Large-Scale Preparation of Synovial Fluid Mesenchymal Stem Cell-Derived Exosomes by 3D Bioreactor Culture

Summary

Here, we present a protocol to produce a large number of GMP-grade exosomes from synovial fluid mesenchymal stem cells using a 3D bioreactor.

Abstract

Exosomes secreted by mesenchymal stem cells (MSCs) have been suggested as promising candidates for cartilage injuries and osteoarthritis treatment. Exosomes for clinical application require large-scale production. To this aim, human synovial fluid MSCs (hSF-MSCs) were grown on microcarrier beads, and then cultured in a dynamic three-dimension (3D) culture system. Through utilizing 3D dynamic culture, this protocol successfully obtained large-scale exosomes from SF-MSC culture supernatants. Exosomes were harvested by ultracentrifugation and verified by a transmission electron microscope, nanoparticle transmission assay, and western blotting. Also, the microbiological safety of exosomes was detected. Results of exosome detection suggest that this approach can produce a large number of good manufacturing practices (GMP)-grade exosomes. These exosomes could be utilized in exosome biology research and clinical osteoarthritis treatment.

Introduction

Osteoarthritis (OA), resulting from joint cartilage and underlying bone breakdown, remains a severe challenge leading to disability^1,2. Without blood and nerve supply, cartilage self-healing ability is minimal once being injured^3,4. In the past decades, therapies based on autologous chondrocyte implantation (ACI) have made some progress in OA treatment⁵. For chondrocyte isolation and expansion, harvesting small cartilage from the OA joint's non-weight bearing area is necessary, causing injuries to the cartilage. Also, the pr....

Protocol

This study was approved by the Human Ethics Committee of Shenzhen Second People's Hospital. A schematic diagram of exosomes isolated from hSF-MSCs in vitro protocol is shown in Figure 1.

1. Human SF-MSCs culture and identification

1. Harvest 20 mL of SF using a syringe and needle from clinical OA patients.
   1. Disinfect the knee joint of the OA patient. Puncture from the quadriceps femoris tendon outside the patella i.......

Discussion

The mesenchymal stem cells have been widely used in regenerative medicine due to their self-renewal, differentiated into tissue cells with specialized functions, and paracrine effects^16,17. Notably, the paracrine effects exerted by exosomes have attracted much attention¹⁸. Exosomes carry the bio-information of MSCs and perform their biological function and overcome MSC shortcomings, such as troublesome storage and shipment. Thus, exosomes .......

Materials

Name	Company	Catalog Number	Comments
BCA assay kit	ThermoFisher	23227	Protein concentration assay
Blood agar plate	Nanjing Yiji Biochemical Technology Co. , Ltd.	P0903	Bacteria culture
CD105 antibody	Elabscience	E-AB-F1243C	Flow cytometry
CD34 antibody	Elabscience	E-AB-F1143C	Flow cytometry
CD45 antibody	BD Bioscience	555483	Flow cytometry
CD63 antibody	Abclonal	A5271	Western blotting
CD73 antibody	Elabscience	E-AB-F1242C	Flow cytometry
CD81 antibody	ABclonal	A5270	Western blotting
CD9 antibody	Abclonal	A1703	Western blotting
CD90 antibody	Elabscience	E-AB-F1167C	Flow cytometry
Centrifuge	Eppendorf	Centrifuge 5810R
CO2 incubator	Thermo		Cell culture
Confocal laser scanning fluorescence microscopy	ZEISS	LSM 800
Cytodex	GE Healthcare		Microcarrier
Dil	ThermoFisher	D1556	Exosome label
EZ-PCR Mycoplasma detection kit	BI	20-700-20	Mycoplasma detection
Flowcytometry	Beckman		MSC identification
Gene Pulser II System	Bio-Rad Laboratories	1652660	Gene transfection
GraphPad Prism 8.0.2	GraphPad Software, Inc.	Version 8.0.2
HLA-DR antibody	Elabscience	E-AB-F1111C	Flow cytometry
Lowenstein-Jensen culture medium	Nanjing Yiji Biochemical Technology Co. , Ltd.	T0573	Mycobacterium tuberculosis culture
MesenGro	StemRD	MGro-500	MSC culture
Nanosight NS300	Malvern	Nanosight NS300	Nanoparticle tracking analysis
NTA 2.3 software	Malvern		Data analysis
Odyssey FC	Gene Company Limited		Fluorescent western blotting
OptiPrep electroporation buffer	Sigma	D3911	Gene transfection
Protease inhibitors cocktail	Sigma	P8340	Proteinase inhibitor
RNase A	Qiagen	158924	Removal of RNA
Sabouraud agar plate	Nanjing Yiji Biochemical Technology Co., Ltd.	P0919	Fungi culture
TEM		JEM-1200EX
The Rotary Cell Culture System (RCCS)	Synthecon	RCCS-4HD	3D culture
Ultracentrifuge	Beckman	Optima XPN-100	Exosome centrifuge