Name: an ex vivo tissue culture model of cartilage remodeling in bovine knee explants
Uploaded: 2019-11-03T13:00:00
Description: Watch this Scientific Journal Video about An Ex Vivo Tissue Culture Model of Cartilage Remodeling in Bovine Knee Explants at JoVE.com

作者感谢北欧生物科学的技术人员对我们的实验室支持，并感谢丹麦研究基金会对我们的研究的普遍支持。

1. 组织隔离
<ol><li>组织采购<ol>
<li>在无菌环境中在层流罩外执行整个组织采购部分。</li>
		<li>从当地的屠宰场，从1.5至2岁的小牛那里获得整个新鲜牛的膝关节。</li>
		<li>通过首先去除多余的肉，揭开小腿、月经、肌腱和肌膜，轻轻解剖小腿膝盖。切割肌腱和肌膜，使关节肢解。取出半月板，露出股骨。</li>
		<li>使用 3 mm 活检打孔机从股骨的承重区域分离出外植物，并通过用平行于和尽可能靠近?...

<ol>
	<li>Cope, P. J., Ourradi, K., Li, Y., Sharif, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+osteoarthritis:+the+good,+the+bad+and+the+promising.">Models of osteoarthritis: the good, the bad and the promising.</a> Osteoarthritis and Cartilage. 27 (2), 230-239 (2018).</li><li>Thysen, S., Luyten, F. P., Lories, R. J. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targets,+models+and+challenges+in+osteoarthritis+research.">Targets, models and challenges in osteoarthritis research.</a> Disease Models &amp; Mechanisms. 8 (1), 17-30 (2015).</li><li>Reker, 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=Articular+cartilage+from+osteoarthritis+patients+shows+extracellular+matrix+remodeling+over+the+course+of+treatment+with+sprifermin+(recombinant+human+fibroblast+growth+factor+18).">Articular cartilage from osteoarthritis patients shows extracellular matrix remodeling over the course of treatment with sprifermin (recombinant human fibroblast growth factor 18).</a> Osteoarthritis and Cartilage. 26, S43 (2018).</li><li>Kjelgaard-Petersen, 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=Synovitis+biomarkers:+ex+vivo+characterization+of+three+biomarkers+for+identification+of+inflammatory+osteoarthritis.">Synovitis biomarkers: ex vivo characterization of three biomarkers for identification of inflammatory osteoarthritis.</a> Biomarkers. 20 (8), 547-556 (2015).</li><li>Henriksen, K., 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=A+specific+subtype+of+osteoclasts+secretes+factors+inducing+nodule+formation+by+osteoblasts.">A specific subtype of osteoclasts secretes factors inducing nodule formation by osteoblasts.</a> Bone. 51 (3), 353-361 (2012).</li><li>Gigout, 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=Sprifermin+(rhFGF18)+enables+proliferation+of+chondrocytes+producing+a+hyaline+cartilage+matrix.">Sprifermin (rhFGF18) enables proliferation of chondrocytes producing a hyaline cartilage matrix.</a> Osteoarthritis and Cartilage. 25 (11), 1858-1867 (2017).</li><li>Reker, 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=Sprifermin+(rhFGF18)+modulates+extracellular+matrix+turnover+in+cartilage+explants+ex+vivo.">Sprifermin (rhFGF18) modulates extracellular matrix turnover in cartilage explants ex vivo.</a> Journal of Translational Medicine. 15 (1), 3560 (2017).</li><li>Karsdal, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction.">Introduction.</a> Biochemistry of Collagens, Laminins and Elastin. , (2016).</li><li>Heineg&#229;rd, D., Saxne, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+cartilage+matrix+in+osteoarthritis.">The role of the cartilage matrix in osteoarthritis.</a> Nature Reviews Rheumatology. 7 (1), 50-56 (2011).</li><li>Karsdal, M. 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=Osteoarthritis&#8211;+a+case+for+personalized+health+care?.">Osteoarthritis&#8211; a case for personalized health care?.</a> Osteoarthritis and Cartilage. 22 (1), 7-16 (2014).</li><li>Karsdal, M. A., Bay-Jensen, A. C., Henriksen, K., Christiansen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathogenesis+of+osteoarthritis+involves+bone,+cartilage+and+synovial+inflammation:+may+estrogen+be+a+magic+bullet?.">The pathogenesis of osteoarthritis involves bone, cartilage and synovial inflammation: may estrogen be a magic bullet?.