Name: preparation of plasma membrane vesicles from bone marrow mesenchymal stem cells for potential cytoplasm replacement therapy
Uploaded: 2017-05-18T15:00:00.000+00:00
Duration: 9 min 34 s
Description: Watch this Scientific Journal Video about Preparation of Plasma Membrane Vesicles from Bone Marrow Mesenchymal Stem Cells for Potential Cytoplasm Replacement Therapy at JoVE.com

这项研究得到了李嘉诚基金会，广东省高校"海洋工业绿色科技"，"中国自然科学基金"项目（http://www.nsfc.gov.cn/资助号:30971665， 81172894,81370925）和广东省教育厅（http://www.gdhed.edu.cn/ Grant No.cxzd1123）。

8至12周龄的BALB / c小鼠购自上海实验动物中心（中国上海），并在特定的无病原菌和空调动物设施中饲养。动物护理和实验程序符合汕头大学实验动物使用和护理指南。 装置装配<ol><li>为了确保无菌，使用前打开组织培养罩的紫外线30分钟。 </li><li>拧下一次性25毫米过滤器单元，并将设备的盖子和底部浸入装有75％乙醇的200毫升玻璃杯中30分钟。该单元由医用级聚丙烯制成。 </li><li>使用镊子拿起装置的盖子和底部，用手摇动剩余的乙醇，并使部件在发动机罩中风干10分钟。 </li><li>在PBS中湿19mm聚碳酸酯膜，并将其小心地放置在机器人的支撑基质的顶部的单位。聚合物膜具有光滑，平坦的表面和轨道蚀刻的3μm孔。 </li><li>通过将盖子紧紧地拧紧到底部来重新组装本机。确保膜不会从中心移开。 </li><li>取出1 mL胰岛素注射器的针头，取1 mL PBS。将注射器连接到过滤器单元上，然后将PBS推入设备中以润湿组件，并测试是否有泄漏。 </li></ol> 2.产生等离子体膜囊泡（PMV） <ol><li>建立一个BMSC文化，Nemeth et al。 15 ，在含有15％FBS和1％青霉素/链霉素的DMEM培养基中。在含有5％CO 2的加湿培养箱中在37℃培养6孔板中的细胞。 </li><li>为了繁殖细胞，取出培养基并用0.5mL无钙PBS冲洗孔。 </li><li>加入0.5mL的0.25％胰蛋白酶-EDTA，并在37℃下孵育细胞3分钟轻轻敲击手掌几次，以促进细胞分离。通过加入1 mL培养基停止消化。 </li><li>将细胞收集在15mL锥形管中，并在台式离心机中以200xg旋转2分钟。将细胞重悬于4mL培养基中，并将细胞等分至6孔板的两个孔中。 注意:细胞通常在约24小时达到汇合。 </li><li>为了产生PMV，通过胰蛋白酶消化从6孔板的一个孔（约5×10 5个细胞）中收获细胞，并将细胞单分散在0.5mL的培养基中。 </li><li>将细胞装入胰岛素注射器，将其连接到过滤器单元，并快速推动柱塞以将细胞挤压通过过滤器。丢弃挤压的介质，因为它内部只应有几个PMV。 </li><li>将另外0.5 mL的培养基装入注射器，并快速将其推过过滤器。收集第二次挤出的介质，其中包含各种尺寸的PMV。 </li><li>将150μL收集的培养基装入35 mm玻璃底盘中，使PMV沉降至底部约10分钟。使用20X物镜在装有CCD相机的倒置相差显微镜下检查PMV。 </li></ol> 3.使用共焦显微镜检查PMV内的内容<ol><li>在BMSCs中进行转染，在50μL无血清DMEM中稀释2μg超螺旋质粒DNA和6μL阳离子转染试剂（ 如 PolyJet）。涡旋混匀并短暂旋转以从管的两侧收集所有液体。 </li><li>将稀释的溶液（从步骤3.1）滴加到稀释的DNA溶液中，强烈旋转1分钟，短暂旋转，并在室温下孵育10分钟。 </li><li>将已经以约30％汇合过夜的BMSCs上的旧培养基替换为2mL新鲜培养基并加入DN逐滴转染混合液。 </li><li>转染12小时后，用2 mL新鲜培养基更换。 </li><li>如上所述，为了追踪细胞质定位的蛋白质，用包含EGFP表达盒（pEGFP-N1）的质粒转染细胞。如上所述，转染48小时后通过胰蛋白酶消化获得PMV生成的细胞。 </li><li>为了追踪线粒体，用37℃的新鲜培养基中的线粒体染料（1μM，MitoTracker）染色细胞30分钟。 </li><li> 为了检测线粒体的膜电位，用花青染料JC-1（5,5&#39;，6,6&#39;-四氯-1,1&#39;，3,3&#39;-四乙基苯并咪唑基碳菁碘化物）染色细胞（10μg / mL）在新鲜培养基中在37℃下培养30分钟。 <ol><li>用0.5mL PBS洗涤细胞三次，然后如上所述通过胰蛋白酶消化收获细胞以产生PMV。 </li><li>加载150μL收集的药物将其放入35毫米玻璃底盘中，使PMV沉降至底部约10分钟。使用100X油物镜在共焦显微镜下检查PMV。 </li></ol></li><li>要检测EGFP或线粒体染料，打开488 nm激光并在505-530 nm收集信号。要检测JC-1，打开488 nm和543 nm激光，并收集505-530 nm和560-615 nm的信号。将针孔值设置为1.4左右。 </li></ol> 4.通过离心制备浓缩的PMV <ol><li>通过胰蛋白酶消化收获细胞，并如上所述在0.5mL培养基中产生PMV。 </li><li>在室温下在台式离心机中以1,200 xg的速度旋转PMV 5分钟。仔细吸取上清液，并将沉淀物重悬于100μL培养基中，轻柔旋转。 </li><li>将50μLPMV加载到35 mm玻璃底盘中，使其在室温下放置约10分钟。检查PMV在共焦显微镜下使用40X油物镜。 </li><li>将一系列稀释的聚乙烯亚胺（PEI）溶液加入到BMSC培养物中1小时，并检查细胞毒性的迹象。 注意:在这里，选择2μg/ mL的PEI，因为在BMSCs中没有检测到毒性迹象。 </li><li>通过胰蛋白酶消化收获细胞，并加入2μg/ mL的PEI 1小时，以充电细胞膜作为防止聚集的手段。如上所述，在0.5mL培养基中产生PMV。浓缩PMV并在共焦显微镜下检查它们，如上所述。 </li></ol> 5.将PMV注入腓肠肌肌肉<ol><li>为了染色膜，将1mL新鲜培养基中的CM-DiI（10mM甲基二碳酰基 - 碳青霉素）染料加入到BMSCs中30分钟。 </li><li>通过胰蛋白酶消化收获细胞（约5×10 5 ），并将其重悬于0.5mL培养基中。加入PEI（2μg/ mL）1H。如上所述，将PMV生成并浓缩在100μL培养基中。 </li><li>禁食12小时后注射巴比妥钠（10mg / kg）来麻醉BALB / c小鼠。 </li><li>用75％乙醇清洗腓肠肌外侧。使用30 G胰岛素注射器将PMV（100μL）慢慢注入腓肠肌。 </li><li>施用麻醉后，在实验的持续时间内将湿纱布放在眼睛上，并用白炽灯温热鼠标直至醒来。将鼠标单独放在个别通风笼中。 </li><li>在术后12小时收获腓肠肌，通过颈椎脱位杀死小鼠。 </li><li>在腓肠肌上涂上75％的乙醇，用剪刀打开皮肤，并切开两端的整个腓肠肌。 </li><li>用PBS冲洗肌肉，并在荧光显微镜下使用20X物镜进行检查。 </li><li>用a修剪肌肉锋利的剃刀刀片去除不带荧光的部件。将强烈的红色荧光部分切成约9毫米3立方体。 </li><li>将每个立方体嵌入最佳切割温度介质（OCT）中，将其浸没在液氮中5分钟，并将其储存在-20°C冷冻器中直到使用。 </li><li>使用低温恒温器将冷冻切片切成20μm厚，并将每个部分粘附到载玻片上。用PBS冲洗一次。 </li><li>用斑点圆圈在该部分周围绘制一圈，并加入100μLDAPI（1μg/ mL）。在黑暗中室温孵育10分钟后吸入DAPI溶液，并用PBS洗涤三次。 </li><li>将样品放在样品上，用玻璃滑盖盖住，并用指甲油密封滑块。使用100X油物镜在共焦显微镜下检查切片。 </li><li>为了检测CM-DiI，打开543nm激光并收集560-615nm的信号。要检测DAPI，请打开405nm激光并在420-480nm收集信号。将针孔值设置为约1.4。 </li></ol>

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Cytoplasm replacement therapy as proposed in this manuscript has unique advantages over other reported approaches such as gene, molecular, and cell therapy. PMVs generated from BMSCs encapsulate not only the products of stemness genes but also intact cellular organelles, which are essential to remedy the ageing phenotypes associated with senescence. When young cytoplasm is delivered to senescent cells, the malfunctioning mechanisms may gain a brief relief; at the same time, the epigenome could be reprogrammed and invigor...

