This work was supported by the Swiss National Foundation Grant CR3I3_140717/1 and the Swiss Paraplegic Foundation.

注:人类骨髓穿刺，从髂嵴从捐助者同意与卢塞恩州的伦理委员会的批准收集。从髂嵴犬骨髓抽吸收集与狗主人的consent.Human（约20ml）和犬（约10毫升）中的骨髓吸出物为抗凝固通过加入停药后立即将15ml 3.8％柠檬酸钠在手术室。将样品转移到实验室环境，用于处理的同一天，撤回。 1.准备尿激酶（此前1日使用） <ol><li>使用根据制造商的说明无菌磷酸盐缓冲盐水（PBS）重组尿激酶:加入10毫升的PBS到隔膜入口烧瓶用10毫升注射器。纷飞获得50000注射单位（U）每毫升溶解干物质。 </li><li>准备500微升等分（每25000 U）到sterile管。商店等分-20℃，并用它需要至少6个月时。 </li></ol> 2.制备步骤（在此之前的每骨髓治疗） <ol><li>解冻尿激酶的一个等分（25,000 U）每凝结骨髓穿刺（抽吸量高达25毫升已使用25,000 U盘单一等分已成功治疗）。 </li><li>预热水浴或摇床至37℃。 </li></ol> 3.酶消化的凝块<ol><li>执行在层流生物安全柜的所有工作，以避免在处理过程中的骨髓样品的微生物污染。当潜在的传染性人力物力的工作，使用防护手套，以及小心处理建议。 </li><li>放置在无菌的50ml管中的顶部上的100微米的细胞过滤网。通过细胞过滤网小心倾骨髓抽吸物，倾斜滤网或周围的凝块材料移动。使用无菌枪头的通过滤网更好的流动。 注:避免任何稀释凝块，例如 ，通过用PBS洗涤凝块。尿激酶间接行为，并要求血清从活检进行有效的消化成分（ 即 ，纤溶酶）。同时继续与凝块材料的步骤，将滤液可以保持在室温，直至在步骤3.6进一步使用。 </li><li>转移骨髓凝块材料到使用无菌镊子空细胞培养皿。切碎片切成小块约2毫米3使用无菌手术刀。 </li><li>传送小块凝块成使用无菌​​镊子50ml的反应管中。确保碎血块有湿的外观。如果设备出现干燥，使用一些滤液从步骤3.1滋润。 </li><li>通过上下吹打使用5ml的微量滴定吸管5次磨碎的凝块。加入1等份尿激酶至样品。温育30分钟，在37℃或者在水浴或摇（轻轻）孵化器。 </li><li>磨碎通过上下吹打使用5ml的吸量管5次。执行此步骤在生物安全柜。孵育另外30分钟，在37℃。培养后，使用5ml的吸量管磨碎再次5次。越过一个新的100微米的细胞过滤到与步骤3.2滤液池。 </li><li>离心机在室温下将细胞悬浮液在500×g下10分钟。弃去上清液。如下所述，或根据自己的实验室进行扩建MSC文化的标准程序重新暂停在媒体的细胞沉淀。 </li></ol> 4.播种扩建细胞培养和CFU板<ol><li>重悬细胞（包括红细胞和单核细胞）的50ml基础培养基:α-MEM中补充有10％胎牛血清（FBS），100U / ml青霉素，100mg / ml的链霉素和2.5mg / ml的两性霉素B 。计数细胞使用Neubauer室使用的台盼蓝溶液稀释1:10河</li><li>板中的细胞在细胞培养瓶中以5×10 7个细胞/ cm 2的密度，并在37℃下孵育，在湿润的培养箱中。如果可能，使用低氧（5％氧气）的条件。对于CFU试验控制，板10 9细胞在直径10cm的细胞培养皿。 </li><li>培养3天后，间质干细胞附着到细胞培养皿，而其他细胞保持在悬浮液中。用补充有5毫微克/毫升碱性成纤维细胞生长因子（bFGF）的基础培养基更换介质。