Die Autoren bedanken sich bei Sally Roberts (University of Birmingham, Birmingham UK) für die freundliche Gabe des Antikörpers E1E4 danken. Diese Arbeit wurde durch ein Stipendium der National Cancer Institute (4R00CA137160-03) unterstützt.

1. Vorbereitung für Organotypische Floßkulturen <ol><li> Die Metall-Gitter viel Floß zuerst mit Chromsäure Schwefelsäure behandelt werden, um alle Rückstände, die mit der Differenzierung können, zu entfernen. Tauchen Sie ein Metallgitter in einem Becherglas mit Schwefelsäure für eine Stunde, dann kontinuierlich über Nacht mit Leitungswasser abgespült. Nach der Nacht spülen, sollte Floß Grids für 3-5 Stunden in doppelt destilliertem Wasser abgespült werden. </li><li> Um die Unterstützung für den Flößen bereitzustellen, biegen drei Seiten des Floßes etwa 0,5 cm in gleichem Abstand voneinander. Die Metallgitter sollten dann autoklaviert werden. </li><li> Bereiten Sie die 10X Rekonstitutionspuffer durch Zugabe von 2,2 g NaHCO3 und 4,8 g HEPES. Gelöst in 100 ml 0,05 M NaOH. Filtern sterilisieren und lagern in 5 ml Aliquots bei -20 ° C. </li><li> Bereiten Sie die 10X DMEM ohne Natriumbicarbonat. Filtern sterilisieren und lagern in 5 ml Aliquots bei -20 ° C. </li><li> Planen Maus epidermalen Wachstumsfaktor: 100 mg von EGF und 10 mg BSA jeweils in 10 ml entionisiertem ddH 2 0. Kombinieren Sie EGF und BSA und fügen Sie zu 80 ml ​​deionisiertem ddH 2 0 für ein Gesamtvolumen von 100 ml. Filter-sterilisiert, Aliquot (5 ml) und Laden in der -20 ° C. Für jeden Liter E-Medium mit 5 ml von 1 mg / ml EGF (Endkonzentration 5 ng / ml). </li></ol> 2. Herstellung von Kollagen-Gelen <ol><li> Ein Kollagengel für jede Reihe erforderlich. Das Kollagen sollten auf Eis, um es von Verfestigung zu verhindern gehalten werden. Jeder Kollagengel erfordert 3 ml Collagen Mischung bestehend aus: 2,4 ml kaltem Rattenschwanz Kollagen Typ I (Endkonzentration 3-4 mg / ml), 0,3 ml 10X Rekonstitutionspuffer, 0,3 ml DMEM 10X and1-2 x 10 6 Maus 3T3 J2 Fibroblasten als Feeder-Zellen. Für hohe Konzentration von Lösungen Rattenschwanzkollagen, in 0,02 M Essigsäure verdünnen, um die korrekte Arbeit Konzentration zu erreichen. </li><li> Bestimmen Sie die Anzahl der Kollagen-Gelen (Flöße) Sie brauchen, und berechnet dann einen Master-Mix für den Betrag von Kollagen, 10X reconstitution Puffer, 10x DMEM und J2 Fibroblasten erforderlich. Einschließen genug in jeder Master-Mix für 3-4 zusätzliche Gele aufgrund der Viskosität des Kollagens. </li><li> Um das Kollagen-Gel vorzubereiten, trypsinieren die J2 Fibroblasten und neutralisieren mit Medium. Kombinieren Sie alle Fibroblasten und in einem 50 ml konischen Röhrchen. Zählen der Zellen, um die Anzahl von Flößen, die gemacht werden können bestimmen. Wiederum sind 1-2x10 6 Fibroblasten pro Kollagengel erforderlich, und somit pro Floß. Spin-Down der Zellen bei niedrigen Drehzahlen. </li><li> Absaugen des Mediums aus dem Fibroblasten-Pellets und Resuspendieren in der entsprechenden Menge der Rekonstitutionspuffer 10X, 10X DMEM und Kollagen für die Anzahl von Flößen berechnet. In der letzten Kollagen, das Gel aus erstarrenden zu schnell zu verhindern. Sobald das Kollagen hinzugefügt wird, schnell mischen, sondern sanft, um die Einführung von Blasen in das Gel zu verhindern. Das Gel sollte eine rötlich-orange Farbe, was auf dem richtigen pH-Wert sein. Wenn das Gel Gemisch ist zu gelb, fügen Sie ein paar Tropfen sterilfiltriert 1NNaOH und vorsichtig mischen, um die richtige Farbe zu erhalten. </li><li> 3 ml des Kollagens: Fibroblasten-Gemisch auf die Vertiefungen einer 6-Well-Kulturschale. Die Kollagen-Mischung sollte langsam die Seite jedes Well pipettiert werden, um zu vermeiden, Erzeugen von Blasen. Die Platte wird in einem 37 ° C Gewebekultur-Inkubator und lassen Sie ihn 30 Minuten lang erstarren. </li><li> Nach 30 Minuten 3 ml Medium E mit EGF an der Spitze des Kollagen-Gel verfestigt und die Platte zurück in den Inkubator. Die Gele bei 37 ° C für mindestens einen Tag aufbewahrt werden, verwendet innerhalb von vier Tagen. Optimale Resultate kommen nach zwei Tagen Inkubation. </li></ol> 3. Vorbereiten Keratinozyten zur Differenzierung <ol><li> Niedrigen Gang Keratinozyten sollte auf etwa 70% Konfluenz gezüchtet werden. Jedes Floß erfordert 1-2x10 6 Keratinozyten. Trypsinieren die Keratinozyten und neutralisieren mit E-Medium mit EGF. Zählen der Zellen, um die Anzahl von Flößen, die gemacht werden können bestimmen. Spin-Down der Zellen bei niedrigen spehrsg. Das Pellet in 1 ml Medium mit EGF-E für jedes Floß Kultur berechnet. </li><li> Absaugen des Mediums aus den Kollagengelen durch Kippen der 6-Well-Platte. 