Name: an enzymatic method to rescue mesenchymal stem cells from clotted bone marrow samples
Uploaded: 2015-04-12T15:00:00.000+00:00
Duration: 8 min 58 s
Description: Watch this Scientific Journal Video about An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples at JoVE.com

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?...

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

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

Research

JoVE Journal

Developmental Biology

9.2K 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

Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, milk and urine. One subset of these RNAs are the posttranscriptional regulators &#8211; microRNAs (miRNAs). To delineate the miRNAs produced by specific cell types, in vitro culture systems can be used to harvest and profile exRNAs derived from one subset of cells. The secreted factors of mesenchymal stem cells are implicated in alleviating numerous diseases and is used as the in vitro model system here. This paper describes the process of collection, purification of small RNA and library generation to sequence extracellular miRNAs. ExRNAs from culture media differ from cellular RNA by being low RNA input samples, which calls for optimized procedures. This protocol provides a comprehensive guide to small exRNA sequencing from culture media, showing quality control checkpoints at each step during exRNA purification and sequencing.

This protocol demonstrates how to purify extracellular microRNAs from cell culture media for small RNA library construction and next generation sequencing. Various quality control checkpoints are described to allow readers to understand what to expect when working with low input samples like exRNAs.

人骨髓间充质干细胞细胞外微 rna 的分离、测序和分析

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, ...

遗传学

Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular cartilage and subchondral bone in recent years led to increased attention to restoration of the entire osteochondral unit. In comparison to chondral lesions the regeneration of osteochondral defects is much more complex and a far greater surgical and therapeutic challenge. The damaged tissue does not only include the superficial cartilage layer but also the subchondral bone. For deep, osteochondral damage, as it occurs for example with osteochondrosis dissecans, the full thickness of the defect needs to be replaced to restore the joint surface 1. Eligible therapeutic procedures have to consider these two different tissues with their different intrinsic healing potential 2. In the last decades, several surgical treatment options have emerged and have already been clinically established 3-6.
Autologous or allogeneic osteochondral transplants consist of articular cartilage and subchondral bone and allow the replacement of the entire osteochondral unit. The defects are filled with cylindrical osteochondral grafts that aim to provide a congruent hyaline cartilage covered surface 3,7,8. Disadvantages are the limited amount of available grafts, donor site morbidity (for autologous transplants) and the incongruence of the surface; thereby the application of this method is especially limited for large defects.
New approaches in the field of tissue engineering opened up promising possibilities for regenerative osteochondral therapy. The implantation of autologous chondrocytes marked the first cell based biological approach for the treatment of full-thickness cartilage lesions and is now worldwide established with good clinical results even 10 to 20 years after implantation 9,10. However, to date, this technique is not suitable for the treatment of all types of lesions such as deep defects involving the subchondral bone 11.
The sandwich-technique combines bone grafting with current approaches in Tissue Engineering 5,6. This combination seems to be able to overcome the limitations seen in osteochondral grafts alone. After autologous bone grafting to the subchondral defect area, a membrane seeded with autologous chondrocytes is sutured above and facilitates to match the topology of the graft with the injured site. Of course, the previous bone reconstruction needs additional surgical time and often even an additional surgery. Moreover, to date, long-term data is missing 12.
Tissue Engineering without additional bone grafting aims to restore the complex structure and properties of native articular cartilage by chondrogenic and osteogenic potential of the transplanted cells. However, again, it is usually only the cartilage tissue that is more or less regenerated. Additional osteochondral damage needs a specific further treatment. In order to achieve a regeneration of the multilayered structure of osteochondral defects, three-dimensional tissue engineered products seeded with autologous/allogeneic cells might provide a good regeneration capacity 11.
Beside autologous chondrocytes, mesenchymal stem cells (MSC) seem to be an attractive alternative for the development of a full-thickness cartilage tissue. In numerous preclinical in vitro and in vivo studies, mesenchymal stem cells have displayed excellent tissue regeneration potential 13,14. The important advantage of mesenchymal stem cells especially for the treatment of osteochondral defects is that they have the capacity to differentiate in osteocytes as well as chondrocytes. Therefore, they potentially allow a multilayered regeneration of the defect.
In recent years, several scaffolds with osteochondral regenerative potential have therefore been developed and evaluated with promising preliminary results 1,15-18. Furthermore, fibrin glue as a cell carrier became one of the preferred techniques in experimental cartilage repair and has already successfully been used in several animal studies 19-21 and even first human trials 22.
The following protocol will demonstrate an experimental technique for isolating mesenchymal stem cells from a rabbit's bone marrow, for subsequent proliferation in cell culture and for preparing a standardized in vitro-model for fibrin-cell-clots. Finally, a technique for the implantation of pre-established fibrin-cell-clots into artificial osteochondral defects of the rabbit's knee joint will be described.