对于CFU试验对照，同样地对待细胞培养皿。 </li><li>继续细胞培养物，共2周，交换媒体，每周三次。如果细胞超过80％汇合，通过拆分胰酶消化。对于CFU试验控制，交换媒体同样地，但不分裂的细胞为两周的过程中。 </li></ol> 5.姬姆萨染色的CFU分析<ol><li>准备好10毫升每盘姬姆萨液:DILUTE原液（7.6克/升姬姆萨甘油:甲醇）1:10在无菌水（总是准备一个新的工作稀释）。 </li><li>除去从细胞培养皿的培养基，用PBS洗细胞。将液体施加到培养皿时，会非常认真注意和避免清洗过的细胞。 </li><li>固定的细胞中的纯甲醇5分钟，在室温。丢弃的甲醇。添加吉姆萨溶液孵育在37℃下在湿润的培养箱中60分钟。 </li><li>用PBS洗两次。空气首先干板头在纸巾上。手工计数的殖民地。这是通过使用在板的背面的标记笔最好地实现。 </li></ol> 6. MSC分化<ol><li>犬干细胞分化 注:犬的MSCs分化成软骨细胞，成骨和成脂肪谱系的刺激与相应的介质四个星期。 </li><li>为 成脂分化，单层在4×10 5的CE文化的MSCsLLS / cm 2的交替两种不同的培养条件如下: <ol><li>培养在含有DMEM +含有GlutaMAX，3％的FBS，100单位/ ml青霉素，100mg / ml的链霉素，2.5微克/毫升两性霉素B和170毫胰岛素脂肪生成维持培养基。 </li><li>培养在脂肪生成诱导培养基与补充有3％FBS，5％兔血清，1μM地塞米松维持培养基，500μM的3-异丁基-1-甲基黄嘌呤，33μM的生物素，5μM罗格列酮和17μM泛酸16。 </li><li>通过用油红O染色陶醉的脂滴。简单地说，修复细胞，10％的甲醛，洗PBS和染色用0.35％油红O异丙醇。 </li></ol></li><li>对于单层7×10 3个细胞/ cm 2的刺激下成骨分化培养干细胞: <ol><li>先进的DMEM（GIBCO）+含有GlutaMAX，5％FBS，100单位/ ml青霉素，100mg / ml的链霉素，2.5微克/毫升两性霉素B，501，M L-抗坏血酸-2-磷酸，10mM的β-甘油和100nM的地塞米松。 </li><li>确定矿化存款冯科萨染色（5％ 硝酸银）。简要地说，固定细胞，用10％的甲醛，洗净用PBS染色，用5％在蒸馏水中的AgNO 3。 </li></ol></li><li>软骨细胞分化<ol><li>从一个海绵状的医疗装置切立方体（每边3毫米），从冻干胶原I型构成，并且用来作为支架材料，以支持单元17。 </li><li>种子的MSC在立方体的顶部以4×10 6个细胞/ ml（〜70000个细胞/立方体）的浓度。 </li><li>在加入的介质，使细胞附着在立方体30分钟。 </li><li>维持MSC-胶原构建体中软骨介质选自由DMEM / F12 +含有GlutaMAX，2.5％FBS，100单位/ ml青霉素，100mg / ml的链霉素，2.5微克/毫升两性霉素B，40纳克/毫升的地塞米松，50μg/ ml的抗坏血酸的酸-2-磷酸，50μg/ ml的L-脯氨酸，1×胰岛素 - 转铁 - 硒X和10纳克/毫升的转化生长因子β1。 </li><li>用阿尔辛蓝染色可视化蛋白聚糖的积累结构部分。简而言之，沾污部分O / N为0.4％阿尔新蓝溶解于0.01％H 2 SO 4和0.5M盐酸胍。接着，洗涤段中40％的DMSO和0.05M MgCl 2的洗涤30分钟。细胞进行复用核固红。 </li></ol></li></ol>

