We gratefully acknowledge the financial support provided by the Next-Generation BioGreen 21 Program (No. PJ007969), Rural Development Administration, and the National Research Foundation (grant no. NRF-2015R1D1A1A01056639) of the Republic of Korea.

动物实验由动物中心生物医学实验在庆尚大学授权。 1.准备动物操作<ol><li>制备成年女性小型猪用于从骨髓和滑液MSCs的非侵入性集合。执行小型猪前一天麻醉和样品采集的临床检查。 <ol><li>为身体检查，包括体温，呼吸率，和心脏速率，以及用于猪的行为和血液状态的观察，使用全血计数测试提供临床正常的动物。 注意:对于临床健康小型猪参数间隔是11 - 29 /对呼吸速率分钟，68 - 98 /对心脏速率分钟，和37 - 38℃的体温。 </li></ol></li><li>除去在6术前禁食期间从笼子里食用的床上用品 - 12小时PRIOR以麻醉的年轻和成年小型猪的管理，禁食3小时应为新生儿进行观察。提供饮用水直到麻醉的时间。 </li></ol> 2.动物麻醉的制备<ol><li>麻醉与乙酰丙嗪（0.1毫克/千克），美托咪定（0.03毫克/千克），阿托品（0.04毫克/千克）给药前用药的小型猪。 <ol><li>注入使用21g的针与成人小型猪至少30毫米的长度的所有药物肌内（IM）。执行IM注射耳后1-3厘米的横向颈肌或进入大腿肌肉。 注:没有引发恐惧或紧张在动物或要求粗糙处理安全注射术前用药执行所有程序。 </li></ol></li><li>放置小型猪在背卧位。 </li><li>术前用药的的行政管理15分钟后，用启动1.5％异氟烷麻醉管理吸入istered。 <ol><li>继续异氟烷（1.5％），通过气管内管的吸入，随后至2.5％的增加的浓度，与氧气（1.5升/分钟）作为载气。 </li></ol></li><li>连续监测麻醉期间小型猪的心脏速率，体温，和经皮血氧饱和度（SPO 2）。在38的温度使用循环水毯 - 39℃，保持体温。应用眼科软膏，以保护眼睛免受麻醉过程中干燥。 </li><li>遵循Guedel的评估麻醉13的深度分类。 </li></ol> 3.筹备细胞分离培养<ol><li>培养从骨髓和滑液衍生的MSC，制成500毫升之先进的Dulbecco补充有10％胎牛血清（FBS）的改良Eagle培养基（ADMEM），1％Glutamax的，10纳克/毫升碱性成纤维细胞生长事实上的R（bFGF）的，和1.0％青霉素 - 链霉素（10,000 IU和10,000微克/毫升，分别青霉素 - 链霉素）。 <ol><li>孵育在38.5℃下的ADMEM中的5％的CO 2的CO 2培养箱内的潮湿气氛中。 </li></ol></li><li>根据制造商的细胞的分离和洗涤说明制成500毫升之Dulbecco磷酸盐缓冲盐水（DPBS）中。 </li></ol> 4.从小型猪采集骨髓<ol><li>选择一个区域约15×15厘米的上髂嵴（ 图1）的大小，并使用无菌安全剃刀或电动推剪此区域移除毛发。一旦小型猪被定位，交替地擦洗过程现场用下面灭菌剂三次:聚维酮碘和70％乙醇。该区域是完全干燥后，将无菌单。 </li><li>预涂层10毫升注射器用100 U / ml肝素通过与3漂洗 - 第4毫升肝素ejec前廷抗凝结剂。 注:执行此步骤，以避免骨髓穿刺过程中注射器凝固。 </li><li>提取最少5毫升从麻醉下的小型猪的髂骨的骨髓，采用了骨髓提取器（3.0×100毫米）紧紧地贴在肝素预涂-10毫升注射器。 </li></ol> 5.分离细胞从骨髓和体外培养提取<ol><li>隔离从提取骨髓细胞。 <ol><li>稀释所提取的骨髓与等体积的DPBS中的15毫升锥形管中，在室温。 </li><li>添加3毫升密度离心介质（菲可 - 帕克）与15毫升锥形管中。 </li><li>稀释骨髓用DPBS，它被放置在密度离心介质上不混合的4ml的层，和离心机它在400×g下在室温下40分钟。 </li><li>收获的单核细胞的层由含有等离子体（顶层）和密度离心培养基（下层）的两层之间的血沉棕黄层（跨国公司）和跨国公司转移入15ml锥形管中。 </li><li>稀释转印跨国公司7 - 8毫升DPBS中，并在500×g下离心它们在室温下10分钟。弃去上清液并用DPBS洗涤细胞两次。 </li><li>暂停在2ml ADMEM的细胞沉淀，并将其转移到35毫米组织培养皿中。孵育在38.5℃下的培养皿中，5％CO 2的潮湿气氛中。 </li></ol></li><li> 在从骨髓中分离细胞的体外培养。 <ol><li> 48小时后，吸与未附着到培养皿的细胞的培养基，并更换2毫升ADMEM的和在5％CO 2的潮湿气氛中，在38.5℃培养了。