This work has been supported by the German Cancer Research Center (DKFZ), the EU-consortium &#8220;Tolerage&#8221;, the Deutsche Forschungsgemeinschaft (SFB 938) and the Landesstiftung Baden-W&#252;rttemberg.

Questo studio è stato approvato dal comitato etico del Regierungspräsidium Karlsruhe. Tutti gli animali sono stati alloggiati in condizioni esenti da organismi patogeni specifici presso il Cancer Research Center tedesco (DKFZ). Per tutti i cuccioli di esperimenti di coltura del mouse che vanno da 1 a 7 giorni di età sono stati utilizzati. 1. Isolamento di mTECs da Timo NOTA: Le seguenti operazioni di digestione sono state eseguite come descritto in precedenza 1 in condizioni sterili con alcune modifiche come segue. <ol><li> Decapitare i cuccioli di topo e rimuovere il timo. Posizionare il thymi su ghiaccio in una piastra di Petri contenenti terreno RPMI 1640 (contenente 5% FCS). </li><li> Tagliare il thymi in sottili, piccoli pezzi e posto in una provetta a fondo tondo con ~ 5-30 ml RPMI media e mescolate delicatamente con un magnete per 10 minuti a temperatura ambiente. </li><li> In seguito, decantare il surnatante contenente principalmente timociti e digerire la sequenza tessuto rimanente con un giro di collagenasé di tipo IV (0,2 mg / ml e 57 U / ml concentartion finale) per 15 minuti ciascuno a 37 ° C, seguita da collagenasi / dispasi (0,2 mg / ml e la concentrazione finale 1,2 U / ml) per 25 min a 37 ° ciascun C in un bagno d&#39;acqua con agitazione magnetica fino alla thymi sono completamente digerito. Usare 1 ml enzimatica per 2-3 thymi. </li><li> Agitare il tessuto volta ogni 7-10 minuti con una pipetta Pasteur. Si riuniscono gli collagenasi / frazioni dispasi e filtrare attraverso una garza di 70 micron. </li><li> Arricchire i mTECs da cellule magnetico ordinamento (MACS). Eseguire la purificazione di mTECs da cellule magnetico classificare come descritto in precedenza 11, e mostrato in Figura 1. NOTA: Per l&#39;ordinamento magnetico di mTECs abbiamo usato i seguenti anticorpi: anti-CD80-PE (16-10A1, uso a diluizione 1: 100) e anti-EpCAM-bio (G8.8, uso a diluizione 1: 100) 12. MTECs immaturi e maturi con MACS sono stati definiti come: CD45 - EpCAM + CD80 - e CD45 - CD80 + respectively. </li><li> Dopo MACS purificazione (purezza del mTECs immaturi = 83,1 ± 6,3% e mTECs maturi = 79.23 ± 3,42%), seminare i mTECs sulle colture organotipiche come descritto di seguito (punto 2.3). </li><li> In alternativa, ordinare mTECs di FACS (utilizzando 100 ugelli micron) dopo CD45 MACS esaurimento utilizzando i seguenti anticorpi: anti-CD45-PerCP (30-F11, l&#39;uso in diluizione 1: 100), anti-Ly51-FITC (6C3, utilizzare a 1 : 100 diluizione), anti-EpCAM-Alexa647 (G8.8, uso in diluizione 1: 500) e l&#39;uso anti-CD80-PE (16-10A1, a diluizione 1: 100). Escludere le cellule morte con ioduro di propidio (1: 5.000) (4 ° giorno). MTECs immaturi e maturi utilizzando FACS sono stati definiti come: PI - CD45 - Ly51 - EpCAM + CD80 - e PI - CD45 - Ly51 - EpCAM + CD80 +, rispettivamente. </li></ol> 2. 