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The authors have nothing to disclose.

常规我们采样骨髓当患者接受手术（在我们的情况下，主要的脊柱外科手术），条件是只有很少的额外的工作，必须进行的人员在手术室的优​​点。即使将样品撤出后立即用柠檬酸钠混合，许多样品时，他们在实验室中进行处理到达被部分凝结。在此阶段，重采样，以取代凝块标本将是一个独立的额外介入再次迫使局部或全身麻醉6。这需要双方的医护人员和捐助的意愿作出贡献，并消耗了大量的资源21。 在这里，我们提供了一个使用重组人尿激酶凝块骨髓样本分离出干细胞多能的协议。我们可以证明尿激酶治疗不损害细胞和分化潜能被保留。该协议是短暂的，因为它只有大约1个半小时，比unclotted样品，同时产生可比细胞库延长来样加工。迄今为止，该协议已经被成功地应用在使用尿激酶从几个许多不同的个体和犬骨髓凝块，并表现出强大的且可重复的成功。除此之外，尿激酶经由活化的样品固有的纤溶酶原作用是间接的。这意味着，尿激酶反应需要的等离子体环境。因此不可取介绍的步骤血块清洗或类似的如与PBS。这会冲淡血清环境并导致酶反应的一个显著放缓。 决定使用尿激酶我们的方法，而不是另一种药物溶栓是微不足道的:我们开发的协议基础上，溶栓药物在我们医院药房一应俱全。对于其他研究小组也可能因此是更容易使用的组织，典型值Ë纤溶酶原激活剂（ 即 ，如阿替普酶，瑞替普酶或替奈普酶不同的药品），如果尿激酶不可用。然而，该药物之间的潜在的重要差异存在:不像组织型纤溶酶原激活剂，尿激酶可以当结合尿激酶型纤溶酶原激活物受体（uPAR的）22触发细胞活化和增殖。结合后，细胞内的信号级联被激活了导致细胞迁移和增殖。在生理情况，这是通过基质金属蛋白酶的活化细胞分泌的进一步支持。然而，在我们的系统中，尿激酶稀释并在扩增培养，而不显示有害作用逐渐除去。尽管如此，相对于预期用途分离的MSC的，需要的任何的溶栓药物的适用性被单独确定。 一般情况下，高剂量的快速和完整的血块摘要建议将MI血块处理过程中不必要的nimize选择。值得注意的是，此前许多研究使用细菌链激酶的消化血液和骨髓血块13,14。然而，这种药物可能会引起不希望的激活和免疫系统，这可能是一个主要的缺点特别是当应用细胞对患者的目的是响应。 因此，我们相信，我们的协议不仅有助于研究人员和医疗专业人员，以节省时间，降低成本。使用尿激酶 - 经批准的酶的药物，而不称为抗原 - 甚至对于许多转化研究实验室针对治疗用MSC制剂提供了一个有价值的工具。

间充质干细胞（MSCs）起到在再生医学和组织工程的主要作用。他们可以迁移，分化成各种细胞类型1和嫁接，这使得他们的理想人选自体疗法2,3。最近，利用干细胞的骨和软骨修复的临床试验，移植物抗宿主病或心脏疾病患者推出了4款。这些干细胞可以从脐带或脂肪组织收获，但最有希望的结果，从骨髓来源的干细胞5中得到。 髂嵴允许收集了相当数量的骨髓，因此作为吸6主站点。然而，抽吸质量而增加骨髓抽出的体积减小。而第一5毫升骨髓抽吸的含有高品质的MSCs，撤回较大体积导致稀释外周血f显示抽吸的ROM的高度血管骨7。因为本巨核细胞和血小板，骨髓抽吸容易凝结，除非抗凝剂被使用。但即使有抗凝血剂，可能发生血栓。 在骨髓，干细胞代表总细胞池8的只有一小部分，并且必须在培养对于大多数组织工程或治疗应用4展开。这种文化的质量在很大程度上取决于初始细胞库， 即 ，多样性和高起点9号。从取款低数量的干细胞可以通过捐助变异的部分原因。另一方面，从低质量样品的MSC需要更长的时间，培养和传代扩大到达细胞的期望数量。在这两种情况下，扩展的传代是细胞衰老的来源，并可能导致分化潜能10的损失。因此，优化的协议，可以最大限度地提高小区Y屈服和防止有害作用必须开发11,12。 当我们开始与犬间充质干工作，我们惊讶地看到，约四分之三的犬骨髓样本中含有血块，而幸运的是凝结人体样本（十分之一）不太频繁。在另一方面这是毫不奇怪，我们观察到凝血样本的MSCs低得多的产量。为了解决凝血样本的反复出现的问题，我们使用重采样的溶栓药物尿激酶，而不是开发的协议。 溶栓疗法可以抵消危及生命的情况下，如血管造成心脏发作，中风或因不必要的凝血栓塞闭塞。它们通过纤维蛋白溶酶由酶和纤维蛋白溶酶原活化剂的酶裂解的工作由凝块的降解。尽管广泛使用的治疗患者中，只有极少数出版物存在使用溶栓活动实验室应用抢救凝结样品，主要是着眼于淋巴细胞。 1987年，尼库等 。描述了使用链激酶溶解导致功能性淋巴细胞13和四年后血块，可见德等人 。延长的使用链激酶从血液和骨髓供流式细胞应用14分离白血病细胞。最近的一个出版物建议使用阿替普酶的癌症诊断15。而使用相同的酶的方法，我们的协议的重点是多能间充质干形式的骨髓中分离，以提供对研究人员在干细胞领域的工具。

