Name: large-scale preparation of synovial fluid mesenchymal stem cell-derived exosomes by 3d bioreactor culture
Uploaded: 2022-07-26T12:30:00
Description: Watch this Scientific Journal Video about Large-Scale Preparation of Synovial Fluid Mesenchymal Stem Cell-Derived Exosomes by 3D Bioreactor Culture at JoVE.com

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。

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
<ol><li>使用注射器和针头从临床OA患者中收获20 mL SF。<ol><li>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。</li>
		<li>提取 10 mL 关节液。用输血棒覆盖穿刺部位并按压5分钟...

<ol>
	<li>Cross, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+global+burden+of+hip+and+knee+osteoarthritis:+estimates+from+the+global+burden+of+disease+2010+study.">The global burden of hip and knee osteoarthritis: estimates from the global burden of disease 2010 study.</a> Annals of the Rheumatic Diseases. 73 (7), 1323-1330 (2014).</li><li>Loeser, R. F., Goldring, S. R., Scanzello, C. R., Goldring, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoarthritis:+a+disease+of+the+joint+as+an+organ.">Osteoarthritis: a disease of the joint as an organ.</a> Arthritis &amp; Rheumatology. 64 (6), 1697-1707 (2012).</li><li>Huey, D. J., Hu, J. C., Athanasiou, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unlike+bone,+cartilage+regeneration+remains+elusive.">Unlike bone, cartilage regeneration remains elusive.</a> Science. 338 (6109), 917-921 (2012).</li><li>Lu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+recruitment+of+endogenous+stem+cells+and+chondrogenic+differentiation+by+a+composite+scaffold+containing+bone+marrow+homing+peptide+for+cartilage+regeneration.">Increased recruitment of endogenous stem cells and chondrogenic differentiation by a composite scaffold containing bone marrow homing peptide for cartilage regeneration.</a> Theranostics. 8 (18), 5039-5058 (2018).</li><li>Ogura, T., Bryant, T., Merkely, G., Mosier, B. A., Minas, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+analysis+of+revision+autologous+chondrocyte+implantation+for+failed+ACI.">Survival analysis of revision autologous chondrocyte implantation for failed ACI.</a> American Journal of Sports Medicine. 47 (13), 3212-3220 (2019).</li><li>Welch, T., Mandelbaum, B., Tom, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+chondrocyte+implantation:+past,+present,+and+future.">Autologous chondrocyte implantation: past, present, and future.</a> Sports Medicine and Arthroscopy Review. 24 (2), 85-91 (2016).</li><li>McGonagle, D., Baboolal, T. G., Jones, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Native+joint-resident+mesenchymal+stem+cells+for+cartilage+repair+in+osteoarthritis.">Native joint-resident mesenchymal stem cells for cartilage repair in osteoarthritis.</a> Nature Reviews Rheumatology. 13 (12), 719-730 (2017).</li><li>Jo, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intra-articular+injection+of+mesenchymal+stem+cells+for+the+treatment+of+osteoarthritis+of+the+knee:+a+proof-of-concept+clinical+trial.">Intra-articular injection of mesenchymal stem cells for the treatment of osteoarthritis of the knee: a proof-of-concept clinical trial.</a> Stem Cells. 32 (5), 1254-1266 (2014).</li><li>Pers, Y. M., Ruiz, M., No&#235;l, D., Jorgensen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+for+the+management+of+inflammation+in+osteoarthritis:+state+of+the+art+and+perspectives.">Mesenchymal stem cells for the management of inflammation in osteoarthritis: state of the art and perspectives.</a> Osteoarthritis Cartilage. 23 (11), 2027-2035 (2015).</li><li>Neybecker, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+potentialities+for+cartilage+repair+from+human+advanced+knee+osteoarthritis+synovial+fluid-derived+mesenchymal+stem+cells.">In vitro and in vivo potentialities for cartilage repair from human advanced knee osteoarthritis synovial fluid-derived mesenchymal stem cells.</a> Stem Cell Research &amp; Therapy. 9 (1), 329 (2018).</li><li>Jia, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic-activated+cell+sorting+strategies+to+isolate+and+purify+synovial+fluid-derived+mesenchymal+stem+cells+from+a+rabbit+model.">Magnetic-activated cell sorting strategies to isolate and purify synovial fluid-derived mesenchymal stem cells from a rabbit model.