Name: promoting 3-d aggregation of facs purified thymic epithelial cells with eak 16-ii/eakiih6 self-assembling hydrogel
Uploaded: 2016-06-27T14:00:00.000+00:00
Duration: 8 min 2 s
Description: Watch this Scientific Journal Video about Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel at JoVE.com

Diese Arbeit wurde zum Teil durch die National Institutes of Health gewährt R21 AI113000 (WSM) und R01 AI123392 (YF) unterstützt.

Alle Tiere in den Experimenten verwendet wurden in der Tierhaltung bei Allegheny-Singer Research Institute im Rahmen des Protokolls überprüft und genehmigt von der Institutional Animal Care und Use Committee des Allegheny Health Network / Allegheny Singer Research Institute untergebracht. 1. Verdauen des Thymus mit Kollagenase <ol><li> Ernte Thymusdrüse. <ol><li> Euthanize 3-5 Wochen alte C57BL6 / J - Mäusen in einem Kohlendioxid (CO 2) Kammer Thymusdrüse zu ernten. Im Einzelnen stellen die Mäuse in der Kammer unter CO 2 Induktion für 3-5 min und Tod bestätigen durch Herz- oder Atemstillstand zu überprüfen. </li><li> Legen Sie die Karkasse auf den Rücken und nass, den Körper mit 70% Ethanol. Machen Sie einen kleinen horizontalen Schnitt mit einem scharfen, geraden Präparierscheren auf seinem Bauch durch die Haut. Ziehen Sie die Haut an beiden Enden (Kopf und Beine), so dass es von innen nach außen gedreht wird. </li><li> Machen Sie einen horizontalen Schnitt mit den Dissektionsscheren knappdie niedrigste Rippe und seitlich durch die Membran geschnitten. Halten Sie die Xiphoidbasis mit einer Pinzette und schneiden Sie die Mitte der Rippen auf beiden Seiten ausgehend nach oben. Ziehen Sie die Rippen / Brustbein nach oben, so dass der Thymus deutlich sichtbar ist. Hinweis: Thymus direkt auf der Oberseite des Herzens liegt, besteht aus zwei Lappen und einer weißen durchscheinenden Farbe. </li><li> Unter Verwendung einer gebogenen Pinzette vorsichtig schöpfen und die Thymus Lappen weg von dem Bindegewebe und den Transfer in Waschlösung ziehen (siehe Materialliste). </li></ol></li><li> Vorbereitung Aufschlußlösung durch Mischen von 9 ml RPMI-1640 (Rpmi-1640), 0,025 mg / ml gereinigter Kollagenase, 10 mM HEPES [4- (2-Hydroxyethyl) -1-piperazinethansulfonsäure] und 0,2 mg / ml DNase I in einem 50 ml Zentrifugenröhrchen. Halten Sie die Aufschlusslösung in 37 ° C warmen Wasserbad bis zur Verwendung. Anmerkung: Die Menge der Aufschlußlösung ausreichend vorbereitet ist für bis zu fünf erwachsenen Maus Thymi, die zusammen Polystyrol rund- in einem 5 ml inkubiert werden können,Unterrohr. </li><li> Übertragen Sie die Thymusdrüse zu einer 60 mm-Gewebekulturschale mit 5-6 ml RPMI-1640. </li><li> Präparieren Sie die Thymusdrüse mit 28 G Insulinspritze Nadel in kleine Stücke (ca. 1 mm 3 Quadrat) die Lymphozyten zu lassen abfließen. </li><li> Spülen Sie die Thymus-Stücke mit RPMI-1640, das mit Glas Pasteur Pipette in der Petrischale ist. Kippen Sie die Schüssel und lassen Sie die Thymus-Fragmente am Boden absetzen. Langsam ansaugen und das RPMI-1640-Überstandes mit einer Glaspipette zu verwerfen. Wiederholen Sie diesen Spülschritt mit 5-6 ml RPMI-1640 für 4-5 mal mit Glaspipette, bis die Lösung weniger undurchsichtig ist. Hinweis: Glaspipetten werden bei diesem Schritt zu empfehlen, da Thymus-Fragmente zu Kunststoff Verkleben neigen, was zu einem übermäßigen Verlust von Thymus-Gewebe. Für den Rest des Verfahrens, verwenden Sie Plastikpipetten. </li><li> Nach dem letzten Spülgang, den Überstand verwerfen, wie in Schritt 1.5 beschrieben. 