这项研究是由赠款，以RLM从澳大利亚国家健康和医学研究委员会（赠款＃606658）。

1。细胞培养（3-7天之前至BBB大会） 1.1。小学的人脑微血管内皮细胞（建筑物能源效益守则） 建筑物能源效益守则分别购得。细胞通过正常人类脑皮质组织的分散酶分解所产生并提供冻结在通道3（&lt;12个群体倍增）。 <ol><li> 基质 :大衣组织培养器皿"附件因子"按照制造商的说明。 </li><li> 电池保养 :保持在含血清的完全培养基的BEC。对于实验，文化的BEC完全无血清培养基。维持细胞在湿润的5％CO 2，21％O 2的培养箱中在37℃下 注意 :作为一个替代专门的试剂，0.1％（重量/体积）明胶溶液可以作为包衣试剂而DMEM/F-12补充有15mM的HEPES，10％（V / V）胎牛血清（FCS）， 2mM的L-谷氨酰胺，50微克/毫升庆大霉素，20微克/毫升肝素和20μg/ ml的内皮细胞生长添加物，可用于BEC维修。 </li><li> 子培养进行试验 :分流汇合的BEC在1:3至1:6的比例，根据期望的时间为下一次使用（3-7天）。改变维持培养基每3天。使用的BEC多达15代。 </li></ol> 1.2。 SVG胎儿星形胶质细胞细胞系 SVG人胎星形胶质细胞22最初来源于人胎脑转化与复制缺陷型猿猴病毒40的原代培养。 <ol><li> 维护 :文化SVG细胞在含Earle的补充有20％（V / V）FCS（用于维持其亲代原代细胞22相同血清百分比），2mM的L-谷氨酰胺，50单位/ ml青霉素和平衡盐溶液中的最小必需培养基50微克/毫升链霉素。维持细胞在湿润的5％CO 2，21％O 2的</子&gt;培养箱中在37℃下</li><li> 子培养进行试验 :分流汇合SVGs以1:5至1:10的比例，根据期望的时间为下一次使用（3-7天）。除去胰蛋白酶的是不是由于血清的培养基中的高含量必需的。改变培养基每3天。使用SVGs多达20次传代。 </li></ol> 2。大会在人类体外血脑屏障模型和共培养（第0天） 评论 : 在体外接触血脑屏障模型的人是根据发布的协议13,23编制，修改。 2.1。刀片的涂层<ol><li>把组织培养插入物（直径6.5毫米与聚酯的多孔膜，3微米孔径）在所提供的24孔板的孔中。刀片的外套腔面<s> </s>隔夜在湿润的37℃培养箱与大鼠Ⅰ型胶原（20微克/厘米2; 50微升的132微克/毫升胶原蛋白I在0.02％乙酸的Milli-Q水（MQH 2 O））。 （无论是从近腔和腔侧）与MQH 2 O洗一次插入物以除去残余的酸。 注:脑毛细血管基底膜不包含I型胶原，但主要是层粘连蛋白，Ⅳ型胶原亚型，nidogens和硫酸乙酰肝素蛋白多糖9。出于这个原因，一些研究人员利用涂层剂，如Ⅳ型胶原，纤连蛋白13，或基质胶（BD公司）16一个更真实的BBB级建模。 </li></ol> 2.2。在底部（近腔）膜表面播种SVGs <ol><li>反转插入，轻轻地适应周围的多孔膜一小段弹性硅胶管的边缘（"外管";〜10mm长8毫米的直径，1.6 mm管壁），创建本质上是一种新的，远高于近腔面（ 图2A）。 </li><li>插入东东的底部DS被密封，以避免介质的泄漏。首先，准备由硅管（称为"内部管道";〜8mm长，3.2毫米的直径，1.6毫米壁; 图2B）的插头（ 图2A和图2B），在其一端带有一个塑料密封锥面密封（〜3mm的长;制备切割的0.2毫升聚合酶链反应（PCR）管的底部; 图2B）。此锥体不仅密封该内部管道腔，也扩大了它的边缘紧密配合到插入。接着，用无菌镊子，将硅酮插头插入腔腔并将其推进，直到它到达向上到约1-2毫米的膜（ 图2A）。 </li><li>接着，播种4×10 4个细胞的SVG在200μl的SVG维持培养基（SECTION 1.2.2以上）直接进入硅氧烷以及上面的腔外表面的膜（ 图2A）。允许SVGs附着在培养箱中在至少4个小时。 注意:为了保持无菌输送组装的插入在2 6孔板（1板倒置在其他; 图2C）之间，以在手术过程中尽量减少暴露在未过滤的空气。硅胶管塞和洗涤的乙醇和高压灭菌后使用。 注意:使用插头（第2.2.2节）只适用于刀片，膜孔径大于1μm。与膜孔直径≤1微米很少泄漏，需要外部硅胶管（2.2.1节），只有配件。此外，一些比其他胶原蛋白涂层剂I（见附注2.1节）可能会阻止泄漏甚至到3微米的小孔。凭经验确定这一点。 </li><li>为了监视你的星形胶质细胞的粘附状态，在种子成平行的星形胶质细胞的细胞悬浮液的一个标准的96孔相同的体积。该井具有相同的表面面积为6.5 mm插入（0.33 平方厘米），并且允许细胞的相差显微镜易于可视化比插入膜。当你观察在控制星形胶质细胞有足够的坚持96孔，星形胶质细胞已充分也坚持到插入膜。 </li><li>当星形胶质细胞已充分地附着在控制96孔，转让插入组件到组织培养罩（见上文附注），并轻轻地取下外部硅胶管和插头。返回插入到正常（ 即竖直）方向到含有800微升/孔的BEC维持液（上述第1.1.2节）所提供的水井。 </li></ol> 2.3。播种建筑物能源效益守则进入管腔室上方的星形胶质细胞种2×10 4的BEC在200μl到胶原包被的腔表面和返回的樱雪RTS进入孵化器。 2.4。共培养实验前，共培养了SVG / BEC种子镶在BEC介质3天没有媒体的变化。 3。实验与人体血脑屏障模型（3天） <ol><li>通过分别与600μL和100μL无血清的BEC介质，取代近腔和管腔媒体刺激体外血脑屏障先洗细胞。 </li><li>再次吸出管腔介质，并替换为100μl的无血清培养基中以刺激剂。 注意:如果近腔室的刺激是必要的（从星形胶质细胞车厢激活体外 BBB），吸近腔中，替换600微升无血清培养基与刺激剂。 </li><li>测试各实验组的一式三份（因此，如果进行管腔的刺激，这需要3×100微升，我们建议稀释重<html>剂到它们的最终浓度在350微升无血清培养基中的每个组，以减少井之间移液误差）。 </li><li> （对于后者我们用异硫氰酸荧光素（FITC）偶联的白蛋白）和/或TEER 13,16,25的测定如先前所述进行标准的细胞旁和/或标记的示踪剂16,24的跨细胞通透性测定。 </li></ol>考虑到在基线渗透性波动的实验之间，分析，以不含细胞的涂层刀片（其用作用于最大导磁率的参考），并利用公式载体治疗组的变化相对导磁率:导磁率（最大值的％）=（媒介物组的平均渗透率值）×100 - 车辆组）/（透过性的插入件坯件的值的平均渗透率值 - 试验刀片的渗透率值。 4。多孔膜细胞可视化NT"&gt; 注释 :未染色的细胞上插入膜是难以相衬或微分干涉对比（DIC）显微镜观察，因为膜是相当不透明的干扰光传输为了解决这个问题，我们开发了细胞的各种染色程序上。插入件，从而大大提高细胞的外观上的膜，通常，染色膜的插入而仍然附着在插入，在该孔中。该膜应该只在用于安装目的的染色过程结束时被删除。 4.1。苏木素染色<ol><li>固定刀片用冰冷的4％（重量/体积）的多聚甲醛（PFA）在磷酸盐缓冲盐水（PBS; 130 mM氯化钠，10毫米的Na 2 HPO 4，10mM的的NaH 2 PO 4，pH 7.2）中20分钟。在PBS洗一次。 </li><li>淹没在插入2分钟，0.1％Mayer氏苏木素液。 </li><li>轻轻地用自来水冲洗刀片（浸没的插件ERT在一个烧杯中含有自来水轻轻吹打多余的水出来）。插入转移到斯科特的自来水溶液（2 g / L的碳酸氢钠3，20 g / L的硫酸镁4）〜20秒，直到所需的蓝色发展。用自来水（4.1.3节）洗一遍插入。一个可选的伊红染色可以在这里介绍通过浸渍在插入10秒后自来水冲洗1％曙红溶液。 </li><li>空气干燥插入膜。使用手术刀（割开膜边缘）去除膜和将其安装在明场成像的载玻片 注:拆卸和安装膜前用100％乙醇随后二甲苯如果二-N-butylphthalateinxylene（DPX）安装介质是利用清洗刀片。 </li></ol> 4.2。扫描电子显微镜（SEM） <ol><li>固定刀片用冰冷的4％（重量/体积）的PFA的PBS 20分钟。在PBS中洗涤3次。 </li><li>后固定在膜中的1％30分钟（w / v的在MQH 2 O），四氧化锇</li><li>于水（4.1.3节）洗净刀片和脱水在不断升级的乙醇梯度（50％，70％，90％，每次5分钟，然后用3倍100％乙醇为每间隔10分钟）。 </li><li>治疗脱水刀片与六甲基二硅氮烷（HMDS）的混合物:乙醇（1:2，然后5分钟，2:1）。 </li><li>治疗插入物具有100％的HMDS 5分钟，干燥的条件下空气干燥和存储。 </li><li>使用手术刀（割开膜边缘）取下膜。用扫描电子显微镜检查膜。 </li></ol> 4.3。免疫荧光<ol><li>固定刀片用冰冷的4％（重量/体积）的PFA的PBS中20分钟，洗一次，在Tris-缓冲盐水（TBS;的50mM的Tris-HCl pH值为7.6，154 mM氯化钠，0.22微米过滤的）。 </li><li>如先前所述26上的插入膜进行细胞的免疫染色。 </li><li>使用手术刀（割开膜边缘）取下膜将其安装在AG姑娘幻灯片荧光封固剂。通过荧光显微镜检查膜。 </li></ol>