改变介质的每第三天。 </li><li>在70 - 80％汇合，洗细胞用DPBS，用1ml的0.25％离解他们胰蛋白酶/ EDTA，并在37℃下进行3孵育他们 - 5分钟，直到细胞分离。 </li><li>收获用10ml ADMEM的细胞，将它们转移到15毫升锥形管，并在300×g下离心它们在室温下5分钟。弃去上清液。 </li><li>暂停收获的细胞在1ml新鲜ADMEM并使用血细胞计数器计数。 </li><li>放置细胞在5 -含4毫升ADMEM的7.5×10 5 / 25T烧瓶中并在5％CO 2的潮湿气氛中，在38.5℃培养它们。改变介质每三天。 </li></ol></li></ol> 6.收集滑膜从小型猪流<ol><li>类似于骨髓穿刺的过程中，选择大约15×10厘米（长/宽）在股关节适当的区域，并用消毒安全剃刀或电动推子除去毛，用消毒过程（步骤4.1）跟随。 </li><li>将3毫升注射器用23​​政进针触诊地标（髌骨和胫骨嵴）后的联合。 <ol><li>适用于温和抽吸到注射器滑液在关节的愿望。从关节收集滑液的近似体积为0.5 - 1毫升，但根据该小型猪或接头的大小这一变化。通过DPBS中的滑膜腔冲洗如果滑液的收集体积太小。 </li><li>立即撤销注射器以避免血液污染。 </li></ol></li></ol> 7.从收集的滑液和体外培养细胞分离<ol><li>隔离从送气滑液细胞。 <ol><li>稀释吸滑液与等体积的DPBS中的15毫升锥形管中，在室温。 </li><li>要清除杂物，使用40微米尼龙细胞滤网过滤稀释的滑液。 </li><li> 10毫升DPBS添加到含有过滤滑膜管流体并在400×g下在室温下10分钟离心它。弃去上清液并用DPBS洗涤细胞两次。 </li><li>暂停在2ml ADMEM的细胞沉淀，将细胞在2 - 3×10 5细胞/ 35mm组织培养皿用2ml ADMEM中，并在5％CO 2的潮湿气氛中，在38.5℃下孵育他们。 </li></ol></li><li>重复步骤5.2隔离滑液间充质干细胞体外培养。 </li></ol>在小型猪的8后过程护理<ol><li>如果需要的话，关闭皮肤在骨髓提取注射部位用一皮肤缝合器的单个缝合，骨髓采集后。 </li><li>在这个步骤完成后，辖恩诺沙星（5毫克/千克）和美洛昔康（0.2毫克/千克）的肌内注射到小型猪每天一次连续3天。 </li><li>在这个步骤完成后，消毒骨髓和滑液采集部位用0.1％聚维酮碘一次，连续3天。仔细观察每天监测并发症的迹象（ 如红肿，脓，或发红）。 </li></ol> 9.细胞表型采用流式细胞仪<ol><li>当干细胞是70 - 在通道3 80％汇合 - 5，用DPBS洗涤细胞并用1ml的0.25％（V / V）胰蛋白酶/ EDTA处理它们。然后，在37℃下进行3孵育他们 - 5分钟，直到细胞分离。 </li><li>收获用10ml DPBS的细胞，将它们转移到15毫升锥形管，并在300×g下离心它们在室温下5分钟。弃去上清液。 </li><li>以固定细胞，悬浮在10毫升3.7％的甲醛溶液的细胞沉淀并保持在4℃下用于分析的前一天。 <ol><li>定影后一天，洗涤细胞用DPBS两次使用离心分离，在300×g下，每次5分钟。弃去上清液。 </li><li>暂停在4ml细胞沉淀的DPBS，其补充有1％（重量/体积）牛血清白蛋白（BSA），并孵育沉淀1小时，在4℃下以阻止非特异性的Fc介导的相互作用。 </li><li>分发的细胞悬液500μl的等分入1.5毫升管中。 </li><li>对于非共轭抗体（CD29和波形蛋白），在300×g下洗涤细胞离心3分钟。弃去上清液。 </li><li>暂停在500微升1的细胞团:100稀释的第一抗体，并在4℃下孵育其1小时。 </li><li>在300×g下离心，用1％牛血清白蛋白洗涤细胞两次，每次5分钟。弃去上清液。 </li><li>暂停在500微升1的细胞团:100稀释的异硫氰酸荧光素（FITC）的缀合的二级抗体并在4℃下孵育其1小时在黑暗中。 </li></ol></li><li>在FITC标记的抗体（同种型，CD34，CD44和CD45），由centrifu洗细胞gation在300×g下3分钟。弃去上清液。 <ol><li>暂停在500微升1的细胞团:100 FITC缀合的抗体，并在4℃下孵育其1小时在黑暗中。 </li><li>对于这两种染色的条件下，在300×g下通过离心洗涤细胞两次用DPBS，每次5分钟。弃去上清液。 </li><li>悬浮细胞于500μl的DPBS，将它们转移到一个5毫升的圆底管中，并使用流式细胞仪8分析结果。 </li></ol></li></ol>

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The authors declare that they have no conflict of interest.