3D organotipiche co-culture (OTC) NOTA: I costrutti 3D-cutanea per la coltivazione organotipica di keratinocytes state preparate come descritto in precedenza 9,13. In tutte le fasi cellule sono state incubate a 37 ° C e 5% CO 2. I farmaci da banco che utilizzano mTECs sono stati preparati con lievi modifiche nel modo seguente. <ol><li> Preparazione di fibroblasti umani NOTA: i fibroblasti dermici umani sono stati ottenuti da colture espianto di de-epidermised derma come descritto in precedenza 9. <ol><li> In breve, tagliare le strisce di pelle umana (~ 5 cm di lunghezza) e trattare con termolisina (0,5 mg / ml in soluzione salina con 10 mM Hepes pH 7,4) O / N a 4 ° C. </li><li> Successivamente, separare l&#39;epidermide dal derma con pinze. </li><li> Tagliare finemente il derma in piccoli pezzi, posto in un piatto 10 centimetri Petri e lasciare asciugare per 1-2 ore sotto un flusso d&#39;aria sterile. Supplemento espianti regolarmente con DMEM contenente 20% FBS. </li><li> Dividere i fibroblasti crescita fuori quando confluenti (di solito circa 3 settimane) con 0,1% tripsina fare in modo che gli espianti continuano ad aderire alla piastra per ulteriori conturni consecutivi di conseguenza. </li><li> Espandere i fibroblasti dalle stesse espianti per un massimo di 3 volte in DMEM con 10% FBS e crio-conservare le 14. Utilizzare lo stesso lotto di fibroblasti per ogni serie sperimentale. NOTA: I fibroblasti non sono stati irradiati per il set-up degli equivalenti dermici. </li></ol></li><li> Preparazione del impalcatura <ol><li> Tagliare i 0,4-0,6 mm viscosa spessore, materiale fibroso non tessuto (dati del prodotto a sostegno foglio Excel) in ambienti ben delimitate con un netto 11 millimetri di diametro puncher metallo per adattarsi esattamente in 12 inserti bene col filtro. Poi inserirla nel pozzo 12 inserto del filtro (poliestere membrana capillare pori, 3 micron dimensione dei pori) da impalcatura. Posizionare la configurazione filtro completo in un 12-pozzetti sterili. </li><li> Preparare il gel di fibrina utilizzando una colla di fibrina-kit per la chirurgia costituito da una combinazione di fibrinogeno e trombina. Pre-diluire il fibrinogeno e la trombina-componente del kit a 8 mg / ml e 10Unità / ml, rispettivamente. Per un singolo pozzetto di una piastra 12 pozzetti procedere come segue. <ol><li> Diluire 100 microlitri fibrinogeno (8 mg / ml) con 100 ml di tampone fosfato salino (PBS) senza Ca 2 + e Mg 2 +, pH 7,0. Diluire 100 ml di trombina (10 unità / ml) con 100 microlitri FCS contenenti 270.000 fibroblasti. </li><li> Dispensare 200 microlitri della trombina contenente fibroblasti cellule preconfezionato sul ponteggio, a cui si aggiungono 200 pl fibrinogeno (miscela 1: 1), ottenendo una concentrazione finale di fibrinogeno di 2 mg / ml e di trombina di 2,5 unità / ml. Mescolare bene e distribuire in modo uniforme su tutta l&#39;area del ponteggio pipettando dolce (giorno 1). NOTA: Dopo 30 minuti a 37 ° C un coagulo che racchiude i fibroblasti sarà formata, riempiendo completamente gli spazi interni del ponteggio e formando una superficie superiore liscia. </li></ol></li></ol></li><li> Co-coltura con mTECs <ol><li> Per la pre-cultura, immergere le colture d&#39;organo aDMEM con 10% FBS, 50 mg / ml di acido L-ascorbico e 1 ng / ml di TGF-β1 con una variazione media ogni altro giorno per 4-5 giorni. </li><li> Il giorno della MTEC semina, sostituire il mezzo attraverso RFAD media (1: 1 DMEM + DMEM / F12) con 10% FBS, 10 -10 M tossina colerica, 0,4 mg / ml idrocortisone, 50 mg / ml di acido L-ascorbico, RANK ligando (0,1 mcg / ml) e 500 unità / ml di aprotinina, impedendo così fibrinolisi precoce da serina proteasi secrete da fibroblasti. </li><li> 7-8 ore più tardi, set-up co-culture seminando 250.000 mTECs (sia mTECs completi, o