<table border="0" cellpadding="0" cellspacing="0" width="674">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Basal Medium Components</td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">PenStrep 100X</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:119px;">15140122</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Human FGF-basic</td>
			<td style="width:227px;">Peprotech</td>
			<td style="width:119px;">100-18B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">MEM Alpha w/ Nucleoside, w/ stable Glutamine</td>
			<td style="width:227px;">Amimed</td>
			<td style="width:119px;">1-23S50-I</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:328px;">FBS Heat Inactivated</td>
			<td style="width:227px;">Amimed</td>
			<td style="width:119px;">2-01F36-I</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Amphotericin B</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A1907</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;"></td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Adipogenic Medium Components</td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">DMEM-HAM F12 + GlutaMAX</td>
			<td style="width:227px;">Amimed</td>
			<td style="width:119px;">1-26F09-I</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Insulin&#160;</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">I5500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Rabbit serum&#160;</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:119px;">16120099</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Dexamethasone</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">D4902</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">3-Isobutyl-1-methylxanthine</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">I5879</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Biotin&#160;</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">B4639</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Rosiglitazone&#160;</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">R2408</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Pantothenate&#160;</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">P5155</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Oil Red-O&#160;</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">O0625</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;"></td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Osteogenic Medium Components</td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">L-ascorbic acid 2-phosphate</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">A8960</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">&#223;-glycerophosphate</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">G9422</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:328px;">Silver nitrate (AgNO3)</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">S6506</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;"></td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Chondrogenic Medium Components</td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Biopad - sponge shaped medical device&#160;</td>
			<td style="width:227px;">Euroresearch</td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">L-proline&#160;</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">P5607</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Insulin-Transferrin-Selenium X</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:119px;">51500056</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Human transforming growth factor-&#946;1&#160;</td>
			<td style="width:227px;">Peprotech</td>
			<td style="width:119px;">100-21</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Alcian Blue 8GX</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">A3157</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Nuclear fast red</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">N8002</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;"></td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Generic</td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Tri-Sodium citrate dihydrate</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A3901</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">PBS</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">964.9100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Urokinase</td>
			<td style="width:227px;">Medac</td>
			<td style="width:119px;">1976826</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">0.5% Trypsin-EDTA</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:119px;">15400054</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Giemsa stain</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A0885</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formaldehyde</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A0877</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sulfuric acid (H2SO4)</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A0655</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A1584</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium chloride (MgCl2)</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A3618</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Guanidine hydrochloride</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A1499</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;"></td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Consumables</td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">50 mL reaction tube</td>
			<td style="width:227px;">Axygen</td>
			<td style="width:119px;">SCT-50ML-25-S</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">10 mL syringe</td>
			<td style="width:227px;">Braun</td>
			<td style="width:119px;">4606108V</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Sterican needle (22G)</td>
			<td style="width:227px;">Braun</td>
			<td style="width:119px;">4657624</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">1.7 mL Microtubes</td>
			<td style="width:227px;">Brunschwig</td>
			<td style="width:119px;">MCT-175-C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">100 &#956;m cell strainer</td>
			<td style="width:227px;">Falcon</td>
			<td style="width:119px;">6.05935</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">sterile forceps</td>
			<td style="width:227px;">Bastos Viegas, SA</td>
			<td style="width:119px;">489-001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">sterile scalpel</td>
			<td style="width:227px;">Braun</td>
			<td style="width:119px;">5518059</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Primaria cell cuture dish</td>
			<td style="width:227px;">Falcon</td>
			<td style="width:119px;">353803</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">C-Chip Neubauer Improved</td>
			<td style="width:227px;">Bioswisstech</td>
			<td style="width:119px;">505050</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">cell culture flask - Flask T300</td>
			<td style="width:227px;">TPP</td>
			<td style="width:119px;">90301</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;"></td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Equipment</td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Microbiological biosafety cabinet class II</td>
			<td style="width:227px;">Skan</td>
			<td style="width:119px;">82011500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">water bath</td>
			<td style="width:227px;">Memmert</td>
			<td style="width:119px;">1305.0377</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Stripettes Serological Pipette 5ml</td>
			<td style="width:227px;">Corning</td>
			<td>4487-200ea</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">microscope</td>
			<td style="width:227px;">Olympus</td>
			<td style="width:119px;">CKX41</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">humidified incubator Heracells 240</td>
			<td style="width:227px;">Thermo scientific</td>
			<td>51026331</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Heraeus Multifuge 1S-R</td>
			<td style="width:227px;">Thermo scientific</td>
			<td style="width:119px;">75004331</td>
		</tr>
	</tbody>
</table>

an enzymatic method to rescue mesenchymal stem cells from clotted bone marrow samples

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug &#8211; clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.