2 ml frisches Medium E mit dem EGF bei jedem Gel. Für jedes Gel, vorsichtig 1 ml des resuspendierten Keratinozyten zur 2 ml E-Medium bereits in dem Bohrloch. Pipette vorsichtig die Keratinozyten plus die 2 ml Medien und dann fallen lassen tropfenweise wieder auf das Gel. Schütteln oder Schwenken der Platte kann zu einer ungleichmäßigen Verteilung der Zellen zu einer Seite des Kollagengels führen. Die Platte wird in der 37 ° C Inkubator. </li><li> Die Zellen sollten bis zur Konfluenz gezüchtet werden und ersetzt das Medium E täglich. Die Zellen konfluent sind, wenn das Medium eine gelbe Farbe einen Tag nach einer Änderung im Medium. Dies dauert in der Regel 2-4 Tage. Wenn das Medium nicht von Tag vier geändert, um dem nächsten Schritt fort. </li></ol> 4. Making the Floßkulturen <ol><li> Verwenden einer sterilen Pinzette, um ein steriles Metall Floß Netz zu stellen, beugte sich hinunter Seiten, In eine 10 cm-Schale. Saugen Sie das Medium aus Kollagen-Gel. Um das Gel von den Seiten des Bohrlochs zu lösen, um den Umfang des Gels gehen mit einem sterilen Spatel unter Verwendung eines Auf-und Abbewegung. Um das Kollagen-Gel entfernen, neigen Sie die Platte leicht und heben, indem Sie einen Spachtel darunter. Legen Sie den Kollagengel Auf dem Gitter ohne Erzeugen von Blasen zwischen dem Gitter und dem Gel. Zwei Gele können auf jedem Raster platziert werden. </li><li> Um eine Luft-/Flüssigkeits-Schnittstelle zu machen, fügen E-Medium ohne EGF langsam auf den Boden der Schale, so das Floß Gitter berührt die Medien, sondern das Kollagengel ist. Die Medien sollten nicht durch die Löcher in dem Gitter kommen. Fügen Sie das Medium langsam zu vermeiden Erzeugen von Blasen unter dem Raster, da diese einheitliche Differenzierung verhindern. Inkubieren der Flöße bei 37 ° C und ändern das Medium alle zwei Tage Aufrechterhaltung der Luft-/Flüssigkeits-Schnittstelle. Ernten Sie die Flöße nach 14 Tagen. </li></ol> 5. Repräsentative Ergebnisse Wenn ter Protokoll richtig mit primären Keratinozyten durchgeführt wird dieses Verfahren in einem gut differenzierten Epithel, wo die verschiedenen Schichten der Haut ergeben sich führen. Diese durch die histologische Untersuchung durch Färbung Abschnitte Floß Kulturen für Hämatoxylin und Eosin (H &amp; E), wie in 1B gezeigt, erreicht werden. H &amp; E-Färbung ist auch nützlich, um die Morphologie der Flöße und die Wirkung von Viren oder antiviralen Verbindungen auf die Fähigkeit der Zellen zur Schichtung und Differenzierung zu untersuchen. Zum Beispiel werden Flöße aus HPV-immortalisierten primären humanen Keratinozyten (HFK-31) machte vor allem dicker auf Flößen aus normalen menschlichen Keratinozyten Vorhaut (HFK) verglichen, was auf eine erhöhte Rate von Proliferation und die Fähigkeit der HPV-Proteine ​​zu erhalten differenzierenden Zellen aktiv im Zellzyklus (1B) 10. Das führt zum Rückstau von Kernen im gesamten Epithel, während die normalen Keratinozyten Verlassen des Zellzyklus auf Differenzierung, was zu einem Zusammenbruch derKernhülle. Um genauer zu untersuchen Differenzierung kann Immunhistochemie durchgeführt, um spezifische Differenzierung Marker, einschließlich Zytokeratine sowie Involucrin und Filaggrin zu untersuchen. Verschiedene Zytokeratine sind in bestimmten Phasen der Differenzierung 7 ausgedrückt. Wie in 2 gezeigt ist, ist Cytokeratin 10 (K10) nicht in der Basalschicht ausgedrückt und wird nur in den Suprabasalschichten, der aus Zellen bestehen schrittweise Differenzierung vorhanden. In HPV-positiven Flöße, wird die Expression des K10 im Vergleich zu der normalen Keratinozyten, wiederum als den Einfluss von HPV-Proteinen auf Differenzierung der Keratinozyten verzögert. Immunhistochemie kann auch verwendet werden, um die Expression von viralen Genen zu untersuchen. In Abbildung 3 ist eine repräsentative Färbung, die Expression der HPV-Protein E1 ^ E4, die eine späte Protein, dessen Expression in die obersten Schichten des Epithels beschränkt ist. Wie erwartet, wird keine Färbung in Floß Kulturen von normalen Huma beobachtetn Vorhaut Keratinozyten. <img alt="1" src="/files/ftp_upload/3668/3668fig1.jpg" /> Abbildung 1. A) Darstellung von Verfahren zur Herstellung von organotypischen Kulturen Floß aus primären Keratinozyten oder epithelialen Zelllinien. B) Hämatoxylin-und Eosin-Färbung von organotypischen Kulturen Floß aus menschlicher Vorhaut Keratinozyten stabil halten HPV-31 (HFK-31) Episomen, oder aus normalen menschlichen Keratinozyten Vorhaut (HFK) generiert. Die einzelnen Epithelschichten identifiziert, sowie die Kollagen-Stecker. <img alt="2" src="/files/ftp_upload/3668/3668fig2.jpg" /> Abbildung 2. Cytokeratin 10 (K10) Ausdruck wird in den suprabasalen Schichten des Epithels beschränkt. Die Immunhistochemie wurde an Querschnitten von organotypischen Raft-Kulturen von HFK-31-Zellen, ebenso wie normale HFKs Verwendung eines Antikörpers gegen K10 erzeugt wird. Zelluläre DNA wurde mit DAPI gegengefärbt. Bilder wurden unter Verwendung confÖcal Fluoreszenzmikroskopie. Die Pfeile zeigen die Basalschicht des Epithels. <img alt="Abbildung 3" src="/files/ftp_upload/3668/3668fig3.jpg" /> Abbildung 3. Der HPV-E1 ^ E4-Protein wird spät in die produktive Phase des viralen Lebenszyklus produziert. Die Immunhistochemie wurde an Querschnitten von organotypischen Raft-Kulturen von HFK-31-Zellen, ebenso wie normale HFKs Verwendung von Antikörpern gegen E1E4 erzeugt wird. Zelluläre DNA wurde mit DAPI gegengefärbt. Bilder wurden unter Verwendung der konfokalen Fluoreszenzmikroskopie. Die Pfeile zeigen die Basalschicht des Epithels.