CD80 e CD80 lo sottoinsiemi hi), in un volume di 100 ml per pozzetto in cima al ponteggio fibroblasti. Contare le cellule utilizzando una camera di Neubauer (4 ° giorno). NOTA: In ogni momento le timo colture organotipiche 3D sono sommerse dai media a differenza OTC della pelle, che sono dotate di aria sollevato. </li><li> Dopo 24 ore di incubazione, di fornire le culture con medio (totale medio di cambio), come detto sopra (punto 2.3.2) ora containing ridotta quantità di Aprotinina, 250 unità / ml (Giorno 5). </li><li> Per valutare l&#39;attività proliferativa delle mTECs, aggiungere EdU (6,7 mM / ml, cioè, 10 mM / pozzetto) per OTCs per 4 ore prima della cessazione delle culture. Eseguire la colorazione di OTC crio-sezioni come descritto nel Kit EdU Imaging combinata con la co-colorazione di cheratina 14. Determinare gli indici proliferative mTECs, da una delle contando cellule K14 + EdU + in due sezioni di ogni esemplare la cultura o di flusso citometria (EdU citometria a flusso, Sezione 1.7 ma invece di Ly51-FITC utilizzano CDR1-PB 15 ad una diluizione 1: 100 e 2.3.6.4). </li><li> Dopo 4-7 giorni di co-coltura, terminare i farmaci da banco e il processo per l&#39;isolamento di RNA, crio-sezionamento o FACS analisi (giorno 8-11). <ol><li> Terminate le culture utilizzando una pinza, separando l&#39;equivalente scaffold / cutanea dal filtro dell&#39;inserto bene. </li><li> Per crio-sezionamento, incorporare l&#39;intero OTC in OCT e congelare in liquid vapori di azoto prima crio-sezionamento. Preparare 5-7 micron di spessore sezioni OTC utilizzando un criostato e conservare a -20 ° C fino al momento dell&#39;uso. Per immunoistochimica di OTC crio-sezione usare anti-cheratina 14 (AF64, uso a 1: 1.000 diluizione), e anti-vimentina (GP53, uso a 1: 100 diluizione) anticorpi. Eseguire la colorazione immunoistochimica indiretta utilizzando i rispettivi anticorpi secondari. </li><li> Per l&#39;isolamento di RNA, aggiungere 1 ml di denaturazione soluzione (contenente fenolo e guanidina tiocianato) in un tappo a vite di un tubo libero 2 ml RNasi. Tagliare l&#39;intera OTC in pezzi con un bisturi e aggiungere nella provetta contenente soluzione denaturazione. Meccanicamente sminuzzare i OTCs con strumento FastPrep due volte per 30 secondi a una velocità di 6,0, inserire tra il ghiaccio per 2 min (il campione può essere conservato a -80 ° C dopo questo punto). Se congelato a -80 ° C, scongelare su ghiaccio. Centrifugare le provette a 11,500-13,000 rpm per 10 minuti a 4 ° C. Trasferire il surnatante in una nuova provetta, incubare per 5 minuti a temperatura ambiente e fprotocolli di isolamento RNA susseguirsi utilizzando guanidinio estrazione tiocianato-fenolo-cloroformio acido come descritto dal produttore. </li><li> Per l&#39;analisi FACS, rimuovere l&#39;impalcatura e separare la membrana della fibrina / fibroblasti / gel MTEC. Finemente tagliare il gel con un bisturi e digerire in un tubo FACS per ~ 20 minuti o fino a quando completamente digerito con 2 ml di collagenasi / dispasi a 37 ° C in un bagno d&#39;acqua con agitazione magnetica. Agitare la soluzione enzimatica con una pipetta Pasteur volta ogni 5 min. Dopo completa digestione, filtrare la sospensione cellulare attraverso un filtro 70 micron; macchiare la sospensione singola cella utilizzando gli anticorpi come descritto in 1.7 e analizzare mediante citometria a flusso. </li></ol></li></ol></li></ol>