</a> Journal of Visualized Experiments: JoVE. (138), (2018).</li><li>Phinney, D. G., Pittenger, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+MSC-derived+exosomes+for+cell-free+therapy.">Concise review: MSC-derived exosomes for cell-free therapy.</a> Stem Cells. 35 (4), 851-858 (2017).</li><li>Phan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+mesenchymal+stem+cells+to+improve+their+exosome+efficacy+and+yield+for+cell-free+therapy.">Engineering mesenchymal stem cells to improve their exosome efficacy and yield for cell-free therapy.</a> Journal of Extracellular Vesicles. 7 (1), 1522236 (2018).</li><li>Dominici, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal+criteria+for+defining+multipotent+mesenchymal+stromal+cells.+The+international+society+for+cellular+therapy+position+statement.">Minimal criteria for defining multipotent mesenchymal stromal cells. The international society for cellular therapy position statement.</a> Cytotherapy. 8 (4), 315-317 (2006).</li><li>Lv, F. -. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+the+surface+markers+and+identity+of+human+mesenchymal+stem+cells.">Concise review: the surface markers and identity of human mesenchymal stem cells.</a> Stem Cells. 32 (6), 1408-1419 (2014).</li><li>Samsonraj, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+Multifaceted+characterization+of+human+mesenchymal+stem+cells+for+use+in+regenerative+medicine.">Concise review: Multifaceted characterization of human mesenchymal stem cells for use in regenerative medicine.</a> Stem Cells Translational Medicine. 6 (12), 2173-2185 (2017).</li><li>Han, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+for+regenerative+medicine.">Mesenchymal stem cells for regenerative medicine.</a> Cells. 8 (8), (2019).</li><li>Zhang, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+derived+from+human+neural+stem+cells+stimulated+by+interferon+gamma+improve+therapeutic+ability+in+ischemic+stroke+model.">Exosomes derived from human neural stem cells stimulated by interferon gamma improve therapeutic ability in ischemic stroke model.</a> Journal of Advanced Research. 24, 435-445 (2020).</li><li>Zhou, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+ability+and+Toll-like+receptor+expression+of+human+mesenchymal+stem+cells+improves+significantly+after+three-dimensional+culture.">Migration ability and Toll-like receptor expression of human mesenchymal stem cells improves significantly after three-dimensional culture.</a> Biochemical and Biophysical Research Communications. 491 (2), 323-328 (2017).</li><li>Cheng, N. C., Wang, S., Young, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+spheroid+formation+of+human+adipose-derived+stem+cells+on+chitosan+films+on+stemness+and+differentiation+capabilities.">The influence of spheroid formation of human adipose-derived stem cells on chitosan films on stemness and differentiation capabilities.</a> Biomaterials. 33 (6), 1748-1758 (2012).</li><li>Guo, L., Zhou, Y., Wang, S., Wu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+changes+of+mesenchymal+stem+cells+in+three-dimensional+(3D)+spheroids.">Epigenetic changes of mesenchymal stem cells in three-dimensional (3D) spheroids.</a> Journal of Cellular and Molecular Medicine. 18 (10), 2009-2019 (2014).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+administration+of+cell-free+exosomes+generated+by+human+bone+marrow+derived+mesenchymal+stem+cells+cultured+under+2D+and+3D+conditions+improves+functional+recovery+in+rats+after+traumatic+brain+injury.">Systemic administration of cell-free exosomes generated by human bone marrow derived mesenchymal stem cells cultured under 2D and 3D conditions improves functional recovery in rats after traumatic brain injury.</a> Neurochemistry International. 111, 69-81 (2017).</li><li>Cao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+culture+of+MSCs+produces+exosomes+with+improved+yield+and+enhanced+therapeutic+efficacy+for+cisplatin-induced+acute+kidney+injury.">Three-dimensional culture of MSCs produces exosomes with improved yield and enhanced therapeutic efficacy for cisplatin-induced acute kidney injury.</a> Stem Cell Research &amp; Therapy. 11 (1), 206 (2020).</li></ol>