<ol>
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作者什么都没有透露。

医学研究脑病变遭受多大的平移难度。在急性缺血性中风的区域，例如，许多药物，在动物模型中表现出极大的承诺未能在诊所38,39。的原因，这些令人失望的结果是多种多样的，包括临床前测试系统的不忠的人中风的情况，并从动物研究21获得的结果夸大。一个策略，以提高临床前研究结果的预测价值临床阶段的额外开发基于人体细胞的体外模型，这些可以提...

血脑屏障是末梢血液循环和中枢神经系统中，关键的是负责大脑止血的维护之间的专用接口。它包括不同的脑微血管内皮细胞（的BEC）这是由几个细胞和非细胞成分（见下文）在功能上的影响，以形成一个紧凑的和动态的网关到大脑。在生理条件下是BBB限制血细胞，血浆成分和有害物质，所有潜在的神经毒性的通道，进入大脑。平行地，血脑屏障选择性交换密钥离子和营养物（葡萄糖和氨基酸）和代谢废物的大脑和循环之间精确地保持大脑环境1,2。近年来它是日益明显的是，血脑屏障的破坏发生在各种慢性脑病变，如神经变性或炎症有关的疾病（ 如阿尔茨海默氏病和含多处Ë硬化，分 ​​别）3，以及在像缺血性中风急性4条件。 脑内皮细胞（的BEC）的独特属性血脑屏障通过脑环境5在很大程度上引起，特别是由星形胶质细胞6,7。有越来越多的了解，其它细胞类型，例如周细胞8，神经元和神经胶质细胞的1,3，以及基底膜9，支持的BEC和一起形成一个功能单元被称为"神经血管单元"（NVU），它同时夫妻神经元代谢需要它们的供应毛细血管10。 血脑屏障在病理情况下参与的基础无数次尝试开发体外 BBB模型，以协助BBB相关的研究11,12。这些模型的目标是模仿尽可能接近体内血脑屏障的特性根据NVU原理。 体外 </em&gt;的BBB模型通常依赖于紧密连接形成的BEC的单层（主要从牛13，人14大鼠15，鼠标器16，和猪17,18起点），在多孔膜上培养连同支持星形胶质细胞（广泛由德利等人，2005年11审阅）。 星形胶质细胞可在非接触条件下生长的组织培养孔的底部，通过用的BEC通过可溶性因子16的培养基还从通信的BEC（栽培在膜的上表面）分离。在更先进的模型的更好类似于血脑屏障的体内的解剖结构，星形胶质细胞被保持在接触的条件和直接邻近的BEC 13,15,17（ 图1）在膜的另一侧进行培养。这种配置使建筑物能源效益守则和星形胶质细胞之间的身体接触，成立时星形胶质细胞项目通过多孔膜的工序。重要的是，对于发生的真实接触的孔隙应≥1μm的直径，由于星形细胞的最终尺不能穿过较小的孔径（ 即 0.4微米）14,15。值得注意的是，接触BBB系统中演示了一些研究要优于他们对他们的跨内皮细胞电阻（TEER）和各种示踪剂13,17,18的内皮通透性值非接触的同行。媒体流的另外一个维度在一些体外 BBB模型剪力适用于内皮细胞为脑血管12,19的接近模拟最近添 ​​加。 一个技术障碍产生接触BBB模型时，克服星形胶质细胞是克服重力在多孔膜的腔外表面的播种。上一页协议13,20，其中星形胶质细胞被简单地播种在媒体上的顶部下降verted插入，只允许短的播种时间（ 即 10分钟，13或2小时20）被发现在我们的手中是不够正常细胞附着。使用这种基本的方法，较长的星形胶质细胞实习期间需要不断的监测插件被频繁打开培养箱的（导致温度，pH值和湿度的波动），也容易出现不均匀的细胞种植由于通过小孔漏的媒体，尤其是如果毛孔大于1μm就业。 在这里，我们描述了一个通用协议，用于制备接触血脑屏障模型。我们的程序包括用于在反相插入，即解决了上面提到的限制细胞接种另一种方法。该方法允许星形胶质细胞的原状附着到腔外表面的膜，在平衡培养箱，对于延长的时间周期。作为一个结果，星形细胞的均匀播种实现这增加屏障质量并最大限度地减少刀片之间的基底透气性的变化。 由于使用人类细胞是人类有关的研究21重要的是，我们另外在这篇文章中展示出一种新颖的首要人权的BEC相结合的具体运用和永生化人星形胶质细胞的建立接触人体血脑屏障模型的高通量筛选能力。由于观察细胞在多孔膜是很困难的，我们也详细的染色技术，可协助合流的决心和细胞形态的多孔膜。最后，我们举例说明如何我们人类血脑屏障模型可以被用来研究组织型纤溶酶原激活剂（T-PA）的影响 - 一个血块破坏酶可作为急性缺血性中风的唯一的治疗选择 - 对血脑屏障。

