3D生物反应器培养大规模制备滑液间充质干细胞来源外泌体
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
Summary
在这里，我们提出了一种使用3D生物反应器从滑液间充质干细胞产生大量GMP级外泌体的方案。
Abstract
间充质干细胞（MSCs）分泌的外泌体已被建议作为软骨损伤和骨关节炎治疗的有希望的候选者。临床应用的外泌体需要大规模生产。为此，在微载体珠上生长人滑液MSC（hSF-MSCs），然后在动态三维（3D）培养系统中培养。通过利用3D动态培养，该方案成功地从SF-MSC培养上清液中获得了大规模的外泌体。通过超速离心收获外泌体，并通过透射电子显微镜、纳米颗粒透射测定和蛋白质印迹法进行验证。此外，还检测了外泌体的微生物安全性。外泌体检测结果表明，该方法可以产生大量良好生产规范（GMP）级外泌体。这些外泌体可用于外泌体生物学研究和临床骨关节炎治疗。
Introduction
由关节软骨和潜在骨分解引起的骨关节炎（OA）仍然是导致残疾的严峻挑战1，2。没有血液和神经供应，软骨一旦受伤，自愈能力极小3，4。在过去的几十年中，基于自体软骨细胞植入（ACI）的疗法在OA治疗中取得了一些进展5。对于软骨细胞的分离和扩增，必须从OA关节的非承重区域收集小软骨，从而导致软骨损伤。此外，该程序将需要第二次手术来植入扩增的软骨细胞6。因此，无软骨损伤的OA治疗的一步疗法正在广泛探索中。
间充质干细胞（MSCs）被认为是OA治疗的有希望的替代品7，8。MSCs起源于多个组织，可以在特异性刺激下分化成软骨细胞。重要的是，间充质干细胞可以通过抗炎 调节 免疫反应9。因此，间充质干细胞通过修复软骨缺陷和调节免疫反应，特别是在炎症环境中，在OA治疗中具有显着优势。对于OA治疗，滑液MSCs（SF-MSCs）因其比其他MSC来源更强的软骨细胞分化能力而最近引起了广泛关注10，11。值得注意的是，在骨科诊所，从关节腔中提取炎性SF是缓解OA患者疼痛症状的常规疗法。提取的炎性SF通常作为医疗废物处理。患者和医生都准备考虑从炎症性SF中分离的自体MSCs作为OA治疗，几乎没有伦理冲突。然而，SF-MSC治疗由于致瘤风险，长期储存和远距离运输障碍而受到损害。
由许多细胞类型（包括MSC）分泌的外泌体携带大部分亲本细胞生物信息。它已被深入研究为无细胞疗法12，13。根据临床试验政府（ClinicalTrials.gov）网站上提供的最新资源，在癌症，高血压和神经退行性疾病的研究领域启动并进行了更广泛的外泌体临床研究。SF-MSC外泌体治疗可能是一项令人兴奋和具有挑战性的应对OA的试验。良好生产规范（GMP）级和大规模外泌体生产对于临床转化至关重要。基于二维（2D）细胞培养的小规模外泌体分离已被广泛使用。然而，大规模外泌体生产策略需要优化。本研究开发了一种基于无异种条件下大量SF-MSC培养的大规模外泌体制造方法。从细胞培养上清液超速离心后，验证了外泌体的安全性和功能。
Protocol
本研究获得深圳市第二人民医院人类伦理委员会批准。从hSF-MSCs体外方案分离的外泌体示意图如图1所示。 1. 人类SF-MSCs培养与鉴定 使用注射器和针头从临床OA患者中收获20 mL SF。对OA患者的膝关节进行消毒。用7#针从髌骨外股四头肌股四头肌肌腱穿刺入关节腔。 提取 10 mL 关节液。用输血棒覆盖穿刺部位并按压5分钟…
Representative Results
流式细胞术用于鉴定SF-MSC的表面标志物，根据国际细胞治疗学会推荐的定义人类MSC的最低标准14，15。流式细胞术分析表明，本研究培养的SF-MSCs符合MSCs的鉴定标准。CD34、CD45和HLA-DR呈阴性（低于3%），CD73、CD90和CD105呈阳性（95%以上）（图2）。 在倒置显微镜下，注意到SF-MSCs在微载体上增殖（图3A…
Discussion
间充质干细胞因其自我更新、分化为具有特殊功能的组织细胞和旁分泌效应而广泛应用于再生医学16，17。值得注意的是，外泌体施加的旁分泌效应引起了人们的广泛关注18。外泌体携带MSCs的生物信息，发挥其生物学功能，克服MSC储存和运输麻烦等缺点。因此，来源于间充质干细胞的外泌体已被用于治疗干预，这在OA治疗中引起了最多?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
国家自然科学基金（第81972116、第81972085、第81772394号）;广东省自然科学基金重点项目（No.2018B0303110003）;广东国际合作项目（No.2021A0505030011）;深圳市科技项目（编号）GJHZ20200731095606019，编号JCYJ20170817172023838，编号JCYJ20170306092215436，编号JCYJ20170413161649437）;中国博士后科学基金（No.2020M682907）;广东省基础与应用基础研究基金（No.2021A1515010985）;深圳市三明医药项目（SZSM201612079）;广东省高水平医院建设专项资金。
Materials
| 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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Cite This Article
Duan, L., Li, X., Xu, X., Xu, L., Wang, D., Ouyang, K., Liang, Y. Large-Scale Preparation of Synovial Fluid Mesenchymal Stem Cell-Derived Exosomes by 3D Bioreactor Culture. J. Vis. Exp. (185), e62221, doi:10.3791/62221 (2022).