小型猪被用于从骨髓及关节液的MSCs建立隔离。为了消除各种生理条件，诸如年龄，性别和疾病，从同基因背景供体小型猪被选为正确地评价单元源依赖表征。猪是已知的解剖学，生理学和基因与人类相似，并且特别地，小型猪可以产生大小匹配器官;因此，它们可以作为一个替代供体物种为异种移植14,15。关于干细胞，吲哚胺2,3双加氧酶（IDO）和一氧化氮合酶（NOS）的最近建议的免疫调节特性用的MSC用于介导的免疫抑制，这是物种特异性的，虽然从猪和人的MSCs具有相同的IDO 9 。因此，猪模型作为干细胞供者可能提供干细胞为人类治疗的免疫反应的重要见解。此外，猪干的研究细胞起着体外过程和人类16,17它们的各种临床前应用的生物学理解的重要作用。骨髓和滑液可以非侵入性地从小型猪收获。整个过程，从麻醉样品采集，降低的全身或局部器官的应力水平;因此，小型猪可以用作接收者自体移植。在这个实验中，干细胞是从骨髓和小型猪的滑液中分离的基础上，以从RA小鼠模型在先前的研究中得出的免疫抑制能力滑膜液衍生的MSC的确认8 从小型猪骨髓采集 在猪，来源于骨髓成体祖细胞是多并已诱导新血管形成和心肌再生。捐赠者的年龄与增加脂肪含量及相关降低增殖和骨髓分化;因此，骨髓不MSC的老年动物的最佳来源。小型猪的髂嵴是骨髓来源的MSC隔离的首选网站，因为它是广泛的，是坐骨神经损伤低风险的程序网站。在中年或超重小型猪的情况下，脂肪丰富厚的肌肉可能会出现在髂嵴。在这种情况下，在皮肤上刺切口是必要的和骨髓活检针必须正确插入，它可以是难以在髂嵴适用。未能找到正确的活检部位因为针变成由骨堵塞或可能不能够达到髂嵴内的骨髓可能导致失败的骨髓愿望。因此，活检针必须正确检查，并且在开始样品收集之前需要软组织的厚度和髂嵴的骨皮质的知识。 滑膜的抽吸从小型猪的关节液 滑液中存在滑膜关节的空腔，和滑液的体积通常在发炎的关节增加。滑液流体的来源的MSC从优选的候选位点用于软骨再生拍摄， 在体内和体外 。此外，它们已被证明在RA 8以引发免疫调节性能。滑液不仅是一个方便的来源，但也有宝贵的间充质特性。使用滑液作为干细胞的来源的限制是基于所述不同尺寸和关节的病理状况，并在使用3毫升注射器用于从关节滑液抽吸难度关节中发现的不同的卷。因此，注射器的大小应5 - - 10毫升高压。从注射器2毫升的DPBS - 此外，冲洗用1滑液的是优选的。通过使用这种方法，可以得到大量的细胞，即使从在关节较小的空间。 小型猪的骨marrow-和滑膜液充质干细胞的表征 在人类中，滑膜流体和滑膜-膜-衍生的MSC的从骨关节炎患者的形态相似，但比骨髓衍生的MSC 12的形态窄。但是， 在体外培养物中，观察这两个滑膜液衍生的MSC和骨髓衍生的MSC的形态是在这个实验中相似。在研究人员的前期研究，滑膜液充质干细胞的增殖能力高，相比于骨髓间充质干8。 的MSC从成年供体的各种组织来源建立;因此，非侵入性地获得的组织源衍生的MSC是第自体干细胞有价值erapy。单元源依赖于干细胞治疗的疾病或目的的表征和能力。在猪，来源于骨髓成体祖细胞是多并已诱导新血管形成和心肌再生18,19。的MSC已经主要从骨髓分离的，但也从骨膜，骨骼肌，滑膜，脐带，和牙齿组织，虽然从这些组织得到的细胞是不容易的，并且可以要求对患者额外的外科手术。然而，滑液的体积通常增加在骨关节炎或RA患者的关节，这些增加的滑液的活检用于诊断和治疗患者。 MSC的数量也增加了在患者的退化的软骨和骨关节炎20滑液。 在这项研究中，骨marrow-和滑膜液衍生的MSC的细胞表型进行评价，一个ð两者的MSCs呈现为细胞表面标志物的表达水平相似。因此，从滑液分离并确认为间充质干细胞具有相似的表型，以在小型猪的骨髓来源的MSC。研究人员的先前研究评估的其它的细胞表面标记物，如CD90和主要组织相容性和复杂II类（MHC II类），并确认一个容量为多谱系分化成骨细胞，脂肪细胞，软骨细胞，滑膜液的神经元从小型猪8派生的MSCs。在人类中，滑膜液衍生的MSC的细胞表面抗原轮廓类似于滑膜的膜2,21和骨髓衍生的MSC的。然而，CD34和CD45的表达并没有从滑液颞下颌关节紊乱患者充质干细胞检测。因此，在关节的微环境可能影响从滑液来源的MSC的细胞表型。在这个实验中，从骨马获得的细胞rrow和使用非侵入性方法小型猪的滑液。这两种类型的体外培养的细胞被确认为干细胞，并且它们也有类似的细胞表型与共享兼容间质特征。然而，滑液流体来源的MSCs表现为不仅软骨和骨再生的自体干细胞疗法，而且对骨关节炎和类风湿性关节炎的免疫抑制更大的潜力。

