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Abstract

Introduction

Protocol

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Materials

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Biology

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

Published: July 26th, 2022

DOI:

10.3791/62221

1Department of Orthopedics, The First affiliated hospital of Shenzhen University, Shenzhen Second People's Hospital, 2Guangdong Provincial Research Center for Artificial Intelligence and Digital Orthopedic Technology, Shenzhen Second People's Hospital, 3Department of Child and Adolescent Psychiatry, Shenzhen Kangning Hospital, Shenzhen Mental Health Center, Shenzhen Key Laboratory for Psychological Healthcare & Shenzhen Institute of Mental Health

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

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.

Osteoarthritis (OA), resulting from joint cartilage and underlying bone breakdown, remains a severe challenge leading to disability1,2. Without blood and nerve supply, cartilage self-healing ability is minimal once being injured3,4. 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....

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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.......

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Flow cytometry was used to identify the surface markers of SF-MSCs, according to the minimal criteria to define human MSCs recommended by the International Society for Cellular Therapy14,15. Flow cytometry analysis revealed that SF-MSCs cultured in this study met the identification criteria of MSCs. They were negative for CD34, CD45, and HLA-DR (below 3%) and positive for CD73, CD90, and CD105 (above 95%) (Figure 2).

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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 effects16,17. 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 .......

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National Natural Science Foundation of China (No. 81972116, No. 81972085, No. 81772394); Key Program of Natural Science Foundation of Guangdong Province (No.2018B0303110003); Guangdong International Cooperation Project (No.2021A0505030011); Shenzhen Science and Technology Projects (No. GJHZ20200731095606019, No. JCYJ20170817172023838, No. JCYJ20170306092215436, No. JCYJ20170413161649437); China Postdoctoral Science Foundation (No.2020M682907); Guangdong Basic and Applied Basic Research Foundation (No.2021A1515010985); Sanming Project of Medicine in Shenzhen (SZSM201612079); Special Funds for the Construction of High-level Hospitals in Guangdong Province.

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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

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