连同74％的犬骨髓样本（N = 54）载有血块，当他们在我们的实验室（ 图1A）到达事实从这些样品下降MSC收益率，使我们相信，有相当多的干细胞被血块内被困。事实上，简单的DAPI染色切片凝块材料证实有核细胞以高密度（ 图1B）的存在。这最终导致低的数字可用于扩增培养MSC的，从而引发我们开发使用尿激酶的协议。血块通常运用我们的协议时基本消失。然而，方法开发过程中，我们解决凝块的问题通过称重前，后消化系统消化。这些实验显示，该未消化余数分别为15％（狗）或初始凝块重量（ 图2A）的9％（人）。 骨髓来源的MSCs一个典型特征是一相容性，坚持以细胞培养皿。研究人员利用这一特性来选择干细胞。其结果是，集落形成试验允许评价在一个简单的，定量的和可靠的方式的小区池的质量。在我们的实验室中，本文所描述的集落形成测定法已经被常规地应用于处理的所有骨髓样本（也非凝结的）。这使我们能够用该测定作为主要标准确定尿激酶消化的功效。以允许直接比较，从凝块样品滤液的细胞（ 即 ，好像凝结样品刚过滤，但尚未酶处理），并消化凝块接种在单独10厘米菜肴然后孵育两周。当可视化菌落形成单位（CFU）用吉姆萨染色（ 如图2B），我们观察到3.8倍以上的CFU比从相应的初始的滤液（ 图2C）犬样品的尿激酶反应。 ALTHough不太突出，人类样本随后用1.6倍以上CFU从样品处理类似的趋势，确认该协议的适用性。的确，消化凝块在图2B中的右排中所示的板对应于典型的结果看出的合并样本。但是，捐助变化可能很容易地从上半年导致CFU数量翻一番什么是描绘。 取菌落大小作为指标的细胞池的质量，更介质（2-5毫米）和大（&gt; 5mm）的菌落出现在平板上，从凝块接种相比，CFU从滤液（ 图2C）。这通常表明，干细胞尿激酶治疗后正常生长。另外，在集料，消化犬标本（N = 21）的比较，以样品无凝块（N = 7），得到总的MSCs用于处理样品的可比数字，虽然大间样品变异是由于观察到的异质施主人口（ 图2D）。 由于适合最后的功能测试，我们从诱发消化骨髓样本来源的MSCs分化。在自体干细胞治疗应用程序是基于MSC分化成软骨，骨或脂肪组织或MSC旁分泌信号18。细胞可以分化朝所希望的路径，通过选择适当的培养条件下分化成脂19，成骨分化20或软骨细胞谱系17被引导。因此，我们比较干细胞从骨髓血块从unclotted骨髓样本来源的间充质干。四周培养后，有可能分化的MSCs为所有三种上述谱系。这是组织学与冯科萨 （成骨细胞系）进行测试， 油红O（脂肪细胞）和阿尔辛蓝（软骨）染色，显示在群体之间分化程度无显着差异（ <str翁&gt;图3）。 <img alt="图1" src="/files/ftp_upload/52694/52694fig1.jpg" /> 图1.骨髓凝块，犬和人的骨髓样本抵达后（A）占部分凝结状态。此外，我们发现，从凝块样品的MSC产率强烈由于大量的细胞的凝块内捕集的降低。高一些的有核细胞是从犬骨髓血块（B）证明DAPI染色一冷冻切片的凝块中存在。比例尺条为200微米。 <img alt="图2" src="/files/ftp_upload/52694/52694fig2.jpg" /> 图2.隔离的消化与尿激酶骨髓间充质干血块的 。 （A）通常情况下，骨髓凝块几乎完全消失后，尿激酶摘要。在方法开发中，我们评估血块权重犬（N = 5，平均值±SD，** P≤0.01）和人体样本（N = 3，平均值±SD，NS =不显著P = 0.10），即强烈缩小后，尿激酶摘要。 （B）的一个简单的CFU试验可以作为一种信息工具用于评估一个MSC制剂的质量。为了评估摘要的功效，进行用于从骨髓抽吸分开的滤液和消化的凝块的CFU试验。两周培养后，10厘米控制板用Giemsa染色和CFU计数。该试验证实，高数量的CFU可以从凝块释放尿激酶消化并继续工作。这也由大菌落（类划分高频证实:&gt; 5毫米黑色，2-5毫米，深灰色和&lt;2毫米的浅灰色误差棒代表总CFU的标准差（n = 5狗中，n = 3人，** P≤0.01，NS =不显著，P = 0.17）。（C）图片从姬姆萨染色板的confirm所示的结果。对于所示消化凝块板对应于一个典型的结果为消化骨髓样本 - 然而，数字可能会有所不同主要是由于供体变异。 （D）在总之，应用扩展培养后的尿激酶消化协议（N = 21）的结果相媲美共MSC收益率自然血块免费样品（n = 7，平均值±SD）。使用学生t检验进行了对整个图2的统计分析。 <img alt="图3" src="/files/ftp_upload/52694/52694fig3.jpg" /> 图3比较的消化与本地unclotted样本分化潜能的。犬干细胞从凝结骨髓尿激酶（上排）和unclotted骨髓（下排）处理分离出分化成骨型和冯科萨 （巴=染色200微米），成脂的表型染色用 油红O（巴= 100微米;在插图较大的脂肪空泡倍），而软骨透露了阿辛蓝染色（巴= 100微米;复染核固红）。 <a href="https://www.jove.com/files/ftp_upload/52694/52694fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。 。</a>