<ol>
	<li>Andrei, G., Duraffour, S., VandenOord, J., Snoeck, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+raft+cultures+for+investigations+of+virus+growth,+pathogenesis+and+efficacy+of+antiviral+agents.">Epithelial raft cultures for investigations of virus growth, pathogenesis and efficacy of antiviral agents.</a> Antiviral Research. 85, 431-449 (2010).</li><li>Asselineau, D., Prunieras, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+'simplified'+skin:+control+of+fabrication.">Reconstruction of 'simplified' skin: control of fabrication.</a> The British Journal of Dermatology. 111, 219-222 (1984).</li><li>Bodily, J., Alam, S., Chen, H., Meyers, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Organotypic epithelial raft cultures and the study of the natural history of human papillomavirus. , (2006).</li><li>Cheng, S., Schmidt-Grimminger, D. C., Murant, T., Broker, T. R., Chow, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation-dependent+up-regulation+of+the+human+papillomavirus+E7+gene+reactivates+cellular+DNA+replication+in+suprabasal+differentiated+keratinocytes.">Differentiation-dependent up-regulation of the human papillomavirus E7 gene reactivates cellular DNA replication in suprabasal differentiated keratinocytes.</a> Genes &#38; Development. 9, 2335-2349 (1995).</li><li>Dollard, S. C., Wilson, J. L., Demeter, L. M., Bonnez, W. R., Reichman, C., Broker, T. R., Chow, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+human+papillomavirus+and+modulation+of+the+infectious+program+in+epithelial+raft+cultures.+OFF.">Production of human papillomavirus and modulation of the infectious program in epithelial raft cultures. OFF.</a> Genes &#38; Development. 6, 1131-1142 (1992).</li><li>Frattini, M. G., Lim, H. B., Laimins, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+synthesis+of+oncogenic+human+papillomaviruses+requires+episomal+genomes+for+differentiation-dependent+late+expression.">In vitro synthesis of oncogenic human papillomaviruses requires episomal genomes for differentiation-dependent late expression.</a> Proceedings of the National Academy of Sciences of the United States of America. 93, 3062-3067 (1996).</li><li>Kopan, R., Traska, G., Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoids+as+important+regulators+of+terminal+differentiation:+examining+keratin+expression+in+individual+epidermal+cells+at+various+stages+of+keratinization.">Retinoids as important regulators of terminal differentiation: examining keratin expression in individual epidermal cells at various stages of keratinization.</a> The Journal of Cell Biology. 105, 427-440 (1997).</li><li>Longworth, M. S., Laimins, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+human+papillomaviruses+in+differentiating+epithelia.">Pathogenesis of human papillomaviruses in differentiating epithelia.</a> Microbiology and Molecular Biology Review. 68, 362-372 (2004).</li><li>Meyers, C., Frattini, M. G., Hudson, J. B., Laimins, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosynthesis+of+human+papillomavirus+from+a+continuous+cell+line+upon+epithelial+differentiation.">Biosynthesis of human papillomavirus from a continuous cell line upon epithelial differentiation.</a> Science. 257, 971-973 (1992).</li><li>Wilson, R., Laimins, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Differentiation of HPV-Containing Cells Using Organotypic Raft Culture or Methylcellulose. , 119-119 (2006).</li><li>zur Hausen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Papillomavirus+infections--a+major+cause+of+human+cancers.">Papillomavirus infections--a major cause of human cancers.</a> Biochimica et Biophysica Acta. 1288, F55-F78 (1996).</li></ol>