Multipotent mesenchymal stem cells (MSCs) can be classified into mesenchymal cell lineages, and MSCs have been established and isolated from various tissue sources, such as from bone marrow, umbilical cords, placentas, adipose tissue, dermal skin, skeletal muscle, hair follicles, synovial membranes, and teeth1-5. Currently, attention has been given to synovial-derived MSCs because these cells may help treat joint diseases, such as bone fraction, osteoarthritis, and rheumatoid arthritis (RA), and due to the regenerative potential of MSCs in damaged cartilage or bone, they may also help treat immune modulation or autoimmune diseases6-8. The majority of research of MSCs derived from synovial sources have employed MSCs from the synovial membrane rather than from synovial fluid2,9; thus, the biological understanding of synovial-fluid-derived MSCs is limited.
Synovial fluid is an easily accessible source of MSCs, which can be aspirated during the diagnoses or treatments of patients and during diagnostic confirmations of various arthritis conditions that do not require invasive procedures, such as osteoarthritis or RA. The population of synovial-fluid-derived MSCs in the synovial cavity is significantly increased in arthritic patients compared to non-arthritic patients10-12. Therefore, synovial fluid is an excellent source candidate for MSCs, particularly for autologous stem cell therapy in patients with inflamed or injured joints. Additionally, synovial-fluid-derived MSCs have a chondrogenic capacity, immunomodulation abilities, and a high rate of proliferation. This study established protocols for bone marrow extraction and synovial fluid aspiration from minipigs using non-invasive methods and for the in vitro expansion of isolated cells and analysis of cell phenotypes.