3 ml Aufschlusslösung und übertragen Sie die Thymusdrüse Stücke und die Lösung in einen 5 ml Rundboden-polystyrene Rohr. </li><li> Das Röhrchen wird auf einem Rohr Rotator und inkubieren bei 37 ° C für 6 min bei einer Rotationsgeschwindigkeit von 12 Umdrehungen pro Minute. </li><li> Nach der Inkubation ließ die Thymus-Fragmente auf den Boden des Rohres durch die Schwerkraft absetzen. Sammeln Sie den Überstand mit Pasteur-Pipette und Überführung in eine 50 ml-Zentrifugenröhrchen mit 10 ml Waschlösung. Zentrifuge bei 300 g für 5 min den Überstand verwerfen und Resuspendieren der Zellen in 10 ml Waschlösung. Halten Sie sich auf dem Eis. </li><li> Wiederholen Sie die Schritte 1,6-1,8 zweimal auf den Thymus-Fragmente in den 5 ml Rundboden-Polystyrolröhrchen. Pool den Überstand in der gleichen 50-ml-Tube. Anmerkung: Die Zentrifugation nicht notwendig, für die zweite und die dritte Waschung. </li><li> Nach der endgültigen Verdauung, pipettieren nach oben und unten mit 2 ml Kunststoff serologischen Pipette auf die Zentrifugenröhrchen 50 ml alle verbleibenden Fragmente in der Röhre und den Transfer zu brechen. </li><li> Zentrifuge das 50 ml-Röhrchen bei 300 xg für 5 min, Überstand absaugen, Resuspendieren in 10 ml Wasch buffer und filtriert durch 100 &amp; mgr; m Zellsieb. Halten Sie sich auf dem Eis. </li></ol> 2. Anreicherung von TECs mit unstetigen Dichtegradienten <ol><li> Vorbereitung 21% Dichtegradient-Lösung von 2,4 ml Dichtegradientenmedium Mischen (60% w / v von Iodixanol in Wasser) und 9,6 ml Waschlösung. </li><li> Zentrifugieren Sie die dissoziierten Thymus-Zellen in der 50 ml Sammelröhrchen aus Schritt 1.11 bei 300 g für 5 min und Resuspendieren in 2,5 ml Waschpuffer. </li><li> 20 ml RPMI-1640-Medium zu der Zellsuspension. </li><li> Füllen 12 ml 21% Dichtegradient-Lösung in einem 10 ml serologische Pipette mit einer elektronischen Pipettiervorrichtung. Die Spitze der serologischen Pipette am Boden der 50-ml-Zentrifugenröhrchen, das Zellen und ermöglichen der Dichtegradient-Lösung auf den Boden des Rohres durch Schwerkraft zu entleeren. <ol><li> Sanft auszustoßen, die zwei Schichten unterschiedlicher Dichte zu beenden die Dichtegradientenlösung aus der Pipette zu erzeugen. </li></ol></li><li> Zentrifuge bei 600 xg for 20 min bei RT in einem Schwenkbecherrotor. Stellen Sie die Verzögerungsrate am langsamsten Niveau. </li><li> Übertragen Sie die obere Schicht und die Schnittstelle zu einem neuen 50-ml-Tube. Hinweis: Die meisten der TECs sind in der Schnittstelle beibehalten. Lymphozyten werden dem Boden des Röhrchens nach der Zentrifugation auszufällen. Wenn diese Zellen für andere Zwecke verwendet werden sollen, spült das Pellet mit 20 ml Waschlösung dreimal. Resuspendieren der Zellen in 10 ml Waschpuffer. </li><li> Hinzufügen Waschlösung in das Röhrchen zu 40 ml bis in Schritt 2.6 und Zentrifuge bei 400 × g für 6 Minuten bei 4 ° C. Zu dem Überstand absaugen mit einer Pipette entfernen. </li><li> Wiederholen Sie das Waschen und die Streben 2 weitere Male, wie in Schritt 2.7 beschrieben. </li><li> Resuspension in 2 ml Waschlösung und der Zellen mit einem Hämozytometer zählen. </li></ol> 3. Isolierkanne TECs mit FACS <ol><li> Einstellen Zellen aus Schritt 2.9 in der Konzentration von 1x10 6 Zellen / 100 &amp; mgr; l mit Waschlösung und Transferdie Zellen in einem 5 ml-Rundbodenrohr (Polypropylen oder Polystyrol, wie durch die Anweisungen des Flusssortierer empfohlen). </li><li> Fügen Sie 2 &amp; mgr; l anti-Fc - Rezeptor - IgG pro 1x10 6 Zellen und Inkubieren bei 4 ° C für 10 min. </li><li> Hinzufügen anti-CD45 (1: 150 Verdünnung, 10 ul pro Probe) und anti-EpCAM (1: 100-Verdünnung, 10 ul pro Probe) Antikörper und Inkubieren bei 4 ° C für mehr als 20 min. </li><li> Waschen der Zellen mit 2 ml Waschlösung und Zentrifugation bei 400 × g für 6 Minuten bei 4 ° C. Saugen Sie den Überstand mit einer Pipette. </li><li> Resuspendieren der Zellen in 1 ml 1x phosphatgepufferter Kochsalzlösung (PBS). </li><li> Man filtriert durch 100 &amp; mgr; m Sieb. </li><li> Anwenden, um die Zellen auf die Sortieranlage. Wählen Sie die Lymphozyten und TEC Bevölkerung mit Ausnahme der kleinsten Zellen auf Seitenstreulicht (SSC) / Vorwärtsstreulicht (FSC) Platte (tote Zellen), dann Tor auf der ausgewählten Population für CD45- EpCAM + 13 Sortierung. </li></ol> 4. Generation von TEC / EAK Aggregates Hinweis: Führen Sie Reagenzzubereitung und Schritten Zelle unter der laminaren Haube zu mischen. <ol><li> Bereiten pA / G Adaptor-Komplexe durch Mischen von 4 &amp; mgr; l anti-His-Tag-IgG, 8 &amp; mgr; l anti-EpCAM IgG und 2,6 ul pA / G in ein steriles 1,5 ml Mikrozentrifugenröhrchen, und Inkubation für 5 min bei RT. </li><li> Passen Sie die sortiert TECs aus Schritt 3,7 bis 5x10 4 Zellen / ml mit Waschlösung, und pipettieren 100 &amp; mgr; l zu einer Vertiefung einer niedrigen Verdampfungs, 96-Well - Rundboden - Gewebekulturplatte. Hinzufügen 14,6 ul pA / G Adaptor-Komplexe zu den Zellen. Legen Sie die Platte auf einer Schwingplattform für 20 min bei 4 ° C. </li><li> Waschen Sie einmal mit 200 &amp; mgr; l Waschlösung. Zentrifugieren Sie die Platte bei 400 × g für 6 min. Entsorgen Sie den Überstand mit einer Mikropipette. </li><li> Wasche einmal mit 200 ul 10% igen Saccharoselösung. Zentrifuge bei 400 × g für 6 min. Entsorgen Sie den Überstand mit einer Mikropipette. </li><li> Resuspendiere Zellen in 30 &amp; mgr; l 10% sterile Saccharoselösung pro Vertiefung. </li><li> Fügen Sie 10 &amp; mgr; l EAKIIH6 (7,5 mg / ml) und Inkubation für 5 min bei RT. </li><li> Geben Sie 20 &amp; mgr; l EAK16-II (10 mg / ml) zu der Mischung TEC. </li><li> Füge 100 &amp; mgr; l vollständigem Medium in das System Gelbildung einzuleiten. Legen Sie die Platte auf einem Plattformschüttler für 15-20 min bei RT. </li><li> Die Platte wird in den Inkubator bei 37 ° C und 5% CO 2. Nach 2-4 Stunden, fügen Sie weitere 100 &amp; mgr; l Vollmedium. </li><li> Ändern Medium jeden zweiten Tag bis zum Gebrauch. <ol><li> Absaugen 100 ul des Mediums von der Oberfläche, ohne das Gel zu stören mit einer Mikropipette und fügen Sie vorsichtig 100 ul vorgewärmte Medium durch die Pipettenspitze an der Wand des Bohrlochs platzieren. </li></ol></li></ol> 5. Charakterisierung der TEC / EAK Aggregates <ol><li> Um die Funktion der TEC / EAK - Aggregate in vivo zu untersuchen, sammeln die TEC / EAK - Hydrogel nach O / N - Kultur und Transplantation unter die Nierenkapsel einer athymischen NacktMaus unter Verwendung von zuvor Prozedur 11,14 beschrieben. </li><li> Überwachen Sie die Entwicklung von T-Zellen in der Peripherie durch Ernten 50-100 ul Blutproben in EDTA-enthaltenden Blutentnahmeröhrchen aus dem nackten Mäusen 4-6 Wochen nach der Transplantation und Flecken mit einem Cocktail aus anti-CD4, -CD8, -CD45 und -CD3 Antikörper 12. </li><li> Analysieren die Prozentsätze von T-Lymphozyten in den peripheren Blutleukozyten mit Durchflusszytometrie (FCM), unter Verwendung von Einzelzellanalyse - Software 12. </li></ol>