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Gli autori dichiarano assenza di conflitto finanziaria o commerciale di interesse.

Accanto RTOCs, l&#39;OTC 3D sono stati di gran lunga superiore in termini di differenziazione TEC e PGE manutenzione / induzione (Tabella 1) rispetto ad altri (i) &#39;culture 3D semplificati&#39; utilizzando - fibroblasti da solo senza il patibolo; (Ii) i sistemi 2D utilizzando - cellule fibroblasti / alimentatore co-coltura con TEC 10, (iii) le cellule 3T3-J2 cui cloni TEC sviluppano, ma PGE è perso, (iv) matrigel o (v) componenti ECM (dati non pubblicati). PGE è stato mantenuto fino a 7 ...

Timociti sviluppo costituiscono circa il 98% del timo, mentre il restante 2% è costituito da una varietà di cellule che compongono collettivamente stroma timico (cioè, cellule epiteliali, cellule dendritiche, macrofagi, cellule B, fibroblasti, cellule endoteliali). Le cellule epiteliali esterni corticali (CTEC) procurano immigrazione di cellule pro-T dal midollo osseo, induzione linea cellulare T in multipotenti cellule pre-T e selezione positiva di auto-MHC limitati timociti immaturi. Le cellule interne midollari timiche epiteliali (mTECs) sono coinvolti nella induzione della tolleranza di questi timociti con un alta affinità TCR per complessi auto-peptide / MHC da uno inducendo selezione negativa o loro deviazione nella linea cellulare T normativo. Nel contesto di induzione della tolleranza centrale, mTECs sono unici in quanto esprimono un ampio spettro di auto-antigeni tissutali-limitato (Tras) rispecchiando così il sé periferica. Questo fenomeno è chiamato espressione genica promiscua (PGE)1,2. La maggior parte degli studi in corso su questo tipo di cellule affascinante si basano su cellule isolate ex vivo, come i vari sistemi di coltura 2D a breve termine invariabilmente portato alla perdita di PGE e molecole regolatore chiave come MHC di classe II, FoxN1 e Aire entro i primi 2 giorni 3-6 . Rimase tuttavia poco chiaro, che particolari componenti e le caratteristiche del reticolo 3D intatto del timo mancavano in modelli 2D. La riaggregazione coltura dell&#39;organo timica (RTOC) è stato finora l&#39;unico sistema 3D che permette lo studio dello sviluppo delle cellule T, da una parte, e la biologia cellule stromali, d&#39;altra parte, in un microambiente timico intatta 7. Tuttavia, RTOCs hanno alcune limitazioni, cioè, essi contengono già una miscela complessa di cellule, richiederà il contributo delle cellule stromali fetali e sopportare un periodo di coltura massima di 5 a 10 giorni. La mancanza di riduzionista nei sistemi di coltura in vitro ha ostacolato lo studio didiversi aspetti di sviluppo delle cellule T e thymic organogenesi non ultima la regolazione molecolare di PGE e il suo rapporto con la biologia dello sviluppo di mTECs. A causa del primo relazionalità dell&#39;organizzazione strutturata delle cellule epiteliali della pelle e del timo, abbiamo optato per un sistema 3D cultura organotipica (OTC), che era stato sviluppato originariamente per emulare la differenziazione dei cheratinociti in vitro e creare un equivalente cutanea così. Il sistema OTC costituito da una matrice inerte scaffold sovrapposti con fibroblasti dermici che sono intrappolati in un gel di fibrina, su cui cheratinociti vengono seminate 8,9. Qui, abbiamo sostituito con cheratinociti mTECs purificati. Pur mantenendo le caratteristiche di base di questo modello, abbiamo ottimizzato alcuni parametri. Nel modello OTC adottato mTECs proliferato, ha subito la differenziazione terminale e mantenuto l&#39;identità MTEC e PGE, quindi mimando in vivo sviluppo mTECs <sup&gt; 10. Questa nota tecnica fornisce un protocollo dettagliato che permette la graduale messa a punto di OTC timo.