<table><tbody><tr><td>Advanced Dulbecco&amp;rsquo;s modified Eagle medium (ADMEM)</td><td>Gibco</td><td>12491-023</td><td>MSC culture medium</td></tr><tr><td>Dulbecco&amp;rsquo;s phosphate-buffered saline (DPBS)</td><td>Gibco</td><td>14190-144</td><td>Cell washing medium, free of Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gibco</td><td>16000-044</td><td>Component of MSCs medium</td></tr><tr><td>Glutamax</td><td>Gibco</td><td>35050-061</td><td>Component of MSCs medium</td></tr><tr><td>Penicillin/streptomycin</td><td>Gibco</td><td>10378-016</td><td>Component of MSCs medium</td></tr><tr><td>Basic fibroblast growth factor (bFGF)</td><td>Simga</td><td>F0291</td><td>Component of MSCs medium</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma</td><td>A6003</td><td>Component of MSCs medium</td></tr><tr><td>Trypsin-EDTA</td><td>Gibco</td><td>25200-072</td><td>Cell dissociation reagent&amp;nbsp;</td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Sigma</td><td>M7522</td><td>Component of MSCs medium</td></tr><tr><td>Isotype antibody</td><td>BD Pharmingen</td><td>BD 550616</td><td>Isotype Control</td></tr><tr><td>CD29 antibody</td><td>BD Pharmingen</td><td>BD 552369</td><td>Integrin beta-1 MSCs marker</td></tr><tr><td>CD44 antibody</td><td>BD Pharmingen</td><td>BD 553133</td><td>Cell surface glycoprotein MSCs marker</td></tr><tr><td>CD45 antibody</td><td>BD Pharmingen</td><td>BD 340664</td><td>Hematopoietic stem cells marker</td></tr><tr><td>CD34 antibody</td><td>BD Pharmingen</td><td>BD 555821</td><td>Hematopoietic stem cells marker</td></tr><tr><td>CD90 antibody</td><td>BD Pharmingen</td><td>BD 555595</td><td>Thy-1 membrane glycoprotein MSCs marker</td></tr><tr><td>MHC Class II antibody</td><td>Santa Cruz</td><td>SC-32247</td><td>Antigen presenting cells marker</td></tr><tr><td>Vimentin antibody</td><td>Sigma</td><td>Sigma-S6389</td><td>Type III intermediate filament MSCs marker</td></tr><tr><td>Alkaline phosphatase</td><td>Promega</td><td>S3841</td><td>Mixture of 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) and nitro blue tetrazolium (NBT)</td></tr><tr><td>Ficoll-Paque</td><td>GE Healthcare</td><td>17-1440-02</td><td>Density gradient centrifugation</td></tr><tr><td>Cell strainer</td><td>BD Falcon</td><td>352340</td><td>40 &amp;micro;m nylon cell strainer</td></tr><tr><td>15-ml polystyrene conical tube</td><td>BD Falcon</td><td>351095</td><td>Sample collection and cell isolation</td></tr><tr><td>35 mm dishes</td><td>Nunc</td><td>153066</td><td>Cell culture dish</td></tr><tr><td>Bone marrow extractor</td><td>GmbH Medizintechnologie, Germany</td><td>1145-1W010</td><td>TrokaBone, 3.0 x 100 mm</td></tr><tr><td>Hematocritchamber</td><td>Marinfeld</td><td>640130</td><td>Cell counting chamber</td></tr><tr><td>Atropine</td><td>Jeil Pharmaceutical Co, South Korea</td><td></td><td>Atropine sulfate Hydrate</td></tr><tr><td>Acepromazine</td><td>Samu Median, South Korea</td><td></td><td>Pre-anaesthetic sedative and antiemetic drug</td></tr><tr><td>Medetomidine</td><td>Pfizer</td><td></td><td>Anesthetic and analgesic drug</td></tr><tr><td>Enrofloxacin</td><td>Bayer Healthcare, Germany</td><td></td><td>Anti-biotics</td></tr><tr><td>Meloxicam</td><td>Over Veterinary Medicine, Argentina</td><td></td><td>Anti-inflammatory and analgesic drug</td></tr><tr><td>Isoflurane</td><td>Hana pharm, South Korea</td><td></td><td>Inhalational anesthesia</td></tr><tr><td>Povidone iodine&amp;nbsp;</td><td>Korea Pharma, South Korea</td><td></td><td>Sterilization agent</td></tr><tr><td>Ethanol</td><td>Sigma</td><td>E7023</td><td>Sterilization agent</td></tr><tr><td>Heparin</td><td>Sigma</td><td>H3393</td><td>Anti-coagulating agent</td></tr><tr><td>Formaldehyde</td><td>Sigma</td><td>F8775</td><td>Cell fixation agent</td></tr><tr><td>Ophthalmologic ointment&amp;nbsp;</td><td>Pfizer</td><td></td><td>Oxytetracycline HCl with polymyxin B sulfate</td></tr><tr><td>Circulating water blanket</td><td>Gaymar Industries</td><td></td><td>Warming system during and after anesthesia</td></tr><tr><td>Skin stapler</td><td>Covidien, USA</td><td></td><td>Suture skin closure</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Thermo Forma</td><td>3111</td><td>Cell culture Equipment</td></tr><tr><td>Flow cytometer</td><td>BD Biosciences</td><td></td><td>Cell analyzer</td></tr><tr><td>Minipig</td><td>PWG Genetics Korea, Ltd.</td><td>T-type</td><td>Miniature pig</td></tr></tbody></table>

isolation and cellular phenotyping of mesenchymal stem cells derived from synovial fluid and bone marrow of minipigs

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, synovial-fluid-derived MSCs were reported to have multi-lineage differentiation potential and immunomodulatory features, which indicates that these cells can be used for tissue engineering and systemic treatments. This study presents a protocol for simple and non-invasive isolation of MSCs derived from the bone marrow and synovial fluid of minipigs to analyze cell surface markers for cell phenotyping and in vitro culturing. Using sexually mature six-month-old minipigs, bone marrow was extracted from the iliac crest bone using a bone marrow extractor, and the synovial fluid was aspirated from the femorotibial joint. Procedures for the collection of samples from both sources were non-invasive. The protocols for effective isolation of MSCs from harvested cell sources and for creating in vitro culture conditions to expand stable MSCs from minipigs and the application of systemic autologous treatments are provided. For cell phenotyping, the cell surface markers of both cells were analyzed using flow cytometry. In the results, the MSCs were isolated from the synovial fluid of the minipigs and showed that synovial-fluid-derived MSCs have a similar morphology and cell phenotype to bone-marrow-derived MSCs. Therefore, non-invasively obtained synovial fluid is a valuable source of MSCs.

