Name: labeling of extracellular vesicles for monitoring migration and uptake in cartilage explants
Uploaded: 2021-10-04T14:00:00
Description: Watch this Scientific Journal Video about Labeling of Extracellular Vesicles for Monitoring Migration and Uptake in Cartilage Explants at JoVE.com

这项研究由卡洛斯三世社会研究所资助，由欧洲社会基金和ERDF欧洲区域发展基金共同资助（MS16/00124;CP16/00124）;由PROJECTA JUNIOR DEL PROYECTO TALENT PLUS，CONSTRUYENDO SALUD，GENERANDO VALOR（JUNIOR01/18）资助，由巴利阿里群岛的可持续旅游税资助;由 Direcció General d'Investigació， Conselleria d'Investigació， Govern Balear （FPI/2046/2017）;由FUTURMed内的FOLIUM博士后计划（FOLIUM 17/01）资助，由巴利阿里群岛的可持续旅游税资助50%，由ESF资助50%;并由巴利阿里银行基金会和基金会委员会（CDI21/03）提供。

注：软骨外植体是在CEI-IB（IB 3656118 PI）对项目进行伦理批准后，根据机构指南从IdISBa生物库（IB 1995/12 BIO）获得的。
1. 色谱柱制备
<ol><li>按照制造商的说明或如下方式平衡色谱柱：<ol>
<li>取下柱帽并平衡柱。通过洗脱取除去储存缓冲液。</li>
		<li>用2.5mL磷酸盐缓冲盐水（PBS）洗涤色谱柱3次。在每次洗涤过程中，等待色谱柱吸收整个体积。 注意：不要让色谱柱干燥。</li>
		...

