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

Esta investigación fue apoyada por el Programa de Investigación Intramural de los Institutos Nacionales de Salud, Instituto Nacional del Cáncer, Centro de Investigación del Cáncer.

Nota: El siguiente protocolo describe los pasos necesarios para eliminar las células específicas utilizando NIR-PIT. Controles y otros detalles acerca de NIR-PIT y la viabilidad celular se pueden encontrar en otro lugar 18. 1. Conjugación del IR700 a anticuerpos monoclonales (mAb) <ol><li> Preparar mAb de interés a 2-5 mg / ml en solución (pH 8,6) 0,1 M Na 2 HPO 4. </li><li> Mezclar 6,8 nmol de mAb con 30,8 nmol de IR700 10 mM en M Na 2 HPO 4 solució...

<ol>
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Se demuestra un método de eliminación de células específica de un cultivo celular mixto 3D sin daño a las células no diana mediante el uso de NIR-PIT. Hasta ahora, no hay ningún método práctico de eliminación de células una vez establecido el tejido o después del trasplante. Por lo tanto, NIR-PIT es un método prometedor para lograr esto. Esta técnica podría utilizarse también in vivo 18,22, ya que las CPA muestran una farmacocinética similar a sí mismo mAb. El tipo...

La eliminación de células específicas sin dañar otras células es extremadamente difícil, especialmente en el tejido establecido, y hay una necesidad urgente de un método de eliminación de células en el campo de la ingeniería de tejidos. Hoy en día en el campo de la medicina regenerativa, cultivos de tejidos utilizando células madre embrionarias (ES), células madre pluripotentes (PSCs), o célula madre pluripotente inducida (iPS) son materiales prometedores de 1 - 3. Aunque esta regeneración de tejidos es prometedor, la contaminación con células no deseadas es una preocupación importante. Por otra parte, hay un problema de seguridad de la tumorigenicidad después del trasplante 4,5. Aunque muchos estudios se han centrado en estos temas para eliminar células específicas, sobre todo en la medicina regenerativa 6-8, ningún método práctico se ha desarrollado. photoimmunotherapy infrarrojo cercano (NIR-PIT) es un tratamiento basado en un anticuerpo conjugat-fotoabsorbentee (APC). Una APC consiste en un anticuerpo específico de células monoclonal (mAb) y un fotoabsorbente, IR700. IR700 es un derivado de sílice-ftalocianina hidrofílica y no induce por sí mismo fototoxicidad 9. IR700 está conjugado covalentemente al anticuerpo a través de los residuos de amida en la cadena lateral de moléculas de lisina. La APC se une moléculas diana en la membrana celular y luego se provoca necrosis celular casi inmediata después de la exposición a la luz NIR a 690 nm. Durante la exposición a la luz NIR, se rompe la membrana celular que conduce a la muerte de 9 celdas - 14. NIR-PIT ha demostrado ser eficaz con múltiples anticuerpos o fragmentos de anticuerpos, incluyendo anti-EGFR, anti-HER2, anti-PSMA, anti-CD25, anti-mesotelina, anti-GPC3, y anti-CEA 15-21. Por lo tanto, NIR-PIT se puede utilizar contra una amplia variedad de moléculas diana. Por otra parte, NIR-PIT es un tratamiento bien controlado que permite el tratamiento selectivo de regiones específicas mediante la restricción de la NIR-light irradiación 18,22. A continuación, presentamos un método de eliminación de células específicas mediante NIR-PIT 3D a partir de cultivos mixtos.

<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.

Para monitorizar ópticamente el efecto de NIR-PIT, la línea celular A431, que sobreexpresa el EGFR, fue modificado genéticamente para expresar también GFP y luciferasa (luc-A431-GFP). Como que no son objeto de NIR-PIT, la línea de células Balb / 3T3 se modificó para expresar ópticamente RFP (3T3-RFP). La APC, panitumumab-IR700 (pan-IR700), se sintetizó. Esferoides mixtos, que se componen de diversas relaciones de células (A431-luc-GFP y 3T3-RFP) se fabricaron de acuerdo con est...

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).

La eliminación selectiva de células de Cultura mezclada 3D mediante técnica de infrarrojo cercano Photoimmunotherapy

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.

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