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?...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BCA assay kit</td>
			<td>ThermoFisher</td>
			<td>23227</td>
			<td>Protein concentration assay</td>
		</tr>
		<tr>
			<td>Blood agar plate</td>
			<td>Nanjing Yiji Biochemical Technology Co. , Ltd.</td>
			<td>P0903</td>
			<td>Bacteria culture</td>
		</tr>
		<tr>
			<td>CD105 antibody</td>
			<td>Elabscience</td>
			<td>E-AB-F1243C</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>CD34 antibody</td>
			<td>Elabscience</td>
			<td>E-AB-F1143C</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>CD45 antibody</td>
			<td>BD Bioscience</td>
			<td>555483</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>CD63 antibody</td>
			<td>Abclonal</td>
			<td>&#160;A5271</td>
			<td>Western blotting</td>
		</tr>
		<tr>
			<td>CD73 antibody</td>
			<td>Elabscience</td>
			<td>E-AB-F1242C</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>CD81 antibody</td>
			<td>ABclonal</td>
			<td>&#160;A5270</td>
			<td>Western blotting</td>
		</tr>
		<tr>
			<td>CD9 antibody</td>
			<td>Abclonal</td>
			<td>&#160;A1703</td>
			<td>Western blotting</td>
		</tr>
		<tr>
			<td>CD90 antibody</td>
			<td>Elabscience</td>
			<td>E-AB-F1167C</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>Centrifuge 5810R</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Thermo</td>
			<td></td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Confocal laser scanning fluorescence microscopy</td>
			<td>ZEISS</td>
			<td>LSM 800</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytodex</td>
			<td>GE Healthcare</td>
			<td></td>
			<td>Microcarrier</td>
		</tr>
		<tr>
			<td>Dil</td>
			<td>ThermoFisher</td>
			<td>D1556</td>
			<td>Exosome label</td>
		</tr>
		<tr>
			<td>EZ-PCR Mycoplasma detection kit</td>
			<td>BI</td>
			<td>20-700-20</td>
			<td>Mycoplasma detection</td>
		</tr>
		<tr>
			<td>Flowcytometry</td>
			<td>Beckman</td>
			<td></td>
			<td>MSC identification</td>
		</tr>
		<tr>
			<td>Gene Pulser II System</td>
			<td>Bio-Rad Laboratories</td>
			<td>1652660</td>
			<td>Gene transfection</td>
		</tr>
		<tr>
			<td>GraphPad Prism 8.0.2</td>
			<td>GraphPad Software, Inc.</td>
			<td>Version 8.0.2</td>
			<td></td>
		</tr>
		<tr>
			<td>HLA-DR antibody</td>
			<td>Elabscience</td>
			<td>E-AB-F1111C</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>Lowenstein-Jensen culture medium</td>
			<td>Nanjing Yiji Biochemical Technology Co. , Ltd.</td>
			<td>T0573</td>
			<td>Mycobacterium tuberculosis culture</td>
		</tr>
		<tr>
			<td>MesenGro</td>
			<td>StemRD</td>
			<td>MGro-500</td>
			<td>MSC culture</td>
		</tr>
		<tr>
			<td>Nanosight NS300</td>
			<td>Malvern</td>
			<td>Nanosight NS300</td>
			<td>Nanoparticle tracking analysis</td>
		</tr>
		<tr>
			<td>NTA 2.3 software</td>
			<td>Malvern</td>
			<td></td>
			<td>Data analysis</td>
		</tr>
		<tr>
			<td>Odyssey FC</td>
			<td>Gene Company Limited</td>
			<td></td>
			<td>Fluorescent western blotting</td>
		</tr>
		<tr>
			<td>OptiPrep electroporation buffer</td>
			<td>Sigma</td>
			<td>D3911</td>
			<td>Gene transfection</td>
		</tr>
		<tr>
			<td>Protease inhibitors cocktail</td>
			<td>Sigma</td>
			<td>P8340</td>
			<td>Proteinase inhibitor</td>
		</tr>
		<tr>
			<td>RNase A</td>
			<td>Qiagen</td>
			<td>158924</td>
			<td>Removal of RNA</td>
		</tr>
		<tr>
			<td>Sabouraud agar plate</td>
			<td>Nanjing Yiji Biochemical Technology Co., Ltd.</td>
			<td>P0919</td>
			<td>Fungi culture</td>
		</tr>
		<tr>
			<td>TEM</td>
			<td></td>
			<td>JEM-1200EX</td>
			<td></td>
		</tr>
		<tr>
			<td>The Rotary Cell Culture System (RCCS)</td>
			<td>Synthecon</td>
			<td>RCCS-4HD</td>
			<td>3D culture</td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman</td>
			<td>Optima XPN-100</td>
			<td>Exosome centrifuge</td>
		</tr>
	</tbody>
</table>