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Authors declare no conflict of interest.

Während TECs die vorherrschende Bevölkerung der Thymus-Stroma und spielen eine entscheidende Rolle für die Struktur und Funktion der Thymusdrüse sind, repräsentieren sie nur etwa 0,1-0,5% des gesamten Thymus Zellularität. Sie sind auch zerbrechlich Zellen als hohe Anteile an Zelltod werden gelegentlich folgenden Kollagenaseverdau beobachtet, dh die Behandlung TECs aus der extrazellulären Matrix (ECM) zu dissoziieren. Ihre Seltenheit (~ 200.000 pro Maus Thymus) und Zerbrechlichkeit machte es eine anspruch...

Der Thymus ist die primäre lymphoiden Organ für die Erzeugung einer vielfältigen Population von pathogen-reaktives, selbst tolerant T-Zellen, die für die Funktion des erworbenen Immunsystems wesentlich ist. Es ist ein dynamisches Organ, in dem die Entwicklung Thymozyten aus dem Knochenmark als Lymphozyten-Vorläuferzellen immigrierte, wandern durch die schwammartige dreidimensionale (3-D) Matrix des Thymus-Stroma, durchlaufen abstammungs Spezifizierung und Differenzierung, und schließlich auswandern als reifen T-Zellen. Der Erfolg dieses gut programmiert Verfahren hängt weitgehend von der Kreuzkopplung zwischen den wandernden Thymozyten und den Wohn Thymus-Epithelzellen (TECs), die vorherrschende Bevölkerung des Thymus-Stroma, die für die Einrichtung und Aufrechterhaltung der Integrität des Thymus Mikroumgebung wesentlich sind. Auf der Grundlage ihrer anatomischen Lage und einzigartige Funktion kann TECs in zwei Untergruppen unterteilt werden: die TECs in der Hirnrinde (CTEC), die Verant sindwortlich Selbst MHC (Major Histocompatibility Complex) beschränkt T-Zellen (positive Auswahl) und die TECs in der Medulla (mTEZ) , die zur Eliminierung autoreaktiver T-Zellen wesentlich sind (negative Selektion) 1,2 für die Auswahl. Viele Faktoren (zB Alterung, Infektion, Bestrahlung, medikamentöse Behandlungen) können irreversible Schäden an der Thymus - Epithel führen, in kompromittierten adaptive Immunität führt. Trotz zahlreicher Versuche hat die Thymusfunktion Wiederherstellung herausfordernd aufgrund der Schwierigkeit, den Thymus-Mikroumgebung zu reproduzieren. Bemerkenswerterweise ist thymic dreidimensionale (3-D) -Konfiguration entscheidend für das Überleben und die Funktion von TECs, während TECs kultiviert in einem 2-D - Umgebung schnell die Expression von Genen kritisch für Thymopoese 3,4 verringern. EAK16-II (AEAEKAKAEAEAKAK) und dessen C-terminale histidinylated analogen EAKIIH6 (AEAEKAKAEAEAKAKHHHHHH) sind niedermolekulare, amphiphile Oligopeptide, die in deionisiertem gegen Ende der löslichenr, sondern gehen Gelieren β-Fibrillen zu bilden, wenn sie Ionenstärke von mehr als 20 mM NaCl (normale Salzkonzentration in der menschlichen Körperflüssigkeit beträgt 154 mM) ausgesetzt. Diese Umwelt reagierende Eigenschaft macht sie vielseitige Bausteine ​​3D-Strukturen zu bilden. Das His-Tag auf EAKII-H6 ein Andockmechanismus, durch welche die anti-His IgGs / Fc-bindenden rekombinantes Protein A / G (αH6: pA / G) -Komplexen als Adapter dienen können Proteinwirkstoffen und anderen Biomolekülen zu verankern ( zB Antikörper) auf dem Hydrogelverbundstoff 5-8. Wir haben zuvor gezeigt , daß 9 bis zu 13 Tage an der Injektionsstelle fluoreszenzmarkierte IgG - Moleküle auf dem Hydrogel verankert können beibehalten werden. Wenn ferner die αH6: pA / G Adapter verankert mit anti-CD4 IgGs an das Hydrogel EAK16-II / EAKIIH6 (EAK) hinzugefügt wurden, wurden CD4 + T - Zellen spezifisch 10 eingefangen. Unter Verwendung ähnlicher Technik haben wir vor kurzem gezeigt, dass 3-D-Aggregation von TECs c(: PA / G: αEpCAM αH6) Ould mit Adapter Komplexe tragen TEC-spezifische anti-EpCAM-Antikörper in EAK Hydrogel gefördert werden. Wenn unterhalb der Nierenkapseln von nackten Mäusen transplantiert, können die TEC - Cluster in EAK Hydrogel eingebettet effektiv die Entwicklung von funktionellen T - Zellen in vivo 11,12 unterstützen. Hier veranschaulichen wir unser Verfahren, um schnell und effektiv reinigen TECs mit fluoreszenzaktivierter Zellsortierung (FACS) und Erzeugen 3-D TEC Aggregate mit dem EAK Hydrogelsystem.