Watch this Scientific Journal Video about An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples at JoVE.com

An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Mesenchymal stem cells are usually obtained from bone marrow and require expansion culture. When samples clot before processing, a protocol using the (enzymatic) thrombolytic drug urokinase can be applied to degrade the clot. Thus, cells are released and available for expansion culture. This protocol provides a rapid and inexpensive alternative to resampling.

酶法从凝块骨髓样本拯救间充质干细胞

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade ...

an-enzymatic-method-to-rescue-mesenchymal-stem-cells-from-clotted

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

9.1K 次观看.  Swiss Paraplegic Research. Mesenchymal stem cells are usually obtained from bone marrow and require expansion culture. When samples clot before processing, a protocol using the (enzymatic) thrombolytic drug urokinase can be applied to degrade the clot. Thus, cells are released and available for expansion culture. This protocol provides a rapid and inexpensive alternative to resampling.

视频: An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Isolation of Human Mesenchymal Stem Cells and their Cultivation on the Porous Bone Matrix

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs bovine bone matrix Nukbone (NKB) as a scaffold. Thus, the application of MSCs in repair and tissue regeneration processes depends principally on the efficient implementation of the techniques for placing these cells in a host tissue. For this reason, the design of biomaterials and cellular scaffolds has gained importance in recent years because the topographical characteristics of the selected scaffold must ensure adhesion, proliferation and differentiation into the desired cell lineage in the microenvironment of the injured tissue. This option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) employs bovine bone matrix as a cellular scaffold and is an efficient culture technique because the cells respond to the topographic characteristics of the bovine bone matrix Nukbone (NKB), i.e., spreading on the surface, macroporous covering and colonizing the depth of the biomaterial, after the cell isolation process. We present the procedure for isolating and culturing MSCs on a bovine matrix.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs the bovine bone matrix Nukbone (NKB) as a scaffold.

人类骨髓间充质干细胞的分离及其培养的多​​孔骨基质

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific ...

科研

发育生物学

Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such cells are not readily available, they can be obtained using different approaches that include, among the many, reprogramming and trans-differentiation, with advantages and limitations that are specific of the different techniques. Here a new strategy for the conversion of an adult mature fibroblast into an insulin-secreting cell, arbitrarily designated as epigenetic converted cells (EpiCC), is described. The method has been developed, based on the increasing understanding of the mechanisms controlling epigenetic regulation of cell fate and differentiation. In particular, the first step uses an epigenetic modifier, namely 5-aza-cytidine, to drive adult cells into a "highly permissive" state. It then takes advantage of this brief and reversible window of epigenetic plasticity, to re-address cells toward a different lineage. The approach is designated "epigenetic cell conversion". It is a simple and robust way to obtain an efficient, controlled and stable cellular inter-lineage switch. Since the protocol does not involve the use of any gene transfection, it is free of viral vectors and does not involve a stable pluripotent state, it is highly promising for translational medicine applications.

Here, a new method that allows the conversion of adult skin fibroblasts into insulin-secreting cells is presented. This technique is based on epigenetic conversion, does not involve the use of retroviral vectors nor the acquisition of a stable pluripotent state. It is therefore highly promising for translational medicine applications.