从骨髓和小型猪的关节液充质干细胞的建立 的MSC已成功地从骨髓和小型猪的关节滑液关节中分离，并在体外扩增（ 图2）。滑液的注射器抽吸简单，并且有可能在原代细胞的体外培养过程中，以获得足够的粘附细胞。滑膜液衍生的MSC的形态类似于骨髓衍生的MSC的，两者的MSC具有成纤维纺锤形且均匀粘附。他们观察到对碱性磷酸酶（AP）染色一阳性表达，所述第三传代培养完成后。 从骨髓及关节液充质干细胞的细胞表型的评价小型猪进行的细胞表面标记物的测定，以创建小型猪的骨髓和滑膜液衍生的MSC的表型。这两种类型的MSCs表达显著量的阳性细胞表面标记，如CD29，CD44，和波形蛋白（≥96 - 阳性细胞的99％），而CD34和CD45的表达为阴性和低（的≤2％阳性细胞）（ 图3）。这些结果与从独特的MSC，包括从滑液分离的细胞获得的那些，并有在这些干细胞之间的细胞表面标记物无显著差异。从骨髓及关节液中分离出的细胞已被确认为在我们以前的研究干细胞8。两者骨髓及关节液，充质干细胞表达干细胞转录因子（的Oct3 / 4，Nanog的，和Sox2）;此外， 在体外分化能力ħ如被证实不但成间充质细胞系（骨细胞，脂肪细胞，软骨细胞），但也神经细胞通过细胞化学染色和细胞特异性基因表达的8的检测。 <img alt="图1" src="/files/ftp_upload/54077/54077fig1.jpg" /> 图1.小型猪的髂嵴的位置。红色箭头代表了用于骨髓采集髂嵴的站点。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54077/54077fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54077/54077fig2.jpg" /> 图2.从骨髓和小型猪的滑液来源的MSC。（A）中的MSC的初级培养物的均一形态揭示了成纤维细胞的形状。 （B）用的混合物染色5-溴-4-氯-3-吲哚磷酸和硝基四氮唑蓝（BCIP / NBT）透露AP活性的干细胞在通道3.比例尺:100微米<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54077/54077fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54077/54077fig3.jpg" /> 图3.从骨髓和小型猪的滑液在通道3来源的MSC的细胞表面标记物使用流式细胞仪分析的流动的MSC的细胞表面标记物鉴定的分析 ，和干细胞被确定为阳性CD29，CD44和波形蛋白和表达CD34和CD45。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54077/54077fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs at JoVE.com

Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs

A protocol establishing mesenchymal stem cells (MSCs) isolated from the synovial fluid and bone marrow of minipigs and non-invasively collected using syringe aspiration is presented. Cellular phenotyping was performed using flow cytometry after cell isolation and in vitro cultivation of bone marrow and synovial fluids derived from MSCs.

从小型猪关节液和骨髓间充质干细胞的分离和细胞表型

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, ...

isolation-cellular-phenotyping-mesenchymal-stem-cells-derived-from

Nanyang Technological University

Research

JoVE Journal

Medicine

10.4K 次观看.  Gyeongsang National University. A protocol establishing mesenchymal stem cells (MSCs) isolated from the synovial fluid and bone marrow of minipigs and non-invasively collected using syringe aspiration is presented. Cellular phenotyping was performed using flow cytometry after cell isolation and in vitro cultivation of bone marrow and synovial fluids derived from MSCs.

视频: Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

隔离和脂肪干细胞分化的猪从皮下脂肪组织

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

科研

发育生物学

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

人牙髓干细胞的分离，鉴定和比较性恒牙使用两种不同的方法，得出

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

医学

Isolation, Expansion, and Differentiation of Mesenchymal Stem Cells from the Infrapatellar Fat Pad of the Goat Stifle Joint

The IFP, present in the knee joint, serves as a promising source of MSCs. The IFP is an easily accessible tissue as it is routinely resected and discarded during arthroscopic procedures and knee replacement surgeries. Additionally, its removal is associated with minimal donor site morbidity. Recent studies have demonstrated that IFP-MSCs do not lose their proliferation capacity during in vitro expansion and have age-independent osteogenic differentiation potential. IFP-MSCs possess superior chondrogenic differentiation potential compared to bone marrow-derived MSCs (BMSCs) and adipose-derived stem cells (ADSCs). Although these cells are easily obtainable from aged and diseased patients, their effectiveness is limited. Hence, using IFP-MSCs from healthy donors is important to determine their efficacy in biomedical applications. As access to a healthy human donor is challenging, animal models could be a better alternative to enable fundamental understanding. Large animals such as dogs, horses, sheep, and goats play a crucial role in translational research. Amongst these, the goat could be a preferred model since the stifle joint of the goat has the closest anatomy to the human knee joint. Moreover, goat-IFP can fulfill the higher MSC numbers needed for tissue regeneration applications. Furthermore, low cost, availability, and compliance with the 3R principles for animal research make them an attractive model. This study demonstrates a simple protocol for isolating IFP-MSCs from the stifle joint of goats and in vitro culture conditions for their expansion and differentiation. The aseptically isolated IFP from the goat was washed, minced, and digested enzymatically. After filtration and centrifugation, the collected cells were cultured. These cells were adherent, had MSCs-like morphology, and demonstrated remarkable clonogenic ability. Further, they differentiated into adipogenic, chondrogenic, and osteogenic lineages, demonstrating their multipotency. In conclusion, the study demonstrates the isolation and expansion of MSCs, which show potential in tissue engineering and regenerative medicine applications.

