这项工作得到中国国家基础研究计划 (2014CB932400)、中国国家自然科学基金 (51525204 号和 U1607206) 和深圳基础研究项目 (no。JCYJ20150529164918735)。此外, 我们还要感谢二价酸福辛普利拉-Allnex 有限公司和 JSR 公司, 为亲切提供聚氨酯和苯乙烯丁橡胶, 分别。

1. 制备 1 wt% 22、66-Tetramethylpiperidin-1-oxyl (速度) 介导的氧化纤维素碳纤维 (TOCN) 溶胶
注: 溶胶被定义为在连续液体介质中非常小的固体颗粒的胶体悬浮。
<ol><li>悬浮66.7 克 Nadelholz 漂白硫酸盐浆 (NBKP, 含12克纤维素) 在700毫升的去离子 (DI) 水与机械搅拌器在 300 rpm 为20分钟。</li>
	<li>将20毫升水速溶液 (含0.15 克速度) 和20毫升水 NaBr 溶液 (含1.5 克 NaBr) 缓慢地添加到上述 NBKP 悬浮14,15。</li>
	<li>将上述悬浮液的 pH 值调整到大约 10.5 (用 pH 值计), 慢慢加入3.0 米氢氧化钠溶液。</li>
	<li>慢慢地添加大约63.8 克水次氯酸钠溶液 (与 6-14 wt% 活性氯) 与吸管到上述混合物开始速度介导的氧化。</li>
	<li>添加次氯酸钠时, 继续添加氢氧化钠溶液, 使系统的 pH 值保持在10.0 到10.5 之间。这个过程大约需要2.5 小时。</li>
	<li>用 di 水冲洗速度介导的氧化纤维素纤维3次 (每次1200毫升的底水), 以除去残留的次氯酸钠、氢氧化钠和其他化学物质。</li>
	<li>用强大的机械搅拌器处理糊状物, 将氧化纤维素纤维分解成纳米纤维。仔细地进行机械处理几次, 加上等量的水。最后, 获得了 1 wt% 速度介导的氧化纤维素碳纤维 (TOCN) 溶胶。TOCNs 的直径为4到 ~ 6 nm, 长度为0.5 到 ~ 2 微米。</li>
	<li>将 1 wt% TOCN 溶胶保存在4摄氏度 (纤维素纤维在室温下趋于腐烂)。</li>
</ol>2. TOCN-苯乙烯丁二烯橡胶 (SBR) 混合溶胶的制备
<ol><li>在11.7 毫升玻璃容器中加入0.21 克 SBR 胶体 (48.5 wt%) 到10克 1 wt% TOCN 溶胶 (步骤 20)。</li>
	<li>搅动上述混合物为3分钟与漩涡搅拌机在功率水平6达到均匀地分散的溶胶. 在使用前将上述混合物溶胶保存在4摄氏度。</li>
</ol>3. TOCN2混合溶胶的制备
<ol><li>在1毫升玻璃容器中添加0.1 克的2纳米粒子 (平均粒径为 20 nm) 到10克的 20 wt% TOCN 溶胶中。</li>
	<li>将上述混合物与均质机搅拌10分钟, 以达到均匀混合的溶胶. 间歇性地执行这10分钟过程, 因为过程中产生了大量的热量, 导致温度升高, 这可能会降低 TOCNs。使用前将混合物溶胶贮存在4摄氏度。</li>
</ol>4. TOCN 表面氧化炭纤维 (SOCF) 混合溶胶的制备
<ol><li>回流1.7 克碳纤维 (300 目, 直径5.5 到 ~ 6.0 微米和长度约50微米) 在150毫升浓硝酸在60摄氏度为 6 h 达到 SOCF16。将上述 SOCF 的0.01 克添加到20毫升玻璃容器内10克的 1 wt% TOCN 溶胶中。</li>
	<li>摇动混合上述混合物, 并超油脂实验混合物5分钟, 以达到均匀混合溶胶. 在使用前将混合物溶胶保存在4摄氏度。</li>
</ol>5. 从 1 wt% TOCN 溶胶 (表示 MHM-TOCN) 制备 Microhoneycomb 巨石