</a> Menopause International. 18 (4), 139-146 (2012).</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 and Rheumatism. 64 (6), 1697-1707 (2012).</li><li>Goldring, M. B., Goldring, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoarthritis.">Osteoarthritis.</a> Journal of Cellular Physiology. 213 (3), 626-634 (2007).</li><li>Karsdal, M. 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=The+coupling+of+bone+and+cartilage+turnover+in+osteoarthritis:+opportunities+for+bone+antiresorptives+and+anabolics+as+potential+treatments?.">The coupling of bone and cartilage turnover in osteoarthritis: opportunities for bone antiresorptives and anabolics as potential treatments?.</a> Annals of the Rheumatic Diseases. 73 (2), 336-348 (2014).</li><li>Genovese, F., Karsdal, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+degradation+fragments+as+diagnostic+and+prognostic+biomarkers+of+connective+tissue+diseases:+understanding+the+extracellular+matrix+message+and+implication+for+current+and+future+serological+biomarkers.">Protein degradation fragments as diagnostic and prognostic biomarkers of connective tissue diseases: understanding the extracellular matrix message and implication for current and future serological biomarkers.</a> Expert Review of Proteomics. 13 (2), 213-225 (2016).</li><li>Gudmann, N. 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=Cartilage+turnover+reflected+by+metabolic+processing+of+type+II+collagen:+a+novel+marker+of+anabolic+function+in+chondrocytes.">Cartilage turnover reflected by metabolic processing of type II collagen: a novel marker of anabolic function in chondrocytes.</a> International Journal of Molecular Sciences. 15 (10), 18789-18803 (2014).</li><li>Madej, W., van Caam, A., Davidson, E. B., Buma, P., van der Kraan, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unloading+results+in+rapid+loss+of+TGF&#946;+signaling+in+articular+cartilage:+role+of+loading-induced+TGF&#946;+signaling+in+maintenance+of+articular+chondrocyte+phenotype?.">Unloading results in rapid loss of TGF&#946; signaling in articular cartilage: role of loading-induced TGF&#946; signaling in maintenance of articular chondrocyte phenotype?.</a> Osteoarthritis and Cartilage. 24 (10), 1807-1815 (2016).</li><li>Kjelgaard-Petersen, C. F., 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=Translational+biomarkers+and+ex+vivo+models+of+joint+tissues+as+a+tool+for+drug+development+in+rheumatoid+arthritis.">Translational biomarkers and ex vivo models of joint tissues as a tool for drug development in rheumatoid arthritis.</a> Arthritis &amp; Rheumatology. 70 (9), 1419-1428 (2018).</li><li>Wang, B., 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=Suppression+of+MMP+activity+in+bovine+cartilage+explants+cultures+has+little+if+any+effect+on+the+release+of+aggrecanase-derived+aggrecan+fragments.">Suppression of MMP activity in bovine cartilage explants cultures has little if any effect on the release of aggrecanase-derived aggrecan fragments.</a> BMC Research Notes. 2 (4), 259 (2009).</li><li>Bay-Jensen, A. 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=Enzyme-linked+immunosorbent+assay+(ELISAs)+for+metalloproteinase+derived+type+II+collagen+neoepitope,+CIIM&#8212;Increased+serum+CIIM+in+subjects+with+severe+radiographic+osteoarthritis.">Enzyme-linked immunosorbent assay (ELISAs) for metalloproteinase derived type II collagen neoepitope, CIIM&#8212;Increased serum CIIM in subjects with severe radiographic osteoarthritis.</a> Clinical Biochemistry. 44 (5-6), 423-429 (2011).</li><li>Lories, R. J., Luyten, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bone-cartilage+unit+in+osteoarthritis.">The bone-cartilage unit in osteoarthritis.</a> Nature Reviews Rheumatology. 7 (1), 43-49 (2011).</li></ol>