Infrapatellar fat pad mesenchymal&#160;stem cells (IFP-MSCs) can be isolated easily from the infrapatellar fat pad of the knee joint. They proliferate well in vitro, form CFU-F colonies, and differentiate into adipogenic, chondrogenic, and osteogenic lineages. Herein, the methodology for the isolation, expansion, and differentiation of IFP-MSCs from goat stifle joint is provided.

山羊窒息关节髌下脂肪垫间充质干细胞的分离、扩增和分化

The IFP, present in the knee joint, serves as a promising source of MSCs. The IFP is an easily accessible tissue as it is routinely resected and discarded ...

生物工程

Isolation and Identification of Porcine Bone Marrow Mesenchymal Stem Cells and their Derived Extracellular Vesicles

With the development of stem cell therapy in translational research and regenerative medicine, bone marrow mesenchymal stem cells (BM-MSCs), as a kind of pluripotent stem cells, are favored for their instant availability and proven safety. It has been reported that transplantation of BM-MSCs is of great benefit to repairing injured tissues in various diseases, which might be related to modulating the immune and inflammatory responses via paracrine mechanisms. Extracellular vesicles (EVs), featuring a double-layer lipid membrane structure, are considered to be the main mediators of the paracrine effects of stem cells. Recognized for their crucial roles in cell communication and epigenetic regulation, EVs have already been applied in vivo for immunotherapy. However, similar to its maternal cells, most of the studies on the efficacy of transplantation of EVs still remain at the level of small animals, which is not enough to provide essential evidence for clinical translation. Here, we use density-gradient centrifugation to isolate bone marrow cells (BMC) from porcine bone marrow at first, and get porcine BM-MSCs (pBM-MSCs) by cell culture subsequently, identified by the results of observation under the microscope, induced differentiation assay, and flow cytometry. Furthermore, we isolate EVs derived from pBM-MSCs in cell supernatant by ultracentrifugation, proved by the techniques of transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting successfully. Overall, pBM-MSCs and their derived EVs can be isolated and identified effectively by the following protocols, which might be widely used in pre-clinical studies on the transplantation efficacy of BM-MSCs and their derived EVs.

<meta charset="utf8"></head><body>猪骨髓间充质干细胞及其衍生的细胞外囊泡的分离和鉴定

This article elaborates a method to isolate and identify porcine bone marrow mesenchymal stem cells (pBM-MSCs) and extracellular vesicles (EVs) derived from them, providing a methodological basis for the pre-clinical evaluation of transplantation efficacy of BM-MSCs and their derived EVs.

猪骨髓间充质干细胞及其衍生的细胞外囊泡的分离和鉴定

With the development of stem cell therapy in translational research and regenerative medicine, bone marrow mesenchymal stem cells (BM-MSCs), as a kind of ...

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

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

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 ...

生物学

大鼠颌骨髓间充质干细胞的基于生态位的分离

Technique for Isolation and Culture of Rat Jaw Bone Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from appendicular bones, BMMSCs derived from the jaw have greater proliferative and osteogenic differentiation ability, gradually becoming important seed cells for jaw defect repair. However, the mandible has a complex bony structure and less cancellous content than appendicular bones. It is difficult to acquire a large number of high-quality jaw-derived marrow mesenchymal stem cells using traditional methods. This study presents a &#39;niche-based approach on stemness&#39; for isolating and culturing rat jaw bone marrow mesenchymal stem cells (JBMMSCs). Primary rat JBMMSCs were isolated and cultured using the whole bone marrow adherent method combined with the bone slice digestion method. The isolated cells were identified as JBMMSCs through cell morphology observation, detection of cell surface markers, and multi-directional differentiation induction. The cells extracted by this method exhibit a &#39;fibroblast-like&#39; spindle shape. The cells are long, spindle-shaped and fibroblast-like. The flow cytometry analysis shows these cells are positive for CD29, CD44, and CD90 but negative for CD11b/c, CD34, and CD45, which is congruent with BMMSCs characteristics. The cells show strong proliferation capacity and can undergo osteogenic, adipogenic, and chondrogenic differentiation. This study provides an effective and stable method for obtaining enough high-quality JBMMSCs with strong differentiation ability in a short time, which could facilitate further studies of the exploration of biological function, regenerative medicine, and related clinical applications.