<ol><li>加载聚丙烯 (PP) 管 (内径为13毫米, 外径为15毫米, 长度为150毫米), 并填充管的底部5厘米部分13。</li>
	<li>负载一定量 (每次不控制量, 但通常大于2.7 毫升, 以确保随后的切割过程) 的 1 wt% TOCN 溶胶进入上述 PP 管含有玻璃珠。 注: TOCN 溶胶直接填充在聚丙烯管中, 不注入玻璃珠, 用于研究单向冻结过程中所涉及的距离效应。在这种情况下, TOCN 溶胶的用量为11毫升。</li>
	<li>小心删除在溶胶加载过程中可能产生的气泡。在使用前, 将含有 TOCN 溶胶的 PP 管保持在4摄氏度。</li>
	<li>将含有 TOCN 溶胶的上述 PP 管附着在用于单向冻结的浸渍机上。设置相关参数并开始将 PP 管浸入到含有液态氮气 (-196 °c) 的热水壶中, 其恒定速度为50厘米 h-1 (图 1)。</li>
	<li>用锯子切割 PP 管部分, 将冷冻 TOCN 溶胶部分分成几个部分。冷冻干燥这些部分与冷冻干燥机在-10 °c 1 天, 然后在-5 °c 1 天, 最后在0°c 1 天。MHM TOCN 以白色石柱 (图 1) 获得。</li>
</ol>6. 从 TOCN-SBR 混合溶胶 (表示 MHM-TOCN/SBR) 和 TOCN2混合溶胶 (表示 MHM-TOCN/Microhoneycomb2 ) 中制备巨石
<ol><li>负载聚丙烯 (PP) 管 (内径为13毫米, 外径为15毫米, 长度为150毫米), 玻璃珠, 填补了底部 5 cm 部分的管。 注: 玻璃珠用于覆盖不稳定冰晶生长的区域, 从而达到样品的均匀形貌。玻璃珠的尺寸和表面性质都不影响结果样品的形貌。</li>
	<li>加载一定量 (每次不控制金额, 但通常大于2.7 毫升, 以确保随后的切割过程) TOCN-SBR 混合溶胶或 TOCN2混合溶胶进入含有玻璃珠的 PP 管中。</li>
	<li>小心删除在溶胶加载过程中可能产生的气泡。在使用前, 将含有上述混合溶胶的 PP 管保持在4摄氏度。</li>
	<li>将含有上述混合溶胶的 PP 管附着在用于单向冻结的浸渍机上。设置相关参数, 并开始将 PP 管浸入一个含有液氮 (-196 °c) 的水箱中, 其恒定速度为20厘米 h-1。</li>
	<li>用锯切 PP 管部分, 将冷冻 TOCN-SBR 混合溶胶部分裂成几部分。</li>
	<li>冷冻干燥这些部分与冷冻干燥机在-10 °c 1 天, 然后在-5 °c 1 天, 最后在0°c 1 天。MHM-TOCN/SBR 和 MHM TOCN/2作为白石柱获得。</li>
</ol>7. TOCN-SOCF 混合溶胶制备 Microhoneycomb 巨石 (指 MHM TOCN/SOCF)
<ol><li>装载聚丙烯 (PP) 管 (内径为13毫米, 外径为15毫米, 长度为150毫米), 玻璃珠, 填充管底部5厘米的部分。</li>
	<li>负载一定量 (每次不控制量, 但通常大于2.7 毫升, 以确保随后的切割过程) 的 TOCN-SOCF 混合溶胶进入上述 PP 管含有玻璃珠。</li>
	<li>小心删除在溶胶加载过程中可能产生的气泡。在使用前, 将含有上述混合溶胶的 PP 管保持在4摄氏度。</li>
	<li>将含有上述混合溶胶的 PP 管附着在用于单向冻结的浸渍机上。设置相关参数, 并开始将 PP 管浸入一个含有液氮 (-196 °c) 的水箱中, 其恒定速度为20厘米 h-1。</li>
	<li>用锯子切割 PP 管部分, 将冷冻 TOCN SOCF 溶胶部分裂成几部分。冷冻干燥这些部分与冷冻干燥机在-10 °c 1 天, 然后在-5 °c 1 天, 最后在0°c 1 天。MHM-TOCN/SOCF 是一个白色灰色的巨石。</li>
</ol>

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作者没有什么可透露的。

实现 MHMs 的最关键步骤是单向冻结步骤, 其间水凝固形成柱状冰晶, 并将分散体推到一边形成框架。单向冻结过程主要涉及前驱体溶胶与冷却剂之间的热传递。在我们的设置中, 使用浸渍机将含有前驱溶胶的 PP 管插入到冷却剂 (液氮) 中, 并具有恒定的速度。由于液态氮一直在蒸发, 因此在氮气液位以上产生脉动温度梯度。在接触氮气液位之前, pp 管在氮气液位以上的冷空气中不可避免地经历了热交换, 导致了聚丙烯管底部温度的波动。此外, 在接触氮气液位时, PP 管底部的温度立即降至接近液态氮的温度 (-196 °c), 相邻部分也很快冷却到液氮的温度。.直到某一位置, 与单向冻结有关的 psudo 稳态传热才开始发生。冷冻后, 聚丙烯管被裂解成部分冷冻干燥。当冰趋于解冻时, 这些剖面立即被转移到冰冷的井中, 从而导致样品的形态退化。此外, 冷冻干燥过程小心地进行了在温度低于0摄氏度, 以避免冰的解冻。在图 2a中, 我们观察到了 MHM TOCN 的不同位置, 如下所示。用 SEM 观察到离底部1-7 厘米的位置 (b h), 如图 2bh所示。从底部1厘米以上的位置 (b) 具有面向批量的中心的形态学 (图 2b)。这类似于从冻结的13中制备的巨石, 它涉及沿基底平面的主导热交换。从底部距2厘米的位置, 得到了一个良好排列的蜂窝状形貌 (图 2ch), 显示了冰晶沿 PP 管的长度方向的单向生长。应该提到的是, microhoneycomb 的大小从 (c) 到 (d) 的位置有明显的增加, 此后保持稳定。这归因于距离作用, 在这样一个低位置作为位置 (c), 一个更高的温度梯度和冰晶的更高的成长的速度17参与了, 因而导致更小的冰晶。然而, 在位置 (d) 等较高位置, 距离效应不再适用, 温度梯度变得相对稳定, 从而导致稳定的通道大小为10微米。MHM-TOCN 的通道尺寸将根据 PP 管的浸渍速度而改变, 但 microhoneycomb 形态学保留13。信道大小可以在10到200微米13的范围内进行调整, 而更大或更小的通道大小只能通过特殊设计实现。图 2i提供沿纵向剖面的 MHM TOCN 的形态学, 显示 MHM TOCN 的单向穿透性质。这与从冰箱冻结的18或液氮淬火19中获得的3维多孔结构有很大区别。
我们的方法论最大的优点是它在控制产生的巨石的组成方面的通用性。我们发现, TOCNs 有一个强大的趋势, 形成 MHM 结构通过 UDF 过程。简单地通过制备各种混合物溶胶, 可以实现一系列复合 MHMs。我们在以前的报告13中显示了许多示例。一个典型的例子是与水溶性聚合物的结合, 我们提出另一个例子-SBR 在这里, 如图 3a所示。这些类型的复合 MHMs 具有平滑的 microhoneycomb 壁, 显示了所含元件的均匀分布。此外, 我们已经确认, MHM-TOCN 可以用作纳米粒子的支持, 如图 3b所示。TOCNs 和2纳米粒子的混合物前体溶胶, 产生了一个有序的 MHM, 它的2纳米粒子附着在 microhomeycomb 壁表面。这可以进一步扩展, 以准备功能 MHMs 包括各种纳米粒子。
最后, 我们的方法可以进一步扩展到新的结构, 在微内部的结构。我们发现, 通过在前体溶胶中引入表面氧化碳纤维 (SOCF), 最终通过 UDF 过程 (图 4) 获得了 SOCFs 桥接相邻 microhoneycomb 壁的复合 MHM。虽然进一步增加的 SOCF 干扰的 psudosteady 增长的冰晶, 导致 MHM, 目前的结果表明, 这一方法的可行性, 用于探索新的结构。一旦达到了具有一定韧性的致密结构, 就可以想象这些材料的各种应用, 如储能。

作为一种全新的材料, microhoneycomb 巨石 (指 MHM) 最近吸引了多学科领域的巨大关注1,2,3,4,5,6,7,8. MHM 最初是由美国 Mukai et . 通过改良的单向冷冻干燥 (UDF) 方法制备的, 它是一个带有蜂窝状剖面9的直微阵列的整体。MHM 具有蜂窝结构的一般优点,即, 高效镶嵌, 高强度-重量比, 低压降。此外, 与较大通道尺寸的蜂窝巨石相比, MHM 具有更大的特定表面积。UDF 方法包括冰晶的单向生长和冻结时的同时相分离。在冰晶体去除之后, 得到了由冰晶塑造的固体成分。相分离形成的形态学取决于前驱体 (溶胶或凝胶) 的固有性质, 在大多数情况下, 薄片10、光纤11和鱼骨12结构很可能形成而不是 MHMs。因此, MHMs 的形成只在有限的前体中报告, 这大大阻碍了它们的化学性质的多样性。我们最近发现, 纤维素纳米纤维具有强大的结构导向功能, 通过 UDF 过程13形成 MHM 结构。只要将纤维素纳米纤维与其他水分散组分混合, 就有可能制备出各种不同化学性质的 MHMs。此外, 它们的外部形状和通道尺寸灵活且易于控制13。因此, MHMs 有望被用作过滤器, 催化剂支持, 流型电极, 传感器和生物材料支架。
本文从纤维素纳米纤维的水分散过程中, 详细介绍了 MHMs 的基本制备技术。此外, 我们还演示了几种不同类型的复合 MHMs 的制备方法。

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microhoneycomb monoliths prepared by the unidirectional freeze-drying of cellulose nanofiber based sols: method and extensions

单片蜂窝结构由于其高强度与重量比, 对多学科领域具有吸引力。特别地, microhoneycomb 石柱 (MHMs) 以微米级的渠道被期望作为高效率的平台为反应和分离由于他们的大表面区域。到目前为止, MHMs 是由单向冷冻干燥 (UDF) 方法, 只有从非常有限的前体制备。在此, 我们报告了一个协议, 其中包括一系列由不同的组件组成的 MHMs 可以得到。最近, 我们发现纤维素纳米纤维作为一个独特的结构导向剂的作用, 通过 UDF 过程形成 MHMs。通过将纤维素纤维与不产生 MHMs 的水溶性物质混合, 可以制备各种复合 MHMs。这极大地丰富了 MHMs 的化学组成对多功能应用。

研究了 MHM-TOCN 沿单向冻结方向的不同位置的形貌, 并在图 2中显示。随着位置远离 MHM-TOCN 的底部, 显示了一个渐进的形态学变化 (图 2,讨论)。通过引入 TOCN 溶胶中的第二组分形成均匀的混合物溶胶, 可以制备各种复合 MHMs。例如, 编写了包括 SBR (图 3a)、MHMs2 (图 3b), 甚至碳纤维 (图 4) 的复合型。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57144/57144fig1.jpg"> 图 1: 单向冷冻干燥法制备 MHM TOCN 的示意图.单向冻结是用在左侧显示的浸渍机执行的。单向冷冻后, 采用冷冻干燥机进行冻干, 以产生 MHM TOCN。此图已从 Pan、z-z 修改。et 。13.<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57144/57144fig1large.jpg" target="_blank">请单击此处查看此图的更大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57144/57144fig2.jpg"> 图 2: MHM-TOCN 的不同位置的形态学特征.(a) 示意图, 标记 MHM-TOCN 的不同位置。(b h)扫描电镜图像的横断面的 MHM-TOCN 与底部 (尖端) 的 MHM-TOCN 1, 2, 3, 4, 5, 6, 7 厘米, 分别。(i) MHM-TOCN 纵断面的 SEM 图像。请注意, 在典型的 UDF 实验中, 玻璃珠总是用来填充 PP 管底部5厘米的部分, 然后再加载溶胶进行单向冻结, 这样就可以实现冰晶的伪稳定生长。然而, 在这里, TOCN 溶胶是直接填充到聚丙烯管没有浇注玻璃珠首先研究的距离效应, 涉及的单向冻结过程。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57144/57144fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57144/57144fig3.jpg"> 图 3: 两个 MHM 复合材料的形态学刻画.(a和b) 分别显示 (a) MHM TOCN/SBR 和 (b) MHM TOCN/2的横断面 SEM 图像。在 (a) 和 (b) 内的右上插入是 MHM-TOCN/SBR 和 MHM TOCN/2的光学图像。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57144/57144fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57144/57144fig4.jpg"> 图 4: MHM TOCN/SOCF 的 SEM 图像.图像显示了 SOCF 连接相邻 microhoneycomb 墙的新型结构, 图像内的嵌入是 MHM-TOCN/SOCF 的光学图像。

Watch this Scientific Journal Video about Microhoneycomb Monoliths Prepared by the Unidirectional Freeze-drying of Cellulose Nanofiber Based Sols: Method and Extensions at JoVE.com

Microhoneycomb Monoliths Prepared by the Unidirectional Freeze-drying of Cellulose Nanofiber Based Sols: Method and Extensions

在这里, 我们提出了一个通用的协议, 以准备各种 microhoneycomb 石柱 (MHMs), 其中流体可以通过与极低的压力下降。MHMs 获得的可作为过滤器, 催化剂支持, 流型电极, 传感器和支架的生物材料。

纤维素碳纤维溶胶单向冷冻干燥制备 Microhoneycomb 石柱: 方法和外延

Monolithic honeycomb structures have been attractive to multidisciplinary fields due to their high strength-to-weight ratio. Particularly, microhoneycomb ...

microhoneycomb-monoliths-prepared-unidirectional-freeze-drying

Nanyang Technological University

Research

JoVE Journal

Bioengineering

8.8K 次观看.  Tsinghua University. 在这里, 我们提出了一个通用的协议, 以准备各种 microhoneycomb 石柱 (MHMs), 其中流体可以通过与极低的压力下降。MHMs 获得的可作为过滤器, 催化剂支持, 流型电极, 传感器和支架的生物材料。

视频: Microhoneycomb Monoliths Prepared by the Unidirectional Freeze-drying of Cellulose Nanofiber Based Sols: Method and Extensions

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

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

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 ...

科研

生物工程

Manufacturing Of Robust Natural Fiber Preforms Utilizing Bacterial Cellulose as Binder

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose and short sisal fibers are dispersed into a water suspension containing bacterial cellulose. The fiber and nanocellulose suspension is then filtered (using vacuum or gravity) and the wet filter cake pressed to squeeze out any excess water, followed by a drying step. This will result in the hornification of the bacterial cellulose network, holding the loose natural fibers together.
Our method is specially suited for the manufacturing of rigid and robust preforms of hydrophilic fibers. The porous and hydrophilic nature of such fibers results in significant water uptake, drawing in the bacterial cellulose dispersed in the suspension. The bacterial cellulose will then be filtered against the surface of these fibers, forming a bacterial cellulose coating. When the loose fiber-bacterial cellulose suspension is filtered and dried, the adjacent bacterial cellulose forms a network and hornified to hold the otherwise loose fibers together.
The introduction of bacterial cellulose into the preform resulted in a significant increase of the mechanical properties of the fiber preforms. This can be attributed to the high stiffness and strength of the bacterial cellulose network. With this preform, renewable high performance hierarchical composites can also be manufactured by using conventional composite production methods, such as resin film infusion (RFI) or resin transfer molding (RTM). Here, we also describe the manufacturing of renewable hierarchical composites using double bag vacuum assisted resin infusion.

We present a novel method of manufacturing rigid and robust short natural fiber preforms using a papermaking process. Bacterial cellulose acts simultaneously as the binder for the loose fibers and provides rigidity to the fiber preforms. These preforms can be infused with a resin to produce truly green hierarchical composites.

制造业的鲁棒天然纤维预制体的利用细菌纤维素为粘结剂

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

全球舒张功能通过静脉流的通过参数化舒张形式主义运动学建模为基础的定量分析

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

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 ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

静电纳米纤维支架与渐变的纤维组织

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

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

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

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 ...

Synthesis of Keratin-based Nanofiber for Biomedical Engineering

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of these porous nanofibers is of great interest due to their unique physiochemical properties. Here we elaborate on the fabrication of keratin containing poly (&#949;-caprolactone) (PCL) nanofibers (i.e., PCL/keratin composite fiber). Water soluble keratin was first extracted from human hair and mixed with PCL in different ratios. The blended solution of PCL/keratin was transformed into nanofibrous membranes using a laboratory designed electrospinning set up. Fiber morphology and mechanical properties of the obtained nanofiber were observed and measured using scanning electron microscopy and tensile tester. Furthermore, degradability and chemical properties of the nanofiber were studied by FTIR. SEM images showed uniform surface morphology for PCL/keratin fibers of different compositions. These PCL/keratin fibers also showed excellent mechanical properties such as Young&#39;s modulus and failure point. Fibroblast cells were able to attach and proliferate thus proving good cell viability. Based on the characteristics discussed above, we can strongly argue that the blended nanofibers of natural and synthetic polymers can represent an excellent development of composite materials that can be used for different biomedical applications.

Electrospun nanofibers have a high surface area to weight ratio, excellent mechanical integrity, and support cell growth and proliferation. These nanofibers have a wide range of biomedical applications. Here we fabricate keratin/ PCL nanofibers, using the electrospinning technique, and characterize the fibers for possible applications in tissue engineering.

基于角蛋白纳米纤维生物医学工程合成

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of ...

Dendrimer-based Uneven Nanopatterns to Locally Control Surface Adhesiveness: A Method to Direct Chondrogenic Differentiation

Cellular adhesion and differentiation is conditioned by the nanoscale disposition of the extracellular matrix (ECM) components, with local concentrations having a major effect. Here we present a method to obtain large-scale uneven nanopatterns of arginine-glycine-aspartic acid (RGD)-functionalized dendrimers that permit the nanoscale control of local RGD surface density. Nanopatterns are formed by surface adsorption of dendrimers from solutions at different initial concentrations and are characterized by water contact angle (CA), X-ray photoelectron spectroscopy (XPS), and scanning probe microscopy techniques such as scanning tunneling microscopy (STM) and atomic force microscopy (AFM). The local surface density of RGD is measured using AFM images by means of probability contour maps of minimum interparticle distances and then correlated with cell adhesion response and differentiation. The nanopatterning method presented here is a simple procedure that can be scaled up in a straightforward manner to large surface areas. It is thus fully compatible with cell culture protocols and can be applied to other ligands that exert concentration-dependent effects on cells.

A method to obtain dendrimer-based uneven nanopatterns that permit the nanoscale control of local arginine-glycine-aspartic acid (RGD) surface density is described and applied for the study of cell adhesion and chondrogenic differentiation.

基于状的不均匀 Nanopatterns 对局部控制表面粘附的研究--一种直接软骨分化的方法

Cellular adhesion and differentiation is conditioned by the nanoscale disposition of the extracellular matrix (ECM) components, with local concentrations ...

A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. Human-induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs), human cardiac fibroblasts (HCFs), and human umbilical vein endothelial cells (HUVECs) are isolated and used to generate a cell suspension with 70% iPSC-CMs, 15% HCFs, and 15% HUVECs. They are co-cultured in an ultra-low attachment "hanging drop" system, which contains micropores for condensing hundreds of spheroids at one time. The cells aggregate and spontaneously form beating spheroids after 3 days of co-culture. The spheroids are harvested, seeded into a novel mold cavity, and cultured on a shaker in the incubator. The spheroids become a mature functional tissue approximately 7 days after seeding. The resultant multilayered tissues consist of fused spheroids with satisfactory structural integrity and synchronous beating behavior. This new method has promising potential as a reproducible and cost-effective method to create engineered tissues for the treatment of heart failure in the future.

This protocol describes a net mold-based method to create three-dimensional scaffold-free cardiac tissues with satisfactory structural integrity and synchronous beating behavior.

基于网模的无支架三维心脏组织创建方法

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.