<table><tbody><tr><td>Pregnant C57BL/6 mice&amp;nbsp;</td><td>Charles River WIGA</td><td></td><td></td></tr><tr><td>LS columns</td><td>Miltenyi Biotec</td><td>130-042-401</td><td></td></tr><tr><td>MS columns</td><td>Miltenyi Biotec</td><td>130-042-201</td><td></td></tr><tr><td>CD45 Microbeads, mouse</td><td>Miltenyi Biotec</td><td>130-052-301</td><td></td></tr><tr><td>Anti-PE Microbeads</td><td>Miltenyi Biotec</td><td>130-048-801</td><td></td></tr><tr><td>Streptavidin Microbeads</td><td>Miltenyi Biotec</td><td>130-048-101</td><td></td></tr><tr><td>EpCAM (G8.8 -Alexa 647 and -biotin)</td><td>Ref. 12</td><td></td><td></td></tr><tr><td>CD80-PE antibody</td><td>BD Pharmingen</td><td>553769</td><td></td></tr><tr><td>CD45-PerCP antibody</td><td>BD Pharmingen</td><td>557235</td><td></td></tr><tr><td>Ly51-FITC antibody</td><td>BD Pharmingen</td><td>553160</td><td></td></tr><tr><td>CDR1-Pacific Blue</td><td>Ref. 15</td><td></td><td></td></tr><tr><td>Keratin 14 antibody</td><td>Covance</td><td>PRB-155P</td><td></td></tr><tr><td>Vimentin antibody</td><td>Progen</td><td>GP58</td><td></td></tr><tr><td>Cy3-conjugated AffiniPure Goat anti-Rabbit IgG (H+L)</td><td>Jackson ImmunoResearch&amp;nbsp;</td><td>111-165-003</td><td></td></tr><tr><td>Alexa 488-conjugated AffiniPure F(ab&#039;)2 Fragment Goat anti-Guinea Pig IgG (H+L)</td><td>Jackson ImmunoResearch&amp;nbsp;</td><td>106-546-003</td><td></td></tr><tr><td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate</td><td>Molecular Probes (Invitrogen GmbH)</td><td>A-11008</td><td></td></tr><tr><td>Click-iT EdU Alexa Fluor 594 Imaging Kit</td><td>Invitrogen</td><td>C10339</td><td></td></tr><tr><td>Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit</td><td>Invitrogen</td><td>C10425</td><td></td></tr><tr><td>12-well filter inserts (thincerts)</td><td>Greiner bio-one</td><td>657631</td><td></td></tr><tr><td>12-well plate</td><td>Greiner</td><td>665180-01</td><td></td></tr><tr><td>Jettex 2005/45</td><td>ORSA, Giorla Minore, Italy</td><td></td><td></td></tr><tr><td>Fibrinogen TISSUECOL-Kit Immuno</td><td>Baxter</td><td></td><td></td></tr><tr><td>Thrombin TISSUECOL-Kit Immuno</td><td>Baxter</td><td></td><td></td></tr><tr><td>PBS</td><td>Serva</td><td>47302.03</td><td></td></tr><tr><td>DMEM</td><td>Lonza</td><td>BE12-604F</td><td></td></tr><tr><td>DMEM/F12</td><td>Lonza</td><td>BE12-719F</td><td></td></tr><tr><td>HEPES</td><td>Gibco</td><td>15630-049&amp;nbsp;</td><td></td></tr><tr><td>FBS Gold</td><td>GE Healthcare</td><td>A11-151</td><td></td></tr><tr><td>Aprotinin (Trasylol)</td><td>Bayer</td><td>4032037</td><td></td></tr><tr><td>Cholera toxin</td><td>Biomol</td><td>G117</td><td></td></tr><tr><td>Hydrocortisone</td><td>Seromed (Biochrom)</td><td>K3520</td><td></td></tr><tr><td>L-ascorbic acid</td><td>Sigma</td><td>A4034</td><td></td></tr><tr><td>TGF-&amp;szlig;1</td><td>Invitrogen</td><td>PHG9214</td><td></td></tr><tr><td>RANKL</td><td>R&amp;amp;D systems</td><td>462-TR-010</td><td></td></tr><tr><td>Thermolysin</td><td>Sigma Aldrich</td><td>&amp;nbsp;T-7902</td><td></td></tr><tr><td>OCT Compound</td><td>TissueTek</td><td>4583</td><td></td></tr><tr><td>Trizol (aka. Denaturing solution - Acid guanidinium thiocyanate-phenol-chloroform extraction)</td><td>Invitrogen</td><td>10296028</td><td></td></tr><tr><td>FastPrep FP120</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Collagenase Type IV&amp;nbsp;</td><td>CellSystems</td><td>LS004189</td><td>0.2 mg/ml and 57U/ml final conc.</td></tr><tr><td>Neutrale Protease (Dispase)</td><td>CellSystems</td><td>LS002104</td><td>0.2 mg/ml and 1.2U/ml final conc.</td></tr><tr><td>DNase I&amp;nbsp;</td><td>Roche</td><td>11 284 932 001</td><td>25 &amp;micro;g/ml final conc.</td></tr></tbody></table>