这里提出的用于分析牛软骨外植的软骨组织周转率的协议可用于描述许多类型药物的治疗效果，包括炎症细胞内通路的抑制剂、抑制药物的蛋白解物酶，或合成代谢生长因子。
该协议描述了两种不同的设置：一种是合成代谢设置，其中外植用胰岛素样生长因子1（IGF-1）刺激;另一种代谢设置，包括TNF-α和Oncostatin M的刺激，其中组织周转率可以使用广谱MMP抑制剂抑制。该方法的?...

软骨细胞产生并维持软骨基质。为了研究软骨细胞的生物学，以及它们和周围的ECM对外部刺激的反应，在它们的原生环境中询问它们1，2是至关重要的。研究软骨组织周转对于增进对关节疾病（如OA）的基本机制的理解非常重要，OA是一种目前尚无疾病改变治疗的疾病。因此，迫切需要更好的翻译模型2。
细胞和组织效应的外体表征对于补充其他临床前模型至关重要，包括体外模式，如软骨细胞单层培养，以及体内，如手术诱导的OA模型或自身免疫胶原蛋白诱发关节炎模型（CIA).许多研究都强调了细胞在2D单层培养和3D结构或其原生组织3、4中行为方式之间的差异。二维层中的许多细胞采用非自然形态，包括细胞极性和组织附着性的差异，导致原生组织细胞的视觉和功能差异5。这些差异在细胞的功能上也很明显，这可能改变蛋白质表达，导致分化模式、调节机制和细胞功能发生深刻变化5，6，7 ，8.
软骨ECM主要由II型胶原蛋白组成，提供基质框架，以及帮助在组织内保留液体的蛋白酶。其他基质分子，如胶原蛋白IV型、VI型、IX型、X型、十一种、十二种、纤维素、软骨寡聚蛋白（COMP）、大脂蛋白、分体素和渗透素也存在9。
虽然OA的病因仍不清楚10，11，疾病的发病被认为是由组织周转和修复过程的不平衡造成的12，13。关节软骨的退化是OA的标志。软骨驻留软骨细胞或周围组织中的细胞增加细胞因子的释放，刺激基质金属蛋白酶 （MmPs） 和 agacanas 等蛋白酶的增产，从而增加软骨 ECM 的降解14.这种降解导致小的独特蛋白质片段的释放，称为新表位，可以在血清、尿液或培养培养基15中量化。在胶原蛋白形成和成熟时，所谓的蛋白酶也被释放;这些可以量化为矩阵生产的度量16。
该协议的目的是建立一个体外软骨模型，以比较刺激和/或药物治疗对ECM组织周转的影响。软骨周转率是通过使用 ELISA：AGNx1（反射阿胶酶活性）、C2M（反映基质 MMP 活性）和 ProC2（反映类型 II 胶原蛋白）直接在条件培养基中测量基质衍生的新表位生物标志物来分析的形成）。这些发现可以通过ECM的组织染色来验证，这也形象化了软骨细胞在个别外植中的组织。所述方案可用于测试新疗法对软骨细胞功能和软骨ECM周转的影响。许多研究使用软骨外植来描述生物过程或干预对细胞因子挑战的外植的影响，使用定量组织学或免疫组织化学方法，mRNA，蛋白质表达，或蛋白质组学2 ，17，18.但是，这些协议超出了当前手稿的范围。