<html>已经投入了大量的努力来建立基因，酶和细胞替代疗法的方法。这导致了巨大的突破，甚至临床应用1，2，3。最近，基于核转移技术的有争议的线粒体替代疗法被应用于老年妇女的体外受精或携带致死线粒体DNA突变4 。在年龄相关疾病中发现的缺陷，包括动脉粥样硬化，2型糖尿病，阿尔茨海默病和癌症，通常是多方面的。已经证明脂滴的积累;沉淀淀粉样蛋白;展开蛋白质在内质网中的保留;缺陷型蛋白酶体，自噬体和线粒体有助于这些疾病的发展或恶化"外套参照"&gt; 5,6,7,8,9,10,11。目前没有可用于直接修复细胞质和细胞器故障的机制，这导致衰老和衰老表型。 我们以前通过机械挤压哺乳动物细胞报道了生物质膜囊泡（PMVs） 12 。除了细胞核外，膜或细胞溶质中的组分（包括蛋白质和RNA）以及细胞器如线粒体都发现在PMV中。基本上，PMV可以被认为是一个微型的去核细胞。更重要的是，PMV与线粒体缺乏的Rho0细胞的融合在正常培养条件下恢复有丝分裂活性。这是关于成立的第一份报告为细胞质替代疗法建立潜在的有效途径。 骨髓间充质干细胞（BMSCs）是通常从骨髓产生并且在培养物中容易扩增的多能祖细胞。胚胎干细胞标记Oct4，Nanog和SOX2在MSCs 13中检测到低水平。端粒酶活性也是可测量的。此外，不存在共刺激分子和人白细胞抗原（HLA）II类分子以及MSC上的低HLA I类表达使其成为同种异体或"现成"使用的理想细胞再生医学和免疫调节应用14 。 在这里，我们描述如何通过挤出通过具有3-μm孔隙的聚碳酸酯膜来制备来自小鼠BMSC的PMV，确定线粒体的存在并使用confoc检查PMV中膜电位的维持通过离心法制备浓缩但不聚集的PMV，并进行PMVs 体内注射到小鼠的腓肠肌中。

<table><tbody><tr><td>IsoporeTM membranes</td><td>Millipore</td><td>TSTP04700</td><td>3 mm pore</td></tr><tr><td>Disposable filter unit</td><td>Xinya, Shanghai, China</td><td>25 mm</td><td>Medical grade polypropylene</td></tr><tr><td>Insulin syringe</td><td>BD</td><td>328446</td><td>1 mL</td></tr><tr><td>pN1-EGFP</td><td>Clontech</td><td>&amp;nbsp;6085-1</td><td></td></tr><tr><td>MitoTracker</td><td>Molecular Probes</td><td>M7514</td><td>Green FM, 1 &amp;mu;M</td></tr><tr><td>JC-1</td><td>Beyotime, Haimen, China</td><td>C2006</td><td>10 mg/mL</td></tr><tr><td>CM-DiI</td><td>Beyotime, Haimen, China</td><td>C1036</td><td>10 mM</td></tr><tr><td>PEI</td><td>Sigma</td><td>P3143</td><td>Mn = 75,000</td></tr><tr><td>Fluorescence Microscope</td><td>Nikon</td><td>Eclipse TE 2000</td><td>With CCD camera</td></tr><tr><td>Confocol Microscope</td><td>Carl Zeiss</td><td>LSM 510 Meta</td><td></td></tr><tr><td>PolyJet</td><td>SigaGen</td><td>SL100688</td><td>For cell transfection</td></tr></tbody></table>

preparation of plasma membrane vesicles from bone marrow mesenchymal stem cells for potential cytoplasm replacement therapy

我们以前报道了通过机械挤压哺乳动物细胞产生质膜囊泡（PMV）。 PMV与线粒体缺陷Rho0细胞的融合在正常培养条件下恢复有丝分裂活性。据报道，动脉粥样硬化，2型糖尿病，阿尔茨海默病和癌症是与各种细胞类型的细胞溶质和细胞器中的多种机械和功能缺陷相关的年龄相关疾病。骨髓间充质干细胞（BMSCs）代表骨髓中独特的细胞群，具有自我更新能力，同时保持其多能性。通过PMV融合补充来自自体骨髓基质干细胞的年轻细胞质的衰老细胞提供了改善或甚至逆转年龄相关表型的有希望的方法。该方案描述了如何通过使用3的聚碳酸酯膜挤出从BMSC制备PMV1; m孔，确定线粒体的存在，并使用共聚焦显微镜检查PMV内膜电位的维持，通过离心浓缩PMV，并进行体内注射PMV至小鼠腓肠肌。

成功准备PMV的关键在很大程度上取决于过滤器单元的正确组装（ 图1 ），可以通过将1mL PBS推过膜来测试。如果发生泄漏，重新组装过滤器单元并再次测试。然而，当细胞被推过膜时，泄漏只能被可靠地测试。如果在常规显微镜下仅使用10X物镜检测到几个PMV，或者如果PMV的大小大多为1μm左右，则表明大多数单元由于组装不正确而被捕获在单元内部。尺寸在约3μ...

