Diese Forschung wurde von der Intramural Research Program der National Institutes of Health, National Cancer Institute, Zentrum für Krebsforschung unterstützt.

Hinweis: Das folgende Protokoll beschreibt die notwendigen Schritte spezifischen Zellen unter Verwendung von NIR-PIT zu beseitigen. Kontrollen und andere Details über NIR-PIT und die Lebensfähigkeit der Zellen kann an anderer Stelle 18 zu finden. 1. Konjugation von IR700 monoklonale Antikörper (mAb) <ol><li> Bereiten Sie mAb von Interesse bei 2-5 mg / ml in 0,1 M Na 2 HPO 4 (pH 8,6) Lösung. </li><li> Mischungs 6,8 nmol von mAb mit 30,8 nmol von 10 mM IR700 in 0,1 M Na 2 HPO 4 -Lösung (pH 8,6) in einem Mikrozentrifugenröhrchen und Inkubieren bei RT für 1 h, mit einer Aluminiumfolie abgedeckt. </li><li> Waschen Sie die PD-10-Säule (siehe Tabelle der Materialien / Ausrüstung) mit 15 ml PBS, zweimal. Legen Sie die Probe aus Schritt 1.2. </li><li> Man reinige das Gemisch über den PD-10-Säule durch Elution PBS entsprechend den Anweisungen des Herstellers. Hinweis: Hier wird die Elution entlang der Bandfarbe von IR700 war. Für eine 300 ul Probe Fraktion PBS Elution liegt typischerweise zwischen 2,5 ml bis 4,4 ml. </li><li> Bestimmen dieProteinkonzentration mit Coomassie - Färbung durch die Absorption bei 595 nm mit einem Spektrophotometer 9 gemessen wird . Bestimmen Sie die Konzentration von IR700 mit Absorption bei 689 nm , die Anzahl der Fluorophormoleküle zu bestätigen , konjugiert an jedem mAb Molekül 9. Hinweis: Es ist wichtig, eine optimale Anzahl von conjugation IR700 Moleküle in 1 mAb mit einem Spektrophotometer bestimmt werden. Im Allgemeinen etwa 3 IR700 Moleküle in 1 mAb - Molekül ist optimal sowohl für in vitro und in vivo arbeiten. HPLC und SDS-PAGE-Methoden können verwendet werden, um zu bestätigen, ob der mAb und IR700 gebunden sind oder nicht. </li><li> Lagerung bei 4 ° C nach der Proteinkonzentration zu bestimmen. </li></ol> 2. Herstellung von Misch 3D-Zellkultur (Mixed Sphäroid) <ol><li> Tragen Sie steriles Wasser (ca. 1 ml) in den Plattenbehälterabschnitt der Platten Tropfen hängen. </li><li> Bereiten Sie verschiedene Verhältnisse der Zelltypen von Interesse - A431-luc-GFP-Zellen und 3T3-RFP-Zellen -mit jeder Probe, die 5000 Zellen insgesamt, in 50 ul Kulturmedium suspendiert. Anmerkung: Bereiten 1: 100, 10: 100, 25: 100, 50: 100 etc. Verhältnisse von Zelltypen von Interesse in Kulturmedien je nach Zelltyp. </li><li> Inkubieren der Mischung für 5-7 Tage in 96-Well-Fallplatte bei 37 ° C und 5% Kohlendioxid in einem befeuchteten Inkubator hängt. Ändern Sie die Kulturmedien alle 2 Tage. Hinweis: Um die 3D-spheroid präzise, ​​handhaben die Platte sanft, als Tropfen machen die Zellen enthalten, fallen leicht vor 3D-Formen zu bilden. </li><li> Achten Sie auf die Morphologie und die Größe der Sphäroiden eines invertierten