large-scale preparation of synovial fluid mesenchymal stem cell-derived exosomes by 3d bioreactor culture

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。

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...

Watch this Scientific Journal Video about Large-Scale Preparation of Synovial Fluid Mesenchymal Stem Cell-Derived Exosomes by 3D Bioreactor Culture at JoVE.com

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

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。

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

Exosomes secreted by mesenchymal stem cells (MSCs) have been suggested as promising candidates for cartilage injuries and osteoarthritis treatment. ...

我们提出了一种协议，使用我们的3D生物反应器从滑液间充质干细胞中产生大量GMP级外泌体。这些外泌体可用于外泌体生物学研究和临床关节炎治疗。演示该程序的将是梁玉杰博士。通过在 1， 500 倍 G 下以 4 摄氏度离心滑液 10 分钟，开始收集人间充质细胞或 MSC。离心后，弃去上清液并用10毫升PBS重悬细胞沉淀。将重悬的细胞在 1， 500 倍 G 下在 4 摄氏度下离心 10 分钟并丢弃 PBS，然后用 10 毫升人 MSC 培养基重悬沉淀，细胞密度为 10 倍至每毫升第四个细胞的五倍，然后将悬浮液铺在 100 毫米培养皿中。将培养皿在含有 37% 二氧化碳的气氛中孵育 5%。两周后，收集细胞培养上清液以使用流式细胞术鉴定滑液间充质干细胞或SF-MSC。为此，通过在 1， 000 倍 G 下在 4 摄氏度下离心五分钟来消化第三代 SF-MSC 的第三代传代。然后，在收集细胞沉淀之前丢弃上清液。接下来，将400微升封闭缓冲液以五乘以10到第四的浓度添加到细胞沉淀中，并使沉淀在室温下静置15分钟。15分钟后，如前所述离心细胞悬液，然后将分离的沉淀重悬于100微升1X PBS中。如手稿中所述，以每管1：100的稀释比例加入1微升单克隆荧光抗体，并将试管置于室温下孵育30分钟。孵育后，用1X PBS洗涤细胞两次，并将细胞重悬于100微升1X PBS中。然后，使用红色和FITC通道在流式细胞仪上检测多达10， 000个细胞中的荧光团。为了制备微载体，在室温下以每克50毫克的浓度在1X DPBS中膨胀并水合0.75克微载体至少三个小时。然后，倒出上清液以在新鲜的DPBS中洗涤微载体五分钟。用每克 50 毫克微载体的新鲜 1X DPBS 替换培养基后，通过在 121 摄氏度下以每平方英寸 15 磅的速度高压灭菌 15 分钟对微载体进行灭菌。然后，在倾析上清液之前让灭菌的微载体沉淀。在室温下以每克50毫克微载体的浓度冲洗培养基中的微载体。当微载体沉降时，弃去上清液。要制备灌注生物反应器，如前所述通过高压灭菌对生物反应器进行灭菌。完成后，计算SF-MSC的数量，以将2.5乘以10分配给第七个SF-MSC，并将微载体分配给注入250毫升GMP级MSC培养基的生物反应器。将生物反应器置于含有5%二氧化碳的培养箱中，温度为37摄氏度，速度为每分钟15转。每六天更换一次培养基。14天后，收集细胞培养上清液和微载体进行进一步分析。将细胞培养上清液在4摄氏度下以300倍G离心10分钟，并在丢弃细胞碎片的同时收集上清液。在4摄氏度下，依次将上清液以2， 000倍G离心10分钟，然后在10， 000倍G下离心30分钟，以丢弃较大的囊泡并收集沉淀。将沉淀重悬于40毫升PBS中后，在4摄氏度下以120， 000倍G离心细胞悬液70分钟。然后，将收集的含有外泌体的沉淀重悬于500微升PBS中。用无菌PBS稀释新鲜分离的外泌体样品至每毫升10至第七至10至第九颗粒的浓度，并在每分钟30微升和24.4至24.5摄氏度的每次运行中注入500微升样品进入纳米颗粒跟踪分析或NTA系统。根据制造商的协议手动设置捕获和分析系统。通过激光散射可视化粒子，并在数字视频上捕获它们的布朗运动。接下来，使用软件分析录制的录像带，每次运行至少跟踪 200 个单独的粒子。对于蛋白质印迹，将300微升裂解缓冲液和蛋白酶抑制剂混合物加入外泌体中，通过上下移液混合进行混合。