Name: selective cell elimination from mixed 3d culture using a near infrared photoimmunotherapy technique
Uploaded: 2016-03-14T14:00:00
Description: Watch this Scientific Journal Video about Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique at JoVE.com

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?...

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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...

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</...

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 ...

The overall goal of this experimental procedure is to demonstrate the elimination of specific cells of interest from a mixed 3D cell culture using near infrared photoimmunotherapy. This method can help answer key questions in the tissue-engineering field about regenerating medicine. The main advantage of this technique that it can be used against a variety of target molecules and that the treatment can be restricted to specific lesions of interest.Thus can minimize the side effect. For IR700 conjugation to monoclonal antibodies, incubate the antibodies of interest with IR700 in Disodium Phosphate in a microcentrefuge tube at room temperature for one hour covered in aluminum foil. At the end of the incubation, wash a PD10 column two times with 15 milliliters of PBS.Then, load the antibody solution onto the column and purify the mixture by PBS elution according to the manufacturer's instructions. To confirm the number of fluoro-4 molecules conjugated to each monoclonal antibody molecule, use a spectrophotometer to determine the monoclonal antibody protein concentration. Then, store the conjugate at 4-degree Celsius.To prepare the mixed 3D cell culture, add 1 milliliter of sterile water into the plate reservoir section of a 96-well hanging drop plate. Then, plate various ratios of the cell types of interest in the appropriate cell culture medium at at least 5, 000 cells per 50 microliters of medium per well for five to seven days in a humidified incubator at 37-degree Celsius and 5%carbon dioxide. Change the culture medium every two days and observe the morphology and size of the spheroids daily with an inverted bright-field microscope at 10 to 40 times magnification.When the spheroids have reached 400 to 600 microns in diameter, replace the hanging drop plate medium with antibody photoabsorber conjugate-containing medium, and return the plate to the incubator for six hours. At the end of the incubation, wash the spheroids two times with fresh phenol red free-culture medium. Then use a 200-microliter pipette tip with a cut-off end to transfer one spheroid at a time into individual glass-bottom 50-millimeter dishes containing 100 microliters of fresh phenol red free-culture medium per dish.Use the inverted microscope to observe changes in the spheroid morphology. To irradiate the spheroids, place a light-emitting diode no higher than 5 inches above the glass-bottom dishes. And use an optical power meter to confirm the emission of a 670 to 710 nanometer wavelength at 2 joules per centimeter squared.After the NIR infrared photoimmunotherapy, transfer the spheroids into a new hanging drop plate with 50 microliters of fresh-culture medium per well for one day in the cell-culture incubator. Then, using another 200-microliter pipette tip with a cut-off end, gently transfer the spheroids into new glass-bottom dishes containing 100 microliters of phenol red free-culture medium, and assess the optical reporter expression under the flourescent microscope. Finally, detect the dead cells by the addition of propidium iodide to the medium.Or by the loss of cytoplasmic GFP flourescence. In this representative experiment, mixed spheroids composed of various ratios of A431-luc-GFP and 3T3-RFP cells were prepared as just demonstrated. Repeated NIR infrared photoimmunotherapy was then performed with the incubation of panitumumab-IR700 as illustrated.And the target cell elimination was monitored through the assessment of the flourescent and luciferase activities within the hanging drop cultures. Once mastered, this technique can be completed in one hour if it is performed properly. While attempting this procedure, it's important to remember that light energy decreases according to inverse-square law.

Research

JoVE Journal

Bioengineering

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

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

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

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

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

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

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 ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

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

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

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 ...

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

3D 动态培养系统构建多层间充质干细胞片材

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

Synthesis of Near-Infrared Emitting Gold Nanoclusters for Biological Applications

生物应用近红外发光金纳米簇的合成

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been ...