<table>
	<tbody>
		<tr>
			<td>45% Iron(III) chloride solution</td>
			<td>Sigma-Aldrich</td>
			<td>12322</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Merck</td>
			<td>1.00056.2500</td>
			<td></td>
		</tr>
		<tr>
			<td>Alamar Blue</td>
			<td>Life tech Invitrogen</td>
			<td>DAL1100</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy processing cassettes &#8211; green</td>
			<td>IHCWORLD</td>
			<td>BC-0109G</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy punch W/Plunger (3 mm)</td>
			<td>Scandidat</td>
			<td>MTP-33-32</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine cartilage (Bovine knees)</td>
			<td>Local slaughterhouse</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C2M</td>
			<td>Nordic Bioscience</td>
			<td></td>
			<td>Fee for service</td>
		</tr>
		<tr>
			<td>Corning 96-well plate</td>
			<td>Sigma-Aldrich</td>
			<td>CLS7007</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover Glass &#216; 13 mm</td>
			<td>VWR</td>
			<td>631-0150P</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12-GlutaMAX Dulbecco&#39;s Modified Eagle Medium/Nutrient Mixture F-12) without HEPES</td>
			<td>Gibco</td>
			<td>31331-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol &#8805;96%</td>
			<td>VWR</td>
			<td>83804.36</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute &#8805;99.5%</td>
			<td>VWR</td>
			<td>83813.36</td>
			<td></td>
		</tr>
		<tr>
			<td>exAGNx1</td>
			<td>Nordic Bioscience</td>
			<td></td>
			<td>Fee for service</td>
		</tr>
		<tr>
			<td>exPRO-C2</td>
			<td>Nordic Bioscience</td>
			<td></td>
			<td>Fee for service</td>
		</tr>
		<tr>
			<td>Fast green</td>
			<td>Sigma-Aldrich</td>
			<td>F7252</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde solution 4%</td>
			<td>Merck</td>
			<td>1004965000</td>
			<td></td>
		</tr>
		<tr>
			<td>GM6001</td>
			<td>Sigma-Aldrich</td>
			<td>M5939-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Sigma-Aldrich</td>
			<td>H3136</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Merck</td>
			<td>30721-M</td>
			<td></td>
		</tr>
		<tr>
			<td>IGF-1</td>
			<td>Sigma-Aldrich</td>
			<td>I3769-50UG</td>
			<td></td>
		</tr>
		<tr>
			<td>Oncostatin M</td>
			<td>Sigma-Aldrich</td>
			<td>O9635-10UG</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin (P/S)</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>Pertex (mounting medium for light microscopy)</td>
			<td>HistoLab</td>
			<td>811</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Safranin O</td>
			<td>Sigma-Aldrich</td>
			<td>S2255</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Standard Scalpels</td>
			<td>Integra Miltex</td>
			<td>12-460-451</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfuric acid</td>
			<td>Sigma-Aldrich</td>
			<td>30743</td>
			<td></td>
		</tr>
		<tr>
			<td>SUPERFROST PLUS Adhesion Microscope Slides</td>
			<td>Thermo scientific</td>
			<td>J1800AMNT</td>
			<td></td>
		</tr>
		<tr>
			<td>TNF-alpha</td>
			<td>R&#38;D Systems</td>
			<td>210-TA-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluene</td>
			<td>Merck</td>
			<td>1.08327.2500</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum Filtration &#34;rapid&#34;-Filtermax</td>
			<td>TPP</td>
			<td>99955</td>
			<td></td>
		</tr>
	</tbody>
</table>