<ol>
	<li>Sutton, S., 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+contribution+of+the+synovium,+synovial+derived+inflammatory+cytokines+and+neuropeptides+to+the+pathogenesis+of+osteoarthritis.">The contribution of the synovium, synovial derived inflammatory cytokines and neuropeptides to the pathogenesis of osteoarthritis.</a> The Veterinary Journal. 179 (1), 10-24 (2009).</li><li>Zyli&#324;ska, B., Silmanowicz, P., Sobczy&#324;ska-Rak, A., Jarosz, &. #. 3. 2. 1. ;., Szponder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+articular+cartilage+defects:+Focus+on+tissue+engineering.">Treatment of articular cartilage defects: Focus on tissue engineering.</a> In Vivo. 32 (6), 1289-1300 (2018).</li><li>Mobasheri, A., Kalamegam, G., Musumeci, G., Batt, M. 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E., 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=Neutrophil-derived+microvesicles+enter+cartilage+and+protect+the+joint+in+inflammatory+arthritis.">Neutrophil-derived microvesicles enter cartilage and protect the joint in inflammatory arthritis.</a> Science Translational Medicine. 7 (315), 1-13 (2015).</li><li>Liu, X., 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+platelet-rich+plasma+present+a+novel+potential+in+alleviating+knee+osteoarthritis+by+promoting+proliferation+and+inhibiting+apoptosis+of+chondrocyte+via+Wnt/&#946;-catenin+signaling+pathway.">Exosomes derived from platelet-rich plasma present a novel potential in alleviating knee osteoarthritis by promoting proliferation and inhibiting apoptosis of chondrocyte via Wnt/&#946;-catenin signaling pathway.</a> Journal of Orthopaedic Surgery and Research. 14 (1), 470 (2019).</li><li>Otahal, A., 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=Characterization+and+chondroprotective+effects+of+extracellular+vesicles+from+plasma-+and+serum-based+autologous+blood-derived+products+for+osteoarthritis+therapy.">Characterization and chondroprotective effects of extracellular vesicles from plasma- and serum-based autologous blood-derived products for osteoarthritis therapy.</a> Frontiers in Bioengineering and Biotechnology. 8 (1), 584050 (2020).</li><li>Penfornis, P., Vallabhaneni, K. 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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=Interfering+with+endolysosomal+trafficking+enhances+release+of+bioactive+exosomes.">Interfering with endolysosomal trafficking enhances release of bioactive exosomes.</a> Nanomedicine: Nanotechnology, Biology, and Medicine. 20, 102014 (2019).</li><li>de Miguel P&#233;rez, D., 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=Extracellular+vesicle-miRNAs+as+liquid+biopsy+biomarkers+for+disease+identification+and+prognosis+in+metastatic+colorectal+cancer+patients.">Extracellular vesicle-miRNAs as liquid biopsy biomarkers for disease identification and prognosis in metastatic colorectal cancer patients.</a> Scientific Reports. 10 (1), 1-13 (2020).</li><li>Morelli, A. 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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=Visualization+and+tracking+of+tumour+extracellular+vesicle+delivery+and+RNA+translation+using+multiplexed+reporters.">Visualization and tracking of tumour extracellular vesicle delivery and RNA translation using multiplexed reporters.</a> Nature Communications. 6, 7029 (2015).</li><li>Takahashi, 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=Visualization+and+in+vivo+tracking+of+the+exosomes+of+murine+melanoma+B16-BL6+cells+in+mice+after+intravenous+injection.">Visualization and in vivo tracking of the exosomes of murine melanoma B16-BL6 cells in mice after intravenous injection.</a> Journal of Biotechnology. 165 (2), 77-84 (2013).</li><li>Askenasy, N., Farkas, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+PKH-labeled+hematopoietic+cells+in+recipient+bone+marrow+in+vivo.">Optical imaging of PKH-labeled hematopoietic cells in recipient bone marrow in vivo.</a> Stem Cells. 20 (6), 501-513 (2002).</li><li>Tamura, R., Uemoto, S., Tabata, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunosuppressive+effect+of+mesenchymal+stem+cell-derived+exosomes+on+a+concanavalin+A-induced+liver+injury+model.">Immunosuppressive effect of mesenchymal stem cell-derived exosomes on a concanavalin A-induced liver injury model.</a> Inflammation and Regeneration. 36, 26 (2016).</li><li>Deddens, J. C., 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=Circulating+extracellular+vesicles+contain+miRNAs+and+are+released+as+early+biomarkers+for+cardiac+injury.">Circulating extracellular vesicles contain miRNAs and are released as early biomarkers for cardiac injury.</a> Journal of Cardiovascular Translational Research. 9 (4), 291-301 (2016).</li><li>Skardelly, 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=Long-term+benefit+of+human+fetal+neuronal+progenitor+cell+transplantation+in+a+clinically+adapted+model+after+traumatic+brain+injury.">Long-term benefit of human fetal neuronal progenitor cell transplantation in a clinically adapted model after traumatic brain injury.</a> Journal of Neurotrauma. 28 (3), 401-414 (2011).</li><li>Protocol guide: Exosome labeling using PKH lipophilic membrane dyes. Sigma-Aldrich Available from: <a target="_blank" href="https://www.sigmaaldrich.com/technical-documents/protocols/biology/cell-culture/exosome-labeling-pkh.html">https://www.sigmaaldrich.com/technical-documents/protocols/biology/cell-culture/exosome-labeling-pkh.html</a> (2021)</li><li>Dehghani, M., Gulvin, S. M., Flax, J., Gaborski, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+evaluation+of+PKH+labelling+on+extracellular+vesicle+size+by+nanoparticle+tracking+analysis.">Systematic evaluation of PKH labelling on extracellular vesicle size by nanoparticle tracking analysis.</a> Scientific Reports. 10 (1), 1-10 (2020).</li><li>Morales-Kastresana, A., 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=Labeling+extracellular+vesicles+for+nanoscale+flow+cytometry.">Labeling extracellular vesicles for nanoscale flow cytometry.</a> Scientific Reports. 7 (1), 1-10 (2017).</li><li>Takov, K., Yellon, D. M., Davidson, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confounding+factors+in+vesicle+uptake+studies+using+fluorescent+lipophilic+membrane+dyes.">Confounding factors in vesicle uptake studies using fluorescent lipophilic membrane dyes.</a> Journal of Extracellular Vesicles. 6 (1), 1388731 (2017).</li><li>Mortati, L., 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+study+of+extracellular+vesicles+migration+in+cartilage-derived+osteoarthritis+samples+using+real-time+quantitative+multimodal+nonlinear+optics+imaging.">In vitro study of extracellular vesicles migration in cartilage-derived osteoarthritis samples using real-time quantitative multimodal nonlinear optics imaging.</a> Pharmaceutics. 12 (8), 1-18 (2020).</li></ol>

EV成像有助于了解EV特性，例如它们的释放和摄取机制。它们的成像允许监测它们的生物分布，并表征它们作为药物载体的药代动力学特性。然而，EV成像和跟踪可能由于其小尺寸而变得困难，尽管已经开发了许多成像设备和标记技术来帮助研究人员在体外和体内条件下监测EV23，24，25。
通过?...

骨关节炎（OA）是一种关节退行性疾病，意味着进行性和不可逆的炎症和关节软骨细胞外基质的破坏1。虽然各种形式的关节炎有许多治疗方法2，3，4，但这些都受到其副作用和有限疗效的限制。使用自体软骨细胞植入的组织工程技术常规应用于早期OA软骨病变中受伤软骨的再生治疗4。基于细胞的疗法受到限制，主要是由于能够有效修复软骨的表型稳定的软骨细胞或软骨祖细胞的数量有限3。因此，开发新的治疗策略来预防疾病进展和再生大软骨病变至关重要。
细胞外囊泡（EV）已被不同的作者建议作为OA的治疗方法5，6。EV是由大多数细胞类型分泌的膜性体，参与细胞间信号传导，并且已被证明可以发挥干细胞的治疗作用7，8，9，因此它们最近引起了对再生医学的兴趣10。来自间充质基质细胞（MSCs）的EV是研究OA的主要治疗EV，尽管其他关节相关细胞已被用作EV来源，例如软骨祖细胞或免疫细胞11，12。
血小板浓缩物，如血小板裂解物（PLs），用于增强不同损伤的伤口愈合，如角膜溃疡13，14，15或肌腱组织再生16，因为假设血小板浓缩物的EV组分可能负责这些影响17.一些与关节相关疾病相关的研究使用血小板衍生的EV（PL-EV）作为改善骨关节炎疾病的治疗方法。PL-EV通过激活Wnt/β-catenin途径18来改善软骨细胞增殖和细胞迁移，促进骨关节炎软骨细胞19中软骨标志物的表达，或者在用PL-EVs18治疗的骨关节炎兔中显示出更高水平的软骨蛋白和更少的囊状异常。
EV含有释放到靶细胞的蛋白质，脂质和核酸，建立细胞间通讯，这是与其治疗应用相关的主要特征20。电动汽车的影响取决于它们到达细胞和随后的货物释放。这种效应可以通过细胞中引起的变化间接证实，例如代谢活动或基因表达修饰。然而，这些方法不允许可视化EV如何到达细胞以发挥其功能。因此，本文提出了一种标记这些PL衍生的EV的方法，以用作炎症驱动的OA软骨外植体的治疗方法。共聚焦显微镜用于监测EV的摄取和进展到外展体中存在的软骨细胞，延时为5小时。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Centrifuge tube</td>
			<td>SPL life sciences</td>
			<td>PLC60015</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL Syringe BD Plastipak</td>
			<td>BD</td>
			<td>303174</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Propanol (Isopropanol)</td>
			<td>Panreac AppliChem</td>
			<td>1.310.901.211</td>
			<td>Prepared at 20% with Milli-Q water</td>
		</tr>
		<tr>
			<td>96-well culture plate</td>
			<td>SPL life sciences</td>
			<td>PLC30096</td>
			<td></td>
		</tr>
		<tr>
			<td>Absolute ethanol Pharmpur</td>
			<td>Scharlab</td>
			<td>ET0006005P</td>
			<td>Used to prepare 96% and 75% ethanol with Milli-Q water</td>
		</tr>
		<tr>
			<td>Biopsy Punch with plunger 3 mm</td>
			<td>Scandidact</td>
			<td>MTP-33-32</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum&#160;Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A7030</td>
			<td>Prepared at 5% with PBS</td>
		</tr>
		<tr>
			<td>Cartilage explants</td>
			<td>IdISBa Biobank</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Concentrating tube 15 mL Nanosep 100 kD Omega</td>
			<td>Pall</td>
			<td>MCP100C41</td>
			<td></td>
		</tr>
		<tr>
			<td>Concentrating tube 500 &#181;L Nanosep 100 kD Omega</td>
			<td>Pall</td>
			<td>OD003C33</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass 24 x 60 mm</td>
			<td>Deltalab</td>
			<td>D102460</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM-F12 -GlutaMAX medium</td>
			<td>Biowest</td>
			<td>L0092</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s PBS (1x)</td>
			<td>Capricorn Scientific</td>
			<td>PBS-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Embedded paraffin tissue blocks</td>
			<td>IdISBa Biobank</td>
			<td></td>
			<td>Fee for service</td>
		</tr>
		<tr>
			<td>Exo-spin mini-HD columns</td>
			<td>Cell guidance systems</td>
			<td>EX05</td>
			<td></td>
		</tr>
		<tr>
			<td>Feather S35 Microtome Blade</td>
			<td>Feather</td>
			<td>43037</td>
			<td></td>
		</tr>
		<tr>
			<td>Filtropur S 0.2 &#181;m syringe filter</td>
			<td>Sarstedt</td>
			<td>83.1826.001</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoroshield with DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>F-6057</td>
			<td></td>
		</tr>
		<tr>
			<td>Oncostatin M Human</td>
			<td>Sigma-Aldrich</td>
			<td>O9635-10UG</td>
			<td>Prepare a stock solution to a final concentration of 0.1 &#181;g/&#181;L diluten in PBS-0.1% BSA</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>8.18715.1000</td>
			<td>Prepared at 4% with PBS and stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution 100x</td>
			<td>Biowest</td>
			<td>L0022</td>
			<td></td>
		</tr>
		<tr>
			<td>PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling</td>
			<td>Sigma-Aldrich</td>
			<td>MINI26</td>
			<td>PKH26 and Dliuent C included</td>
		</tr>
		<tr>
			<td>Sodium citrate dihydrate</td>
			<td>Scharlab</td>
			<td>SO019911000</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus Microscope Slides</td>
			<td>Thermo Scientific</td>
			<td>J1800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>TNF&#945;</td>
			<td>R&#38;D systems</td>
			<td>210-TA-005</td>
			<td>Prepare a stock solution to a final concentration of 0.01 &#181;g/&#181;L diluted in PBS-0.1% BSA</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>Used to prepare a 0.1% Triton-0.1% sodium citrate solution with Milli-Q water</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Scharlab</td>
			<td>XI0050005P</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5430 R</td>
			<td>Eppendorf</td>
			<td>5428000210</td>
			<td>F-45-48-11 rotor</td>
		</tr>
		<tr>
			<td>NanoSight NS300</td>
			<td>Malvern</td>
			<td>NS300</td>
			<td>Device with embedded laser at &#955;= 532 nm and camera sCMOS</td>
		</tr>
		<tr>
			<td>Shandon Finesse 325 Manual Microtome</td>
			<td>Thermo Scientific&#8482;</td>
			<td>A78100101</td>
			<td></td>
		</tr>
		<tr>
			<td>TCS-SPE confocal microscope</td>
			<td>Leica Microsystems</td>
			<td>5200000271</td>
			<td></td>
		</tr>
	</tbody>
</table>

labeling of extracellular vesicles for monitoring migration and uptake in cartilage explants

细胞外囊泡（EV）用于不同的研究，以证明其作为无细胞治疗的潜力，因为它们的货物来自其细胞来源，如血小板裂解物（PL）。当用作治疗时，EV有望进入靶细胞并从中产生反应。在这项研究中，PL衍生的EV已被研究为骨关节炎（OA）的无细胞治疗。因此，建立了一种方法来标记EV并测试它们在软骨外植体上的摄取。PL衍生的EV用亲脂性染料PKH26标记，通过柱洗涤两次，然后在通过纳米颗粒跟踪分析（NTA）进行颗粒定量后 在体外 炎症驱动的OA模型中测试5小时。每小时，将软骨外植体固定，石蜡化，切成6μm切片以安装在载玻片上，并在共聚焦显微镜下观察。这允许验证EV在此期间是否进入靶细胞（软骨细胞）并分析其直接影响。

EV标签和车载监测的原理图如图 1所示。 表1 中NTA检测到的颗粒浓度和EV大小表明，由于在用色谱柱标记后执行两次纯化步骤，EV浓度在加工过程中降低。然而，获得的量在用于处理的颗粒数量的最佳范围内。该颗粒浓度用于计算用于治疗骨关节炎软骨外植体的PKH-PL-EV和对照的体积。
一旦软骨外植体用EV或对照组处理，它们就会固定在不同的?...

Watch this Scientific Journal Video about Labeling of Extracellular Vesicles for Monitoring Migration and Uptake in Cartilage Explants at JoVE.com

Labeling of Extracellular Vesicles for Monitoring Migration and Uptake in Cartilage Explants

在这里，我们提出了一种标记血小板裂解物衍生的细胞外囊泡的方案，以监测它们在软骨外植体中的迁移和摄取，这些外植体用作骨关节炎的模型。

标记细胞外囊泡以监测软骨外植体的迁移和摄取

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their ...

这种简单的协议允许人们标记EV并通过共聚焦显微镜对其进行监测。例如，当EV成像用于治疗时，它可能有助于评估EV分布。该技术允许人们轻松监测软骨外植体中的EV迁移和摄取，使用共聚焦显微镜，没有任何特殊功能。相同的方案可用于EV进行体外或体内实验。确保事先准备好色谱柱，并且EV的初始体积足以达到所需的最终颗粒浓度，考虑到在纯化步骤中将损失约10%的颗粒。首先浓缩细胞外囊泡或EV样品和对照。将样品置于15毫升或500微升浓缩管中，具体取决于EV样品的起始体积。根据制造商的说明离心管，直到获得几乎干燥的样品。用200微升重新悬浮浓缩的EV样品，用100微升稀释剂C重新悬浮对照组，并将其转移到新的1.5毫升离心管中。将EV样品分成两个等分试样，每个等分试样各100微升。用染料标记其中一个等分试样，并将其用作处理。将另一个保留为未标记，但像EV样品一样处理它，并使用它通过纳米颗粒跟踪分析来量化EV浓度。制备2x染料溶液，使得稀释剂C中所得的PKH26溶液的浓度为8微摩尔。在样品中每125微升稀释剂C混合一微升1毫摩尔PKH26接头。准备以1：1的比例添加到样品中所需的体积。将2x染料溶液加入PKH血小板裂解物衍生的EV中，并以1：1的比例控制样品，以达到1x染料浓度和4微摩尔PKH26浓度。将相同体积的PBS加入NTA血小板裂解物衍生的EV样品中。在室温下孵育五分钟。以1：1的比例向样品中加入5%牛血清白蛋白-PBS溶液，并确保最终体积约为400微升。继续将标记的EV与未结合的染料和与色谱柱的非特异性染料相互作用分离。从色谱柱上取下盖子，加入400微升PKH血小板裂解物衍生的EV，NTA血小板裂解物衍生的EV，或对照并丢弃所有洗脱的液体。等待样本完全进入列，然后再继续下一步。加入600微升PBS并丢弃所有洗脱的液体。等待 PBS 完全进入列，然后再继续执行下一步。加入600微升PBS，并在1.5毫升离心管中收集600微升的一小部分。对于新色谱柱，重复涉及方案开始时提到的样品浓度的步骤。向色谱柱中加入600微升先前洗脱的EV，并丢弃洗脱的体积。然后加入400微升PBS并丢弃所有洗脱体积。向色谱柱中加入600微升PBS，并在1.5毫升离心管中收集600微升的馏分。用PBS清洗软骨两次，并在无菌条件下使用直径为3毫米的活检孔将其切除。将外植体置于具有DMEM-F12培养基的96孔培养板中，在37摄氏度，5%CO2和80%湿度下补充1%青霉素链霉素。为了建立炎症驱动的OA模型，用每毫升10纳克的癌抑素M和每毫升2纳克的肿瘤坏死因子-α补充细胞培养基。在补充了癌抑素M和肿瘤坏死因子-α的细胞培养基中，用每孔PKH血小板裂解物衍生的EV或对照物1倍10至第9个颗粒处理外植体。从含有软骨外植体的96孔细胞培养板中取出培养基。如手稿中所述，向每个孔中加入200微升的细胞培养基。每小时停止一次体外检测，从零小时到五小时。用200微升PBS洗涤含有软骨外植体的细胞培养孔两次。向组织中加入100微升4%多聚甲醛，在四摄氏度下固定三小时。除去PFA，加入100微升PBS，将固定组织储存在4摄氏度，并在48小时内处理样品。在不同时间点拍摄的图像显示了EV如何进入组织，直到它们到达软骨细胞，并随着时间的推移进入软骨细胞。经过一小时的孵育后，标记的EV已经局限于软骨细胞周围。确保EV的初始体积足以达到所需的最终颗粒浓度。要记住的另一件重要事情是在接下来的24小时内使用带有标签的电动汽车。如果在24小时内不使用，可能会降低荧光。这项技术将帮助研究人员改进电动汽车成像，并在生物学和治疗学中研究电动汽车。

Research

JoVE Journal

Bioengineering

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

非接触，无标签使用拉曼光谱研究细胞和细胞外基质的监测

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

科研

生物工程

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

质量流式细胞术：协议，每日调整和执行细胞的样品上一个CyTOF的质量流式细胞仪

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Quantification and Size-profiling of Extracellular Vesicles Using Tunable Resistive Pulse Sensing

定量和大小分析外囊泡的使用可调电阻脉冲检测

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have ...

Paper-based Devices for Isolation and Characterization of Extracellular Vesicles

基于纸张的设备进行隔离外囊泡与表征

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain ...

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

3D水凝胶支架的关节软骨细胞培养及软骨生成

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

上皮细胞复育和肾组织的发展准备啮齿动物细胞外基质的支架

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

3D磁干细胞聚集和生物反应器为成熟软骨再生

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

Real-time Visualization and Analysis of Chondrocyte Injury Due to Mechanical Loading in Fully Intact Murine Cartilage Explants

全完整型小鼠软骨外种机械负荷对软骨细胞损伤的实时可视化与分析

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread ...

Evaluation of the Storage Stability of Extracellular Vesicles

细胞外囊储存稳定性的评价

Extracellular vesicles (EVs) are promising targets in current research, to be used as drugs, drug-carriers, and biomarkers. For their clinical ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

用于研究基质细胞代谢串扰的人类3D细胞外基质-脂肪培养模型

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...