<table><tbody><tr><td>&lt;strong&gt;TEC Isolation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>70% Ethanol</td><td>Decon Laboratories</td><td>2701(1 Gallon)</td><td>Ethanol 200 Proof, deionized water</td></tr><tr><td>Dissecting scissors, straight</td><td>Fine Science Tools, Inc.</td><td>91460-11</td><td></td></tr><tr><td>Graefe forceps, straight</td><td>Fine Science Tools, Inc.</td><td>11053-10</td><td></td></tr><tr><td>Graefe forceps, curved</td><td>Fine Science Tools, Inc.</td><td>11052-10</td><td></td></tr><tr><td>Washing solution</td><td></td><td></td><td>1x PBS, 0.1% BSA, 2&amp;nbsp;mM EDTA</td></tr><tr><td>1x PBS (Phosphate buffered saline)</td><td>Gibco</td><td>10010-023</td><td></td></tr><tr><td>BSA (Bovine serum albumin)</td><td>Sigma</td><td>A1470-100G</td><td></td></tr><tr><td>EDTA (ethylenediaminetetraacetic acid)</td><td>Invitrogen</td><td>15575-038</td><td></td></tr><tr><td>Digestion solution</td><td></td><td></td><td>9 ml RPMI-1640, 0.025&amp;nbsp;mg/ml Liberase TM Research grade, 10&amp;nbsp;mM HEPES, 0.2&amp;nbsp;mg/ml DNaseI</td></tr><tr><td>RPMI-1640</td><td>Gibco</td><td>11879-020</td><td></td></tr><tr><td>Liberase TM Research Grade</td><td>Roche</td><td>05 401 127 001</td><td>referred as &quot;purified collagenase&quot;</td></tr><tr><td>1&amp;nbsp;M HEPES</td><td>Lonza</td><td>17-737E</td><td></td></tr><tr><td>Dnase I</td><td>Roche</td><td>10 104 159 001</td><td></td></tr><tr><td>50&amp;nbsp;ml Centrifuge tube</td><td>Corning</td><td>430290</td><td></td></tr><tr><td>60&amp;nbsp;mm tissue culture dish</td><td>Falcon</td><td>353002</td><td></td></tr><tr><td>1/2&amp;nbsp;cc U-100 Insulin syringe 28&amp;nbsp;1/2&amp;nbsp;G</td><td>Becton Dickinson</td><td>329461</td><td></td></tr><tr><td>5&amp;nbsp;ml Polystyrene round-bottom tube</td><td>Falcon</td><td>352058</td><td></td></tr><tr><td>5&amp;nbsp;ml glass pipet</td><td>Fisher Healthcare</td><td>13-678-27E</td><td>Use for rinsing the thymic fragments. Thymic fragments tend to stick to the wall with plastic pipets.</td></tr><tr><td>MACSmix tube rotator</td><td>Miltenyi</td><td>130-090-753</td><td></td></tr><tr><td>100&amp;nbsp;&amp;micro;m Cell strainer</td><td>Falcon</td><td>352360</td><td></td></tr><tr><td>Density gradient medium: OptiPrep</td><td>Axis-Shield</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Cell Sorting&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>5&amp;nbsp;ml Polypropylene round-bottom tube</td><td>Falcon</td><td>352063</td><td></td></tr><tr><td>Anti-mouse CD16/CD32 (Fc Block)</td><td>BD Biosciences</td><td>553142</td><td>Use as undiluted, 2&amp;nbsp;&amp;micro;l per sample</td></tr><tr><td>Anti-mouse CD45-PercpCy5.5</td><td>eBioscience</td><td>45-0451-80</td><td>Use at 1:150, 10&amp;nbsp;&amp;micro;l per sample</td></tr><tr><td>Anti-mouse CD326 (EpCAM)-PE</td><td>eBioscience</td><td>12-5791-82</td><td>Use at 1:100, 10&amp;nbsp;&amp;micro;l per sample</td></tr><tr><td>BD Influx</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>Single cell analysis software</td><td>FlowJo</td><td></td><td></td></tr><tr><td>&lt;strong&gt;EAK Gel Assembly&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anti-His-Tag</td><td>AnaSpec</td><td>29673</td><td>&quot;anti-His-Tag IgG&quot;</td></tr><tr><td>Purified anti-mouse CD326 (EpCAM)</td><td>BioLegend</td><td>118202</td><td>&quot;anti-EpCAM IgG&quot;</td></tr><tr><td>Recombinant protein A/G</td><td>Pierce Biotechnology</td><td></td><td></td></tr><tr><td>1.5&amp;nbsp;ml Safe-Lock Tubes, Biopur, Sterile</td><td>Fisher Healthcare</td><td>05-402-24B</td><td>referred as &quot;1.5&amp;nbsp;ml microcentrifuge tube&quot;</td></tr><tr><td>96-well, Tissue culture plate, Round-bottom with low evaporation lid</td><td>BD Falcon</td><td>353917</td><td></td></tr><tr><td>Rocking platform: Nutator Mixer no.1105</td><td>BD Clay Adams</td><td></td><td></td></tr><tr><td>10% sucrose</td><td>Sigma</td><td>S0389</td><td>Prepare with sterile distilled water</td></tr><tr><td>EAK16-II (AcNH-AEAEAKAKAEAEAKAK-CONH&lt;sub&gt;2&lt;/sub&gt;)</td><td>American Peptide Company&amp;nbsp;</td><td></td><td>custom synthesized, 10&amp;nbsp;mg/ml</td></tr><tr><td>EAKIIH6 (AcNH-AEAEAKAKAEAEAKAKHHHHHH-CONH&lt;sub&gt;2&lt;/sub&gt;)</td><td>American Peptide Company&amp;nbsp;</td><td></td><td>custom synthesized, 7.5&amp;nbsp;mg/ml</td></tr><tr><td>Complete medium</td><td></td><td></td><td>RPMI-1640, 10% FBS, 1% Pen/Strep, 1% L-glutamine, 1% NEAA, 5&amp;nbsp;mM HEPES, 50&amp;nbsp;&amp;micro;M 2-Mercaptoethanol</td></tr><tr><td>RPMI-1640</td><td>Gibco</td><td>11879-020</td><td></td></tr><tr><td>FBS (Fetal Bovine Serum)</td><td>Atlanta Biologicals</td><td>S11150</td><td>Heat inactivated before use</td></tr><tr><td>Pen/Strep</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>L-glutamine 200&amp;nbsp;mM (100x)</td><td>Gibco</td><td>25030-081</td><td></td></tr><tr><td>NEAA (non-essential amino acid) 100x</td><td>Gibco</td><td>11140-050</td><td></td></tr><tr><td>1&amp;nbsp;M HEPES</td><td>BioWhittaker</td><td>17-737E</td><td></td></tr><tr><td>2-Mercaptoethanol (100x)</td><td>Millipore</td><td>ES-007-E</td><td></td></tr><tr><td>Platform shaker: The Belly Dancer</td><td>Stovall Life Sciences Inc.</td><td>model: USBDbo</td><td></td></tr></tbody></table>

promoting 3-d aggregation of facs purified thymic epithelial cells with eak 16-ii/eakiih6 self-assembling hydrogel

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing thymus is crucial to maintain effective T cell-mediated acquired immune response against invading pathogens. However, thymus regeneration and revitalization proved to be challenging, largely due to the difficulties of reproducing the unique 3D microenvironment of the thymic stroma that is critical for the survival and function of thymic epithelial cells (TECs). We developed a novel hydrogel system to promote the formation of TEC aggregates, based on the self-assembling property of the amphiphilic EAK16-II oligopeptides and its histidinylated analogue EAKIIH6. TECs were enriched from isolated thymic cells with density-gradient, sorted with fluorescence-activated cell sorting (FACS), and labeled with anti-epithelial cell adhesion molecule (EpCAM) antibodies that were anchored, together with anti-His IgGs, on the protein A/G adaptor complexes. Formation of cell aggregates was promoted by incubating TECs with EAKIIH6 and EAK16-II oligopeptides, and then by increasing the ionic concentration of the medium to initiate gelation. TEC aggregates embedded in EAK hydrogel can effectively promote the development of functional T cells in vivo when transplanted into the athymic nude mice.

Um die Wirksamkeit der Verwendung der Dichtegradiententrennung Protokoll untersuchen die CD45- Stromazellen zu bereichern, geerntete Zellen, sowohl von der Oberfläche und den ausgefällten Lymphozyten Pellets wurden mit anti-CD45 und anti-EpCAM-Antikörper. Sowohl anti-Ulex Europaeus Agglutinin 1 (UEA1) und Anti-MHC-Klasse II-Antikörper wurden auch in der Färbungs Cocktail aufgenommen, um die cTEC und mTEC Untergruppen identifizieren. Wie in Abbildung 1 gezeigt, konnt...

Watch this Scientific Journal Video about Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel at JoVE.com

Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel

This video demonstrates a protocol to enrich thymic epithelial cells (TECs) with density gradient for FACS isolation. It also shows the use of EAK16-II/EAKIIH6 peptides to promote the TEC aggregate formation. The microenvironments of EAK16-II/EAKIIH6 hydrogel provide the 3-D configuration necessary to maintain the survival and function of the TECs.

Die Förderung der 3-D-Aggregation von FACS Purified Thymusepithelzellen mit EAK 16-II / EAKIIH6 Selbstaufbau Hydrogels

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing ...

promoting-3-d-aggregation-facs-purified-thymic-epithelial-cells-with

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

6.5K Aufrufe.  Allegheny Health Network. This video demonstrates a protocol to enrich thymic epithelial cells (TECs) with density gradient for FACS isolation. It also shows the use of EAK16-II/EAKIIH6 peptides to promote the TEC aggregate formation. The microenvironments of EAK16-II/EAKIIH6 hydrogel provide the 3-D configuration necessary to maintain the survival and function of the TECs.