an ex vivo tissue culture model of cartilage remodeling in bovine knee explants

外体培养系统涵盖广泛的实验，致力于在原生环境中研究组织和细胞功能。软骨是一种独特的组织，对结膜关节的正常运作非常重要，由密集的细胞外基质（ECM）组成，富含蛋白酶和II型胶原蛋白。软骨细胞是软骨中唯一的细胞类型，广泛存在且数量相对较少。改变的外部刺激和细胞信号可导致ECM成分的变化和恶化，这是骨关节炎（OA）和风湿性关节炎等疾病的重要病理特征。
前软骨模型允许1）分析软骨细胞介导的软骨组织周转改变，2）可视化软骨ECM组合，和3）软骨细胞直接在组织中重新排列。分析这些改变对刺激或治疗的反应在软骨生物学的各个方面非常重要，并补充在分离的软骨细胞体外实验，或在实验条件的活体动物中更复杂的模型更难控制。
软骨外植植物提供了一种翻译和易于访问的方法，用于在可控设置下评估软骨 ECM 中的组织重塑。在这里，我们描述了一个分离和培养活牛软骨外植植物的协议。该方法使用来自牛膝的组织，从当地的屠宰场很容易获得。可以分析外植和条件培养基，以研究组织周转、ECM组成和软骨细胞功能，从而分析ECM调制。

牛全深度外植被隔离、培养和治疗3周（图1）。培养基因每周增加3次治疗而改变。每周一次，通过resazurin测定测量代谢活性。ECM周转的生物标志物在从培养板收获的上清液中测量，每周3次。外植被分为4组进行治疗：1）青花青丁丁和TNF+（O+T）;2） O+T = GM6001 （GM6001）;3） 胰岛素，如生长因子-1（IGF-1）;4） 无处理的控件（w/o）。
代谢活动。</p...

Watch this Scientific Journal Video about An Ex Vivo Tissue Culture Model of Cartilage Remodeling in Bovine Knee Explants at JoVE.com

An Ex Vivo Tissue Culture Model of Cartilage Remodeling in Bovine Knee Explants

在这里，我们提出一个协议，描述分离和培养软骨外植从牛膝盖。这种方法提供了一个简单易懂的工具，用于描述组织变化，以响应生物刺激或针对关节的新型治疗。

牛膝外生软骨重塑的外骨组织培养模型

Ex vivo culture systems cover a broad range of experiments dedicated to studying tissue and cellular function in a native setting. Cartilage is a unique ...

