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

Denna forskning stöddes av Intramural forskningsprogram National Institutes of Health, National Cancer Institute, Center for Cancer Research.

Obs: Följande protokoll beskriver de åtgärder som krävs för att undanröja specifika celler med hjälp av NIR-PIT. Kontroller och andra detaljer om NIR-PIT och cellviabilitet kan hittas någon annanstans 18. 1. Konjugering av IR700 till monoklonala antikroppar (mAb) <ol><li> Förbered mAb av intresse vid 2-5 mg / ml i 0,1 M Na 2 HPO 4 (pH 8,6) lösning. </li><li> Blanda 6,8 nmol av mAb med 30,8 nmol 10 mM IR700 i 0,1 M Na 2 HPO 4-lösning (pH 8,6) i e...

<ol>
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Vi visar en metod för specifik eliminering cell från en blandad 3D cellkultur utan att skada icke-målceller med hjälp av NIR-PIT. Hittills finns det ingen praktisk metod för eliminering cell när vävnaden är etablerad eller efter transplantation. Sålunda är NIR-PIT en lovande metod för att åstadkomma detta. Denna teknik kan också användas in vivo 18,22, eftersom APC visar liknande farmakokinetik som mAb själv. Typen målcellen kan anpassas med olika APC. Olika antikroppar eller...

Eliminera specifika celler utan att skada andra celler är extremt svårt, särskilt i etablerade vävnad, och det finns ett brådskande behov av en eliminering cellmetod i tissue engineering fältet. Nuförtiden inom området regenerativ medicin, vävnadskulturer med användning av embryonala stamceller (ES), pluripotenta stamceller (PSC) eller inducerade pluripotenta stamceller (iPS) är lovande material 1 - 3. Även om denna vävnadsregenerering är lovande, är kontaminering med oönskade celler ett stort problem. Dessutom finns det en oro för säkerheten av tumörbildning efter transplantation 4,5. Även om många studier har fokuserat på dessa frågor för att eliminera specifika celler, särskilt inom regenerativ medicin 6-8, har ingen praktisk metod utvecklats. Nära infraröd photoimmunotherapy (NIR-PIT) är en metod som bygger på en antikropp-photoabsorber conjugate (APC). En APC består av en cellspecifik monoklonal antikropp (mAb) och en photoabsorber, IR700. IR700 är en hydrofil kiseldioxid-ftalocyanin-derivat och inducerar inte fototoxicitet i sig nio. IR700 är kovalent konjugerad till antikroppen via amid-rester på sidokedjan i lysin-molekyler. APC binder målmolekyler på cellmembranet och sedan inducerar nästan omedelbar cellnekros efter exponering för NIR-ljus vid 690 nm. Under exponering för NIR-ljus, det cellulära membranet brister som leder till celldöd 9-14. NIR-PIT har visat sig vara effektivt med flera antikroppar eller antikroppsfragment, inklusive anti-EGFR, anti-HER2, anti-PSMA, anti-CD25, anti-mesotelin, anti-GPC3, och anti-CEA 15-21. Därför kan NIR-PIT användas mot ett stort antal olika målmolekyler. Dessutom är NIR-PIT en väl kontrollerad behandling som tillåter selektiv behandling av specifika regioner genom att begränsa NIR-light bestrålning 18,22. Här presenterar vi en metod för specifik cell eliminering med hjälp av NIR-PIT från blandade 3D kulturer.

<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.

Att optiskt övervaka effekterna av NIR-PIT, A431 cellinje som överuttrycker EGFR var genetiskt modifierad för att också uttrycka GFP och luciferas (A431-luc-GFP). Som en icke-mål av NIR-PIT, var Balb / 3T3 cellinje optiskt modifierad för att uttrycka RFP (3T3-RFP). APC, panitumumab-IR700 (pan-IR700), syntetiserades. Blandade sfäroider, som består av olika förhållanden mellan celler (A431-luc-GFP och 3T3-RFP) tillverkades enligt detta protokoll (Figur 1). Upprep...

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).

Selektiv Cell Eliminering från Mixed 3D kultur med hjälp av en Near Infrared Photoimmunotherapy Technique

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

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