An experimental technique for the treatment of osteochondral defects in the rabbit's knee joint is described. The implantation of allogeneic mesenchymal stem cells into osteochondral defects provides a promising development in the field of tissue engineering. The preparation of fibrin-cell-clots in vitro offers a standardized method for implantation.

异基因骨髓间充质干细胞植入兔子的膝关节软骨缺损治疗纤维蛋白凝块

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular ...

医学

Isolation and Enrichment of Rat Mesenchymal Stem Cells (MSCs) and Separation of Single-colony Derived MSCs

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and can be easily separated from the hematopoietic stem cells (HSCs) due to their plastic adherence. This protocol describes how to isolate MSCs from rat femurs and tibias. The isolated cells were further enriched against two MSCs surface markers CD54 and CD90 by magnetic cell sorting. Expression of surface markers CD54 and CD90 were then confirmed by flow cytometry analysis. HSC marker CD45 was also included to check if the sorted MSCs were depleted of HSCs. MSCs are naturally quite heterogeneous. There are subpopulations of cells that have different shapes, proliferation and differentiation abilities. These subpopulations all express the known MSCs markers and no unique marker has yet been identified for the different subpopulations. Therefore, an alternative approach to separate out the different subpopulations is using cloning cylinders to separate out single-colony derived cells. The cells derived from the single-colonies can then be cultured and evaluated separately.

Isolation and Enrichment of Rat Mesenchymal Stem Cells MSCs and Separation of Single-colony Derived MSCs

Rat MSCs were isolated from femurs and tibias and then enriched by magnetic cell sorting. Sorted cells were confirmed for the expression of surface markers by flow cytometry. These cells were also cultured at clonal density to form single colonies and then these colonies were separated by cloning cylinders.

分离和富集大鼠间质干细胞（MSCS）及衍生的干细胞分离单细胞集落

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and ...

生物学

Mesenchymal Stromal Cell Culture and Delivery in Autologous Conditions: A Smart Approach for Orthopedic Applications

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on potential biohazard and inflammation risks exerted by xenogeneic nutrients for their culture. Human derivatives or recombinant materials are the first choice candidates to reduce these reactions. Therefore, culture supplements and materials of autologous origin represent the best nutrients and the safest products.
Here, we describe a new protocol for the isolation and culture of bone marrow hMSCs in autologous conditions &#8212;&#160;namely,&#160;patient-derived serum as a supplement for the culture medium and fibrin as a scaffold for hMSC administration. Indeed, hMSC/fibrin clot constructs could be extremely useful for several clinical applications. In particular, we focus on their use in orthopedic surgery, where the fibrin clot derived from the donor&#39;s own blood allowed effective cell delivery and nutrient/waste exchanges. To ensure optimal safety conditions, it is of the utmost importance to avoid the risks of hMSC transformation and tissue overgrowth. For these reasons, the approach described in this paper also indicates a minimally ex vivo hMSC expansion, to reduce cell senescence and morphologic changes, and short-term osteo-differentiation before implantation, to induce osteogenic lineage specification, thus decreasing the risk of subsequent uncontrolled proliferation.

Culturing human Mesenchymal Stromal Cells (hMSCs) with autologous serum, reduces the risks of rejection by xenogeneic material and other negative effects. It also allows for the recovery of a subset of mesodermal progenitors, which can deliver fresh hMSCs. Embedding hMSCs in an autologous fibrin clot enables easy handling and effective surgical implantation.

间质干细胞培养和交付自体条件:用于骨科应用程序的智能方法

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on ...

生物工程

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80&#8211;90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).