后生转换为安全和简单的方法来获得成人皮肤成纤维细胞胰岛素分泌细胞

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

从人胎盘组织源分离和间充质干扩张/基质细胞

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel cell population specifically selected for growth in human serum supplemented medium. These cells are characterized by morphological, phenotypic, and molecular features distinct from MSCs and we named them Mesodermal Progenitor Cells (MPCs). MPCs are round, with a thick highly refringent core region; they show strong, trypsin resistant adherence to plastic. Failure to expand MPCs directly revealed that they are slow in cycling. This is as also suggested by Ki-67 negativity. On the other hand, culturing MPCs in standard medium designed for MSC expansion, gave rise to a population of exponentially growing MSC-like cells. Besides showing mesenchymal differentiation capacity MPCs retained angiogenic potential, confirming their multiple lineage progenitor nature. Here we describe an optimized highly reproducible protocol to isolate and characterize hBM-MPCs by flow cytometry (CD73, CD90, CD31, and CD45), nestin expression, and F-actin organization. Protocols for mesengenic and angiogenic differentiation of MPCs are also provided. Here we also suggest a more appropriate nomenclature for these cells, which has been re-named as "Mesangiogenic Progenitor Cells".

Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow

Here we describe an optimized, highly reproducible protocol to isolate Mesodermal Progenitor Cells (MPCs) from human bone marrow (hBM). MPCs were characterized by flow cytometry and nestin expression. They showed the ability to give rise to exponentially growing MSC-like cell cultures while retaining their angiogenic potential.

从人骨髓分离Mesangiogenic祖细胞（多用途储值卡）

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel ...

Temporal Ordering of Dynamic Expression Data from Detailed Spatial Expression Maps

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a strict species-specific periodicity. The periodicity of the process is regulated by a molecular oscillator, known as the "segmentation clock," acting in the PSM cells. This clock drives the oscillatory patterns of gene expression across the PSM in a posterior-anterior direction. These so-called clock genes are key components of three signaling pathways: Wnt, Notch, and fibroblast growth factor (FGF). In addition, Notch signaling is essential for synchronizing intracellular oscillations in neighboring cells. We recently gained insight into how this may be mechanistically regulated. Upon ligand activation, the Notch receptor is cleaved, releasing the intracellular domain (NICD), which moves to the nucleus and regulates gene expression. NICD is highly labile, and its phosphorylation-dependent turnover acts to restrict Notch signaling. The profile of NICD production (and degradation) in the PSM is known to be oscillatory and to resemble that of a clock gene. We recently reported that both the Notch receptor and the Delta ligand, which mediate intercellular coupling, themselves exhibit dynamic expression at both the mRNA and protein levels. In this article, we describe the sensitive detection methods and detailed image analysis tools that we used, in combination with the computational modeling that we designed, to extract and overlay expression data from distinct points in the expression cycle. This allowed us to construct a spatio-temporal picture of the dynamic expression profile for the receptor, the ligand, and the Notch target clock genes throughout an oscillation cycle. Here, we describe the protocols used to generate and culture the PSM explants, as well as the procedure to stain for the mRNA or protein. We also explain how the confocal images were subsequently analyzed and temporally ordered computationally to generate ordered sequences of clock expression snapshots, hereafter defined as "kymographs," for the visualization of the spatiotemporal expression of Delta-like1 (Dll1) and Notch1 throughout the PSM.

The segmentation clock drives oscillatory gene expression across the pre-somitic mesoderm (PSM). Dynamic Notch activity is key to this process. We use imaging and computational analyses to extract temporal dynamics from spatial expression data to demonstrate that Delta ligand and Notch receptor expression oscillate in the vertebrate PSM.

从详细的空间表达图谱的动态表达数据的时间排序

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

从人类脐带和胎盘胎儿分离和鉴定间充质干细胞

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

Preparation of Plasma Membrane Vesicles from Bone Marrow Mesenchymal Stem Cells for Potential Cytoplasm Replacement Therapy

We have previously reported on the generation of plasma membrane vesicles (PMVs) through the mechanical extrusion of mammalian cells. The fusion of PMVs with mitochondrial deficient Rho0 cells restored mitotic activity under normal culture conditions. Atherosclerosis, type 2 diabetes, Alzheimer&#39;s disease, and cancer are age-related diseases that have been reported to be associated with multiple mechanical and functional defects in the cytosol and organelles of a variety of cell types. Bone marrow mesenchymal stem cells (BMSCs) represent a unique cell population from the bone marrow that possess self-renewal capabilities while maintaining their multipotency. The supplementation of senescence cells with young cytoplasm from autologous BMSCs via the fusion of PMVs provides a promising approach to ameliorate or even reverse age-associated phenotypes. This protocol describes how to prepare PMVs from BMSCs via extrusion through a polycarbonate membrane with 3 &#181;m pores, determine the existence of mitochondria and examine the maintenance of membrane potential within PMVs using a confocal microscope, concentrate PMVs by centrifugation, and carry out the in vivo injection of PMVs into the gastrocnemius muscle of mice.