<table border="0" cellpadding="0" cellspacing="0" width="863">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Attachment Factor</td>
			<td style="width:193px;">Cell-Systems Corporation</td>
			<td style="width:171px;">4Z0-210</td>
			<td style="width:243px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Brain microvascular endothelial cells (BECs), primary, human</td>
			<td style="width:193px;">Cell-Systems Corporation</td>
			<td style="width:171px;">ACBRI 376</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Complete medium</td>
			<td style="width:193px;">Cell-Systems Corporation</td>
			<td style="width:171px;">4Z0-500</td>
			<td style="width:243px;">Supplemented with CSC JetFuel</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Complete serum-free medium</td>
			<td style="width:193px;">Cell-Systems Corporation</td>
			<td style="width:171px;">SF-4Z0-500</td>
			<td style="width:243px;">Supplemented with CSC RocketFue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">DMEM/F-12 with 15 mM HEPES</td>
			<td style="width:193px;">Life Technologies</td>
			<td style="width:171px;">11330-032</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Endothelial cells growth supplement (from bovine origin)</td>
			<td style="width:193px;">Sigma-Aldrich</td>
			<td style="width:171px;">E2759-15MG</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">External silicone tubing</td>
			<td style="width:193px;">Watson-Marlow</td>
			<td style="width:171px;">913.A080.016</td>
			<td style="width:243px;">Pumpsil brand, 8 mm internal diameter, 1.6 mm wall</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Foetal calf serum</td>
			<td style="width:193px;">Lonza</td>
			<td style="width:171px;">14-501F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Gentamycin sulfate</td>
			<td style="width:193px;">Life Technologies</td>
			<td style="width:171px;">15750-060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Heparin sodium</td>
			<td style="width:193px;">Pfizer</td>
			<td style="width:171px;"></td>
			<td style="width:243px;">1,000 U/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Hexamethyldisilazane (HMDS)</td>
			<td style="width:193px;">Sigma-Aldrich</td>
			<td style="width:171px;">H4875</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Human brain microvascular endothelial cells</td>
			<td style="width:193px;">Cell-Systems Corporation</td>
			<td style="width:171px;">ACBRI 376</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Internal silicone tubing</td>
			<td style="width:193px;">Watson-Marlow</td>
			<td style="width:171px;">913.A032.016</td>
			<td style="width:243px;">Pumpsil brand, 3.2 mm internal diameter, 1.6 mm wall</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">L-Glutamine</td>
			<td style="width:193px;">Life Technologies</td>
			<td style="width:171px;">25030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Mayer&#8217;s hematoxylin solution</td>
			<td style="width:193px;">Amber Scientific</td>
			<td style="width:171px;">MH</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Minimum essential medium with Earle&#8217;s balanced salt solution</td>
			<td style="width:193px;">HyClone Laboratories</td>
			<td style="width:171px;">SH30244.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Penicillin/Streptomycin</td>
			<td style="width:193px;">Life Technologies</td>
			<td style="width:171px;">15140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Rat collagen I</td>
			<td style="width:193px;">Trevigen</td>
			<td style="width:171px;">3440-100-01</td>
			<td style="width:243px;">Cultrex brand</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:257px;">Tissue culture inserts</td>
			<td style="width:193px;">Corning Life Sciences</td>
			<td style="width:171px;">3472</td>
			<td style="width:243px;">Transwell brand, 6.5 mm in diameter, with polyester porous membrane, 3 &#181;m pore-size</td>
		</tr>
	</tbody>
</table>

improved method for the preparation of a human cell-based, contact model of the blood-brain barrier

血 - 脑屏障（BBB）包括不可渗透的，但适应性脑毛细血管紧紧控制大脑环境。血脑屏障的破坏已经隐含在许多脑病变的病因，这就需要对人类的发展在体外 BBB模型，以协助临床相关研究。之间迄今所描述的众多BBB模型，静态（无流量），与血脑屏障模型，其中星形胶质细胞和脑血管内皮细胞（的BEC）的共培养在多孔膜的相对侧上，成为一种简单但真实的系统来模拟BBB与高通量筛选能力。然而这种模式的产生提出了一些技术上的挑战。在这里，我们描述了一个协议，用于制备一种利用一种新型的初级人类的BEC和永生化星形胶质细胞的组合接触人体血脑屏障模型。具体来说，我们为细节上的倒插入细胞播种一种创新的方法以及指定插入背板T染色技术和体现我们如何使用我们的模型为BBB-的相关研究。

为了建立一个人，接触BBB模型，我们不得不培养于多孔膜SVGs和的BEC具有3微米的孔隙大小，显示允许的星形胶质细胞的最终英尺通道，用于与内皮细胞14,15,27,28接触。完整的接触模型的示意图如图1（左图）。通过接触系统呈现的主要技术挑战是需要的种子的星形胶质细胞对抗重力在膜腔外表面。我们已通过主要产生一个可移动的硅氧烷以及在近腔膜表面的顶部上，同时防止介?...

