笔者衷心感谢，并承认医学研究理事会资助的这项研究 - MRC-DPFS金码G1000788-98812。

1.Scaffold制作和消毒<ol><li>溶解的PCL在1,1,1,3,3,3-六氟异丙醇，得到10％w / v的浓度。如所描述的在博斯沃思等人 ，9静电纺丝聚合物溶液（参数：20千伏1毫升/小时，有20厘米）。并收集在旋转的心轴（600转）的边缘排列的纤维。用解剖刀除去收集纤维的色带，然后切成较短的长度 - 3厘米（对于槽和旋转容器）和4厘米（用于细胞培养插入）的长度。 </li><li>用细镊子浸入蒸馏水中个别条和删除。 </li><li>拿着拇指和食指之间的两端;手动拧条，直到它类似于线程。 </li><li>简要地浸没在蒸馏水此线状支架和放置在干净的，非纤维状的卡晾干。 </li><li>一旦干燥，代替单独的干净的微量离心管中，并在蒸馏水中加入1毫升50％体积/体积乙醇。关闭盖子，并留下24小时。 注意：执行下层流下列步骤： </li><li>放置离心管在层流柜和吸出50％的v / v溶液。用1ml 70％体积/体积乙醇蒸馏水，靠近盖更换和离开24小时。 </li><li>重复的90％和在蒸馏水水（1ml体积）100％体积/体积乙醇。 </li><li>洗涤用磷酸盐缓冲盐溶液（PBS）中，每次洗涤24小时，（2×1毫升）脚手架两次。 </li><li>除去PBS中，并添加1 ml的细胞培养介质。注：脚手架已经准备好使用后的24小时。 </li></ol> 2。确定脚手架面积和单元数<ol><li>使用光学显微镜和图像处理软件，测量沿其长度的静电纱线的直径，以确定一个平均值。 </li><li>假设纱是一个圆柱棒和近似使用表面积： </li><li>其中，A =表面积，R =半径和H =长度 注意：表面积计算为18902，800微米2。此外，应该指出的是，实际表面积比该计算更大的纱是由数百个细纤维，这将增加的表面积的。因此细胞的更大数目应能附着到支架上。然而，这并不影响正在测试组之间的直接比较。 </li><li>确定细胞可以通过附着到暴露的支架表面的最大数量： 注意：使用光学显微镜和图像处理软件，人类间质干细胞的直径被确定为20微米（假设细胞是圆的），因此在这种情况下，细胞= 60200的数。 </li></ol> 3.脚手架设置 - 细胞培养插入（图1A） <ol><li>在层流中，打开无菌的6孔细胞培养插入，并从更广泛的环状体中分离的短环带齿。 </li><li>取环带齿朝上。悬垂1所述4厘米支架在环使中心的两侧确保它重叠。取环形体和位置上的带齿环和支架和向下推确保将支架保持在适当的位置，并通过位于细胞培养插管的中心。 </li><li>将细胞培养插管与支架成6孔，低结合板的孔。 </li><li>加入10 mL培养基的脚手架。 </li></ol> 4.脚手架建立 - 槽（图1B） <ol><li>下的层流，代替聚（四氟乙烯）（PTFE）波谷成单独的培养皿和培养介质加入10毫升的低谷。 </li><li>使用镊子披覆3厘米支架进料槽中的一个，并确保其长度位于平行于槽的长边缘。 </li></ol> 5.脚手架建立 - 生物反应器容器（图1C） <ol><li>在层流，将10毫升无菌PBS通过生物反应器容器的主要港口，并留下了10分钟。 </li><li>移除PBS并更换用8ml培养基。 </li><li>使用镊子，经由主端口插入3厘米支架之一到容器。 </li><li>关闭掉的主要港口。 </li></ol> 6.细胞计数<ol><li>从骨髓，根据制造商的协议到通道4之前收获来自培养人间充质干细胞（hMSC的）。 </li><li>从75厘米吸媒体2瓶含有从骨髓（第4代，80％融合）得到的hMSCs。 </li><li>用10ml无菌PBS和抽吸洗细胞。 </li><li>加入3 ml胰蛋白酶孵育烧瓶在37℃，5％的CO 2，直到细胞已经从烧瓶表面脱落。 </li><li>添加7毫升培养基使酶失活，这总体积（10毫升）转移到一个离心管中。 </li><li>通过上下吹打数次重悬该细胞悬浮液，以均匀分散中的细胞媒体和限制细胞团块（10毫升吸管）。 </li><li>取出20微升细胞悬液，转移到一个血球。 </li><li>放置在血细胞计数X10客观光显微镜下的图像。 </li><li>聚焦网格线和计数在网格的每个角落的细胞，在4×4方块数和落入正方形内和那些穿过右手或底部边界线。