然后，让其在冰上静置 20 分钟。将混合物在4摄氏度下以9，391倍G离心10分钟后，使用蛋白质测定试剂盒测量上清液中的蛋白质浓度。测定后，用100微升4X蛋白质上样缓冲液在100摄氏度下加热样品10分钟。接下来，以每毫升10毫克的浓度加载15微升蛋白质，通过凝胶电泳在120伏下运行70分钟，并在100伏特下在4摄氏度下电印迹60分钟。通过荧光蛋白质印迹检测非外泌体特异性标志物，例如calnexin和外泌体生物标志物，例如CD9，CD63和CD81。流式细胞术分析显示，培养的SF-MSCsCD34、CD45和HLA-DR阴性，CD73、CD90和CD105阳性，符合MSCs鉴定标准。在倒置显微镜下，注意到SF-MSCs在微载体上增殖。与2D培养相比，SF-MSC从6天开始在3D培养中增殖更快。使用蛋白质印迹法鉴定来自2D和3D培养的SF-MSC的外泌体，发现外泌体表达CD63，CD9和CD81，而钙连接蛋白呈阴性。纳米细胞分析表明，2D和3D外泌体的直径约为120纳米。透射电子显微镜分析揭示了2D和3D外泌体的形态，大致呈球状囊泡。使用NTA分析，分析了30至160纳米尺寸颗粒的浓度。3D培养后，颗粒浓度为每毫升10至6的4.0倍。然而，在2D培养后，相同大小颗粒的浓度为每毫升10至6的2.5倍。此外，3D培养比2D培养产生更多的外泌体蛋白。代表性分析显示原代软骨细胞内化了Dil标记的外泌体。Dil标记的外泌体进入原代软骨细胞，在三小时时达到峰值。体外递送研究表明，外泌体可以将磺基-花青3标记的microRNA-140递送至软骨细胞。电镀细胞，在生物反应器中开始反应以及上清液是该协议中最重要的步骤。3D培养通常用于贴壁细胞的大规模培养。其他方法，如自旋柔性也可用于外泌体的大规模生产。我们的方法提高了外泌体的产量，从而实现了基于MSC外泌体的临床前应用。

Research

JoVE Journal

Biology

Large-Scale Screens of Metagenomic Libraries

宏基因组库的大型屏幕

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation.  Generating a ...

科研

生物学

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

成人内皮细胞集落形成祖细胞的分离和大型规模扩张

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Large Scale Zebrafish-Based In vivo Small Molecule Screen

斑马鱼为基础的大型在体内小分子屏幕

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to ...

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

一个培养人关节软骨细胞的三维系统在滑液

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not ...

Infinium Assay for Large-scale SNP Genotyping Applications

Infinium检测的大型SNP基因分型应用

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of ...

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

敏感，大规模定量代谢组学的策略

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

透射电镜下体的样品制备与成像

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

从兔模型中分离纯化滑液源性间充质干细胞的磁活化细胞分选策略

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

赫连图斯安努斯大型热成像筛选装置新稳压器的鉴定

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water ...

A Semiautomated ChIP-Seq Procedure for Large-scale Epigenetic Studies

用于大规模表观遗传学研究的半自动ChIP-Seq程序

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is a powerful and widely used approach to profile chromatin DNA associated with specific ...