Watch this Scientific Journal Video about Preparation of Plasma Membrane Vesicles from Bone Marrow Mesenchymal Stem Cells for Potential Cytoplasm Replacement Therapy at JoVE.com

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

与年龄相关的疾病与细胞质组分中的多种缺陷有关。在这里，我们提出了从骨髓间充质干细胞制备质膜囊泡的方案。这种技术可能被用作细胞质替代疗法的手段，以改善或甚至逆转年龄相关表型。

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 ...

preparation-plasma-membrane-vesicles-from-bone-marrow-mesenchymal

Research

JoVE Journal

Developmental Biology

7.5K 次观看.  Shantou University. 与年龄相关的疾病与细胞质组分中的多种缺陷有关。在这里，我们提出了从骨髓间充质干细胞制备质膜囊泡的方案。这种技术可能被用作细胞质替代疗法的手段，以改善或甚至逆转年龄相关表型。 

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

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

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.

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 ...

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

在干细胞移植在体外模拟缺血/再灌注模型

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

医学

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 ...

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. Currently, efforts are being made to regenerate damaged cartilage with ex vivo expanded chondrocytes or bone marrow-derived mesenchymal stem cells (BMSCs). However, the restricted viability and instability of these cells limit their application in cartilage reconstruction. Human induced pluripotent stem cells (hiPSCs) have received scientific attention as a new alternative for regenerative applications. With unlimited self-renewal ability and multipotency, hiPSCs have been highlighted as a new replacement cell source for cartilage repair. However, obtaining a high quantity of high-quality chondrogenic pellets is a major challenge to their clinical application. In this study, we used embryoid body (EB)-derived outgrowth cells for chondrogenic differentiation. Successful chondrogenesis was confirmed by PCR and staining with alcian blue, toluidine blue, and antibodies against collagen types I and II (COL1A1 and COL2A1, respectively). We provide a detailed method for the differentiation of cord blood mononuclear cell-derived iPSCs (CBMC-hiPSCs) into chondrogenic pellets.

Here, we propose a protocol for chondrogenic differentiation from cord blood mononuclear cell-derived human induced pluripotent stem cells.

脐带血诱导的多能干细胞的软骨形成颗粒形成

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

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

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

重组胶原蛋白 i. 肽载体在生物反应器中的应用及其作为细胞传递系统的潜力

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

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 ...

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.

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.

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

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological regulators of organismal homeostasis and important indicators of pathologies; in the meantime, their immense potential to establish accessible and controllable disease therapeutics is emerging. Mesenchymal stem cells (MSCs) can release large amounts of EVs in culture, which have shown promise to jumpstart effective tissue regeneration and facilitate extensive therapeutic applications with good scalability and reproducibility. There is a growing demand for simple and effective protocols for collecting and applying MSC-EVs. Here, a detailed protocol is provided based on differential centrifugation to isolate and characterize representative EVs from cultured human MSCs, exosomes, and microvesicles for further applications. The adaptability of this method is shown for a series of downstream approaches, such as labeling, local transplantation, and systemic injection. The implementation of this procedure will address the need for simple and reliable MSC-EVs collection and application in translational research.

The present protocol describes the differential centrifugation for isolating and characterizing representative EVs (exosomes and microvesicles) from cultured human MSCs. Further applications of these EVs are also explained in this article.

培养的人间充质干细胞细胞外囊泡的分离、表征和治疗应用

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological ...

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

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

酶法从凝块骨髓样本拯救间充质干细胞

在干细胞移植在体外模拟缺血/再灌注模型

从人骨髓分离Mesangiogenic祖细胞（多用途储值卡）

脐带血诱导的多能干细胞的软骨形成颗粒形成

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

重组胶原蛋白 i. 肽载体在生物反应器中的应用及其作为细胞传递系统的潜力

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

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

培养的人间充质干细胞细胞外囊泡的分离、表征和治疗应用

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

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

酶法从凝块骨髓样本拯救间充质干细胞

在干细胞移植在体外模拟缺血/再灌注模型

从人骨髓分离Mesangiogenic祖细胞（多用途储值卡）

脐带血诱导的多能干细胞的软骨形成颗粒形成

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

重组胶原蛋白 i. 肽载体在生物反应器中的应用及其作为细胞传递系统的潜力

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

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

培养的人间充质干细胞细胞外囊泡的分离、表征和治疗应用

人类骨髓间充质干细胞的分离及其培养的多孔骨基质