计数为每组4×4平方（每格4计数）。 </li><li>重复再悬浮和细胞计数三次（步骤6.6-6.9）。 </li><li>计算平均细胞计数，并确定所需的细胞再悬浮（细胞浓度60200在200μl）介质的体积。例如，确定从血球的平均细胞数，然后确定细胞从三个独立计数的总体平均数。由1×10 4乘以此，得到每毫升的细胞数，然后乘以细胞悬浮液的总体积，得到以细胞的数量TAL。使用下面的公式来确定所需的悬浮介质体积： </li><li>离心细胞悬浮液在241×g离心5分钟。 </li></ol> 7.细胞种植<ol><li>从离心管中吸出介质离开细胞沉淀，并替换为所计算出的介质体积。 </li><li>重悬的细胞和培养基对偶数混合。 </li><li>使用P200的吉尔森吸管，缓缓地分配200μl的细胞悬浮液在每个支架通过运行沿该支架的长度和介质液面下的移液管的尖端。离开原状20分钟。 </li><li>对于生物反应器的船只，免除200微升细胞悬液通过主要港口。补足其余2毫升培养经由注射器端口媒体，得到为10ml的总体积。 </li></ol> 8.实验开始<ol><li>转让生物反应器船只到农村信用社，4DQ生物反应器，并设置9的转速旋转。 </li><li>陈德良sfer所述孔板与细胞培养插入和波谷到摇动板，并设置以30rpm旋转。 </li><li>传送孔板与细胞培养插入和波谷至细胞培养箱设定为37℃，5％的CO 2（静态培养）的货架。 </li></ol> 9. DNA检测<ol><li>制备溶液 - 裂解缓冲液，1×TE缓冲液，DNA的标准和细胞DNA工作液 - 按照制造商的说明。 </li><li> 4小时后，从孵化器中取出样品，并在层流放置。 </li><li>所有样品取出介质分数，并放置在单独标示离心管。离心管241 XG。去除上清，加3毫升裂解液。悬浮溶液分手细胞沉淀。 </li><li>使用镊子取下支架并将其放置在装有3毫升裂解液（用于生物反应器内的血管支架离心管，取出支架，用来吸取我之前直径）。用于细胞培养插入物内保持支架，第一释放该支​​架通过切割所述支架附近使用解剖刀刀片的边缘。 </li><li>加入3毫升裂解液到每个支架插座和刮去表面（大力鼓吹的生物反应器的船只）。除去裂解缓冲液，并放置在单独标示离心管中。 </li><li>涡旋每个离心管约1分钟，以确保细胞的足够的搅拌和缓冲，并鼓励细胞膜裂解。 </li><li>在一个黑色的96孔板，加入100微升的裂解液对每个样品部分 - 脚手架，媒体和好（副本）。 </li><li>在黑暗中，加入100微升细胞DNA溶液到含有裂解缓冲液的所有孔中，并轻轻混匀。 </li><li>包括孔用不含DNA和细胞的DNA溶液中以提供一个阴性和阳性对照的孔板裂解缓冲液。 </li><li>使用荧光板阅读器，测量孔的吸光度S使用485nm激发和520 nm发射。 </li><li>与从DNA标准按照制造商的指示生成的标准曲线进行比较的数据。 </li></ol> 10.扫描电子显微镜（SEM）的固定<ol><li>执行以下步骤在层流：4小时后，从他们的插座和地点取出一个新的6孔板（不同孔）内的所有支架。 </li><li>用PBS洗两次支架。 </li><li>执行以下步骤打开板凳上：每孔加入2毫升的PBS 1.5％V / V戊二醛，确保支架完全覆盖。 </li><li>离开板至少30分钟，在4℃，进行细胞固定。 </li><li>除去固定液，用PBS洗两次支架。 </li><li>脱水支架随着乙醇浓度的蒸馏水开始用50％体积/体积，随后70％体积/体积至90％体积/体积。对于每一个浓度，充分浸没在solutio支架N（2ml）中，并离开3分钟。丢弃的解决方案和重复。 </li><li>通过完全浸没在溶液中的支架（2毫升）中，并静置5分钟，脱水在100％乙醇。丢弃的解决方案和重复。 </li><li>化学干燥使用六甲基二（HMDS）在通风橱支架。沉浸在HMDS的支架（2毫升），并留下5分钟。取出HMDS和重复。 </li><li>除去HMDS，并允许支架进行干燥。安装在市售的SEM存根的支架（在此情况下的不锈钢存根与粘合剂碳片）。 </li><li>到缓解的SEM观察中，涂层用金溅射镀膜机进行2分钟的样品，以确保一个薄而均匀的覆盖。 </li><li>内SEM和地方样本使用5千电子伏的电子束可视化细胞接种支架。 </li></ol>

