Perform the process of removing cell deb....

Results

Morphological characterization of sEVs
In the final stage of concentration, the wasted fluid was also collected as described. The concentrated medium and wasted fluid were ultracentrifuged, respectively. We collected the precipitations for transmission electron microscopy (TEM) analysis. As anticipated, a significant number of cup-shaped nanovesicles were observed in the concentrated medium group (Figure 2A,B). However,.......

Discussion

Traditional methods for the isolation of sEVs include differential ultracentrifugation, size exclusion chromatography, and PEG precipitation, each with its own merits and demerits. While amalgamation of these disparate techniques may enhance the yield or purity of sEVs, additional steps often introduce more opportunities for sample contamination. There are integrated systems claiming to extract sEVs in bulk and adhere to GMP standards have emerged on the market²¹. However, their widespread adoptio.......

Materials

Name	Company	Catalog Number	Comments
Anti-CD63	SBI System Biosciences	EXOAB-CD63A-1	1:1000 dilution
Anti-CD9	SBI System Biosciences	EXOAB-CD9A-1	1:1000 dilution
Anti-HSP70	SBI System Biosciences	EXOAB-Hsp70A-1	1:1000 dilution
Bicinchoninic Acid Protein Assay Kit	Beyotime	P0012
Bloodlines	Fresenius Medical Care	AP16641
Bovine serum albumin 5%	Solarbio	9048-46-8
Cell culture supplement	Helios	HPCPLCGL05	5% (v/v) in cell culture media
Copper grid	Precise	RGRS GP-SMPG-1
Dialyzer	Helixone	FX8	100 kDa MWCO
Drainage bag	CZRUIDE	YLD-01
Goat Anti-Rabbit HRP	SBI System Biosciences	EXOAB-CD63A-1	1:10000 dilution
Goat Anti-Rabbit HRP	SBI System Biosciences	EXOAB-CD9A-1	1:10000 dilution
Goat Anti-Rabbit HRP	SBI System Biosciences	EXOAB-Hsp70A-1	1:10000 dilution
Mesenchymal Stem Cell Basal Medium (MSCBM)	Dakewe	DKW34-BM20500
Microfiltration membrane	shanghaixingya	WKLM-50-10	0.45 μm and 0.22 μm
Parafilm	Fisher Scientific	1337416
Peristaltic pump	LongerPump	YZ1515x
Phosphate buffer saline	Solarbio	P1022-500ml
Immun-Blot PVDF Membrane	BIO-RAD	1620177
SDS-PAGE Gel Quick Preparation Kit	Beyotime	P0012AC
SDS-PAGE Sample Loading Buffer	Beyotime	P0015A
Super ECL Plus Western Blotting Substrate	BIOGROUND	BG0001
TBST buffer	Solarbio	T1081
Ultracentrifuge tubes 38.5 mL	Beckman	344058
Bio-Rad ChemiDoc MP Imaging System	BIO-RAD
Hemodialyzer	NIKKISO	DBB-27
Nanoparticle Tracking Analysis	ZetaView	PMX120	To measure particle size distribution and particle concentration
Transmission Electron Microscopy	JEOL	JEM-1400PLUS	Recommended settings：Exposure: 1.0 s, HT Voltafe 100.00 kV, Beam Curr: 50 μA, Spot Size: 1, Mode: TEM.
Ultracentrifuge	BECKMAN COULTER	OPTIMA XPN-100	SW 28Ti SwingingBucket Rotor