这项工作得到了联合大豆委员会拨款1940-352-0701-C和美国农业部\林务局的支持。我们感谢AES的Phil Humphrey的支持和详细信息。

1. 基材的制备
<ol><li>使用适合应用的基材表面。对于木材，请使用可靠生产商提供的约 0.6 至 0.8 毫米厚的切片单板，因为这些单板用于胶合板和层压单板木材 （LVL） 制造。这些是从单板供应商处获得的，厚度为 0.6 至 0.8 毫米，侧面切成 305 毫米。一致的基材是硬枫（Acer saccharum）面单板，因为它的表面光滑度和一致的厚度，它是一种弥漫多孔和高模量硬木。枫木面贴面通常用于橱柜结构，通常没有缺陷。</li>
	<li>使用前，将木材在21°C和50%相对湿度（RH）下调理至少一天。避免使用过度波浪形、表面不平整且包含变色等缺陷的贴面。 注意:其他木材种类可用于了解粘合剂与这些树种的粘合性能。然而，建议使用弥漫多孔硬木和具有渐变早木到晚木过渡的软木，因为它们的均匀性。要小心，因为木材可以是酸性的或碱性的，或者表面上有提取物，可以改变粘合剂固化过程。此外，树木从切割到单板生产的加工可以改变粘合强度12，13。由于ABES使用少量木材，因此受其他测试（例如木材水分含量和单板检查深度）发生的木材变化的影响较小。</li>
	<li>确保单板的侧面沿边缘没有任何松散的纤维，并且粘合产品没有任何明显的粘合剂挤出，因为这些会高估粘合强度，因为样品没有粘合后改性。</li>
</ol>2. 标本的制备
<ol><li>将木材试样在21°C和50%RH下调节至少一天。切割试样时，检查单板是否有任何裂缝、变色或颗粒不规则。</li>
	<li>确保气动驱动的试样切割装置可操作。</li>
	<li>使用特殊的模切机，将所需的试样尺寸从 0.6 至 0.8 毫米厚的枫木单板切割 20 mm x 117 mm（图 1， 材料表）。<ol><li>在切割刀片下方放置一块至少 150 mm x 300 mm 的单板，使单板纹理与长方向平行，然后按下气压按钮以切割每块 20 mm x 117 mm 的木材。</li>
		<li>将切割刀片下方的单板移动到未切割的区域，然后再次按下按钮以切割另一块木头。继续直到单板完全切成碎片。 注意:如果试样的长方向与纹理方向不平行，则在测试期间，远离粘合部分的木材可能会发生早期断裂。</li>
	</ol></li>
	<li>对于木材以外的材料，请使用适当的技术切割试样。如果无法用试样切割器切割材料，请使用任何可以切割的材料将其切割成所需的尺寸。由于粘合面积小，切割必须准确，试样边缘和粘合表面没有碎屑，这一点很重要。</li>
</ol>3. 设备的可操作性
<ol><li>对于粘合过程，请确保ABES设备按照标准操作程序11正常运行。ABES装置正面用于粘接和破碎样品的设置为:LP压力机0.2 MPa，HP压力机0.2 MPa，拉力0.65 MPa和冷空气0.2 MPa。</li>
	<li>使用至少 0.62 MPa （90 psig） 的供气压力，因为压力过低会导致夹具和压盘在样品上关闭得太慢或不均匀，从而导致粘合强度不正确（图 2，顶部）。</li>
	<li>清洁从先前样品中挤出的任何粘合剂的压盘。将压盘的温度调节到所需温度，并在粘合样品之前进行平衡。</li>
	<li>要粘合木材，请在21°C和50%RH的房间内操作设备。如果无法做到这一点，请将经过处理的试样放在塑料袋中，直到粘合，因为试样的尺寸很小，木材水分会迅速变化。</li>
	<li>为了获得动力学固化数据，请按照ASTM D7998-1911中所述，设计使机械和电子速度足以准确收集数据的方法。</li>
</ol>4. 试样与粘合剂的粘合
注意:粘合剂的应用对于木材粘合剂来说是一个关键问题，因为从胶合板中的层压粘合剂到粘合剂应用的可喷涂粘合剂，粘度和固体百分比的变化很大。木材粘合剂通常是水性的，因此蒸发只是一个小问题。然而，水浸入多孔木材中很重要。
<ol><li>将5mg正在研究的粘合剂涂抹在0.5cm的末端，足以覆盖粘合区域并转移到另一个样品上，但不要过度挤出。为了获得相对恒定的粘合剂铺展速率，请在天平上对木材试样进行去皮，并在粘合剂应用后重新称重。</li>
	<li>在分配粘合剂、重叠试样并确保两个试样对齐时要格外小心，因为使用小的粘合区域，强度确定为粘合区域的拉力（图 2 底部）。可以使用不同的粘合区域，但由于搭接剪切试验力学的变化，强度不一定具有可比性。 注意:文献推荐了几种将粘合剂涂在木材上的方法，具体取决于粘合剂的稠度。最初推荐的粘合剂应用方法使用专门设计的微喷涂装置10，但发现这很杂乱，速度慢，并且非常依赖于粘合剂的流变性。尽管这种方法将粘合剂作为离散点应用于刨花板和定向刨花板的粘合剂应用，但印刷方法似乎更可靠14。该微量移液器施用方法可以提供可重复体积的粘合剂10，但是要均匀分布有些困难。刮刀法对于使粘合剂在粘合区域均匀分布效果最好，建议使用微量天平来获得测量量11。</li>
	<li>最终强度数据<ol><li>将试样在120°C下粘合2分钟，并在21°C和50%RH下调节过夜，因为粘合过程中的热压会使木材变干。要粘合木材，请关闭ABES测试仪上的夹具，确保样品与测试仪对齐，将样品锁定到位。然后按下机器上的启动按钮，让120°C压盘压在重叠部分2分钟，然后缩回压盘并松开夹具，以便取出样品。 注意:固化的时间和温度由应用和粘合剂化学决定。应优化粘接温度和时间，通过使用不同的粘合温度和时间来确定最大强度的条件，使强度达到最高平台。对于木粘结，测试干剪切强度很有价值，但湿测试通常对于确定粘合剂耐久性更为关键，并且需要将样品浸泡在水中4小时室温。</li>
		<li>对于测试，通过关闭ABES测试仪上的夹具来锁定样品，确保样品与测试仪对齐。然后，通过按下启动按钮，仪器通过伺服驱动器拉动一端，而样品的另一端拉动连接到夹具的称重传感器。这种拉动一直持续到键断裂。计算机记录样品可以承受的最大力，记录为粘合强度。<ol><li>对干燥和水浸泡的样品使用相同的程序。在测量断裂力时，请注意确保握把紧握住木材，因为如果粘合剂非常牢固，木材可能会打滑。如果样品在粘合区域之外破裂，请丢弃该值，因为这是在测量木材强度，而不是粘合剂。</li>
		</ol></li>
	</ol></li>
	<li>动能强度开发<ol><li>确定粘合剂的强度发展速度，以估计大规模产品所需的压制时间。遵循与步骤 4.3 中相同的步骤，但改变温度和时间。在100°C压板温度下开始强度测试，使用10，30，60，90，120，150，180和210秒的粘合时间。随后，将温度升高10°C，并重复粘合时间，直到在低粘合时间下不再有任何强度与时间的线性部分。</li>
		<li>粘合后，缩回压盘并使用ABES的风冷功能将样品冷却到接近室温，然后测量样品强度。通过从低压榨时间开始，首先增加后续样品的时间，收集强度与时间的数据，直到增加时间导致强度增加很少或没有增加。然后在较高温度下执行相同的序列将产生强度随时间的关系图和固化速率作为斜率的结果（图 3）。 注意: 图3a10 中的酚醛粘合剂数据显示了不同时间温度对强度发展的影响。 图3b 显示了回归的等温强度发展速率与温度的关系。为了获得等温强度的发展，在测试前冷却样品。一些粘合剂，如脲醛15，在开始降解之前具有最佳的粘合时间和温度。此方法可以检测此问题并确定最佳条件。</li>
	</ol></li>
	<li>耐热性<ol><li>如果产品需要满足一定的耐温性，请将粘合的样品夹入ABES单元中。将压盘加热到该温度后，例如220°C，高于该温度，木材开始降解，将它们关闭在预粘合样品上2分钟，然后打开以测量粘合强度，如4.3.2所示，以确定粘合剂与120°C的粘合温度相比的任何热软化。</li>
		<li>重复此测试，除了将压板关闭在样品上30分钟，然后测试强度以确定粘合剂是否热降解的强度。压盘的释放和测试强度将决定样品的耐热性与加热前的值相比。这种类型的程序用于测试木材粘合剂16。由于ABES使用快速加热，并且可以在热时测量强度，而无需将样品移动到另一台机器，因此可用于区分两种故障模式（即热软化或降解）。热软化在加热后立即产生强度损失，并且通常是可以恢复的。在高温下，化学降解会随着时间的推移逐渐发生，并且在冷却时不会恢复机械强度。 注意:粘合剂制造商需要区分强度损失是由热软化还是化学降解引起的，因为这些问题需要不同的解决方案。有许多方法可以测量软化转变，包括其他热分析，但它们不能区分机械性能和化学结构的变化。</li>
	</ol></li>
</ol>5. 失效粘接面的图像分析
<ol><li>由于主要目的是确定粘合强度或内聚强度发展速率，因此请确保故障发生在粘合剂内部，而不是粘附在基材上（图4）或基材失效。如果发生基材故障，则粘合剂具有足够的强度。或者，本体粘合剂中的内聚失效表明粘合剂薄弱。然而，在粘附和粘合剂相间失效之间做出选择可能很困难17.已经开发了多种木材分析方法18.</li>
</ol>

<ol>
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作者没有什么可透露的。

程序中的关键步骤如下:基材的选择、样品的制备、设备的可操作性和样品的粘合。
基材必须坚固，缺陷最小（光滑，平整，无裂纹和变色）。未打磨，旋转切割的橱柜面单板为漫反射多孔硬木，首选糖枫（Acer saccharum）。打磨会产生不那么平坦和更破碎的表面7.在21°C和50%RH下调节单板至少一天后，切割20毫米乘117毫米的条带。通常将 5 毫克粘合剂均匀...

木材粘接是最大的单一粘合剂市场，并导致森林资源的有效利用。几个世纪以来，除家具结构外，实木被用于大多数应用，除了产品使用耐久性外，没有测试标准。然而，粘合木制品变得更加普遍，从胶合板和胶合木梁开始，使用生物基粘合剂1，2。虽然这些产品在当时令人满意，但用含有甲醛的合成粘合剂代替大豆、酪蛋白和血胶，改善了性能。这些新型粘合剂的更高性能导致了明确的测试标准，与大多数生物基粘合剂相比，具有更高的性能期望。合成粘合剂还可以粘合颗粒，包括形成刨花板的锯末、形成不同密度纤维板的纤维、提供定向刨花板和平行刨花材的木片、生产胶合板和层压单板木材的单板，以及指接木材、胶合木、交叉层压木材和木工字托梁3。这些产品中的每一个都有自己的测试标准4.因此，开发新型粘合剂可能需要大量的配方工作和广泛的测试，以确定是否有潜力开发足够的强度。这种耗时的测试以及木材性能和木材粘合的复杂性5限制了新型粘合剂的开发。此外，木材粘合剂在木材表面之间固化时的机械性能可能与整齐6不同。与木材接触固化允许粘合剂中的水和低分子量组分逸出，此外粘合剂与木材的复杂界面和化学相互作用3，7。
自动粘接评估系统（ABES）的开发对于了解木材粘合剂的强度发展非常有帮助，因为它快速且易于使用8，9，10。该系统是一个整体单元，用于粘合搭接剪切样品，然后测量断裂粘合所需的张力。它的实用性导致了使用该系统的ASTM方法D7998-19的开发11。虽然该系统最初设计用于测量粘合剂强度随温度和时间的变化，但它也可以测量固化粘合剂的耐热性，以及常规的粘合强度评估。尽管ABES测试是一种非常有用的初步筛选工具，但与任何测试一样，它有其局限性，并不能取代所有特定的产品强度和耐久性测试。
虽然有许多方法可以测量粘合剂的固化特性，从凝胶时间流变仪到差示扫描量热法、动态力学分析和多种类型的光谱法，但只有ABES方法测量机械强度的发展。这需要一台严格控制的仪器，用于加热、冷却和原位拉伸测试11。

<table>
	<tbody>
		<tr>
			<td>Adhesive</td>
			<td>Supplied by user</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Balance</td>
			<td>Normal supply house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mark II Automated Bonding Evaluation System (ABES-II)</td>
			<td>Adhesive Evaluation Systems Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pneumatically driven sample cutting device</td>
			<td>Adhesive Evaluation Systems Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Regular spatula</td>
			<td>Normal supply house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wood supply &#8211; Hard maple</td>
			<td>Besse Forest Products Group</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

standard test method astm d 7998-19 for the cohesive strength development of wood adhesives

固化木材胶粘剂的性能由于水分和其他组分对木材的损失，木材对胶粘剂固化的影响以及粘合剂渗透对木材界面的影响而难以研究;因此，对纯胶膜进行正常测试通常没有用。大多数木材粘合剂粘合强度的测试都是缓慢的，费力的，可能受到木材的强烈影响，并且不能提供有关固化动力学的信息。然而，测试方法ASTM D 7998-19可用于快速评估木材粘结强度。使用光滑、均匀和坚固的木材表面（如枫木面饰面）和足够的粘合压力可降低粘附力和木材强度对粘合强度的影响。此方法有三个主要应用。首先是提供一致的粘结强度发展数据。第二种是测量粘结搭接剪切样品的干湿强度。第三是通过快速评估热灵敏度并区分热软化和热降解来更好地了解粘合剂的耐热性。

该程序已广泛用于林产品实验室的蛋白质粘合剂研究。已经发现，低于2 MPa的湿粘合强度不足以保证进一步的木材粘合剂测试，而大于3 MPa是进一步测试的有希望的结果19。它已被证明可用于证明木材加工条件的敏感性12，13。更多的例子可以在弗里哈特的出版物中找到 7.该方法的精密度和偏差已经确定（研究报告RR:D14-...

Watch this Scientific Journal Video about Standard Test Method ASTM D 7998-19 for the Cohesive Strength Development of Wood Adhesives at JoVE.com

Standard Test Method ASTM D 7998-19 for the Cohesive Strength Development of Wood Adhesives

我们提出了一种程序，ASTM D7998-19，用于快速，更一致地评估木材上粘合剂的干强度和湿强度。该方法还可用于提供强度发展信息，作为温度和时间的函数或高达250°C的强度保持。

用于木材粘合剂内聚强度发展的标准测试方法 ASTM D 7998-19

The properties of cured wood adhesives are difficult to study because of the loss of water and other components to the wood, the influence of wood on the ...

standard-test-method-astm-d-7998-19-for-cohesive-strength-development

Research

JoVE Journal

Bioengineering

2.8K 次观看.  USDA, Forest Service. 我们提出了一种程序，ASTM D7998-19，用于快速，更一致地评估木材上粘合剂的干强度和湿强度。该方法还可用于提供强度发展信息，作为温度和时间的函数或高达250°C的强度保持。

视频: Standard Test Method ASTM D 7998-19 for the Cohesive Strength Development of Wood Adhesives

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

卵泡在蛋白质降解的3维系统的封装和文化的一种方法

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

科研

生物工程

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

一个简单的悬滴细胞生成三维球体文化协议

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

Measurement of Aggregate Cohesion by Tissue Surface Tensiometry

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium. We have developed tissue surface tensiometry (TST) specifically to measure the surface free energy of interaction between cells. The biophysical concepts underlying TST have been previously described in detail1,2. The method is based on the observation that mutually cohesive cells, if maintained in shaking culture, will spontaneously assemble into clusters. Over time, these clusters will round up to form spheres. This rounding-up behavior mimics the behavior characteristic of liquid systems. Intercellular binding energy is measured by compressing spherical aggregates between parallel plates in a custom-designed tissue surface tensiometer. The same mathematical equation used to measure the surface tension of a liquid droplet is used to measure surface tension of 3D tissue-like spherical aggregates. The cellular equivalent of liquid surface tension is intercellular binding energy, or more generally, tissue cohesivity. Previous studies from our laboratory have shown that tissue surface tension (1) predicts how two groups of embryonic cells will interact with one another1-5, (2) can strongly influence the ability of tissues to interact with biomaterials6, (3) can be altered not only through direct manipulation of cadherin-based intercellular cohesion7, but also by manipulation of key ECM molecules such as FN8-11 and 4) correlates with invasive potential of lung cancer12, fibrosarcoma13, brain tumor14 and prostate tumor cell lines15. In this article we will describe the apparatus, detail the steps required to generate spheroids, to load the spheroids into the tensiometer chamber, to initiate aggregate compression, and to analyze and validate the tissue surface tension measurements generated.

We describe a method of measuring binding energy, expressible as tissue surface tension, between cells within 3D tissue-like aggregates. Differences in tissue surface tension have been demonstrated to correlate with invasiveness of lung, muscle, and brain tumors, and are fundamental determinants of establishing spatial relationships between different cell types.

总结凝聚力测量组织表面Tensiometry

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium.  We ...

Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming

Correlating gene expression with cell behavior is ideally done at the single-cell level. However, this is not easily achieved because the small amount of labile mRNA present in a single cell (1-5% of 1-50pg total RNA, or 0.01-2.5pg mRNA, per cell 1) mostly degrades before it can be reverse transcribed into a stable cDNA copy. For example, using standard laboratory reagents and hardware, only a small number of genes can be qualitatively assessed per cell 2. One way to increase the efficiency of standard laboratory reverse transcriptase (RT) reactions (i.e. standard reagents in microliter volumes) comprising single-cell amounts of mRNA would be to more rapidly mix the reagents so the mRNA can be converted to cDNA before it degrades. However this is not trivial because at microliter scales liquid flow is laminar, i.e. currently available methods of mixing (i.e. shaking, vortexing and trituration) fail to produce sufficient chaotic motion to effectively mix reagents. To solve this problem, micro-scale mixing techniques have to be used 3,4. A number of microfluidic-based mixing technologies have been developed which successfully increase RT reaction yields 5-8. However, microfluidics technologies require specialized hardware that is relatively expensive and not yet widely available. A cheaper, more convenient solution is desirable. The main objective of this study is to demonstrate how application of a novel "micromixing" technique to standard laboratory RT reactions comprising single-cell quantities of mRNA significantly increases their cDNA yields. We find cDNA yields increase by approximately 10-100-fold, which enables: (1) greater numbers of genes to be analyzed per cell; (2) more quantitative analysis of gene expression; and (3) better detection of low-abundance genes in single cells. The micromixing is based on acoustic microstreaming 9-12, a phenomenon where sound waves propagating around a small obstacle create a mean flow near the obstacle. We have developed an acoustic microstreaming-based device ("micromixer") with a key simplification; acoustic microstreaming can be achieved at audio frequencies by ensuring the system has a liquid-air interface with a small radius of curvature 13. The meniscus of a microliter volume of solution in a tube provides an appropriately small radius of curvature. The use of audio frequencies means that the hardware can be inexpensive and versatile 13, and nucleic acids and other biochemical reagents are not damaged like they can be with standard laboratory sonicators.

We describe a novel method for increasing cDNA yield from single-cell quantities of mRNA in otherwise standard laboratory reverse transcription reactions. The novelty resides in the use of a micromixer, which utilizes the phenomenon of acoustic microstreaming, to mix fluids at microliter scales more effectively than shaking, vortexing or trituration.

增加从单细胞的数量标准实验室逆转录反应的mRNA的cDNA产量，使用声Microstreaming

Correlating gene expression with cell behavior is ideally done at the single-cell level.  However, this is not easily achieved because the small amount of ...

Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor microfabrication.1-10 Recently, thermal inkjet printing has also been used for gene transfection.8,9 The thermal inkjet printing process was shown to temporarily disrupt the cell membranes without affecting cell viability. The transient pores in the membrane can be used to introduce molecules, which would otherwise be too large to pass through the membrane, into the cell cytoplasm.8,9,11
The application being demonstrated here is the use of thermal inkjet printing for the incorporation of fluorescently labeled g-actin monomers into cells. The advantage of using thermal ink-jet printing to inject molecules into cells is that the technique is relatively benign to cells.8, 12 Cell viability after printing has been shown to be similar to standard cell plating methods1,8. In addition, inkjet printing can process thousands of cells in minutes, which is much faster than manual microinjection. The pores created by printing have been shown to close within about two hours. However, there is a limit to the size of the pore created (~10 nm) with this printing technique, which limits the technique to injecting cells with small proteins and/or particles. 8,9,11
A standard HP DeskJet 500 printer was modified to allow for cell printing.3, 5, 8 The cover of the printer was removed and the paper feed mechanism was bypassed using a mechanical lever. A stage was created to allow for placement of microscope slides and coverslips directly under the print head. Ink cartridges were opened, the ink was removed and they were cleaned prior to use with cells. The printing pattern was created using standard drawing software, which then controlled the printer through a simple print command. 3T3 fibroblasts were grown to confluence, trypsinized, and then resuspended into phosphate buffered saline with soluble fluorescently labeled g-actin monomers. The cell suspension was pipetted into the ink cartridge and lines of cells were printed onto glass microscope cover slips. The live cells were imaged using fluorescence microscopy and actin was found throughout the cytoplasm. Incorporation of fluorescent actin into the cell allows for imaging of short-time cytoskeletal dynamics and is useful for a wide range of applications.13-15

A description of the methods used to convert an HP DeskJet 500 printer into a bioprinter. The printer is capable of processing living cells, which causes transient pores in the membrane. These pores can be utilized to incorporate small molecules, including fluorescent G-actin, into the printed cells.

创建瞬态细胞膜毛孔，使用标准的喷墨打印机

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor ...

The Portable Chemical Sterilizer (PCS), D-FENS, and D-FEND ALL: Novel Chlorine Dioxide Decontamination Technologies for the Military

There is a stated Army need for a field-portable, non-steam sterilizer technology that can be used by Forward Surgical Teams, Dental Companies, Veterinary Service Support Detachments, Combat Support Hospitals, and Area Medical Laboratories to sterilize surgical instruments and to sterilize pathological specimens prior to disposal in operating rooms, emergency treatment areas, and intensive care units. The following ensemble of novel, &#8216;clean and green&#8217; chlorine dioxide technologies are versatile and flexible to adapt to meet a number of critical military needs for decontamination6,15. Specifically, the Portable Chemical Sterilizer (PCS) was invented to meet urgent battlefield needs and close critical capability gaps for energy-independence, lightweight portability, rapid mobility, and rugged durability in high intensity forward deployments3. As a revolutionary technological breakthrough in surgical sterilization technology, the PCS is a Modern Field Autoclave that relies on on-site, point-of-use, at-will generation of chlorine dioxide instead of steam. Two (2) PCS units sterilize 4 surgical trays in 1 hr, which is the equivalent throughput of one large steam autoclave (nicknamed &#8220;Bertha&#8221; in deployments because of its cumbersome size, bulky dimensions, and weight). However, the PCS operates using 100% less electricity (0 vs. 9 kW) and 98% less water (10 vs. 640 oz.), significantly reduces weight by 95% (20 vs. 450 lbs, a 4-man lift) and cube by 96% (2.1 vs. 60.2 ft3), and virtually eliminates the difficult challenges in forward deployments of repairs and maintaining reliable operation, lifting and transporting, and electrical power required for steam autoclaves.

The Portable Chemical Sterilizer PCS, D-FENS, and D-FEND ALL: Novel Chlorine Dioxide Decontamination Technologies for the Military

The Portable Chemical Sterilizer (PCS) is a revolutionary, energy-independent, almost waterless sterilization technology for Army medical units. The PCS generates chlorine dioxide from dry reagents mixed with water on-site, at-will, and at point-of-use (PoU) in a plastic suitcase. The Disinfectant-sprayer for Foods and ENvironmentally-friendly Sanitation (D-FENS) and the Disinfectant for ENvironmentally-friendly Decontamination, All-purpose (D-FEND ALL) produce aqueous chlorine dioxide in a collapsible spray bottle and other potential embodiments. These versatile decontamination technologies kill microbes in myriad diverse Dual-use applications for military and civilian consumers.

便携式化学消毒（PCS），D-沼泽，和D-FEND ALL:新型二氧化氯去污技术用于军事

There is a stated Army need for a field-portable, non-steam sterilizer technology that can be used by Forward Surgical Teams, Dental Companies, Veterinary ...

Fabrication of Carbon Nanotube High-Frequency Nanoelectronic Biosensor for Sensing in High Ionic Strength Solutions

The unique electronic properties and high surface-to-volume ratios of single-walled carbon nanotubes (SWNT) and semiconductor nanowires (NW) 1-4 make them good candidates for high sensitivity biosensors. When a charged molecule binds to such a sensor surface, it alters the carrier density5 in the sensor, resulting in changes in its DC conductance. However, in an ionic solution a charged surface also attracts counter-ions from the solution, forming an electrical double layer (EDL). This EDL effectively screens off the charge, and in physiologically relevant conditions ~100 millimolar (mM), the characteristic charge screening length (Debye length) is less than a nanometer (nm). Thus, in high ionic strength solutions, charge based (DC) detection is fundamentally impeded6-8.
We overcome charge screening effects by detecting molecular dipoles rather than charges at high frequency, by operating carbon nanotube field effect transistors as high frequency mixers9-11. At high frequencies, the AC drive force can no longer overcome the solution drag and the ions in solution do not have sufficient time to form the EDL. Further, frequency mixing technique allows us to operate at frequencies high enough to overcome ionic screening, and yet detect the sensing signals at lower frequencies11-12. Also, the high transconductance of SWNT transistors provides an internal gain for the sensing signal, which obviates the need for external signal amplifier.
Here, we describe the protocol to (a) fabricate SWNT transistors, (b) functionalize biomolecules to the nanotube13, (c) design and stamp a poly-dimethylsiloxane (PDMS) micro-fluidic chamber14 onto the device, and (d) carry out high frequency sensing in different ionic strength solutions11.

We describe the device fabrication and measurement protocol for carbon nanotube based high frequency biosensors. The high frequency sensing technique mitigates the fundamental ionic (Debye) screening effect and allows nanotube biosensor to be operated in high ionic strength solutions where conventional electronic biosensors fail. Our technology provides a unique platform for point-of-care (POC) electronic biosensors operating in physiologically relevant conditions.

制备碳纳米管高频纳电子生物传感器的传感高离子强度的解决方案

The unique electronic properties and high surface-to-volume ratios of single-walled carbon nanotubes (SWNT) and semiconductor nanowires (NW) 1-4 make them ...

Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is strongly regulated by the surrounding flow and nutritional environment. Biofilm growth also increases the heterogeneity of the local microenvironment by generating complex flow fields and solute transport patterns. To investigate the development of heterogeneity in biofilms and interactions between biofilms and their local micro-habitat, we grew mono-species biofilms of Pseudomonas aeruginosa and dual-species biofilms of P. aeruginosa and Escherichia coli under nutritional gradients in a microfluidic flow cell. We provide detailed protocols for creating nutrient gradients within the flow cell and for growing and visualizing biofilm development under these conditions. We also present protocols for a series of optical methods to quantify spatial patterns in biofilm structure, flow distributions over biofilms, and mass transport around and within biofilm colonies. These methods support comprehensive investigations of the co-development of biofilm and habitat heterogeneity.

Biofilms have complex interactions with their surrounding environment. To comprehensively investigate biofilm-environment interactions, we present here a series of methods to create heterogeneous chemical environment for biofilm development, to quantify local flow velocity, and to analyze mass transport in and around biofilm colonies.

方法表征的共同发展生物膜和生境异质性

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is ...

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

定义人工程心脏组织建设探讨心肌细胞疗法的机理

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

Development of an In Vitro Ocular Platform to Test Contact Lenses

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic physiological tear volumes.7 The traditional model also lacks the natural tear flow component and the blinking reflex, both of which are defining factors of the ocular environment. The development of a novel model is described in this study, which consists of a unique 2-piece design, eyeball and eyelid piece, capable of mimicking physiological tear volume. The models are created from 3-D printed molds (Polytetrafluoroethylene or Teflon molds), which can be used to generate eye models from various polymers, such as polydimethylsiloxane (PDMS) and agar. Further modifications to the eye pieces, such as the integration of an explanted human or animal cornea or human corneal construct, will permit for more complex in vitro ocular studies. A commercial microfluidic syringe pump is integrated with the platform to emulate physiological tear secretion. Air exposure and mechanical wear are achieved using two mechanical actuators, of which one moves the eyelid piece laterally, and the other moves the eyeballeyepiece circularly. The model has been used to evaluate CLs for drug delivery and deposition of tear components on CLs.