Wir haben nichts zu offenbaren.

Wir beschreiben hier eine Methode, die verwendet werden, um epitheliale Differenzierung im allgemeinen studieren kann, kann aber auch einfach an viralen Pathogenese, sowie die Wirksamkeit von potenziellen Therapeutika zu studieren. Viele Viren Ziel Epithelzellen entweder als primäre Ort der Infektion, wie im Fall von HPV, oder an irgendeinem Punkt in dem viralen Lebenszyklus, wie bei Herpesviren. Obwohl wachsende organotypischen Kulturen Floß ist zeitaufwendig, die Fähigkeit, treu in vivo epithelialen Differ...

<table border="1"><tbody><tr><td> Name des Reagenz </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> Rattenschwanzkollagen Typ 1 (4-5mg/ml) </td><td> BD Biosciences </td><td> 354236 </td></tr><tr><td> DMEM ohne NaHC0 3 </td><td> Invitrogen </td><td> 12100-061 </td></tr><tr><td> NaHC0 3 </td><td> Sigma </td><td> S5761 </td></tr><tr><td> Hepes </td><td> Calbiochem </td><td> 391338 </td></tr><tr><td> Edelstahl-Metallgitter </td><td> Williams und Co. Mettle </td><td> CR-03063-040-100-S </td></tr><tr><td> E Medium 11 </td><td></td><td></td></tr><tr><td> Epidermal Growth Factor </td><td> BD Biosciences </td><td> 354010 </td></tr></tbody></table>