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The authors declare that they have no competing financial interests.

从生物聚合物制成电纺纤维基质经常用于支持细胞附着和增殖为生物材料和/或组织工程应用11,12。在这些情况下，基质常常薄板纤维容易覆盖细胞培养井板的整个基础，从而在与接种的细胞可提高细胞附着完全接触的。然而，如果生物材料支架不完全覆盖所述孔板的底部，有一个高的机会，该接种的细胞的相当大的比例不会停留在与支架接触，并最终将不能附着。该研究调查了几种不同的方法对种子细胞到支架不覆盖孔板的底部，以确定可能被推荐用于未来的基于细胞的实验的优化技术。 五个不同的调校进行了研究（ 图1）：scaffo使用低绑定孔板内的细胞培养插入LDS（静电纱）举行，要么保持静态条件下或摇动30转;支架置于狭窄的PTFE槽内，并保持静态或动摇在30转;和安置生物反应器内旋转的船只9转支架。确定细胞，通过DNA分析附着在静电纱线的数量显示出附件的所有接种设置窗口（ 图2）低的百分比;这进一步从扫描电子显微照片（SEM）（ 图3）确认。最大的细胞附着 - 30％〜18060细胞-was观察举行了细胞培养插入内，进行连续运动的纱线。有趣的是，最低细胞附着（15％），用于通过细胞培养插入持有，但在静态条件下保持的纱线，这将表明，包含径向运动对保持细胞与支架接触的积极效果达到了。然而，但是应当注意的是，媒体的流量连续旋绕可能负责从SEM图像中观察到的细胞团块。该摇动板被设置在它的最低设置 - 30rpm下 - 这可能是一个限制到这种设置。使用较慢的径向运动可有助于预防或减少细胞凝集，也可以提高细胞附着作为细胞将经历较小的力。未来的实验应该着力优化的理想摇床速度提高细胞附着。掺入运动对于保持槽内没有导致了类似的趋势纱线，与这两种情况下，得到18％的附着性（〜10836个细胞）;虽然这可能是由于在槽内的支架的部分浮起（观察放置在摇动器板上的槽），因为它们并没有固定在基座上。支架的局部浮将防止已经从进入与材料和粘附接触下沉到槽的底部的任何细胞。在t他的特殊设置中，该槽被装在一个培养皿中，并共10毫升体积的介质中加入。槽的尺寸小意味着大多数媒体内的培养皿本，如果有任何的运动，细胞可能漂离槽到培养皿并保持完全脱离支架接触。为了克服这些限制，进一步的实验应该包括在该协议中，从而使支架的端部被固定到用无菌细针槽的底部的额外步骤，因为这应当防止其浮起和运动（特别是对于暴露于支架径向运动），这最终应导致附接至所述支架单元的数量增加。细胞的16％的已附连在旋转容器内存在的纱线。尽管是一个行之有效的技术为3D的文化，并出现从船只的主要港口拆除支架，这可能导致松散ATTAC问题丢失HED细胞。容器可以完全打开将消除这个问题;这些是可以购买的，但都显着高于在该研究中使用的一次性容器更昂贵。 这项研究表明，目前的问题播种支架不包括标准的细胞培养孔板的整个基。播种一个已知的细胞数目导致小于三分之一附着到所述支架，尽管支架的表面积，允许所有的细胞附着。这可能在其他基于细胞的试验，可能评估生物相容性和细胞材料/细胞 - 细胞的行为和相互作用与该支架作为一个潜在的未来医疗装置的有害后果。该研究的进一步限制可能包括4小时的时间点-尽管是足够长的时间，以确保初始细胞接种（细胞已被证明牢固附着到基板30分钟13,14,15内），它可能是合理的，在vestigate稍后的时间点，提供细胞较长的时间帧期间不增殖，因为这本来歪斜的起始细胞数。还原的介质体积，在此情况下10毫升，还可以改善细胞和支架之间的接触，并最终增加细胞附着。未来的研究也应考虑细胞活力作为细胞接种的过程中可能会导致细胞损伤和/或细胞死亡16。细胞的DNA分析不可行的和非存活细胞区分，因为这样的活/死测定法，例如，将突出生存能力的水平。 本次调查提高认识到细胞附着在支架的实际数量，尽管播种一个已知量。对于依赖于细胞的起始数的研究，这是非常重要的是，研究人员知道究竟有多少该数字做实际上附着于感兴趣的底物。

