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

이 연구는 암 연구를위한 국립 보건원 (National Institutes of Health)의 교내 연구 프로그램, 국립 암 연구소, 센터에 의해 지원되었다.

참고 : 다음 프로토콜은 NIR-PIT를 사용하여 특정 세포를 제거하는 데 필요한 단계를 설명합니다. 컨트롤 및 NIR-PIT 및 세포 생존에 대한 기타 세부 사항은 다른 곳에서 18 찾을 수 있습니다. 단일 클론 항체에 IR700 1. 활용 (단클론 항체) <ol><li> 2-5 밀리그램 / 0.1 M 나 2 HPO 4 (PH 8.6) 용액 ㎖의이자의 단클론 항체를 준비합니다. </li><li> microcentrifuge 관에 0.1 M 나 2 HPO <s...

<ol>
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우리는 NIR-PIT를 사용하여 비 - 표적 세포의 손상없이 혼합 3D 세포 배양으로부터 특정 세포를 제거하는 방법을 보여준다. 지금까지 실제적인 세포 제거 방법 조직이 설립되면 또는 이식 후가 없습니다. 따라서, NIR-PIT이 작업을 수행 할 수있는 유망한 방법이다. 장갑차는 단클론 항체 자체와 유사한 약물 동태를 보여 때문에이 기술은도 18, 22 생체 내에서 이용 될 수있다. 표적 ?...

다른 세포에 손상을주지 않고 특정 세포를 제거하는 것은 특히 설립 조직에서는 매우 곤란하며, 조직 공학 분야에서 세포 제거 방법이 절실히 요구되고있다. 3 - 현재 재생 의학 분야에서, 배아 줄기 세포 (ES), 다 능성 줄기 세포 (PSC를) 또는 유도 된 다 능성 줄기 세포 (IPS)를 이용하여 조직 배양 유망한 재료 1이다. 이러한 조직 재생 유망하지만, 원치 않는 세포 오염의 주요 관심사이다. 또한, 이식 4,5 후 종양 유발의 안전 우려가있다. 많은 연구는 이러한 문제에 집중하고 있지만, 특히 재생 의료 6, 특정 세포를 제거 - (8) 실제적인 방법이 개발되지 않았다. 근적외선 photoimmunotherapy (NIR-PIT)는 항체 - photoabsorber의 conjugat에 따라 치료E (APC). APC는 세포 특이 단일 클론 항체 (단클론 항체)와 photoabsorber, IR700로 구성되어 있습니다. IR700은 친수성 실리카 - 프탈로시아닌 유도체이며, 자체 9 광독성을 유발하지 않습니다. IR700는 공유 라이신 분자의 측쇄상의 아미드 잔기를 통해 항체에 접합된다. APC는 세포막을 표적 분자에 결합하고 690 nm에서 NIR 광에 노출 후 거의 즉각적으로 세포 괴사를 유도한다. 14 - NIR 광, 세포 사멸 (9)에 이르는 세포 멤브레인 파열로 노광시. 21 - NIR-PIT는 항 EGFR, 항 HER2, PSMA 방지, 항 CD25, 항 mesothelin, 항 GPC3 및 안티 CEA (15)를 포함한 다수의 항체 또는 항체 단편으로 효과적인 것으로 입증되었다. 따라서, NIR-PIT는 표적 분자의 다양한 대해 사용될 수있다. 또한, NIR-PIT은 NIR-조명 관리를 제한하여 특정 지역의 선택적 치료를 할 수있는 잘 조절 치료입니다t 조사 18, 22. 여기서는 3D 혼합 배양에서 NIR-PIT를 사용하여 특정 세포를 제거하는 방법을 제시한다.

<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.

광학 EGFR 과발현 NIR-PIT, A431 세포주의 효과를 모니터링하기 위해 유전자 또한 GFP와 루시 페라 제 (A431 뤽-GFP)을 표현하기 위해 수정되었습니다. NIR-PIT의 비 표적으로의 Balb / 3T3 세포주 광학적 RFP (3T3-RFP)를 표현하기 위해 변경되었다. APC, panitumumab-IR700 (팬-IR700)를 합성 하였다. 세포 (A431 뤽-GFP 및 3T3-RFP)의 다양한 비율로 구성 된 혼합 타원체는이 프로토콜 (그림 1)에</stron...

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).

근적외선 Photoimmunotherapy 기술을 사용하여 혼합 된 3D 문화에서 선택적 세포 제거

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

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Bioengineering

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

3D Spheroids의 생성을위한 간단한 한깅 드롭 세포 배양 프로토콜

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

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

인간 세포 배양을위한 회전 세포 배양 시스템 : 모델로 인간 Trophoblast 셀

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

3D 섬유소의 준비는 줄기 세포 배양 용도 공사장 공중 발판

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

견인 힘 현미경의 세포 배양 표면 근처 지역화 기자 형광 구슬을위한 새로운 방법

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

위한 도구로 리포좀 캡슐화 된 근적외선 형광의 형광 담금질<em&gt; 생체</em&gt; 광학 이미징

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

3 차원 세포 배양을위한 폐 세포 외 매트릭스에서 조정 히드로 겔

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

연골과 피부 조직 아날로그 소설 수동 Bioink Precellularization에 대 한 단위 기술 혼합을 이용 하 여 Bioprinting

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

셀 시드를 위한 사용자 지정 Agarose 우물 및 자기 조립 금형 3D 인쇄를 사용 하 여 조직 반지 제작

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

3 차원 동적 문화 시스템과 다층된 중간 엽 줄기 세포 시트의 건설

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

생물학적 응용 을 위한 근적외선 방출 금 나노클러스터의 합성

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