This protocol illustrates a safe and efficient procedure to modify CD133+ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.

MicroRNA 移植成人骨髓造血干细胞促进细胞工程与磁性靶向结合的协议

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

表型分析和分离小鼠造血干细胞和传承的承诺祖

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

An Enzyme-free Method for Isolation and Expansion of Human Adipose-derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. As a clinically relevant cell type, optimal methods are needed to isolate and expand these cells in vitro. Most methods to isolate adipose-derived MSCs (ADSCs) rely on harsh enzymes, such as collagenase, to digest the adipose tissue. However, while effective at breaking down the adipose tissue and yielding a high ADSC recovery, these enzymes are expensive and can have detrimental effect on the ADSCs &#8212; including the risks of using xenogeneic components in clinical applications. This protocol details a method to isolate ADSCs from fresh lipoaspirate and abdominoplasty samples without enzymes. Briefly, this method relies on mechanical disassociation of any bulk tissue followed by an explant-type culture system. The ADSCs are permitted to migrate out of tissue and onto the tissue culture plate, after which the ADSCs can be cultured and expanded in vitro for any number of research and/or clinical applications.

This protocol provides an enzyme-free method for isolating mesenchymal stem cells from abdominoplasty and lipoaspirate samples using an explant method. The absence of harsh enzymes or centrifugation steps provides for clinically relevant stem cells that can be used for studies in vitro or transferred back to the clinic.

一种分离和扩增人类脂肪衍生的美源性干细胞的无酶方法

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

从人骨骼肌隔离血管源性多能前体

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

优化从胸腺和骨髓中分离基质细胞

Stromal Cell Isolation From Hematopoietic Organs

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens through which to study previously unresolved heterogeneity at steady state, as well as changes in cell type representation induced by extrinsic stresses or during aging. Here, we present step-wise protocols for the isolation of high-quality stromal cell populations from murine and human thymus, as well as murine bone and bone marrow. Cells isolated through these protocols are suitable for generating high-quality single-cell multiomics datasets. The impacts of sample digestion, hematopoietic lineage depletion, FACS analysis/sorting, and how these factors influence compatibility with single-cell sequencing are discussed here. With examples of FACS profiles indicating successful and inefficient dissociation and downstream stromal cell yields in post-sequencing analysis, recognizable pointers for users are provided. Considering the specific requirements of stromal cells is crucial for acquiring high-quality and reproducible results that can advance knowledge in the field.

作者聚焦：使用基质细胞分离进行单细胞测序推进造血研究

Here we present protocols that enable isolation of stromal cells from murine bone, bone marrow, thymus and human thymic tissue compatible with single-cell multiomics.

从造血器官中分离基质细胞

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens ...

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

人骨髓间充质干细胞细胞外微 rna 的分离、测序和分析

异基因骨髓间充质干细胞植入兔子的膝关节软骨缺损治疗纤维蛋白凝块

分离和富集大鼠间质干细胞（MSCS）及衍生的干细胞分离单细胞集落

间质干细胞培养和交付自体条件:用于骨科应用程序的智能方法

MicroRNA 移植成人骨髓造血干细胞促进细胞工程与磁性靶向结合的协议

表型分析和分离小鼠造血干细胞和传承的承诺祖

一种分离和扩增人类脂肪衍生的美源性干细胞的无酶方法

从人骨骼肌隔离血管源性多能前体

从造血器官中分离基质细胞

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

人骨髓间充质干细胞细胞外微 rna 的分离、测序和分析

异基因骨髓间充质干细胞植入兔子的膝关节软骨缺损治疗纤维蛋白凝块

分离和富集大鼠间质干细胞（MSCS）及衍生的干细胞分离单细胞集落

间质干细胞培养和交付自体条件:用于骨科应用程序的智能方法

MicroRNA 移植成人骨髓造血干细胞促进细胞工程与磁性靶向结合的协议

表型分析和分离小鼠造血干细胞和传承的承诺祖

一种分离和扩增人类脂肪衍生的美源性干细胞的无酶方法

从人骨骼肌隔离血管源性多能前体

从造血器官中分离基质细胞

人类骨髓间充质干细胞的分离及其培养的多孔骨基质