支架例行正在开发和研究的生物材料和组织工程应用。因此，它们通常被接种细胞及其在体外行为通过确定细胞增殖和细胞数，例如测定法表征。进行实验，如这些，当务之急是初始小区数量是已知的，并研究经常述在每毫升或厘米2细胞的数目而言播种浓度。虽然这是很好的做法，特别是对于按比例放大的目的，不考虑细胞附着在支架的表面（其也依赖于生物材料表面1的粘结性）的实际数量。这对于支架不覆盖细胞培养井板的整个基更是如此作为细胞可以从构建体都消失，而且，由于实验的通常的静态性质，可能永远不会回到与英特的材料接触休息。电纺纤维纱是一个支架未覆盖的井（ 图1A）的基部的一个很好的例子。在这种情况下，低结合孔板，但没有经表面处理的应用，以防止细胞附着在板的表面，因此扭曲的任何公基测定法的结果。 孔板容易地用于细胞接种到支架的，但它们不是唯一可用的方法。旋转细胞培养系统，一种生物反应器由生命科学部于上世纪80年代开发的NASA，同样可以用于在与模拟微重力三维（3D）环境种子支架。这种类型的生物反应器仍然是一个流行的选择与世界各地的研究人员和研究中已经纳入了细胞信号2,3，干细胞4,5和组织工程6,7。是什么让旋转生物反应器最好孔板是维修的3D环境中，这有助于防止分化的细胞，从去分化，这是常有的情况下，当常规的2D条件8内进行培养。 本文研究不同的技术来作为制作博斯沃思等人 ，9，以便最大限度地附连到这些支架一个4小时期间内的细胞的初始数量上静电聚（ε -己内酯）纤维丝接种人类间质干细胞。对于2D培养，纱线牢固孔板或定做的聚（四氟乙烯）（PTFE）波谷内保持与静态条件下保持，或摇动以30rpm。对于3D的文化，纱线和细胞生物反应器旋转的船只在9转速范围内举行。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Distilled water</td>
			<td>in-house supply</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Merck</td>
			<td>1117271000</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline solution</td>
			<td>Life Technologies</td>
			<td>70013016</td>
			<td></td>
		</tr>
		<tr>
			<td>Human mesenchymal stem cells</td>
			<td>PromoCell GmbH</td>
			<td>C-12974</td>
			<td></td>
		</tr>
		<tr>
			<td>MSC culture media</td>
			<td>PromoCell GmbH</td>
			<td>C-28010B</td>
			<td>Warmed to 37 oC before use</td>
		</tr>
		<tr>
			<td>Supplement mix</td>
			<td>PromoCell GmbH</td>
			<td>C-39810</td>
			<td>Add to culture media</td>
		</tr>
		<tr>
			<td>Antibiotic/antimyotic mix</td>
			<td>Sigma-Aldrich</td>
			<td>A5955</td>
			<td>Add to culture media</td>
		</tr>
		<tr>
			<td>Trypsin (0.05 %) EDTA (0.02 %)</td>
			<td>Sigma-Aldrich</td>
			<td>59417C</td>
			<td>Warmed to 37 oC before use</td>
		</tr>
		<tr>
			<td>Cell culture flasks (T75)</td>
			<td>Becton Dickinson Ltd</td>
			<td>353110</td>
			<td></td>
		</tr>
		<tr>
			<td>Low binding 6-well plates</td>
			<td>Costar Corning</td>
			<td>3471</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well CellCrowns</td>
			<td>Scaffdex</td>
			<td>C00003S</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri-dish 50 ml deep</td>
			<td>Sterilin</td>
			<td>124</td>
			<td></td>
		</tr>
		<tr>
			<td>PTFE troughs</td>
			<td>in-house production</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable RCCS vessels 10 ml</td>
			<td>Synthecon</td>
			<td>D-410</td>
			<td></td>
		</tr>
		<tr>
			<td>4 Vessel Rotary Cell Culture System bioreactor</td>
			<td>Synthecon</td>
			<td>RCCS-4DQ</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaker plate</td>
			<td>Stuart</td>
			<td>SSM1</td>
			<td>Mini Orbital Shaker</td>
		</tr>
		<tr>
			<td>Haemocytometer</td>
			<td>Digital Bio</td>
			<td>DHC-F01</td>
			<td>Disposable C-Chip</td>
		</tr>
		<tr>
			<td>Centrifuge tube</td>
			<td>Deltalab</td>
			<td>352096, 429901</td>
			<td>15 ml and 50 ml</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Hettich</td>
			<td>Rotafix 32 A</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 3 ml</td>
			<td>Shield Medicare Ltd</td>
			<td>3039820</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes</td>
			<td>Sterilin</td>
			<td>40305, 47310, 40125</td>
			<td>5, 10 and 25 ml</td>
		</tr>
		<tr>
			<td>Gilson pipettes</td>
			<td>SLS</td>
			<td>F144801, F144802, F123600, F123601, F123602</td>
			<td>P2 - P1000</td>
		</tr>
		<tr>
			<td>Pipette tips</td>
			<td>SLS</td>
			<td>PIP7852, PIP7834, PIP7840</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro test tube 1.5 ml</td>
			<td>Eppendorf</td>
			<td>30125.15</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>9002-93-1</td>
			<td></td>
		</tr>
		<tr>
			<td>PicoGreen Assay</td>
			<td>Invitrogen</td>
			<td>P7589</td>
			<td>Assay set-up in the dark</td>
		</tr>
		<tr>
			<td>Black 96-well plate</td>
			<td>Greiner Bio One</td>
			<td>655086</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent plate-reader</td>
			<td>BGM Labtech</td>
			<td>FLUOstar Optima</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde 25 %</td>
			<td>TAAB Laboratories</td>
			<td>G002</td>
			<td>Made to a concentration of 1.5 % v/v in PBS</td>
		</tr>
		<tr>
			<td>Hexamethyldisilazane (HMDS)</td>
			<td>Sigma</td>
			<td>999-97-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminium stubs (SEM)</td>
			<td>Agar Scientific</td>
			<td>G301</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon tabs (SEM)</td>
			<td>Agar Scientific</td>
			<td>G3347N</td>
			<td></td>
		</tr>
		<tr>
			<td>Gold sputter coater</td>
			<td>Edwards</td>
			<td>S150B</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscope (SEM)</td>
			<td>Phenom World</td>
			<td>Phenom Pro</td>
			<td></td>
		</tr>
	</tbody>
</table>