Hell- Mikroskop bei 10X - 40-facher Vergrößerung. Hinweis: Obwohl es von Zelltypen und der Größe des hängenden Tropfen abhängig ist, sicherzustellen, dass der Durchmesser des Sphäroids herum ist 400-600 &amp; mgr; m nach 7 Tagen Inkubation in der 96-Well-Platte hängenden Tropfens. </li></ol> 3. In - vitro - NIR-PIT für 3D gemischten Zellkultur <ol><li> Ändern Sie die Medien des harn de Tropfenplatten 10 ug / ml Antikörper-Konjugat Photoabsorptionsmittel (APC) enthaltenden Medium und in einem befeuchteten Inkubator bei 37 ° C und 5% Kohlendioxid für 6 Stunden inkubiert. </li><li> Nach 6-stündiger Inkubation, waschen Sie die Sphäroid zweimal mit frischem Kulturmedien (Phenolrot frei). übertragen Sie vorsichtig die Sphäroid mit einem Glasboden-50-mm-Schale mit 100 &amp; mgr; l frisch Phenolrot freien Kulturmedien mit einer sterilen 200 ul Pipettenspitze mit der Spitze abgeschnitten. Legen Sie ein Sphäroid in jedem Gericht. </li><li> Beachten Sie die Sphäroid mit einem invertierten Hellfeldmikroskop die Änderung der Morphologie zu erkennen. Zur Beobachtung der optischen Reporter (zB GFP und RFP) verwenden Fluoreszenzmikroskop mit folgenden Filtereinstellungen: GFP - 469 nm Anregungsfilter und 525 nm Emissionsfilter; RFP - 559 nm Anregungsfilter und 630 nm Emissionsfilter. </li><li> Setzen Sie die Leuchtdiode (LED) über dem Glasbodenschale für die Bestrahlung. Hinweis: Sphäroide können NIR ausgesetzt werdenLicht entweder auf dem Mikroskop oder in der laminaren Abzugshaube. <ol><li> Messen Sie die Leistungsdichte des NIR-Licht mit einem optischen Leistungsmesser 9. Gemäß dieser Messung bestrahlen NIR-Licht über Leuchtdioden (LEDs) , welche Licht emittieren bei Wellenlängen von 670 bis 710 nm bei 2 J / cm 2. Hinweis: LED-Licht kann eine maximale Tiefe von etwa 5 Zoll eindringen. Zytotoxischen Wirkungen von NIR-PIT abhängig ist nur auf bestimmten Energie unabhängig von der Leistungsdichte und Dauer der Exposition 23. </li></ol></li><li> Nach der Bestrahlung übertragen das Sphäroid in eine neue hängenden Tropfens Platte mit 50 ul frischem Kulturmedien und für 1 Tag in einem befeuchteten Inkubator bei 37 ° C und 5% Kohlendioxid inkubiert. </li><li> übertragen Sie vorsichtig die Sphäroid auf einem Glasbodenschale mit 100 ml frisches Kulturmedien (Phenolrot frei) mit einer sterilen 200 ul Pipettenspitze mit der Spitze abgeschnitten werden. Beachten Sie die Sphäroid mit einem Fluoreszenzmikroskop 1 Tag nach NIR-PIT mit fiFiltereinstellungen in Schritt 3.3 beschrieben. <ol><li> Erkennen toten Zellen Propidiumiodid auf das Medium in einer Endkonzentration von 2 &amp; mgr; g / ml, oder durch den Verlust von cytoplasmatischen GFP-Fluoreszenz mit einem Fluoreszenzmikroskop (2B) durch Zugabe 14,18,22. </li></ol></li><li> Wiederholen Sie die Schritte 3,1-3,6 wenn Zielzellen nicht vollständig eliminiert. </li></ol>

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Die Autoren haben nichts zu offenbaren.

Wir veranschaulichen ein Verfahren zur spezifischen Zellelimination aus einer gemischten 3D-Zellkultur ohne Schaden an Nicht-Zielzellen durch NIR-PIT verwenden. Bisher gibt es keine praktische Methode der Zellelimination, wenn das Gewebe hergestellt wird oder nach der Transplantation. So NIR-PIT ist eine vielversprechende Methode, dies zu erreichen. Diese Technik könnte auch in vivo 18,22 verwendet werden, da APCs eine ähnliche Pharmakokinetik wie mAb selbst zeigen. Die Zielzelltyp kann mit verschiedenen APC angepasst werden. Verschiedenen Antikörpern oder Antikörperfragmenten, einschließlich anti-EGFR, anti-PSMA, Anti-Mesothelin - und Anti-CEA wurden bereits als APCs verwendet , 15-21. Zusätzlich kann der Bereich der NIR-Bestrahlung ändert minimieren die behandelte Region. Hier mit Quantifizierung der Luciferase-Aktivität und Fluoreszenz auf Zielzellen, die vollständige Beseitigung von spezifischen Zellen wurde bestätigt. NIR-PIT ist eine leichte Therapie, damit ein kritischer Punkt ist ter Abstand zwischen der NIR-Lichtquelle und dem Ziel, da die Lichtenergie und damit Verringerung Zellnekrosen gemäß einer inversen Quadratgesetz (Schritt 3.4). Um die 3D genau Sphäroid, sollte die Platte behandelt und so schonend wie möglich inkubiert werden. Die Tropfen enthaltenden Zellen werden leicht zerstört oder durch einen kleinen Schock fallen, bevor 3D-Formen zu bilden. NIR-PIT verfügt über mehrere einzigartige Vorteile. Zuerst NIR-PIT APCs zeigen ähnliche intravenöse Pharmakokinetik parentalen nicht-konjugierten Antikörper, wegen der hydrophilen Eigenschaften von IR700, die Bindung an Zielzellen und Nichtziel minimal Akkumulation zu hochspezifischen führen. Somit kann, selbst nach der Transplantation des Gewebes, NIR-PIT können Zielzellen durch intravenöse Injektion der durch Belichtung der Region von Interesse NIR gefolgt APC behandeln. Zweitens NIR-Licht viel tiefer in das Gewebe als UV- oder sichtbares Licht durchdringen kann daher NIR-PIT konnte nicht nur an der Oberfläche zu behandeln, sondern auch die entire Dicke des transplantierten Gewebes durch Belichtung an der Oberfläche NIR. Schließlich können NIR-PIT wiederholt Zielzellen verwendet werden, ohne Beschränkung zu beseitigen. Dieses Verfahren ist nützlich , wenn die Zielzellen ausreichend Kopien von Zielmolekülen auf der Zelloberfläche 18 auszudrücken. Es gibt jedoch einige Einschränkungen dieser Technik. Zunächst wird die Durchlässigkeit der APC begrenzt, da es sich um ein großes Molekül ist. Um dieses Problem zu überwinden, wiederholt NIR-PIT Behandlung oder Antikörperfragmente 18,19,22 ausgenutzt werden kann. Eine weitere Einschränkung ist das Eindringen von Licht. Obwohl das NIR-Licht kann in vitro Kultur, Übersetzung in vivo Behandlung richtig durchdringen würde angepasst Ansätze erfordern , wie beispielsweise Magen - Endoskopie, Bronchoskopie, oder direkte Thorakoskopie mit Faseroptik , die Lichtquelle näher an dem Zielbereich zu bewegen. Weitere Anpassungen der NIR-PIT wird sowohl für die Anwendung dieser Methode erlauben lokale und specific Zellelimination in vielen Bereichen, wie beispielsweise Immunmodulation oder Tumor - Mikroumgebung 24-26.

Die Beseitigung bestimmter Zellen, ohne dass andere Zellen zu schädigen ist extrem schwierig, vor allem in den etablierten Gewebe, und es besteht ein dringender Bedarf für eine Zelle Eliminationsverfahren im Tissue-Engineering-Bereich. Heute auf dem Gebiet der regenerativen Medizin, Gewebekulturen embryonale Stammzellen (ES), pluripotente Stammzellen (PSCs) oder induzierte pluripotente Stammzellen (iPS) sind vielversprechende Materialien 1 unter Verwendung von - 3. Obwohl diese Geweberegeneration vielversprechend ist, eine Kontamination mit unerwünschten Zellen ist ein wichtiges Anliegen. Darüber hinaus gibt es ein Sicherheitsproblem von tumorigenicity nach der Transplantation 4,5. Obwohl viele Studien zu diesen Themen konzentriert haben spezifische Zellen zu beseitigen, vor allem in der regenerativen Medizin 6 - 8, hat keine praktische Methode entwickelt worden. Nah-Infrarot-photoimmunotherapy (NIR-PIT) ist eine Behandlung, basierend auf einem Antikörper-Photoabsorptions conjugate (APC). Eine APC besteht aus einem zellspezifischen monoklonalen Antikörper (mAb) und einem Photoabsorptionsmittel, IR700. IR700 ist ein hydrophiles Derivat Siliciumdioxid-Phthalocyanin und nicht induziert Phototoxizität von selbst 9. IR700 ist kovalent an den Antikörper über Amidreste an der Seitenkette von Lysin-Moleküle konjugiert. Die APC bindet Zielmoleküle auf der Zellmembran und induziert dann fast sofort Zellnekrose nach Einwirkung von NIR-Licht bei 690 nm. Während der Einwirkung von NIR-Licht, die Zellmembran reißt , was zu Zelltod 9-14. NIR-PIT hat sich mit mehreren Antikörpern oder Antikörperfragmente , um wirksam zu sein, einschließlich anti-EGFR, anti-HER2, Anti-PSMA, anti-CD25, anti-Mesothelin, Anti-GPC3 und Anti-CEA 15-21. Daher NIR-PIT kann gegen eine Vielzahl von Zielmolekülen verwendet werden. Darüber hinaus ist NIR-PIT eine gut kontrollierte Behandlung, die durch die Einschränkung der NIR-ligh selektive Behandlung bestimmter Regionen erlaubtt Bestrahlung 18,22. Hier stellen wir ein Verfahren zur spezifischen Zelleliminierung mittels NIR-PIT aus gemischten 3D-Kulturen.