Watch this Scientific Journal Video about Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier at JoVE.com

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

成立血脑屏障（BBB）人体模型的受益研究与BBB级故障相关的大脑条件。我们在这里描述的改进的技术，用于制备接触BBB模型，它允许在多孔膜的相对侧上的人星形胶质细胞和脑血管内皮细胞的共培养的。

改进方法对血脑屏障的人体细胞为主，接触模型的制备

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

improved-method-for-preparation-human-cell-based-contact-model-blood

Research

JoVE Journal

Bioengineering

16.2K 次观看.  Monash University. 成立血脑屏障（BBB）人体模型的受益研究与BBB级故障相关的大脑条件。我们在这里描述的改进的技术，用于制备接触BBB模型，它允许在多孔膜的相对侧上的人星形胶质细胞和脑血管内皮细胞的共培养的。 

视频: Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform to study protein-protein and protein-lipid interactions3, monitor drug delivery4,5, and encapsulation4. Phospholipids naturally create curved lipid bilayers, distinguishing itself from a micelle.6 Liposomes are traditionally classified by size and number of bilayers, i.e. large unilamellar vesicles (LUVs), small unilamellar vesicles (SUVs) and multilamellar vesicles (MLVs)7. In particular, the preparation of homogeneous liposomes of various sizes is important for studying membrane curvature that plays a vital role in cell signaling, endo- and exocytosis, membrane fusion, and protein trafficking8. Several groups analyze how proteins are used to modulate processes that involve membrane curvature and thus prepare liposomes of diameters &lt;100 - 400 nm to study their behavior on cell functions3. Others focus on liposome-drug encapsulation, studying liposomes as vehicles to carry and deliver a drug of interest9. Drug encapsulation can be achieved as reported during liposome formation9. Our extrusion step should not affect the encapsulated drug for two reasons, i.e. (1) drug encapsulation should be achieved prior to this step and (2) liposomes should retain their natural biophysical stability, securely carrying the drug in the aqueous core. These research goals further suggest the need for an optimized method to design stable sub-micron lipid vesicles.
Nonetheless, the current liposome preparation technologies (sonication10, freeze-and-thaw10, sedimentation) do not allow preparation of liposomes with highly curved surface (i.e. diameter &lt;100 nm) with high consistency and efficiency10,5, which limits the biophysical studies of an emerging field of membrane curvature sensing. Herein, we present a robust preparation method for a variety of biologically relevant liposomes.
Manual extrusion using gas-tight syringes and polycarbonate membranes10,5 is a common practice but heterogeneity is often observed when using pore sizes &lt;100 nm due to due to variability of manual pressure applied. We employed a constant pressure-controlled extrusion apparatus to prepare synthetic liposomes whose diameters range between 30 and 400 nm. Dynamic light scattering (DLS)10, electron microscopy11 and nanoparticle tracking analysis (NTA)12 were used to quantify the liposome sizes as described in our protocol, with commercial polystyrene (PS) beads used as a calibration standard. A near linear correlation was observed between the employed pore sizes and the experimentally determined liposomes, indicating high fidelity of our pressure-controlled liposome preparation method. Further, we have shown that this lipid vesicle preparation method is generally applicable, independent of various liposome sizes. Lastly, we have also demonstrated in a time course study that these prepared liposomes were stable for up to 16 hours. A representative nano-sized liposome preparation protocol is demonstrated below.

This protocol describes an extrusion method for preparing lipid vesicles of sub-micron sizes with a high degree of homogeneity. This method uses a pressure-controlled system with controlled nitrogen flow rates for liposome preparation. The lipid preparation1,2, liposome extrusion, and size characterization will be presented herein.

恒压控制的挤压方法制备纳米脂质体

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform ...

科研

生物工程

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

通过胎盘的外来物质和纳米材料的运输率测定<em&gt;体外</em&gt;人胎盘灌注型号

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

对于I型胶原蛋白的制备皮肤方法的改进

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific molecular mechanisms are identified, new therapeutic strategies can be developed to treat abnormalities in specific behaviors caused by degenerative diseases or aging of the nervous system. The marine snail Aplysia californica is well suited for the investigations of cellular and molecular basis of behavior because neural circuitry underlying a specific behavior could be easily determined and the individual components of the circuitry could be easily manipulated. These advantages of Aplysia have led to several fundamental discoveries of neurobiology of learning and memory. Here we describe a preparation of the Aplysia nervous system for the electrophysiological and molecular analyses of individual neurons. Briefly, ganglion dissected from the nervous system is exposed to protease to remove the ganglion sheath such that neurons are exposed but retain neuronal activity as in the intact animal. This preparation is used to carry out electrophysiological measurements of single or multiple neurons. Importantly, following the recording using a simple methodology, the neurons could be isolated directly from the ganglia for gene expression analysis. These protocols were used to carry out simultaneous electrophysiological recordings from L7 and R15 neurons, study their response to acetylcholine and quantitating expression of CREB1 gene in isolated single L7, L11, R15, and R2 neurons of Aplysia.