作者聚焦：高纯度颚骨骨髓间充质干细胞在组织工程中的应用

Mesenchymal stem cells from the jaw bone marrow have significant functions in diverse differentiation, self-renewal, and immune modulation. They have emerged as a crucial reservoir of precursor cells in gene therapy, tissue engineering, and regenerative medicine. Here, we present a unique method for isolating jaw bone marrow mesenchymal stem cells in rats.

大鼠颌骨骨髓间充质干细胞的分离和培养技术

Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from ...

<meta charset="utf8"></head><body>BAMBI高MFGE8高 UC-MSC 亚群的功能分析

Isolation of Human BAMBIhighMFGE8high Umbilical Cord-Derived Mesenchymal Stromal Cells

Umbilical cord-derived mesenchymal stromal/stem cells (UC-MSCs) present low immunogenicity and potent immunomodulatory effects for treating various diseases. Human UC-MSCs are a heterogeneous population consisting of three main subpopulations with different cell shapes, proliferation rates, differentiation abilities, and immune regulatory functions. Previously, BAMBIhighMFGE8high UC-MSCs, the first subgroup successfully isolated from UC-MSCs were found to fail to alleviate lupus nephritis. Hence, the function and underlying mechanism of this subgroup in MSC therapy for diseases remains unknown. It is necessary to isolate and further investigate BAMBIhighMFGE8high UC-MSCs in terms of their phenotype, metabolism, and function to completely understand the nature of this MSC subgroup. In this protocol, we describe a detailed method for isolating the BAMBIhighMFGE8high subpopulation from human UC-MSCs. The subpopulation of UC-MSCs is labeled with two surface markers, BAMBI and MFGE8, by flow cytometry sorting. The isolated cells are cultured and verified by flow cytometry analysis. The specific genes expressed in the BAMBIhighMFGE8high UC-MSCs are identified by RT-qPCR. This protocol results in highly efficient and pure cell sorting and describes the marker profiles of the BAMBIhighMFGE8high UC-MSCs.

<meta charset="utf8"></head><body>人 BAMBI高MFGE8高 脐带来源的间充质基质细胞的分离

The isolation of BAMBIhighMFGE8high MSCs, one of the three main subgroups constituting heterogeneous human UC-MSCs, is helpful for fully understanding the characteristics and functions of this subtype for its future application to improve clinical efficacy in specific diseases. Here, we present a method for sorting BAMBIhighMFGE8high UC-MSCs.

人 BAMBI高MFGE8高 脐带来源的间充质基质细胞的分离

Umbilical cord-derived mesenchymal stromal/stem cells (UC-MSCs) present low immunogenicity and potent immunomodulatory effects for treating various ...

从小型猪关节液和骨髓间充质干细胞的分离和细胞表型

摘要

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隔离和脂肪干细胞分化的猪从皮下脂肪组织

人牙髓干细胞的分离，鉴定和比较性恒牙使用两种不同的方法，得出

山羊窒息关节髌下脂肪垫间充质干细胞的分离、扩增和分化

猪骨髓间充质干细胞及其衍生的细胞外囊泡的分离和鉴定

猪骨髓间充质干细胞及其衍生的细胞外囊泡的分离和鉴定

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

大鼠颌骨骨髓间充质干细胞的分离和培养技术

大鼠颌骨骨髓间充质干细胞的分离和培养技术

大鼠颌骨骨髓间充质干细胞的分离和培养技术

人 BAMBI^高MFGE8^高脐带来源的间充质基质细胞的分离

从小型猪关节液和骨髓间充质干细胞的分离和细胞表型

摘要

探索更多视频

隔离和脂肪干细胞分化的猪从皮下脂肪组织

人牙髓干细胞的分离，鉴定和比较性恒牙使用两种不同的方法，得出

山羊窒息关节髌下脂肪垫间充质干细胞的分离、扩增和分化

猪骨髓间充质干细胞及其衍生的细胞外囊泡的分离和鉴定

猪骨髓间充质干细胞及其衍生的细胞外囊泡的分离和鉴定

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

大鼠颌骨骨髓间充质干细胞的分离和培养技术

大鼠颌骨骨髓间充质干细胞的分离和培养技术

大鼠颌骨骨髓间充质干细胞的分离和培养技术

人 BAMBI高MFGE8高 脐带来源的间充质基质细胞的分离

人 BAMBI^高MFGE8^高脐带来源的间充质基质细胞的分离