3d organotypic co-culture model supporting medullary thymic epithelial cell proliferation, differentiation and promiscuous gene expression

Lo sviluppo delle cellule T Intra-timico richiede un reticolo tridimensionale complessa composta da varie cellule stromali, cioè cellule non-T. Timociti attraversano questo patibolo in un ordine temporale e spaziale altamente coordinato, mentre in sequenza passa punti di controllo obbligatori, cioè, l&#39;impegno linea cellulare T, seguita da generazione repertorio recettore delle cellule T e la selezione prima della loro esportazione nella periferia. I due principali tipi di cellule che formano residente questa impalcatura sono cellule epiteliali del timo corticali (CTEC) e midollari (mTECs). Una caratteristica fondamentale di mTECs è la cosiddetta espressione promiscua di numerosi antigeni tissutali limitato. Questi antigeni tissutali con restrizioni vengono presentati immaturi timociti direttamente o indirettamente da mTECs o cellule dendritiche del timo, causando rispettivamente in self-tolleranza. Adatto in vitro modelli che emulano i percorsi di sviluppo e le funzioni di CTEC e mTECs sono attualmente lacre. Questa mancanza di adeguati modelli sperimentali è per esempio ostacolato l&#39;analisi dell&#39;espressione genica promiscua, che è ancora poco conosciuta a livello cellulare e molecolare. Abbiamo adattato una organotipica modello di co-coltura 3D per la cultura ex vivo mTECs isolati. Questo modello è stato originariamente concepita per coltivare cheratinociti in modo tale da generare un equivalente pelle in vitro. Il modello 3D conservato caratteristiche funzionali chiave di MTEC biologia: (i) la proliferazione e la differenziazione terminale di CD80 ecco, Aire-negativo in CD80 hi, Aire-positivi mTECs, (ii) la risposta a RANKL, e (iii) espressione sostenuta di FoxN1, Aire e geni tessuto-limitato a CD80 mTECs hi.

Abbiamo adottato un organotipica co-coltura modello 3D (3D OTC) che era stato originariamente sviluppato per la cultura a lungo termine in vitro dei cheratinociti 9. MTECs MACS-arricchiti (vedi MACS arricchimento schema Figura 1) sono state seminate su un ponteggio composto da un gel di fibrina e fibroblasti intrappolati. I fibroblasti forniscono la matrice extracellulare essenziale (ECM) sostegno mTECs in vitro. MTECs sono state coltivate in OTC per 4-14 giorni, in presenza...

Watch this Scientific Journal Video about 3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression at JoVE.com

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

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 organotipica Co-cultura Modello Sostenere midollare timica epiteliale proliferazione, differenziazione e promiscua genica

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

3d-organotypic-co-culture-model-supporting-medullary-thymic

Research

JoVE Journal

Developmental Biology

10.6K Views.  German Cancer Research Center (DKFZ). 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. 

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

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a promising source for adoptive T cell therapy (ACT). However, classical in vitro methods for producing regenerated T cells from iPSC result in either innate-like or terminally differentiated T cells, which are phenotypically and functionally distinct from na&#239;ve T cells. Recently, a novel three-dimensional (3D) thymic culture system was developed to generate a homogenous subset of CD8&#945;&#946;+ antigen-specific T cells with a na&#239;ve T cell-like functional phenotype, including the capacity for proliferation, memory formation, and tumor suppression in vivo. This protocol avoids aberrant developmental fates, allowing for the generation of clinically relevant iPSC-derived T cells, designated as iPSC-derived thymic emigrants (iTE), while also providing a potent tool to elucidate the subsequent functions necessary for T cell maturation after thymic selection.

This article describes a novel method to generate tumor antigen-specific induced pluripotent stem cell-derived thymic emigrants (iTE) by a three-dimensional (3D) thymic culture system. iTE are a homogenous subset of T cells closely related to na&#239;ve T cells with the capacity for proliferation, memory formation, and tumor suppression.

Un sistema di coltura timica tridimensionale per generare emigrati timici derivati da murine

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

Ricerca

Ricerca sul cancro

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.

Reaggregate Culture Thymus

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

Biologia

Preparation of 2-dGuo-Treated Thymus Organ Cultures

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo can limit analysis of individual cellular components and particular stages of development. In vitro culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

This video demonstrates the dissection and removal of the fetal thymus as well the preparation of ex vivo cultures of 2-dGuo-treated thymus.

La preparazione di 2-dGuo-trattati Organ Culture Timo

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

Tessuti epiteliali Ingegneria tridimensionali integrati all&#39;interno di matrice extracellulare

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Bioingegneria

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

Generazione di organoidi mammari mosaici per provepsinizzazione differenziale

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

Biologia dello sviluppo

Extra Cellular Matrix-Based and Extra Cellular Matrix-Free Generation of Murine Testicular Organoids

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed in order to create testicular organoids. Many of these methods rely upon extracellular matrix (ECM) to promote de novo tissue assembly, however, there are differences between methods in terms of biomimetic morphology and function of tissues. Moreover, there are few direct comparisons of published methods. Here, a direct comparison is made by studying differences in organoid generation protocols, with provided outcomes. Four archetypal generation methods: (1) 2D ECM-free, (2) 2D ECM, (3) 3D ECM-free, and (4) 3D ECM culture are described. Three primary benchmarks were used to assess the testicular organoid generation. These are cellular self-assembly, inclusion of major cell types (Sertoli, Leydig, germ, and peritubular cells), and appropriately compartmentalized tissue architecture. Of the four environments tested, 2D ECM and 3D ECM-free cultures generated organoids with internal morphologies most similar to native testes, including the de novo compartmentalization of tubular versus interstitial cell types, the development of tubule-like-structures, and an established long-term endocrine function. All methods studied utilized unsorted, primary murine testicular cell suspensions and used commonly accessible culture resources. These testicular organoid generation techniques provide a highly accessible and reproducible toolkit for research initiatives into testicular organogenesis and physiology in vitro.

Here, four methods for generating testicular organoids from primary neonatal murine testicular cells are described i.e., extracellular matrix (ECM) and ECM-free 2D and 3D culture environments. These techniques have multiple research applications and are especially useful for studying testicular development and physiology in vitro.

Generazione extra di organoidi testicolari murini basati su matrice cellulare ed extra cellulare

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed ...

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.

Preparazione e applicazioni di colture organotipiche timica Slice

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

Immunologia e infezioni

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids and patient-derived xenograft organoids are pathophysiologically relevant, they are costly to propagate, difficult to manipulate, and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. 
Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. The advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these heteromulticellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells (THP-1), and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. 
Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent with the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, heteromulticellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in heteromulticellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression.

There is a critical need for 3D cancer models that capture heterocellular crosstalk to study cancer metastasis. Our study presents the generation of heteromulticellular stromal-epithelial in a scaffold and scaffold-free environment that can be used to study invasion and cellular spatial distributions.

Cellule stromali eteromulticellulari in colture 3D prive di scaffold di cellule tumorali epiteliali per guidare l'invasione

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully ...

<meta charset="utf8"></head><body>Ricreare il microambiente timico con organoidi derivati da iPSC

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>Autore in primo piano: Avanzamento della ricerca sulle cellule epiteliali timiche e sulle cellule T con organoidi timici umani

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.

Formazione di organoidi di timo umano in idrogel di fibrina tridimensionali

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

3D organotipica Co-cultura Modello Sostenere midollare timica epiteliale proliferazione, differenziazione e promiscua genica

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Capitoli in questo video

Un sistema di coltura timica tridimensionale per generare emigrati timici derivati da murine

Reaggregate Culture Thymus

La preparazione di 2-dGuo-trattati Organ Culture Timo

Tessuti epiteliali Ingegneria tridimensionali integrati all'interno di matrice extracellulare

Generazione di organoidi mammari mosaici per provepsinizzazione differenziale

Generazione extra di organoidi testicolari murini basati su matrice cellulare ed extra cellulare

Preparazione e applicazioni di colture organotipiche timica Slice

Cellule stromali eteromulticellulari in colture 3D prive di scaffold di cellule tumorali epiteliali per guidare l'invasione

Formazione di organoidi di timo umano in idrogel di fibrina tridimensionali

Formazione di organoidi di timo umano in idrogel di fibrina tridimensionali