optimizing attachment of human mesenchymal stem cells on poly(&#949;-caprolactone) electrospun yarns

研究生物材料和组织工程通常包括基于细胞的体外研究，这需要的起始细胞数初始知识。尽管研究人员通常引用其接种密度，这并不一定表示已附着于有问题的材料的细胞的实际数量。这是特别为材料或支架，其不包括标准的细胞培养孔板的底部的情况。本研究探讨接种在一个已知的数上静电纺丝的聚（ε - 己内酯）纱线在培养4小时后，自人骨髓间充质干细胞的初始附着。静电纱内几个不同的调校举行，包括生物反应器容器旋转9的转速，位于低结合孔板和聚四氟乙烯（PTFE）的槽放置在培养皿中的细胞培养插入物。后两者经受静态条件或定位在振荡器上板（30 <spañ风格=“字体大小：14px的;线高度：28px;”&gt; RPM）。后4小时在37℃，5％的CO 2中 ，接种细胞的位置是由细胞的DNA分析测定。从容器中取出，放入裂解液支架。媒体馏分类似地除去并离心 - 上清液丢弃，沉淀捣碎用裂解缓冲液。裂解缓冲液加入每个容器，或良好，并且刮下以释放任何细胞可以存在。细胞的DNA分析测定细胞在支架内时，媒体和井馏分的百分比。细胞附着率较低为所有的实验室设备，具有最大的附着（30％）的细胞培养插入物内保持并进行摇动运动的纱线。这项研究提出认识到附加到脚手架不管通过上述细胞接种密度的细胞的实际数量。