<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.

Um optisch die Wirkung von NIR-PIT überwachen, die A431-Zelllinie, die EGFR überexprimiert wurde genetisch modifiziert, um auch GFP und Luciferase (A431-luc-GFP) exprimieren. Als Nicht-Ziel von NIR-PIT wurde die Balb / 3T3-Zelllinie optisch modifiziert RFP (3T3-RFP) zum Ausdruck bringen. Die APC, Panitumumab-IR700 (pan-IR700), synthetisiert. Misch Sphäroide, die aus verschiedenen Verhältnissen von Zellen (A431-luc-GFP und 3T3-RFP) zusammengesetzt wurden , wurden gemäß diesem Protokoll (Figur 1) hergestellt. Wiederholte NIR-PIT wurde mit der Inkubation von pan-IR700 (siehe Schema in 2A) durchgeführt. Zielzellelimination aus dieser gemischten 3D - Kultur wurde mit Fluoreszenz und Luciferase - Aktivitäten (2B, C) ​​erreicht und überwacht. Auf der anderen Seite weiterhin Nicht-Zielzellen zu wachsen. <img alt="Abbildung 1" src="/files/ftp_upload/53633/53633fig1.jpg" /> <strOng&gt; Abbildung 1. Repräsentative Bilder von verschiedenen Verhältnissen von gemischten Sphäroiden. Repräsentative Bilder von gemischten Sphäroiden sind verschiedene Verhältnisse von A431-luc-GFP - Zellen und 3T3-RFP - Zellen am Tag 7 gezeigt. Maßstabsbalken = 200 &amp; mgr; m. Diese Zahl wurde von 16 geändert. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53633/53633fig1large.jpg" target="_blank">Bitte klicken Sie hier</a> , um <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53633/53633fig1large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> <img alt="Figur 2" src="/files/ftp_upload/53633/53633fig2.jpg" /> Figur 2. Zielzellelimination in 3D - Zell Sphäroide (mixed Sphäroide von A431-luc-GFP und 3T3-RFP - Zellen). (A) NIR-PIT (2 J / cm 2) Schema gezeigt. (B) Wiederholte NIR-PIT vollständig eliminiert Zielzellen (A431-luc-GFP) ohne Schaden für Nicht-Zielzellen (3T3-RFP), in einem gemischten 3D Sphäroid. Bar = 200 &amp; mgr; m. (C) Quantifizierung der Luciferase - Aktivitäten (RLE - Verhältnis)zeigte vollständige Beseitigung von Zielzellen (n = 10 Sphäroide in jeder Gruppe). Diese Zahl wurde von 18 geändert. Die Daten werden als Mittelwert ± SEM <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53633/53633fig2large.jpg" target="_blank">Bitte hier klicken</a> , um <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53633/53633fig2large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</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).

Selektive Zellelimination aus Mixed 3D Culture Mit einem Nah-Infrarot-Technik Photoimmunotherapy

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 Aufrufe.  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). 

Serie: 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.

A Simple hängenden Tropfen Cell Culture Protokoll zur Generierung von 3D-Sphäroide

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

Forschung

Biotechnik

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.

Rotierende Cell Culture Systems für Human Cell Culture: Human Trophoblastzellen als Modell

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.

Erstellung von 3D-Fibrin-Gerüste für Stem Cell Culture Anwendungen

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.

Eine neue Methode zur Lokalisierung Reporter fluoreszierende Kügelchen In der Nähe der Zellkulturoberfläche für 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 ...

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.

Fluoreszenz-Abschrecken eines Liposomenverkapseltes NIR-Fluorophore als Instrument zur<em&gt; In-vivo-</em&gt; 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 ...

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.

Mit Adhesive Patterning 3D Paper Mikrofluidik-Vorrichtungen bauen

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.

Mikrostrukturierung und Montage von 3D-Micro-Ader

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.

Tunable Hydrogele aus Pulmonary Extrazellulärmatrix für 3D-Zellkultur

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.

Bioprinting von Knorpel und Haut-Gewebe-Analoga nutzen eine neuartige Passive Einheit Mischtechnik für Bioink Precellularization

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.

Herstellung von benutzerdefinierten Agarose Brunnen für die Aussaat von Zelle und Gewebe Ring Selbstmontage mit 3D-Druck Formen

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