Serie: Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel

3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse this scaffold in a highly coordinated temporal and spatial order while sequentially passing obligatory check points, i.e., T cell lineage commitment, followed by T cell receptor repertoire generation and selection prior to their export into the periphery. The two major resident cell types forming this scaffold are cortical (cTECs) and medullary thymic epithelial cells (mTECs). A key feature of mTECs is the so-called promiscuous expression of numerous tissue-restricted antigens. These tissue-restricted antigens are presented to immature thymocytes directly or indirectly by mTECs or thymic dendritic cells, respectively resulting in self-tolerance.
Suitable in vitro models emulating the developmental pathways and functions of cTECs and mTECs are currently lacking. This lack of adequate experimental models has for instance hampered the analysis of promiscuous gene expression, which is still poorly understood at the cellular and molecular level. We adapted a 3D organotypic co-culture model to culture ex vivo isolated mTECs. This model was originally devised to cultivate keratinocytes in such a way as to generate a skin equivalent in vitro. The 3D model preserved key functional features of mTEC biology: (i) proliferation and terminal differentiation of CD80lo, Aire-negative into CD80hi, Aire-positive mTECs, (ii) responsiveness to RANKL, and (iii) sustained expression of FoxN1, Aire and tissue-restricted genes in CD80hi mTECs.

Studying medullary thymic epithelial cells in vitro has been largely unsuccessful, as current 2D culture systems do not mimic the in vivo scenario. The 3D culture system described herein - a modified skin organotypic culture model - has proven superior in recapitulating mTEC proliferation, differentiation and maintenance of promiscuous gene expression.

3D Organotypische Cokulturmodell Unterstützung Mark Thymusepithel Zellproliferation, Differenzierung und Genexpression Promiscuous

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse ...

Forschung

Entwicklungsbiologie

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

Kultivierung humaner neuronaler Vorläuferzellen in einer 3-dimensionalen Self-Montage Peptide Hydrogel

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Biotechnik

Generation and Recovery of &beta;-cell Spheroids From Step-growth PEG-peptide Hydrogels

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional microenvironment with tissue-like elasticity and high permeability for culturing therapeutically relevant cells or tissues. Hydrogels prepared from poly(ethylene glycol) (PEG) derivatives are increasingly used for a variety of tissue engineering applications, in part due to their tunable and cytocompatible properties. In this protocol, we utilized thiol-ene step-growth photopolymerizations to fabricate PEG-peptide hydrogels for encapsulating pancreatic MIN6 b-cells. The gels were formed by 4-arm PEG-norbornene (PEG4NB) macromer and a chymotrypsin-sensitive peptide crosslinker (CGGYC). The hydrophilic and non-fouling nature of PEG offers a cytocompatible microenvironment for cell survival and proliferation in 3D, while the use of chymotrypsin-sensitive peptide sequence (CGGY&darr;C, arrow indicates enzyme cleavage site, while terminal cysteine residues were added for thiol-ene crosslinking) permits rapid recovery of cell constructs forming within the hydrogel. The following protocol elaborates techniques for: (1) Encapsulation of MIN6 &beta;-cells in thiol-ene hydrogels; (2) Qualitative and quantitative cell viability assays to determine cell survival and proliferation; (3) Recovery of cell spheroids using chymotrypsin-mediated gel erosion; and (4) Structural and functional analysis of the recovered spheroids.

Generation and Recovery of &#946;-cell Spheroids From Step-growth PEG-peptide Hydrogels

The following protocol provides techniques for encapsulating pancreatic &#x03B2;-cells in step-growth PEG-peptide hydrogels formed by thiol-ene photo-click reactions. This material platform not only offers a cytocompatible microenvironment for cell encapsulation, but also permits user-controlled rapid recovery of cell structures formed within the hydrogels.

Erzeugung und Verwertung von β-Zellen Spheroids Von Step-Wachstum PEG-Peptid-Hydrogele

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional  microenvironment with tissue-like elasticity and high permeability for  ...

Reaggregate Thymus Cultures

Stromal cells within lymphoid tissues are organized into three-dimensional structures that provide a scaffold that is thought to control the migration and development of haemopoeitic cells. Importantly, the maintenance of this three-dimensional organization appears to be critical for normal stromal cell function, with two-dimensional monolayer cultures often being shown to be capable of supporting only individual fragments of lymphoid tissue function. In the thymus, complex networks of cortical and medullary epithelial cells act as a framework that controls the recruitment, proliferation, differentiation and survival of lymphoid progenitors as they undergo the multi-stage process of intrathymic T-cell development. Understanding the functional role of individual stromal compartments in the thymus is essential in determining how the thymus imposes self/non-self discrimination. Here we describe a technique in which we exploit the plasticity of fetal tissues to re-associate into intact three-dimensional structures in vitro, following their enzymatic disaggregation. The dissociation of fetal thymus lobes into heterogeneous cellular mixtures, followed by their separation into individual cellular components, is then combined with the in vitro re-association of these desired cell types into three-dimensional reaggregate structures at defined ratios, thereby providing an opportunity to investigate particular aspects of T-cell development under defined cellular conditions. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

In this video the preparation of 2-dGuo-treated reaggregate thymus cultures is demonstrated.

