作者们希望从国家研究基金会（NRF -NRFF2011-01和NRF2015NRF-POC0001-19），国家医学研究理事会（NMRC / CBRG / 0005/2012），和南洋理工大学NJC感谢支持

在亲水性固体支持1组的支持的脂质双层<ol><li>制备10毫克/毫升1,2- dioleoyl- SN -glycero -3-磷酸胆碱脂质储备溶液（DOPC）和1毫克/毫升1,2- dioleoyl- SN -glycero -3- phosphoethanolamine- -N - （丽丝胺若丹明乙磺酰基）（铑-PE）通过将相应的脂质粉末（适当地预先用分析质量平衡）的异丙醇溶液称重。 </li><li>稀释和混合储备溶液在异丙醇中，以在最终浓度制备所需的脂质混合物。为荧光显微镜和FRAP的实验中，0.5重量％的Rh-PE的应包括在脂质混合物。 </li><li>注入在异丙醇脂质混合物到微流体通道中，直到它被充满。 </li><li>孵育在玻璃表面上的脂质混合物约10分钟。 </li><li>使用蠕动泵以非常低的流速逐渐取代与水或缓冲溶液的脂质溶液（10-50微升/分钟）。可替代地，代替通过反复移液脂质混合物。 </li><li>用过量的缓冲液彻底冲洗通道以去除残留的异丙醇。 </li></ol> 2，形成的胆固醇富集支持膜注:固体表面（SiO 2）的支持囊泡融合，但膜组成（高胆固醇）抑制囊泡融合，因为胆固醇富集的囊泡具有较高的抗弯刚度30。 <ol><li>制备含有10毫克/毫升DOPC脂质，10毫克/毫升胆固醇，和1毫克/毫升的Rh-PE通过首先将各粉末的异丙醇母液。 </li><li>使用步骤2.1准备的储液重复步骤1.2-1.6。 </li></ol> 3.膜流动性分析<ol><li>沉浸在十二烷基硫酸钠（SDS）溶液（1％）10分钟的载玻片上。 </li><li>使用去离子水彻底冲洗的幻灯片和冲洗机智^ h乙醇。 </li><li>吹干使用温和氮气流幻灯片。 </li><li>露出滑动到氧等离子体进行30秒，在氧等离子体室最大射频功率。 </li><li>取出使用镊子保护膜涂层的深不见底的商业微流控室（ 图1A）和载玻片附着在室（ 图1B）的粘性面。 </li><li>组装连接器和管导入腔室的入口和出口的位置，并放置在显微镜台（图1C）的微流体通道。 </li><li>形成在微流体通道中的荧光标记的支持的脂质双层[使用所需的脂类组合物重复步骤1（例如，0.5毫克/毫升DOPC），其包括0.5重量％的Rh-PE的在有机溶剂中。 </li><li>定位，以捕捉图像与60X油浸物镜（NA 1.49）的双层飞机。 </li><li>取两个预漂白图像，然后光漂白301;米宽圆光点。一个532纳米，100毫瓦的激光束。漂白前，热身激光，直到其强度趋于稳定。立即漂白后，为了遵循荧光强度的恢复在漂白点捕获的一系列图像的每1秒2分钟。 </li></ol> 4.胆固醇定量分析<ol><li>露出二氧化硅被覆石英晶体传感器芯片以氧等离子体进行30秒，在氧等离子体室最大射频功率。除去从室的芯片和立即装入在测量室中的芯片。 </li><li>实验前，先获得在空气中的传感器芯片的频率和耗散，以便确保正确安装。 <ol><li>对于商用Q-检测E1或E4 QCM-D仪器，运行Q-Soft软件程序，并单击"获取"，然后选择"设置测量"。 </li><li>在出现的新窗口中，检查3，5，7，9，11和13的泛音，然后点击查找，并以检查共振光谱运行。设定在24的温度下 C。如果一个或多个谐振频率不与预期值一致，检查芯片安装和直到达成协议重新执行此步骤。 </li></ol></li><li>启动蠕动泵和流动缓冲溶液（10mM的Tris，150mM的氯化钠，pH值7.5）导入测量室以100微升/分钟的流速。 </li><li>在软件程序中，点击"捕获"，并以记录的谐振频率和耗能信号选择"重新启动测量"。直到一个稳定的基线的频率和功耗的变化得到重复此步骤。注意:对于步骤以后，将有可被记录为时间的函数的其他频率和损耗变化。 </li><li>注入异丙醇（无脂）10分钟。 </li><li>注入DOPC脂质/胆固醇的混合物在期望的摩尔比为0.5毫克/毫升的异丙醇进行10分钟的总脂质浓度。 </li><li>注入缓冲器20分钟以100微升/分钟的流速。 </li><li>注入1mM的在缓冲液（流速100微升/分钟），直到频率信号达到稳定的值制备甲基β环糊精（MβCD）中的溶液。 </li><li>测量即引起所述MβCD处理步骤的相对正的频率偏移和通过利用索尔布雷方程[ΔM=转换ΔF 町和ΔFDOPC值质量值计算的胆固醇和DOPC脂质的质量- （C / N）× ΔF，其中C = 17.7毫微克/厘米2，N:泛音。 </li><li>计算中的胆固醇的SLB的摩尔分数，考虑到DOPC脂质（786.1克/摩尔）和胆固醇（三百八十六点六克/摩尔）的分子量。 </li></ol>

The authors have no competing financial interests to disclose.