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

Marine snail Aplysia californica has been widely used as a neurobiology model for the studies on cellular and molecular basis of behavior. Here a methodology is described for exploring the nervous system of Aplysia for the electrophysiological and molecular analyses of single neurons of identified neural circuitry.

阿普利西亚 单一神经元电生理学和分子分析的甘利亚制备

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific ...

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

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(&#949;-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37&#160;oC, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged &#8211; the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.

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 ...

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., flasks or dishes). In fact, it is widely recognized that cells grown in flasks or dishes tend to de-differentiate and lose specialized features of the tissues from which they were derived. Currently, there are mainly two types of 3-D culture systems where the cells are seeded into scaffolds mimicking the native extracellular matrix (ECM): (a) static models and (b) models using bioreactors. The first breakthrough was the static 3-D models. 3-D models using bioreactors such as the rotating-wall-vessel (RWV) bioreactors are a more recent development. The original concept of the RWV bioreactors was developed at NASA&#39;s Johnson Space Center in the early 1990s and is believed to overcome the limitations of static models such as the development of hypoxic, necrotic cores. The RWV bioreactors might circumvent this problem by providing fluid dynamics that allow the efficient diffusion of nutrients and oxygen. These bioreactors consist of a rotator base that serves to support and rotate two different formats of culture vessels that differ by their aeration source type: (1) Slow Turning Lateral Vessels (STLVs) with a co-axial oxygenator in the center, or (2) High Aspect Ratio Vessels (HARVs) with oxygenation via a flat, silicone rubber gas transfer membrane. These vessels allow efficient gas transfer while avoiding bubble formation and consequent turbulence. These conditions result in laminar flow and minimal shear force that models reduced gravity (microgravity) inside the culture vessel. Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells and fibroblasts cultured under microgravity provided by the RWV bioreactor.

Cells growing in a three-dimensional (3-D) environment represent a marked improvement over cell cultivation in 2-D environments (e.g., flasks or dishes). Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa cultured under microgravity provided by rotating-wall-vessel (RWV) bioreactors.

生长在微重力人体肠道黏膜的多细胞三维器官模型的建立

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., ...

In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

Brain microvascular endothelial cells, supported by pericytes and astrocytes endfeet, are responsible for the low permeation of large hydrosoluble drugs through the blood-brain barrier (BBB), causing difficulties for effective pharmacological therapies. In recent years, different strategies for promoting brain targeting have aimed to improve drug delivery and activity at this site, including innovative nanosystems for drug delivery across the BBB. In this context, an in vitro approach based on a simplified cellular model of the BBB provides a useful tool to investigate the effect of nanoformulations on the trans-BBB permeation of molecules. This study describes the development of a double-layer BBB, consisting of co-cultured commercially available primary rat brain microvascular endothelial cells and astrocytes. A multiparametric approach for the validation of the model, based on the measurement of the transendothelial electrical resistance and the apparent permeability of a high molecular weight dextran, is also described. As proof of concept for the employment of this BBB model to study the effect of different nanoformulations on the translocation of fluorescent molecules across the barrier, we describe the use of fluorescein isothiocyanate (FITC), loaded into ferritin nanoparticles. The ability of ferritins to improve the trans-BBB permeation of FITC was demonstrated by flux measurements and confocal microscopy analyses. The results suggest this is a useful system for validating nanosystems for delivery of drugs across the BBB.

In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

A method to establish an in vitro model of blood-brain barrier based on a co-culture of rat brain microvascular endothelial cells and astrocytes is described and validated. This system proved to be a valid tool to study the effect of nanoformulation on the trans-barrier permeation of fluorescent molecules.

跨越大鼠血脑屏障FITC加载铁蛋白体外渗透:一个模型来研究Nanoformulated分子的交付

Brain microvascular endothelial cells, supported by pericytes and astrocytes endfeet, are responsible for the low permeation of large hydrosoluble drugs ...