Reaggregieren Thymus Kulturen

Stromal cells within lymphoid tissues are organized into three-dimensional structures that provide a scaffold that is thought to control the migration and ...

Biologie

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

Cellular Encapsulation in 3D Hydrogele für Tissue Engineering

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

Produktion von Elastin-Like Protein Hydrogele für Kapselung und Immunostaining Zellen in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

Initial 3D Cell Cluster Control in a Hybrid Gel Cube Device for Repeatable Pattern Formations

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its restrictions. Producing few repeatable results of pattern formation deteriorates the analysis of the mechanisms underlying the self-organization. Reducing variation in initial culture conditions, such as the cell density and distribution in the extracellular matrix (ECM), is crucial to enhance the repeatability of a 3D culture. In this article, we demonstrate a simple but robust procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain highly repeatable pattern formations. A micromold with a desired shape was fabricated by using photolithography or a machining process, and it formed a 3D pocket in the ECM contained in a hybrid gel cube (HGC). Highly concentrated cells were then injected in the pocket so that the cell cluster shape matched with the fabricated mold shape. The employed HGC allowed multi-directional scanning by its rotation, which enabled high-resolution imaging and the capture of the entire tissue structure even though a low-magnification lens was used. Normal human bronchial epithelial cells were used to demonstrate the methodology.&#160;

We present a procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain a repeatable pattern formation. A cubic device containing two different hydrogels is employed to achieve multi-directional imaging for tissue pattern formation.

Erste 3D Cluster Zellkontrolle in einem Hybrid Gel Cube Gerät für wiederholbare Muster Formationen

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its ...

<meta charset="utf8"></head><body>Nachbildung der Thymus-Mikroumgebung mit iPSC-abgeleiteten Organoiden

Formation of Human Thymus Organoids in Three-Dimensional Fibrin Hydrogels

Generation of a functional and self-tolerant T cell repertoire is a complex process dependent on the thymic microenvironment and, primarily, on the properties of its extracellular matrix (ECM). Thymic epithelial cells (TECs) are crucial in thymopoiesis, nurturing and selecting developing T cells by filtering self-reactive clones. TECs have been empirically demonstrated to be particularly sensitive to physical and chemical clues supplied by the ECM and classical monolayer cell culture leads to a quick loss of functionality until their death. Because of this delicate maintenance combined with relative rarity, and despite the high stakes in modeling thymus biology in vitro, models able to faithfully mimic the TEC niche at scale and over time are still lacking. Here, we describe the formation of a multicellular human thymic organoid model, in which the TEC compartment is derived from human induced pluripotent stem cells (iPSC) and reaggregated with primary early thymocyte progenitors in a three-dimensional (3D) fibrin-based hydrogel. This model answers current needs for a scalable culture system that reproduces the thymic microenvironment ex vivo and demonstrates functionality, i.e., the ability to produce T cells and to support thymus organoid growth over several weeks. Thus, we propose a practical in vitro model of thymus functionality through iPSC-derived organoids that would benefit research on TEC biology and T cell generation ex vivo.

<meta charset="utf8"></head><body>Autor im Rampenlicht: Weiterentwicklung von Thymus-Epithelzellen und T-Zell-Forschung mit humanen Thymus-Organoiden

Here, we describe a protocol for the formation of human iPSC-derived thymus organoids grown in 3D fibrin hydrogels aiming to support thymic epithelial cell (TEC) maturation and prolonged maintenance, as well as thymopoiesis in vitro.

Bildung humaner Thymus-Organoide in dreidimensionalen Fibrin-Hydrogelen

Generation of a functional and self-tolerant T cell repertoire is a complex process dependent on the thymic microenvironment and, primarily, on the ...

Die Förderung der 3-D-Aggregation von FACS Purified Thymusepithelzellen mit EAK 16-II / EAKIIH6 Selbstaufbau Hydrogels

Zusammenfassung

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Bildung humaner Thymus-Organoide in dreidimensionalen Fibrin-Hydrogelen

Bildung humaner Thymus-Organoide in dreidimensionalen Fibrin-Hydrogelen

Bildung humaner Thymus-Organoide in dreidimensionalen Fibrin-Hydrogelen