在这项工作中，溶剂交换协议被提出，其中脂类在醇（异丙醇，乙醇或正丙醇）温育与固体载体上，然后将醇逐渐取代用含水缓冲溶液中，以驱动一系列相变的最终生产层状相脂质双层24。它表明，该方法使得能够支持的脂质双层的制造在表面上，例如金，它是顽固性的囊泡融合方法。 最佳的脂质浓度范围（0.1 - 0.5毫克/毫升）已被确定为完整双层形成在标准实验格式测试迄今。在低于0.1毫克/毫升，离散的，微观双层的补丁血脂浓度而形成的。另一方面，在浓度高于0.1毫克/毫升和低于0.5毫克/毫升，一个完整而均匀的双层形成。在高于该范围血脂浓度，流体双层仍形成为的核查通过FRAP分析编，但是，荧光显微镜揭示的额外脂质结构上该双层的顶部的存在。引人注目的是，这些附加的脂质结构的形态，如由QCM-D分析确定，取决于在其上在培养步骤中使用的醇。在乙醇的情况下，相对高的Δf和Δð移类似于用于吸附囊泡层得到的QCM-D签名。当异丙醇或正丙醇代替所使用的，Δτf为比预期的双层（-30至-40赫兹最终ΔF）的值略高，而ΔD为相当高的。这样的QCM-D响应将预期用于扩展脂质结构（例如，蠕虫状胶束）从膜表面向外突出（如通过在某些情况下，荧光显微镜可见）。 溶剂交换的速率可以是临界的另一个重要参数，ESPECially当低血脂浓度（ 例如，0.1毫克/毫升）使用。在低浓度的脂质快速溶剂交换可导致不完全的双层的形成。在用于本文的QCM-D测量（Q-感E4测量室），约100微升/分钟的流速的标准测量室，均适宜高度可重复的完整双层形成。对于流细胞与其他的几何形状和体积，最佳流速可以变化，并且基于本文提出的步骤必须根据经验确定。 除了形成支持的脂质双层上的表面是难治到囊泡融合，所述SALB可以被用来规避需要脂质囊泡可破裂，从而打开门支持膜具有复杂的组合物的制造。作为说明性实例组合物，与胆甾醇的高分数的脂质的混合物进行了研究。胆固醇是mamm的重要组成部分阿莲细胞膜，它的分数可能接近45-50％（摩尔）的膜脂组成（如红细胞）。因此，脂质双层的代表人体细胞膜即使一个简单的模型应该包括胆固醇。 而小泡的融合可以用于制造仅含有10-15％的胆固醇的流体脂质双层，该SALB方法能够形成含胆固醇的高分数的流体脂质双层（高达57％（摩尔），作为量化的QCM-D测量）36。但是，当胆甾醇的水平进一步升高（最多63摩尔％），条纹形状结构域观察到37。共存的结构域是液体，使人联想起在空气 - 水界面中的β区观察到胆固醇/磷脂单层的相图。 总体而言，SALB方法证明是一种简单而有效的方法，以形成支持的脂质双层，尤其是我n个个案超出常规的囊泡融合方法的范围。迄今为止，QCM-D技术和荧光显微镜主要用来表征由SALB方法形成的支持的脂质双层。展望未来，范围广泛的表面敏感分析测量技术，包括表面等离子共振（SPR）38，原子力显微镜（AFM）39,40，傅立叶变换红外光谱41，X射线42和中子反射率43，可以可用于进一步表征和研究简单和复杂的双层结构准备由SALB方法。这些新兴的功能，打开大门，更多的科学家谁可以采取一个简单而强大的实验方案的优势，探索人工细胞膜。

固体支持的脂质双层1（SLB）是一种多功能的平台，其保留生物膜的基本特性，如双层厚度，二维脂质扩散率，和主机膜相关的生物分子的能力。由于天然细胞膜的复杂性，这个简单的平台已经显示用作用于体外研究膜相关的处理的诸如筏形成2，蛋白质的高效平台结合3，病毒和病毒样颗粒的结合4,5-和细胞信号6。形成在靠近固体支持物，所述SLB平台是与一系列的表面敏感的测量技术相容如全内反射显微镜（TIRF），石英晶体微天平耗散（QCM-D）和阻抗谱。 几种方法已经被开发，以生产不同类型的SLBs的，包括气泡崩7和蘸笔纳米光刻8为亚微米尺寸的脂质斑点，旋涂为双层堆叠和LB膜（LB）10和囊泡融合（VF）11，用于全跨越，单脂质双层涂层9。在VF方法包括小单层囊泡吸附到固体支持物和随后的自发破裂和熔化，形成连续的脂质双层。然而，在生理条件下，自发囊泡破裂主要限于硅系材料，如二氧化硅，玻璃，和云母。此外，泡囊破裂不会发生自发为复合脂质组合物的囊泡，例如含有胆甾醇或带负电荷的脂质的高级分。根据不同的系统，囊泡破裂可通过进一步剪裁实验条件，如温度12，溶液的pH值13，和盐度14，渗透压休克15被诱导</s向上&gt;或压力16，或添加二价离子如钙离子的17，另外，在膜的活性AH肽可以破坏的层吸附囊泡被引入，导致小泡破裂和双层形成在一系列表面18-22。 此外，成功的双层形成需要制备的小单层囊泡，可以是耗时且难以实现的某些膜的组合物良好控制的人口。因此，尽管其高效率的最佳情况下（例如，后囊23的广泛冻融预处理），囊泡融合的一般应用由合适的底物和膜组合物的范围的限制。 的溶剂辅助脂质双层（SALB）方法24-28是一种替代的制造技术，它不需要的脂质囊泡。该方法是基于沉积Ô˚F脂质分子上在与水混溶的有机溶剂后用此溶剂与以触发SLB形成缓冲水溶液逐渐交换的存在下的固体表面。在溶剂交换步骤，脂质，有机溶剂的三元混合物，和水进行一系列相变而增加水部分，这导致层状相的结构在本体溶液的形成和SLB固体基材上。重要的是，该自组装的路线绕过需要囊泡破裂​​，这通常是用于吸附囊泡转​​变成一个SLB限制步骤。该协议是适用于多种表面，包括二氧化硅，氧化铝，铬，铟锡氧化物，和金的。在本文和在所附的视频，脂质沉积由SALB和囊泡融合方法进行了比较，提出。特别是，实验参数的影响，包括脂浓度，流量，和水混溶性有机溶剂的选择，由所述SALB法形成的双层的质量进行了讨论。所制造的SLBs的分析表征由QCM-D，荧光显微镜，荧光漂白恢复（FRAP）技术之后进行。 QCM-D监测是，由于由Keller和Kasemo 29进行的开创性工作，已被广泛用于定量研究双层形成一个表面感光质量测量的技术。荧光显微镜允许膜均一性的检查以及膜结构域的可视化。的FRAP技术是一个标准的工具，以确定在SLB，这是流体膜的一个重要性质脂质分子的横向流动。 该研究的第一部分涉及SALB应用到试图对二氧化硅和金双层形成的QCM-D分析和囊泡融合的方法。在第二部分中，的制备方法和含有一个范围胆固醇浓度与SALB方法的支持膜表征演示了，结果与那些由囊泡融合方法获得了比较。

<table border="0" cellpadding="0" cellspacing="0" width="1459">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:719px;">QCM-D silicon dioxide-coated substrates</td>
			<td style="width:356px;">QSense AB,&#160; Sweden</td>
			<td style="width:217px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QCM-D gold-coated substrates</td>
			<td style="width:356px;">QSense AB,&#160; Sweden</td>
			<td style="width:217px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Q-Sense E4 module</td>
			<td>QSense AB,&#160; Sweden</td>
			<td style="width:217px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Plasma Cleaner, PDC-32G</td>
			<td>Harrick Plasma, Ithaca, NY</td>
			<td>PDC-001 (115V)&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:719px;">1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)</td>
			<td>Avanti Polar Lipids</td>
			<td>850375P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt) (Rh-PE)</td>
			<td>Avanti Polar Lipids</td>
			<td>810150P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cholesterol</td>
			<td>Avanti Polar Lipids</td>
			<td style="width:217px;">700000P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methyl-&#946;-cyclodextrin</td>
			<td style="width:356px;">Sigma&#160;</td>
			<td style="width:217px;">C4555</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isopropanol</td>
			<td style="width:356px;">Sigma&#160;</td>
			<td align="right">673773</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:719px;">Ethanol</td>
			<td style="width:356px;">Sigma&#160;</td>
			<td align="right" style="width:217px;">459844</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:719px;">n-propanol</td>
			<td style="width:356px;">Sigma&#160;</td>
			<td align="right">279544</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:719px;">Sticky-Slide I 0.1 Luer</td>
			<td style="width:356px;">IBIDI</td>
			<td align="right" style="width:217px;">81128</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:719px;">Male elbow 1/8&#8221;</td>
			<td style="width:356px;">Cole-Parmer</td>
			<td style="width:217px;">30505-70</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:719px;">Silicon tubing 1.6mm ID</td>
			<td style="width:356px;">IBIDI</td>
			<td align="right" style="width:217px;">10842</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:719px;">Glass coverslip No. 1.5H, 25 mm x 75 mm</td>
			<td style="width:356px;">IBIDI</td>
			<td align="right" style="width:217px;">10812</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:719px;">Reglo Digital M2-2/12 Peristaltic Pump</td>
			<td style="width:356px;">Ismatec</td>
			<td style="width:217px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:719px;">Sodium dodecyl sulfate</td>
			<td style="width:356px;">Sigma&#160;</td>
			<td align="right" style="width:217px;">71725</td>
			<td></td>
		</tr>
	</tbody>
</table>

biomembrane fabrication by the solvent-assisted lipid bilayer (salb) method

为了模仿细胞膜，所支持的脂质双层（SLB）是一个有吸引力的平台使在膜相关过程的体外调查而赋予的生物相容性和生物功能，以固体基质。磷脂小泡的自发吸附和破裂是最常用的方法来形成的SLBs。然而，在生理条件下，囊泡融合（VF）被限制在脂质组合物和固体载体的一个子集。这里，我们描述为了形成SLBs的，它不需要囊泡一个称为溶剂辅助脂质双层（SALB）的形成方法的一个步骤的一般方法。所述SALB方法以触发SLB形成包括脂质分子上的沉积，在水混溶的有机溶剂（例如，异丙醇）中，用缓冲水溶液后续溶剂交换的存在下的固体表面。连续的溶剂交换步骤使应用程​​序的方法在适合于监视双层形成和使用范围广泛的表面敏感的生物传感器进一步修改的流通配置。所述SALB方法可被用于制造SLBs的就广泛亲水固体表面，包括那些棘手到囊泡融合的。此外，它使SLBs的脂质组合物的构成不能使用的囊泡融合方法制备的制造。在此，我们比较有两个说明性的亲水性的表面，二氧化硅和黄金SALB和常规囊泡融合方法得到的结果。为了优化实验条件用于制备经由SALB方法制备高品质双层，各种参数，包括有机溶剂在沉积步骤的类型，溶剂交换的速率，和脂质浓度的效果连同故障排除提示讨论。含胆固醇高的分数支持膜的形成也是魔鬼trated与SALB方法，突出SALB技术的技术能力，适用范围广的膜配置。

制造亲水性基材支持脂质双层。 的音频和SALB形成方法进行了尝试对二氧化硅和金并在实时的QCM-D测量技术监测的形成过程。在大规模的吸附的振荡压电石英晶体到晶体的表面的QCM-D测量仪器中的共振频率（ΔF）的变化。此外，QCM-D仪器测量以表征所述吸附层的粘弹性（刚性和柔软性）的振荡能量的消耗。泡融合实验如前所述31进行。简要地说，一个基线最初成立于水性缓冲液〔10毫摩尔Tris，150mM的氯化钠，pH值7.5的频率和耗散信号; （图2A 和B）。接着，在相同的缓冲液小单层DOPC脂质囊泡分别在投影在 t = 10分钟（箭头1）到二氧化硅（ 图2A）和金（图2B）。有关二氧化硅囊泡融合，两步吸附动力学，观察在-26（±1）赫兹频率和能量耗散和0.3最终改变（±0.2）×10 - ，6分别。这些值是用支持的脂质双层29的形成是一致的。 如图2B所示，除了囊泡溶液到金表面的导致了同时减少和增加Δf和ΔD信号 ，分别直到它们的值达到-150（±10）Hz和（7.5±2） ×10 - 6。这些值对应于被吸附的囊泡层的形成。因此，正如所料，一个SLB没有通过囊泡融合法形成黄金。 QC对于双层形成于二氧化硅和金由SALB方法的MD分析示于图2C和 D。上二氧化硅，的-25.6（±0.55）最终Δf和Δð移位赫兹和0.4（±0.27）×10 - 6，分别实现，并且这些值表示形成一个SLB的。 Δf和 Δ 的D类似范围（Δ˚F 金 :-27.3±2.7赫兹，Δð 金 :0.48（±0.26）×10 - 6），观察其对黄金。这些结果支持了SALB方法能够在其上防止囊泡破裂面形成支持的脂质双层。 溶剂交换流量对支持的脂质双层的质量。 为了确定最佳条件为高品质的支持脂双层六制造一个所述SALB方法，脂质浓度，溶剂交换速率和有机溶剂的选择的影响进行了研究。 图3显示了在SALB协议的最后步骤中的QCM-D频率的变化，如在两个不同的流速（100和600微升/分钟）和两种不同的脂质浓度（0.125和0.5mg / ml）的执行上二氧化硅。 当0.5毫克/毫升DOPC脂质，使用（图3A），双层形式并不受流动速率和大约-26赫兹在两个流速得到最终ΔF移位。 相反，当一个低脂质浓度使用，吸附脂质后完成溶剂交换的量显著受流动速率（ 图3B）。以100微升/分钟的平均流动速率，双层形成完成（约-26赫兹ΔF）。然而，在6倍较高流速（600微升/分钟），一个完整的双层不形成（大约-17赫兹ΔF）。这些结果提供了指导选择合适的实验条件，使用方法SALB用异丙醇作为首选的有机溶剂成功的双层形成。 从各种醇溶液不同血脂浓度形成支持的脂质双分子层的表征。 脂浓度是影响SLBs中的通过使用SALB方法获得的质量的另一参数。荧光显微镜显示，在0.05毫克/毫升脂质浓度，只有分离的，亚微观脂质结构形成（图4A）。而增大SALB过程中所使用的脂质浓度，脂质结构的荧光强度变得更加均匀。以0.1mg / ml的脂质浓度，有微观脂质斑块虽然结构没有跨越整个F跨越的视图ield（图4B）。然而，当为0.25mg / ml的脂质浓度为所使用的，均匀的脂质双层形成（图4C）。因此，存在以形成一个完整的，跨越SLB所需的最小的脂质浓度。 在SALB实验的最后结果的脂质浓度的影响也检查在更宽的脂质浓度范围（0.01至5mg / ml）和在不同的有机溶剂（异丙醇，乙醇和正丙醇）。的Δf和对应于SALB过程中的最后一步ΔD取值 示于图5。 我们定义双层形成基于-25和-30赫兹和小于0.5×10 -6，分别之间最终的变化频率和能量耗散。根据这些标准，最佳脂质浓度范围，以形成一个支持的脂质双层被确定为在0.1和0.5mg/毫升很大程度上独立的有机溶剂的类型。在所获取的Δf和ΔD中的偏差偏移的上述范围以外是由于附加质量（例如，双层叠层），的非双层形貌的发生（例如，水疱，蠕虫状胶束）的存在，和零散双层岛与整个基板不完整的形态存在。 而SALB过程来得到支持的脂双层是比较稳健的，高品质的双层形成的最佳脂质浓度可能需要调谐取决于具体的实验配置。基本上，存在不仅是一个最小的脂质浓度，但也需要经由SALB方法最佳双层形成一个最大的脂质浓度。最佳浓度范围取决于流速和可能也受基板和脂质组合物。根据经验，在许多情况下，我们有found即0.5毫克/毫升和100微升的流速的脂质浓度/ min是用于均质支持的脂质双层的形成条件的最佳集合。然而，根据不同的脂类组合物和流动池几何后者影响期间脂质浓度的溶剂交换进一步优化流动轮廓可能是必要的。因此，我们建议您使用0.5毫克/毫升脂浓度和评价使用QCM-D或荧光显微镜技术的双层质量进行试点SALB实验。如果双层出现不完整的，则所述脂质浓度应在10％的递增增加，直到满意的成果得以实现。如果该双层似乎具有附加脂质结构共存，则脂质浓度应在10％的递增下降，直至满意的疗效得以实现。 支持的脂质双层与各种脂质组合物以及d的制造ifferent胆固醇分数。 接着，SALB和VF方法被采用，以形成胆固醇-富集的SLBs;图6示出了包含胆固醇编写的SALB方法支持膜的代表性荧光图像（100×100微米）。这种膜由通过特征的统一的荧光亮度的连续相包围的圆形染料排除在外域。暗域面积的增加而增加胆固醇分数前体脂质混合物。接着，FRAP测量为了检查脂质双层膜的流动性被执行。该FRAP测量显示在周围阶段几乎完全荧光恢复，说明脂质的横向流动，从而形成一个单一的脂质双层的。由于的Rh-PE隔板优先进入流体相，暗域最有可能由密集的胆固醇丰富的结构。 </P&gt; 为了比较，使用VF方法DOPC /胆固醇双层膜的制造还尝试。 DOPC泡囊随着胆固醇部分（10 - 40摩尔％），制备囊泡挤出方法。的Rh-PE脂质（0.5重量％）用作荧光标记进行成像。 图7示出在玻璃基板与含有胆固醇的囊泡的孵化产生结构的代表性荧光图像（100×100微米）。 FRAP分析表明使用含有20摩尔％澈或更少囊泡的流体脂质双层的形成。然而，样品制备用囊泡具有更高胆固醇馏分并没有表现出恢复，这表明吸附的但是未破裂囊泡的存在。 胆固醇将其最终掺入支持脂质双层的馏分量化为胆固醇馏分已被包括在该前体唇的函数的id混合物在SALB方法或在音频方法水溶液囊泡有机溶剂。使用QCM-D技术，双层形式进行了监测，然后MβCD加入以专门从支持的脂质双层32提取澈。质量损失由于除去胆固醇导致了频移的绝对值减少与SLB关联（| |＆Delta F）。所造成的MβCD处理步骤的相对正频移示于图8A。胆固醇的摩尔分数基础上的频移进行了计算，如在图8B呈现。 掺入编写的SALB方法支持的脂质双层中的胆固醇百分率为几乎线性正比于前体的脂质混合物中的胆固醇含量。有趣的是，编写的VF方法在双层的胆固醇部分（小泡含至多20摩尔％澈）比包含在前体囊泡低得多。事实上，胆固醇通过VF方法得到的最高分数仅约为10摩尔％。 条纹上层建筑中胆固醇的磷脂的β两相共存区域观察支持的脂质双层。 SALB实验使用的脂质混合物与胆固醇的一个更高的级分进一步进行。当4:使用DOPC和胆固醇的混合物6，均匀的液相逐渐分层成两个共存相形象化为在黑暗（染料不含）背景明亮的条形结构域观察到（图9）。经确定的Rh-PE被排除富含胆固醇的结构域33，并因此而出现作为背景的主导暗域是胆固醇富集区域。微米大小的亮域在一个黑暗的背景，在形成喜GH胆固醇级分（&gt; 50摩尔％）是具有在胆固醇/磷脂的混合物34,35单层相图的β区一致。此外，条纹的域，从弱线张力而出现的形成，表明混合物接近混溶性临界点。 <img alt="图1" src="/files/ftp_upload/53073/53073fig1.jpg" /> 图1.微流控室SALB形成一个合适的配置荧光显微镜。（一）商业微流控室，（B）附着于室的粘合面玻璃盖玻片，（C）完成安装与管显微镜支架连接成腔室的入口和出口， 和 （D）蠕动泵用于控制溶剂交换的速率。脂质溶解在异丙醇注入测量chamb呃与蠕动泵的帮助。 <a href="https://www.jove.com/files/ftp_upload/53073/53073fig1large.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/53073/53073fig2.jpg" /> 的囊泡融合和SALB实验上的二氧化硅和金基板图2的QCM-D分析。QCM-D频率（ΔF，蓝色）和耗散用于第三谐波（ΔD，红色）反应（N = 3）记录为一个时间在脂质吸附（A和C）二氧化硅，（B和D），黄金的功能。板a和b给出的囊泡融合方法。 DOPC脂质囊泡在 t = 10分钟注射（箭头1）。板c和d对应SALB形成方法。箭头指示在注射缓冲液（1），异丙醇（2），脂质混合物[0.5毫克/毫升DOPC在异丙醇脂质; （3）]和浅黄色呃交易所（4）。在面板B中的虚线曲线对应于一个控制实验，其中脂质不注入。指定ΔF与△D的每个表面的最终值。这些原理图给出建议组装脂质结构从最终的频率和功耗的变化推断。从参考24改编，并与美国化学协会的许可使用。 <a href="https://www.jove.com/files/ftp_upload/53073/53073fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53073/53073fig3.jpg" /> 图上的SALB形成过程中的溶剂汇率3.影响 。 QCM-D的频率偏移（ΔF）对应于最终的步骤（见箭头4在图2C）在SALB法以两种不同的汇率，100和600微升/分钟，U上测量二氧化硅<html>唱（A）中为0.5mg / ml和异丙醇（B）的 0.125毫克/毫升DOPC脂质。最终ΔF值也指定相比，在水性缓冲溶液中的测量基准。从参考24改编，并与美国化学协会的许可使用。 <a href="https://www.jove.com/files/ftp_upload/53073/53073fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53073/53073fig4.jpg" /> 图4.阈值脂浓度的完整的二级行政边界形成的使用（A）0.05毫克/毫升制备SALB二氧化硅脂质层荧光显微镜。 （B）的0.1毫克/毫升; 和 （C）0.25毫克/毫升脂质浓度。从参考26适于和与美国化学学会的授权使用。/files/ftp_upload/53073/53073fig4large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图5" src="/files/ftp_upload/53073/53073fig5.jpg" /> 由SALB方法图5.影响脂质浓度和上支持的脂质双层形成的有机溶剂。在QCM-D中的最后的变化（A）的频率和 （B）消能使用各种有机溶剂SALB实验，作为脂质的函数浓度。虚线绿线对应于预期的频率和耗散移的完整双层（-30赫兹&lt;ΔF&lt;-25赫兹和ΔD&lt;1×10 -6）。从参考26改编，并与美国化学协会的许可使用。 <a href="https://www.jove.com/files/ftp_upload/53073/53073fig5large.jpg" target="_blank">请点击此处查看该网络的放大版本</a>古尔。 <img alt="图6" src="/files/ftp_upload/53073/53073fig6.jpg" /> 漂白支持的脂质双层的分析具有不同胆固醇制备在玻璃基板上的方法SALB馏分之后，图6的荧光恢复 。双层的（AE）荧光显微照片使用各种胆固醇馏分中前体混合物制备。图像进行漂白后立即（上）和1分钟（中）记录下来。在图像中心的暗斑对应于光漂白区域。该比例尺为20微米。每个样品中的单独的染料排除结构域的表面积直方图还提出（底部）。从参考36改编，并与美国化学协会的许可使用。 <a href="https://www.jove.com/files/ftp_upload/53073/53073fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a><img alt="图7" src="/files/ftp_upload/53073/53073fig7.jpg" /> 图含胆固醇编写的囊泡融合方法支持双层7. FRAP分析 。双层的（AD）的荧光显微照片使用各种胆固醇部分（10〜40摩尔％）中制备，在该前体囊泡。图像进行漂白后立即（上）和1分钟（底部）记录。在图像中心的暗斑对应于光漂白区域。该比例尺为20微米。从参考36改编，并与美国化学协会的许可使用。 <a href="https://www.jove.com/files/ftp_upload/53073/53073fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图8" src="/files/ftp_upload/53073/53073fig8.jpg" /> 图8.曲胆固醇部分的支持脂双层antification。 （A）中的阳性QCM-D频移注射后的1毫MβCD到所支持的脂质双层具有不同的胆固醇的摩尔分数中的前体混合物（在0和50摩尔％）。 （B）的胆固醇从编写的SALB和囊泡融合方法作为前体的混合物或囊泡的胆固醇分数的函数的双层耗尽的摩尔％。从参考36改编，并与美国化学协会的许可使用。 <a href="https://www.jove.com/files/ftp_upload/53073/53073fig8large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图9" src="/files/ftp_upload/53073/53073fig9.jpg" /> 6摩尔比CONTA:荧光显微结构的DOPC /胆固醇（4支持双层图9.时间依赖演变进不去0.5％罗丹明-PE）编写的SALB方法。均匀相逐渐相分离成在完整的溶剂交换的液-液共存区域。从参考37改编，并与美国化学协会的许可使用。 <a href="https://www.jove.com/files/ftp_upload/53073/53073fig9large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method at JoVE.com

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

我们提出了一个试验性协议，形成对固体基质支持的脂质双层不使用脂质囊。我们展示了一个一步法，以形成具有各种生物应用胆固醇富集域上的二氧化硅和金脂质双层以及支持膜。

生物膜制作的溶剂辅助脂质双分子层（SALB）方法

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

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Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

14.8K 次观看.  Nanyang Technological University. 我们提出了一个试验性协议，形成对固体基质支持的脂质双层不使用脂质囊。我们展示了一个一步法，以形成具有各种生物应用胆固醇富集域上的二氧化硅和金脂质双层以及支持膜。 

视频: Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

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

Microfluidic Chip Fabrication and Method to Detect Influenza

Fast and effective diagnostics play an important role in controlling infectious disease by enabling effective patient management and treatment. Here, we present an integrated microfluidic thermoplastic chip with the ability to amplify influenza A virus in patient nasopharyngeal (NP) swabs and aspirates. Upon loading the patient sample, the microfluidic device sequentially carries out on-chip cell lysis, RNA purification and concentration steps within the solid phase extraction (SPE), reverse transcription (RT) and polymerase chain reaction (PCR) in RT-PCR chambers, respectively. End-point detection is performed using an off-chip Bioanalyzer (Agilent Technologies, Santa Clara, CA). For peripherals, we used a single syringe pump to drive reagent and samples, while two thin film heaters were used as the heat sources for the RT and PCR chambers. The chip is designed to be single layer and suitable for high throughput manufacturing to reduce the fabrication time and cost. The microfluidic chip provides a platform to analyze a wide variety of virus and bacteria, limited only by changes in reagent design needed to detect new pathogens of interest.

An integrated microfluidic thermoplastic chip has been developed for use as a molecular diagnostic. The chip performs nucleic acid extraction, reverse transcriptase, and PCR. Methods for fabricating and running the chip are described.

微流控芯片的制备及方法检测甲型流感

Fast  and effective diagnostics play an important role in controlling infectious  disease by enabling effective patient management and treatment. Here, we ...

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are heterogeneous mixtures of many types of lipids, contain membrane-bound proteins and are decorated with carbohydrates. In some experiments, it is desirable to decouple the biophysical or biochemical properties of the lipid bilayer from those of the natural membrane. Such cases call for the use of model systems such as giant vesicles, liposomes or supported lipid bilayers (SLBs). Arrays of SLBs are particularly attractive for sensing applications and mimicking cell-cell interactions. Here we describe a new method for forming SLB arrays. Submicron-diameter SiO2 beads are first coated with lipid bilayers to form spherical SLBs (SSLBs). The beads are then deposited into an array of micro-fabricated submicron-diameter microwells. The preparation technique uses a "squeegee" to clean the substrate surface, while leaving behind SSLBs that have settled into microwells. This method requires no chemical modification of the microwell substrate, nor any particular targeting ligands on the SSLB. Microwells are occupied by single beads because the well diameter is tuned to be just larger than the bead diameter. Typically, more 75% of the wells are occupied, while the rest remain empty. In buffer SSLB arrays display long-term stability of greater than one week. Multiple types of SSLBs can be placed in a single array by serial deposition, and the arrays can be used for sensing, which we demonstrate by characterizing the interaction of cholera toxin with ganglioside GM1. We also show that phospholipid vesicles without the bead supports and biomembranes from cellular sources can be arrayed with the same method and cell-specific membrane lipids can be identified.

Supported lipid bilayers and natural membrane particles are convenient systems that can approximate the properties of cell membranes and be incorporated in a variety of analytical strategies. Here we demonstrate a method for preparing microarrays composed of supported lipid bilayer-coated SiO2 beads, phospholipid vesicles or natural membrane particles.

生物膜的微阵列用刮墨刀型组合方式形成

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are ...

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies1,2. Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation3. The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting.

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

脂质双层囊泡代使用微流控喷射

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

Polymeric Microneedle Array Fabrication by Photolithography

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a photomask consisting of embedded micro-lenses. Embedded micro-lenses were found to influence MN geometry (sharpness). Robust MN arrays with tip diameters ranging between 41.5 &#181;m &#177; 8.4 &#181;m and 71.6 &#181;m &#177; 13.7 &#181;m, with two different lengths (1,336 &#181;m &#177; 193 &#181;m and 957 &#181;m &#177; 171 &#181;m) were fabricated. These MN arrays may provide potential applications in delivery of low molecular and macromolecular therapeutic agents through skin.

Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography.

聚合物微针阵列制造用光刻

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

固体脂质纳米粒（前哨淋巴结）细胞内定位的应用

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for biosensor applications. However, conventional BLM formation techniques have several drawbacks and they often require specific expertise and laborious processes. In particular, conventional BLMs suffer from low formation success rates and inconsistent membrane formation time. Here, we demonstrate a storable and transportable BLM formation system with controlled thinning-out time and enhanced BLM formation rate by replacing conventionally used films (polytetrafluoroethylene, polyoxymethylene, polystyrene) to polydimethylsiloxane (PDMS). In this experiment, a porous-structured polymer such as PDMS thin film is used. In addition, as opposed to conventionally used solvents with low viscosity, the use of squalene permitted a controlled thinning-out time via slow solvent absorption by PDMS, prolonging membrane lifetime. In addition, by using a mixture of squalene and hexadecane, the freezing point of the lipid solution was increased (~16 &#176;C), in addition, membrane precursors were produced that can be indefinitely stored and readily transported. These membrane precursors have reduced BLM formation time of &#60; 1 hr and achieved a BLM formation rate of ~80%. Moreover, ion channel experiments with gramicidin A demonstrated the feasibility of the membrane system.

We demonstrate a storable, transportable lipid bilayer formation system. A lipid bilayer membrane can be formed within 1 hr with over 80% success rate when a frozen membrane precursor is brought to ambient temperature. This system will reduce laborious processes and expertise associated with ion channels.

自动化脂双层膜的形成使用聚二甲基硅氧烷薄膜

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for ...

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological studies. In the past years, it has become increasingly clear that living cells respond, not only to the biochemical nature of the molecules presented to them but also to the way these molecules are presented. Creating protein micro-patterns is therefore now standard in many biology laboratories; nano-patterns are also more accessible. However, in the context of cell-cell interactions, there is a need to pattern not only proteins but also lipid bilayers. Such dual proteo-lipidic patterning has so far not been easily accessible. We offer a facile technique to create protein nano-dots supported on glass and propose a method to backfill the inter-dot space with a supported lipid bilayer (SLB). From photo-bleaching of tracer fluorescent lipids included in the SLB, we demonstrate that the bilayer exhibits considerable in-plane fluidity. Functionalizing the protein dots with fluorescent groups allows us to image them and to show that they are ordered in a regular hexagonal lattice. The typical dot size is about 800 nm and the spacing demonstrated here is 2 microns. These substrates are expected to serve as useful platforms for cell adhesion, migration and mechano-sensing studies.

We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies.

在支撑脂双层配体纳米簇阵列

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

A Simple and Scalable Fabrication Method for Organic Electronic Devices on Textiles

Today, wearable electronics devices combine a large variety of functional, stretchable, and flexible technologies. However, in many cases, these devices cannot be worn under everyday conditions. Therefore, textiles are commonly considered the best substrate to accommodate electronic devices in wearable use. In this paper, we describe how to selectively pattern organic electroactive materials on textiles from a solution in an easy and scalable manner. This versatile deposition technique enables the fabrication of wearable organic electronic devices on clothes.

In this paper, we present a protocol to selectively deposit organic materials on textiles, which allows for the direct integration of organic electronic devices with wearables. The fabricated devices can be fully integrated in textiles, respecting their mechanical appearance and enabling sensing capabilities.

关于纺织品有机电子设备的简单和可扩展的制作方法

Today, wearable electronics devices combine a large variety of functional, stretchable, and flexible technologies. However, in many cases, these devices ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

脱碳软骨衍生基质支架的制备

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...