软骨植入物是一种可转换、易于访问的方法，用于询问软骨基质中的组织重塑。该方法可能提供早期洞察潜在的新疗法如何影响关节结构。该技术的主要优点是，软骨保留在本机 3D 环境中，允许使用生物标志物分析细胞与周围基质之间的相互作用。该方法的原理并非软骨所独有。外植物可以从其他组织（如卵巢、肝脏和肺）中提取，并使用基质衍生的生物标志物进行询问。演示这个程序的将是阿玛莉·恩斯特罗姆，一个来自我们实验室的研究生。在无菌环境中，在层流罩之外执行整个组织采购过程。从当地的屠宰场，从1.5至2岁的小腿获得整个新鲜的牛皮骨股关节。然后，通过先去除多余的肉，发现凹凸、半月板、肌腱和连体膜，温柔地解剖小腿膝盖。切割肌腱和突触膜，使关节被肢解。取出半月板以露出股骨。使用三毫米活检打孔机将外植物从股骨的承重区域分离，然后通过用与子骨平行且尽可能靠近子骨的手术刀切割，将其从动脉表面释放。立即在含有 1% 青霉素和链霉素的 DMEM F12 谷氨酸培养培养基中储存和混合出植物，在 50 毫升管或培养皿中。要开始组织培养，请将外植转移到层流罩中的无菌 96 井板中，对角线放置每个组内的所有复制，以尽量减少蒸发引起的变异。将 PBS 添加到培养板的外孔中，以进一步避免上干液的蒸发。然后，在培养介质或 PBS 中清洗外植三次。培养每井200微升培养培养物，直到实验开始，在37摄氏度的孵化器中用5%的二氧化碳进行长达10周。如果应用任何治疗，在改变培养媒介之前，通过稀释在培养介质中稀释，将这些处理准备到外植井中想要的浓度。每两到三天在层流罩中更换培养介质。轻轻地从每一个井中卸下上一液，并将其转移到一个新的 96 井板中存放。立即在每井中添加200微升的新鲜培养或处理。不要让外植在介质变化过程中干燥，并确保所有外植完全淹没在新的介质中。用密封胶带密封新板，并将其存放在零下20摄氏度，用于组织周转和蛋白质表达的生物标志物分析。要开始雷萨祖林试验，请对含有10%雷沙祖林的培养介质进行溶液。如先前所述，将上一提液收获，并在37摄氏度的10%resazurin溶液中浸入10%的再祖林溶液中，三小时或直到超自然者变成紫色。然后，将条件控制和背景控制再祖林溶液转移到黑色微刺激板中，在540纳米的激发和590纳米的发射时测量荧光。在此之后，将外植在洗涤介质中淹没5至10分钟，以让雷萨祖林完全扩散出来，并添加新的培养基或治疗，如果使用。继续测量代谢活动一次，作为细胞生存能力的间接测量。在这项研究中，牛全深度外植被隔离、培养和治疗。外植被分为四组进行治疗，包括使用肿瘤坏死因子α治疗，使用肿瘤坏死因子α治疗的组，使用GM 6001治疗的肿瘤坏死因子α组，使用胰岛素生长因子1治疗的组，以及无治疗控制治疗组。代谢活动是相对稳定的，在整个三个星期的所有四组。与控制相比，IGF-1呈上升趋势，O加T组呈减少趋势。O 加 T 然后每周应用于培养井三次，以调查 O 加 T 中导软骨降解。O 加 T 被认为会增加第 7 天至 21 天的第二类胶原蛋白降解，与对照组相比，从第 3 天到 14 天增加二型胶原蛋白降解。添加 GM 6001 与 O plus T 结合阻止 O 加 T 中继 C2M 的发布，但对 AGN X1 的影响有限，在第 10 天与 O plus T 组类似的水平。胰岛素如生长因子1每周应用三次，用于牛的全深度外植，以调查合成代谢刺激如何模块软骨 ECM 周转。IGF-1对软骨外植的影响主要观察对二型胶原蛋白形成测量的预期合成代谢刺激。使用IGF-1治疗时，pro-C2水平比对照组观察到的要低，表明IGF-1从第7天起刺激第二类胶原蛋白形成。随着时间的推移，外植可能会恶化，因此，在所有处理和洗涤步骤中，小心移液至关重要，以便不丢失或破坏不同介质交换步骤中的外植。使用此模型生成的结果不应单独产生，而应受人体组织以及细胞和较新的模型中生成的数据的支持，以进一步验证结果。为了揭示组织中的基本机制，而不仅仅是结果，可以使用其他分子生物学技术（如基因和蛋白质表达分析、免疫组织化学或其他永久性分子生物学技术）进一步分析组织和超自然物质。

Research

JoVE Journal

Biology

Method for Culture of Early Chick Embryos ex vivo (New Culture)

Method for Culture of Early Chick Embryos ex vivo New Culture

文化早期鸡胚体外（新文化）的方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

科研

生物学

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

前初级人类输卵管上皮细胞的体外培养

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

一种新型的<em&gt;体外</em小鼠胚胎心脏的培养方法

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

小鼠胚胎皮肤和Live成像黑素细胞迁移的体外培养

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

An Ex vivo Culture System to Study Thyroid Development

An Ex vivo Culture System to Study Thyroid Development

一个<em&gt;体外</em&gt;企业文化系统来研究甲状腺发展

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

在c-fos蛋白免疫组织学检测：一个有用​​的工具，中央通路在特定的生理反应所涉及的标记<em&gt;在体内</em&gt;和<em&gt;离体</em

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

测量的刚度<em&gt;离体</em&gt;鼠标主动脉用原子力显微镜

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

A Tissue Culture Model of Estrogen-producing Primary Bovine Granulosa Cells

雌激素原代牛颗粒细胞的组织培养模型

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca ...

Bovine Ovarian Cortex Tissue Culture

牛卵巢皮质组织培养

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors ...

An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

眼部微血管血管生成前维沃胆球状芽分析

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell ...