这项研究是由美国国立卫生研究院院内研究计划，国家癌症研究所，癌症研究中心的支持。

注意:以下协议描述必要的步骤来消除使用NIR-PIT特定细胞。控制和有关NIR-PIT和细胞活力等细节可以在其他地方18中找到。 1.共轭IR700，以单克隆抗体（MAB） <ol><li>在2-5毫克/毫升的0.1M 的 Na 2 HPO 4（pH为8.6）的溶液制备的兴趣的mAb。 </li><li>混合mAb的6.8纳摩尔用10mM IR700 30.8纳摩尔在0.1M 的 Na 2 HPO 4在微量离心管中液（pH 8.6），并在室温孵育1小时，用铝箔覆盖。 </li><li>用15ml的PBS洗涤PD-10柱（参见材料/设备的数据表），两次。从步骤1.2装载样品。 </li><li>根据制造商的说明通过PBS洗脱纯化经由PD-10柱的混合物。 注:在这里，洗脱沿IR700的带颜色。对于300微升样品，PBS洗脱分数毫升〜4.4毫升通常为2.5之间。 </li><li>确定蛋白质浓度用考马斯染色，通过测量在595nm处的吸收用分光光度计9。确定IR700与吸收的浓度在689纳米到确认缀合每种mAb分子9荧光团分子的数目。 注意:确定在1单抗IR700分子的最佳偶联次数用分光光度计是很重要的。通常，在1单抗分子约3 IR700分子是最适用于在体外和体内的工作。 HPLC和SDS-PAGE方法可以用于确认单抗和IR700是否可能会或没有。 </li><li>测定蛋白质浓度后储存于4℃。 </li></ol> 2.准备混合3D细胞培养（混合球体） <ol><li>应用无菌水（约1ml）中成悬滴板的板储部分。 </li><li>制备的细胞类型的利益的各种比例 - A431-LUC-GFP细胞和3T3-RFP细胞 -用含有5,000个细胞总数，悬浮于50μl培养基的每一个样品。 注意:根据细胞类型的培养基中的细胞类型的兴趣的比率100 等 :准备1:100，10:100; 25:100，50。 </li><li>在37℃和5％二氧化碳孵育在96孔悬滴板5-7天的混合物在加湿培养箱中。改变培养基，每2天。注:为了使3D球体准确，轻拿轻放盘，如含有细胞滴形成3D形状之前，容易脱落。 </li><li> 40X放大率 - 使用倒置亮视野显微镜在10倍观察球体的形态和尺寸。注意:尽管这取决于细胞类型和悬滴的大小，确保该球体的直径后，在96孵育7天后的孔悬滴板周围400-600微米。 </li></ol> 3. 在体外 NIR-PIT混合3D细胞培养<ol><li>更改HA媒体nging滴板，以10微克/毫升抗体 - 光吸收剂结合物（APC）含有介质，并且在37℃和5％二氧化碳的潮湿培养箱孵育6小时。 </li><li>后6小时温育后，用新鲜的培养基（无酚红）洗球体的两倍。轻轻球体用100微升新鲜无酚红培养基用无菌200微升枪头，转移到玻璃底50毫米菜尖切断。放在每道菜1球体。 </li><li>观察球体用倒置明显微镜检测形态的变化。观察光学记者（如 GFP和RFP）使用具有以下过滤器设置荧光显微镜:GFP - 469 nm激发过滤器和525 nm发射滤波器; RFP - 559 nm的激发滤光片和630nm的发射滤光器。 </li><li>放置照射玻璃底培养皿上方的发光二极管（LED）。 注:球体​​可以接触到近红外光无论是在显微镜或在层流罩。 <ol><li>测量近红外光的功率密度与光功率计9。根据该测定，通过发光二极管（LED），其在在2J / cm 2的670至710纳米的波长的发射光照射近红外光。 注:LED灯可以穿透大约5英寸最大深度。不管功率密度和曝光23的持续时间的近红外PIT的细胞毒性作用是只依赖于给定的能量。 </li></ol></li><li>照射后，转移球体到一个新的悬滴板用50μl新鲜培养基，并在37℃和5％二氧化碳的潮湿培养箱孵育1天。 </li><li>轻轻球体用100微升使用带切断尖端无菌200微升枪头，新鲜培养基（无酚红）的转移到玻璃底菜。观察与NIR-PIT使用网络连接后，荧光显微镜1天球体在步骤3.3说明过滤器设置。 <ol><li>通过，或通过使用荧光显微镜（ 图2B）14,18,22细胞质的GFP荧光的损失以2微克/毫升的最终浓度加入碘化丙啶至媒体检测死细胞。 </li></ol></li><li>如果靶细胞没有完全消除重复步骤3.1-3.6。 </li></ol>

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作者什么都没有透露。

我们证明特定细胞消除从混合三维细胞培养物的方法，而不使用近红外-PIT损害对非靶细胞。到目前为止，存在一旦组织被建立或细胞消除无实际方法移植后。因此，NIR-PIT是实现这个很有前途的方法。这种技术也可在体内 18,22利用，因为的APCs显示出类似的药代动力学为单克隆抗体本身。靶细胞类型可适于与各种的APC。各种抗体或抗体片段，其中包括抗EGFR，抗PSMA，抗间皮素和抗CEA已经用作的APC 15 - 21。此外，改变NIR照射的区域可以最小化处理的区域。这里，以萤光素酶活性的定量和荧光对靶细胞，特定的细胞完全消除了确认。 NIR-PIT是一种基于光治疗，因而临界点为t他的近红外光源和目标之间的距离，由于光能量并根据一平方反比定律（步骤3.4），因此细胞坏死减少。使三维球体确切地说，板应处理并轻轻孵育越好。含滴细胞很容易被破坏，或形成3D形状面前有点惊世骇俗脱落。 NIR-PIT有一些独特的优势。首先，NIR-PIT的APCs展示出相似静脉内药代动力学给父母的非共轭的抗体，由于IR700的亲水性，导致高度特异性结合到靶细胞和最小非目标累积。因此，即使在组织移植后，NIR-PIT可通过在APC随后感兴趣近红外区域的曝光的静脉注射治疗的靶细胞。第二，近红外光可以穿透更深入组织比紫外线或可见光，因此，近红外PIT可以治疗不仅表面而且ENTI再通过暴露于NIR表面处的移植组织的厚度。最后，近红外PIT可反复使用，以消除靶细胞而没有限制。当靶细胞在细胞表面表达18靶分子足够的副本，此方法非常有用。 不过，也有这种技术有一些限制。首先，在APC的渗透性被限制，因为它是一个大的分子。为了克服这个问题，反复NIR-PIT治疗或抗体片段可以利用18,19,22。另一个限制是光线穿透。虽然近红外光可正确地穿透体外培养，翻译到体内治疗需要适于的方法，如消化内镜，支气管镜，或直接胸腔镜与光导纤维移动光源更靠近目标区域。 NIR-PIT的进一步修改将允许在本地和规范使用这种方法的IFIC细胞消灭在许多领域，如免疫调节或肿瘤微环境中24 - 26。

消除特定细胞而不损害其他细胞是非常困难的，特别是在建立组织，并且存在用于在组织工程领域中的细胞消除方法的迫切需要。现今在再生医学领域中，使用胚胎干细胞（ES），多能干细胞（的PSCs），或诱导的多能干细胞（IPS）的组织培养物是有前途的材料1 - 3。 虽然这个组织再生是有前途的，具有不需要的细胞污染是一个大问题。此外，有移植4,5-之后致瘤性的安全问题。虽然许多研究都集中在这些问题上，以消除特定细胞，尤其是在再生医学6 - 8，没有实际的方法已被开发出来。 近红外photoimmunotherapy（NIR-PIT）是一种基于抗体 - 光吸收剂conjugat的处理E（APC）。一个APC由细胞特异性单克隆抗体（mAb）和光吸收剂，IR700的。 IR700是亲水性二氧化硅的酞菁衍生物和本身并不9诱发光毒性。 IR700是通过赖氨酸分子的侧链酰胺残基共价结合到抗体上。 APC的结合细胞膜上的靶分子，然后诱导暴露于NIR光在690纳米的近后立即细胞坏死。在暴露于近红外光，细胞膜破裂，导致细胞死亡9 - 14。 NIR-PIT已被证明是具有多个抗体或抗体片段，其中包括抗EGFR，抗HER2，抗PSMA，抗CD25，抗间皮素，抗GPC3，和抗CEA 15有效- 21。因此，近红外PIT可对多种靶分子使用。此外，近红外PIT是良好控制的治疗，通过限制近红外二极管灯允许特定区域的选择性治疗ŧ照射18,22。 这里，我们目前采用NIR-PIT从混合3D文化特异性细胞清除的方法。

<table border="0" cellpadding="0" cellspacing="0" width="1083">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:424px;">IRDye 700DX Ester Infrared Dye</td>
			<td style="width:364px;">LI-COR Bioscience (Lincoln, NE, USA)</td>
			<td style="width:191px;">929-70011</td>
			<td style="width:104px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4</td>
			<td>SIGMA-ALDRICH (St. Louis, MO, USA)</td>
			<td>S9763</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sephadex G25 column (PD-10)&#160;</td>
			<td>GE Healthcare (Piscataway, NJ, USA)</td>
			<td>17-0851-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Coomassie (bradford) Plus protein assay</td>
			<td>Thermo Fisher Scientific Inc (Waltham, MA, USA)</td>
			<td>PI-23200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Perfecta3D 96-Well hanging Drop Plates</td>
			<td>3D Biomatrix Inc (Ann Arbor, MI, USA)</td>
			<td>HDP1096-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Optical power meter</td>
			<td>Thorlabs (Newton, NJ, USA)</td>
			<td>PM100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LED: L690-66-60</td>
			<td>Marubeni America Co. (Santa Clara, CA, USA)</td>
			<td>L690-66-60</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vectibix (panitumumab)</td>
			<td>Amgen (Thousand Oaks, CA, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35mm glass bottom dish, dish size 35mm, well size 10mm</td>
			<td>Cellvis (Mountain View, CA, USA)</td>
			<td>D35-10-0-N</td>
			<td></td>
		</tr>
	</tbody>
</table>

selective cell elimination from mixed 3d culture using a near infrared photoimmunotherapy technique

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem cell (iPS) emerged as a new method in regenerative medicine. Although this tissue regeneration is promising, contamination with unwanted cells during tissue cultures is a major concern. Moreover, there is a safety concern regarding tumorigenicity after transplantation. Therefore, there is an urgent need for eliminating specific cells without damaging other cells that need to be protected, especially in established tissue. Here, we present a method for specific cell elimination from a mixed 3D cell culture in vitro with near infrared photoimmunotherapy (NIR-PIT) without damaging non-targeted cells. This technique enables the elimination of specific cells from mixed cell cultures or tissues.

以光学监控NIR-PIT中，A431细胞系，其过表达EGFR的效果，被遗传修饰以也表达GFP和荧光素酶（A431-LUC-GFP）。作为近红外PIT的一个非目标，所述的Balb / 3T3细胞系的光学修饰以表达RFP（3T3-RFP）。 APC的，帕尼单抗-IR700（泛IR700），合成。混合球状体，其中分别组成细胞（A431-LUC-GFP和3T3-RFP）的各种比例的是按照此协议（ 图1）制成。反复NIR-PIT与泛IR700（参见图2A方案）的孵化执行。从这种混合三维培养靶细胞消除达到与荧光和荧光素酶的活动（ 图2B，C）进行监测。另一方面，非靶细胞持续增长。 <img alt="图1" src="/files/ftp_upload/53633/53633fig1.jpg" /> <str翁&gt;图1.混合球状体的各种比例的代表性图像。混合球状体的代表性图像A431-LUC-GFP细胞和在第7天3T3-RFP细胞各种比例被示出。比例尺= 200微米。这个数字已经从16修改。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53633/53633fig1large.jpg" target="_blank">请点击此处查看本图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53633/53633fig2.jpg" /> 在三维细胞球体图2.靶细胞消除（A431-LUC-GFP和3T3-RFP细胞混合球体）。（A）的近红外-PIT（2J / cm 2）的方案中示出。 （B）的重复NIR-PIT完全消除，没有损害非靶细胞（3T3-RFP）靶细胞（A431-LUC-GFP）中的混合三维球体。栏= 200微米。荧光素酶的活动（C）定量（RLU比）靶细胞（每组10球体）的证明完全消除。这个数字已经从18修改。数据表示为平均值±SEM <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53633/53633fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique at JoVE.com

Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, yet there is an urgent need for a cell elimination method in the tissue engineering field. Here, we present a method for specific cell elimination from a mixed 3D cell culture using near infrared photoimmunotherapy (NIR-PIT).

从混合三维培养使用近红外Photoimmunotherapy技术选择性细胞消除

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem ...

selective-cell-elimination-from-mixed-3d-culture-using-near-infrared

Nanyang Technological University

Research

JoVE Journal

Bioengineering

7.8K 次观看.  National Cancer Institute. Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, yet there is an urgent need for a cell elimination method in the tissue engineering field. Here, we present a method for specific cell elimination from a mixed 3D cell culture using near infrared photoimmunotherapy (NIR-PIT). 

视频: Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

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 ...

科研

生物工程

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

旋转的人类细胞培养的细胞培养系统:作为一种模式的滋养细胞

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

制备纤维蛋白三维支架干细胞培养应用

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

新方法本地化记者荧光珠近细胞培养表面的牵引力显微镜

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

荧光淬一个脂质体包封的近红外荧光团的作为一个工具<em&gt;在体内</em&gt;光学成像

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

使用粘性图案构建3D纸微流体装置

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

缩微和3D微血管大会

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

可调水凝胶由肺细胞外基质的三维细胞培养

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioink Precellularization 无源混合装置打印软骨及皮肤组织类似物的应用

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

用 3 d-印刷模具制作细胞播种和组织环自组装用的自定义琼脂糖井

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...