generation of organotypic raft cultures from primary human keratinocytes

Die Entwicklung von organotypischen Kulturen epithelialen Floß hat die Forscher mit einer effizienten In-vitro-System zur Verfügung gestellt, der naturgetreu rekapituliert epithelialen Differenzierung. Es gibt viele Einsatzmöglichkeiten für dieses System. So hat die Fähigkeit zur dreidimensionalen organotypischen Floß Kulturen von Keratinozyten wachsen war ein wichtiger Meilenstein in der Erforschung des humanen Papillomavirus (HPV) 1. Der Lebenszyklus des HPV ist eng mit der Differenzierung von Plattenepithel 2 verknüpft. Organotypische epithelialen Floß Kulturen wie hier gezeigt reproduzieren das gesamte Papillomavirus Lebenszyklus, einschließlich Virus-Produktion 3,4,5. Darüber hinaus weisen diese Floß Kulturen Dysplasien der vergleichbar, die sich auf In-vivo-Infektion mit HPV. Daher kann dieses System auch verwendet werden, um Epithelzellen Krebsarten, sowie die Wirkung von Medikamenten auf Epithelzellendifferenzierung im allgemeinen zu untersuchen. Zitat von Asselineau und Prunieras entwickelt <sbis&gt; 6 und modifiziert durch Kopan et al. 7, der organotypischen Kultur epithelialen Floß-System in einer allgemeinen, relativ leicht Kultur Modell, das das Wachstum von Zellen auf Kollagen umfasst reifen Stecker gehalten bei einem Luft-Flüssigkeits-Grenzfläche (1A). Im Laufe von 10-14 Tagen, die Zellen Schichtung und Differenzierung, die Bildung eines vollständigen Dicke Epithel, das differenzierungsspezifische Cytokeratine produziert. Geerntet Flöße können histologisch untersucht werden, sowie von Standard-molekularen und biochemischen Techniken. In diesem Artikel beschreiben wir eine Methode zur Erzeugung von Raft-Kulturen aus primären humanen Keratinozyten. Die gleiche Technik kann mit bestimmten epithelialen Zelllinien verwendet werden, und kann leicht für die Verwendung mit Epithelgewebe von normalen oder kranken Biopsien 8 angepasst werden. Viele Viren zielen entweder auf die Haut oder Schleimhaut Epithel als Teil ihrer replikativen Lebenszyklus. In den vergangenen Jahren mit der Machbarkeit von organotypischen Floß Kultnahmen als Methode zur Untersuchung Virus-Wirt-Zell-Wechselwirkungen seit mehreren Herpesviren, sowie Adenoviren, Parvoviren, und Pockenviren 9 gezeigt. Organotypische Floß Kulturen können somit an viralen Pathogenese zu untersuchen, und sind die einzigen Mittel, um neuartige antivirale Mittel für jene Viren, die nicht kultivierbaren in permanenten Zelllinien zu testen.

Watch this Scientific Journal Video about Generation of Organotypic Raft Cultures from Primary Human Keratinocytes at JoVE.com

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

Ein<em&gt; In-vitro-</em&gt; Verfahren zu imitieren<em&gt; In-vivo-</em&gt; Epithelialen Differenzierung beschrieben. Viele Viren zielen Epithelzellen als Teil ihrer viralen Lebenszyklus, und diese Methode liefert ein Mittel zur Prüfung Virus: Wirt-Interaktionen, die ähnelt mehr dem, was kommt<em&gt; In-vivo-</em&gt;. Diese Technik kann mit primären Keratinozyten, etablierte Zelllinien, sowie normaler oder kranker Biopsie Gewebe verwendet werden.

Generierung von Organotypische Floßkulturen aus primären humanen Keratinozyten

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

generation-organotypic-raft-cultures-from-primary-human

Research

JoVE Journal

Immunology and Infection

19.1K Aufrufe.  University of North Carolina-Chapel Hill. Ein In-vitro- Verfahren zu imitieren In-vivo- Epithelialen Differenzierung beschrieben. Viele Viren zielen Epithelzellen als Teil ihrer viralen Lebenszyklus, und diese Methode liefert ein Mittel zur Prüfung Virus: Wirt-Interaktionen, die ähnelt mehr dem, was kommt In-vivo-. Diese Technik kann mit primären Keratinozyten, etablierte Zelllinien, sowie normaler oder kranker Biopsie Gewebe verwendet werden.