Age-related diseases are associated with multiple defects in components of the cytoplasm. Here, we present a protocol to prepare plasma membrane vesicles from bone marrow mesenchymal stem cells. This technique could potentially be used as a means of cytoplasm replacement therapy to ameliorate or even reverse age-associated phenotypes.

从骨髓间充质干细胞制备血浆膜囊泡用于潜在的细胞质替代治疗

We have previously reported on the generation of plasma membrane vesicles (PMVs) through the mechanical extrusion of mammalian cells. The fusion of PMVs ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

人类骨髓微环境在小鼠中的生物工程及其通过实时成像的可视化

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

小鼠骨髓基质细胞与骨破骨祖的分离、培养及分化

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

Isolation and Characterization of Cardiac Mesenchymal Stromal Cells from Endomyocardial Bioptic Samples of Arrhythmogenic Cardiomyopathy Patients

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The isolation of these cells offers the possibility of studying their involvement in cardiac diseases, and, in addition, provides a useful primary cell model to investigate biological mechanisms.
Here, the method for the isolation of C-MSC from arrhythmogenic cardiomyopathy patients&#39; bioptic samples is described. The endomyocardial biopsy sampling is guided in the right ventricular areas adjacent to the scar visualized by electro-anatomical mapping. The digestion of the biopsies in collagenase and their plating on a plastic dish in culture medium to allow C-MSC growth is described. The isolated cells can be expanded in culture for several passages. To confirm their mesenchymal phenotype, the description of immuno-phenotypical characterization is provided. C-MSC are able to differentiate into several cell types like adipocytes, chondrocytes, and osteoblasts: in the context of ACM, characterized by adipocyte deposits in patients&#39; hearts, the protocols for the adipogenic differentiation of C-MSC and the characterization of lipid droplet accumulation are described.

In this article, a method to isolate cardiac mesenchymal stromal cells from endomyocardial bioptic samples of arrhythmogenic cardiomyopathy patients is provided. Their characterization and the protocol to boost their adipogenic differentiation are described.

致心律失常心肌病患者心内膜心肌活检样间质基质细胞的分离与鉴定

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The ...

酶法从凝块骨髓样本拯救间充质干细胞

摘要

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此视频中的章节

人类骨髓间充质干细胞的分离及其培养的多孔骨基质

后生转换为安全和简单的方法来获得成人皮肤成纤维细胞胰岛素分泌细胞

从人胎盘组织源分离和间充质干扩张/基质细胞

从人骨髓分离Mesangiogenic祖细胞（多用途储值卡）

从详细的空间表达图谱的动态表达数据的时间排序

从人类脐带和胎盘胎儿分离和鉴定间充质干细胞

从骨髓间充质干细胞制备血浆膜囊泡用于潜在的细胞质替代治疗

人类骨髓微环境在小鼠中的生物工程及其通过实时成像的可视化

小鼠骨髓基质细胞与骨破骨祖的分离、培养及分化

致心律失常心肌病患者心内膜心肌活检样间质基质细胞的分离与鉴定

酶法从凝块骨髓样本拯救间充质干细胞

摘要

探索更多视频

此视频中的章节

人类骨髓间充质干细胞的分离及其培养的多​​孔骨基质

后生转换为安全和简单的方法来获得成人皮肤成纤维细胞胰岛素分泌细胞

从人胎盘组织源分离和间充质干扩张/基质细胞

从人骨髓分离Mesangiogenic祖细胞（多用途储值卡）

从详细的空间表达图谱的动态表达数据的时间排序

从人类脐带和胎盘胎儿分离和鉴定间充质干细胞

从骨髓间充质干细胞制备血浆膜囊泡用于潜在的细胞质替代治疗

人类骨髓微环境在小鼠中的生物工程及其通过实时成像的可视化

小鼠骨髓基质细胞与骨破骨祖的分离、培养及分化

致心律失常心肌病患者心内膜心肌活检样间质基质细胞的分离与鉴定

人类骨髓间充质干细胞的分离及其培养的多孔骨基质