Lucifer Yellow - A Robust Paracellular Permeability Marker in a Cell Model of the Human Blood-brain Barrier

The blood-brain barrier BBB consists of endothelial cells that form a barrier between the systemic circulation and the brain to prevent the exchange of non-essential ions and toxic substances. Tight junctions (TJ) effectively seal the paracellular space in the monolayers resulting in an intact barrier. This study describes a LY-based fluorescence assay that can be used to determine its apparent permeability coefficient (Papp) and in turn can be used to determine the kinetics of the formation of confluent monolayers and the resulting tight junction barrier integrity in hCMEC/D3 monolayers. We further demonstrate an additional utility of this assay to determine TJ functional integrity in transfected cells. Our data from the LY Papp assay shows that the hCMEC/D3 cells seeded in a transwell setup effectively limit LY paracellular transport 7 days-post culture. As an additional utility of the presented assay, we also demonstrate that the DNA nanoparticle transfection does not alter LY paracellular transport in hCMEC/D3 monolayers.

We present a fluorescence assay to demonstrate that Lucifer Yellow (LY) is a robust marker to determine the apparent paracellular permeability of hCMEC/D3 cell monolayers, an in vitro model of the human blood-brain barrier. We used this assay to determine the kinetics of a confluent monolayer formation in cultured hCMEC/D3 cells.

路西法黄 - 人类血脑屏障细胞模型中一个健壮的细胞渗透标记

The blood-brain barrier BBB consists of endothelial cells that form a barrier between the systemic circulation and the brain to prevent the exchange of ...

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically been studied in vivo in animal models showing characteristic patterns of cell migration. However, in vivo models have several limitations, including intercellular mediators that are difficult to access and analyze, as well as the inability to directly analyze human neutrophils. Because of these limitations, there is a need for an in vitro platform that studies swarming with human neutrophils and provides easy access to the molecular signals generated during swarming. Here, a multistep microstamping process is used to generate a bioparticle microarray that stimulates swarming by mimicking an in vivo infection. The bioparticle microarray induces neutrophils to swarm in a controlled and stable manner. On the microarray, neutrophils increase in speed and form stable swarms around bioparticle clusters. Additionally, supernatant generated by the neutrophils was analyzed and 16 proteins were discovered to have been differentially expressed over the course of swarming. This in vitro swarming platform facilitates direct analysis of neutrophil migration and protein release in a reproducible, spatially controlled manner.

This protocol generates bioparticle microarrays that provide spatially controlled neutrophil swarming. It provides easy access to the mediators that neutrophils release during migration and allows for quantitative imaging analysis.

人体神经细胞在体外热的化学和分子分析的生物粒子微阵列

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically ...

Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between animal and clinical trials. Numerous animal and cell models have been used to examine the effects of drugs, yet these responses often differ in humans. Human cardiac slices offer an advantage for drug testing in that they are directly derived from viable human hearts. In addition to having preserved multicellular structures, cell-cell coupling, and extracellular matrix environments, human cardiac tissue slices can be used to directly test the effect of innumerable drugs on adult human cardiac physiology. What distinguishes this model from other heart preparations, such as whole hearts or wedges, is that slices can be subjected to longer-term culture. As such, cardiac slices allow for studying the acute as well as chronic effects of drugs. Furthermore, the ability to collect several hundred to a thousand slices from a single heart makes this a high-throughput model to test several drugs at varying concentrations and combinations with other drugs at the same time. Slices can be prepared from any given region of the heart. In this protocol, we describe the preparation of left ventricular slices by isolating tissue cubes from the left ventricular free wall and sectioning them into slices using a high precision vibrating microtome. These slices can then either be subjected to acute experiments to measure baseline cardiac electrophysiological function or cultured for chronic drug studies. This protocol also describes dual optical mapping of cardiac slices for simultaneous recordings of transmembrane potentials and intracellular calcium dynamics to determine the effects of the drugs being investigated.

This protocol describes the procedure for sectioning and culturing human cardiac slices for preclinical drug testing and details the use of optical mapping for recording transmembrane voltage and intracellular calcium signals simultaneously from these slices.

人体器官性心脏切片双电压和钙光学映射的临床前心脏电生理评估

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between ...