该结果强调以下4小时后播种每种实验设置的细胞的位置的影响。 图2A表明细胞在此期间已附着在支架的表面的百分比。 8.5皮克/细胞A转换因子是用来测量的DNA含量为细胞数转换，从而确定电池10的比例。对于所有的播种调校研究，细胞附着的百分比相对较低，具有最大的细胞粘附（30％）的细胞培养插入物内保持并摇动以30rpm（插入振荡器）的支架。坚持最低（15％）是在细胞培养插入内举行脚手架，并保持静态条件（将静态）下。 大量细胞是媒体级分（ 图2B）内时，最明显的是对于低结合板（插入静态）和旋转式容器内保持细胞培养插入物为50％和51％的分别。槽内保持支架证明细胞保持器本身内本的凸起数 - 48％的细胞为槽振荡器和用于槽的静态的50％。 扫描电子显微镜所允许的细胞接种支架的视觉评估（ 图3）。代表图像强调不论接种建立细胞的有限的存在在纤维表面上，。然而，更大数目的细胞和细胞聚集体是存在于细胞培养插入物内保持并摇动以30rpm（插入振荡器）的支架。 <img alt="图1" src="/files/ftp_upload/52135/52135fig1.jpg" /> 图1.实验调校，其中静电纺丝纱内举行; （A）的细胞培养物的插入和低结合孔板，（B）聚（四氟乙烯）（PTFE）和波谷培养皿;和（C）生物反应器容器。 <a href="https://www.jove.com/files/ftp_upload/52135/52135fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52135/52135fig2.jpg" /> 图2.位置细胞4小时后播种到PCL电纺的纱线采用不同播种调校的。 （A）的演示已经附连到的PCL支架的细胞的百分比（平均值±标准偏差）;（B）的突出位置细胞的百分比传播三个馏分内-媒体，以及与支架（n = 4时，数据表示为平均值）。 <a href="https://www.jove.com/files/ftp_upload/52135/52135fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" sRC =“/文件/ ftp_upload / 52135 / 52135fig3.jpg”/&gt; 图3.代表性的扫描电子显微镜对PCL电纺的纱线与人骨髓间充质干细胞，采用不同的实验调校初始播种后4小时 （以1,000倍的放大倍率的所有图像，比例尺= 130微米。） <a href="https://www.jove.com/files/ftp_upload/52135/52135fig3highres.jpg" target="_blank">请点击这里查看更大的版本这个数字。</a>

Watch this Scientific Journal Video about Optimizing Attachment of Human Mesenchymal Stem Cells on PolyÎµ-caprolactone Electrospun Yarns at JoVE.com

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&amp;#949;-caprolactone) Electrospun Yarns

This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

优化人骨髓间充质干细胞附着在聚（ε己内酯）静电纺纱线

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

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Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

9.6K 次观看.  The University of Manchester. This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

视频: Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

人类血管周围干细胞用于骨再生

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

科研

生物工程

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

开发，扩展，<em&gt;在体内</em&gt;从人类胚胎干细胞（hESCs）监测人类NK细胞和诱导多能干细胞（iPS细胞）

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

在肌肉再生的老鼠模型中移植诱导多能干细胞衍生的中生代细胞样肌原体

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Electrospun Fibrous Scaffolds of Poly(glycerol-dodecanedioate) for Engineering Neural Tissues From Mouse Embryonic Stem Cells

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among the various fabrication methods, electrospinning is the most attractive one due to its simplicity and versatility. Additionally, electrospun nanofibers mimic the size of natural extracellular matrix ensuring additional support for cell survival and growth. This study showed the viability of the fabrication of long fibers spanning a larger deposit area for a novel biodegradable and biocompatible polymer named&#160;poly(glycerol-dodecanoate) (PGD)1 by using a newly designed collector for electrospinning. PGD exhibits unique elastic properties with similar mechanical properties to nerve tissues, thus it is suitable for neural tissue engineering applications. The synthesis and fabrication set-up for making fibrous scaffolding materials was simple, highly reproducible, and inexpensive. In biocompatibility testing, cells derived from mouse embryonic stem cells could adhere to and grow on the electrospun PGD fibers. In summary, this protocol provided a versatile fabrication method for making PGD electrospun fibers to support the growth of mouse embryonic stem cell derived neural lineage cells.

Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells

Synthesis and fabrication of electrospun long fibers spanning a larger deposit area via a newly designed collector from a novel biodegradable polymer named poly(glycerol-dodecanoate) (PGD) was reported. The fibers were able to support the growth of cells derived from mouse pluripotent stem cells.

保利的静电纤维支架（甘油 - 十二双）工程神经组织从小鼠胚胎干细胞

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.
Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs.
Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)2D3]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

人肺泡骨膜间充质干细胞的分离及维生素 D 对骨膜衍生细胞成骨活性的影响

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Exploring the Potential of Mesenchymal Stem Cell Sheet on The Development of Hepatocellular Carcinoma In Vivo

An in vivo animal model that mimics human cancer could have various applications that deliver significant clinical information. The currently used techniques for the development of in vivo cancer models have considerable limitations. Therefore, in this study, we aim to implement cell sheet technology to develop an in vivo cancer model. Hepatocellular carcinoma (HCC) is successfully developed in nude rats using cell sheets created from HCC cell line cells. The cancer cell sheets are generated through intracellular adhesion and the formation of a stratified structure, controlled by the extracellular matrix. This allows for the HCC sheet transplantation into the liver and the creation of a tumor-bearing animal model within a month. In addition, the role of mesenchymal stem cells (MSC) in the development of this cancer model is investigated. In addition to the HCC cell line sheet, another two cell sheets are created: a sheet of HCC cells and bone marrow MSCs (BMMSCs) and a sheet of HCC cells and umbilical cord MSCs (UCMSCs). Sheets that have a combination of both HCC cells and MSCs are also capable of producing a tumor-bearing animal. However, the addition of MSCs reduces the size of the formed tumor, and this adverse effect on tumor development varies depending on the used MSCs' source. This indicates that a cell sheet made of certain MSC subtypes could be utilized in tumor management and control.

Exploring the Potential of Mesenchymal Stem Cell Sheet on The Development of Hepatocellular Carcinoma In Vivo

Here, we present a protocol to develop an in vivo cancer model using cell sheet technology. Such a model could be very useful for the evaluation of anticancer therapeutics.

骨髓间充质干细胞片对肝癌体外发育的潜能探讨

An in vivo animal model that mimics human cancer could have various applications that deliver significant clinical information. The currently used ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

人骨髓间充质干细胞吸收含有法卡林多醇的新型脂质包覆纳米颗粒

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

人类诱导多能干细胞衍生心肌细胞中的单细胞光学作用潜在测量

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

维持静止人类造血干细胞的文化方法

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. These include the limited number of cadaveric pancreas donors and the long-term use of immunosuppressants. Mesenchymal stem cells (MSCs) have been considered to be a potential candidate as an alternative source of islet-like cell generation. Our previous reports have successfully illustrated the establishment of induction protocols for differentiating human dental pulp stem cells (hDPSCs) to insulin-producing cells (IPCs). However, the induction efficiency varied greatly. In this paper, we demonstrate the comparison of hDPSCs pancreatic induction efficiency via integrative (microenvironmental and genetic manipulation) and non-integrative (microenvironmental manipulation) induction protocols for delivering hDPSC-derived IPCs (hDPSC-IPCs). The results suggest distinct induction efficiency for both the induction approaches in terms of 3-dimensional colony structure, yield, pancreatic mRNA markers, and functional property upon multi-dosage glucose challenge. These findings will support the future establishment of a clinically applicable IPCs and pancreatic lineage production platform.

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

This protocol presents a comparison between two different induction protocols for differentiating human dental pulp stem cells (hDPSCs) toward pancreatic lineages in vitro: the integrative protocol and the non-integrative protocol. The integrative protocol generates more insulin producing cells (IPCs).

体外 人体牙浆干细胞向胰腺血统的诱导

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. ...