Serie: Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

Eine einfache und effiziente Methode, um Makrophagen aus Mixed primären Kulturen von adulten Leber Zellen zu isolieren

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

Forschung

Immunologie und Infektion

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

Generation der induzierte regulatorische T-Zellen aus primären humanen naiven und Gedächtnis-T-Zellen

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

Primäre Mikroglia Isolation vom gemischten Zellkulturen von Gliazellen Neonatal Rattenhirngewebe

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

piggyBac Transposon-System Modifikation von primären menschlichen T-Zellen

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

Generation of Human Alloantigen-spezifischen T-Zellen aus peripherem Blut

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

Erzeugung von rekombinanten humanen IgG monoklonalen Antikörpern aus Immortalisierte Sortiert B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Preparation and Applications of Organotypic Thymic Slice Cultures

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell repertoire. In vitro models to study T lineage commitment and development have provided valuable insights into this process. However, these systems lack the complete three-dimensional thymic milieu necessary for T cell development and, therefore, are incomplete approximations of in vivo thymic selection. Some of the challenges related to modeling T cell development can be overcome by using in situ models that provide an intact thymic microenvironment that fully supports thymic selection of developing T cells. Thymic slice organotypic cultures complement existing in situ techniques. Thymic slices preserve the integrity of the thymic cortical and medullary regions and provide a platform to study development of overlaid thymocytes of a defined developmental stage or of endogenous T cells within a mature thymic microenvironment. Given the ability to generate ~20 slices per mouse, thymic slices present a unique advantage in terms of scalability for high throughput experiments. Further, the relative ease in generating thymic slices and potential to overlay different thymic subsets or other cell populations from diverse genetic backgrounds enhances the versatility of this method. Here we describe a protocol for the preparation of thymic slices, isolation and overlay of thymocytes, and dissociation of thymic slices for flow cytometric analysis. This system can also be adapted to study non-conventional T cell development as well as visualize thymocyte migration, thymocyte-stromal cell interactions, and TCR signals associated with thymic selection by two-photon microscopy.

We describe the preparation of thymic slices that, in combination with flow cytometry, can be used to model positive and negative selection of developing T cells. Thymic slices can also be adapted for the in situ analysis of thymocyte migration, localization, and signaling via immunofluorescence and two-photon microscopy.

Herstellung und Bedeutung von Organotypischen Thymic Scheibe Kulturen

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Herstellung von menschlichen Monozyten abgeleiteten Dendritischen Zellen aus Vollblut

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the uremic milieu, oxidative stress, endotoxemia and the decreased clearance of circulating proinflammatory cytokines by producing a large number of acute-phase reactants. Experimental tools to study inflammation and the underlying role of hepatocytes are crucial to understand the regulation and contribution of hepatic cytokines to a systemic acute phase response and a prolonged pro-inflammatory scenario, especially in an intricate setting such as chronic kidney disease. Since studying complex mechanisms of inflammation in vivo remains challenging, resource-intensive and usually requires the usage of transgenic animals, primary isolated hepatocytes provide a robust tool to gain mechanistic insights into the hepatic acute-phase response. Since this in vitro technique features moderate costs, high reproducibility and common technical knowledge, primary isolated hepatocytes can also be easily used as a screening approach. Here, we describe an enzymatic-based method to isolate primary murine hepatocytes, and we describe the assessment of an inflammatory response in these cells using ELISA and quantitative real-time PCR.

Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR.

Die Induktion einer entzündlichen Reaktion in der Primär Hepatozyten-Kulturen von Mäusen

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated from na&#239;ve CD4+ T cells in vitro by activating them in the presence of the polarizing cytokines IL-1&#946;, IL-6, IL-23, and TGF&#946;. Th17 cells orchestrate immunity against extracellular bacteria and fungi, but their aberrant activity has also been associated with several autoimmune and inflammatory diseases. Th17 cells are identified by the chemokine receptor CCR6 and defined by their master transcription factor, ROR&#947;t, and characteristic effector cytokine, IL-17A. Optimized culture conditions for Th17 cell differentiation facilitate mechanistic studies of human T cell biology in a controlled environment. They also provide a setting for studying the importance of specific genes and gene expression programs through RNA interference or the introduction of microRNA (miRNA) mimics or inhibitors. This protocol provides an easy to use, reproducible, and highly efficient method for transient transfection of differentiating primary human Th17 cells with small RNAs using a next generation electroporation device.

Next generation electroporation is an efficient method for transfecting human Th17 cells with small RNAs to alter gene expression and cell behavior.

Kleine RNA Transfektion in primären humanen Th17-Zellen durch Next Generation Elektroporation

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated ...