Siamo grati ai nostri assistenti tecnici: U. Bröder e H. Shkarlet, che ci hanno aiutato durante le procedure sperimentali. Questo lavoro è stato parzialmente finanziato dalla UE sussidi (UniVax, contratto n. 601738 e TRANSVAC2, contratto n. 730964) e una sovvenzione di Associazione Helmholtz (know-IDR). Le fonti di finanziamento non hanno influenzato la ricerca di design, generazione del manoscritto o decisione di inviarla per la pubblicazione.

Tutti i topi utilizzati in questo studio erano dallo sfondo C57BL/6. Tutti gli animali erano tenuti in condizioni esenti da patogeni. Tutti gli esperimenti sono stati eseguiti secondo la normativa della legge tedesca di protezione animale (TierSchG BGBl. I S 1105; 25.05.1998) e sono stati approvati dal Comitato Bassa Sassonia l'etica di esperimenti di animale e l'ufficio statale (Bassa Sassonia stato ufficio della protezione dei consumatori e la sicurezza alimentare), sotto il numero di permesso 33,4-42502-04-13/1281 e 162280.
1. CFSE macchiatura di OT-I T cellule e trasferimento adottivo
Nota: OT-I topi sono animali transgenically generati che esprimono un recettore delle cellule T (TCR) con fisso α e β catene che insieme riconoscono il peptide di immuno-dominante di ovuli, SIINFEKL10,11. Di conseguenza, questi topi hanno un numero considerevolmente elevato di CD8 specifici SIINFEKL+ T cellule (97%)12 rispetto ai topi normali o OVA-vaccinati (≤ 1%)13.
<ol><li>Isolamento di CD8 tracciabili+ T cellule da OT-I topi che esprimono specifici del linfocita T Thy1un (Thy1.1) allele:<ol>
<li>Eutanasia 6-9 settimane vecchio OT-I topi da inalazione di CO2 seguirono da dislocazione cervicale14. Sezionare la milza e linfonodi principali (solo per coppie di inguinale, ascellare e cervicale) tagliando la pelle con forbici chirurgiche, staccando la pelle dal corpo con l'aiuto di pinze chirurgiche e forcipe e metterli in capsule di Petri (60 x 15 mm) ognuno dei quali contiene un 100 µm poro maglia Coppa per rilascio di macinazione e delle cellule del tessuto.</li>
		<li>Mantenere di Petri (ciascuno con una mesh Coppa) in 3-5 mL di terreno RPMI completo (RPMI 1640, 10% v/v FCS, 100 U/mL penicillina, 50 µ g/mL di streptomicina) tenuta su ghiaccio.</li>
		<li>Schiacciare gli organi con l'aiuto di un stantuffo della siringa o un analogo strumento sterile (previo taglio non è necessario) e raccogliere la sospensione singola cella risultante in provette per centrifuga da 15 mL.</li>
		<li>Centrifugare la sospensione cellulare a 300 x g per 10 min a 4 ° C. Scartare il surnatante. Lavare le cellule in 10 mL di PBS freddo mediante centrifugazione e lisare gli eritrociti dalla milza sospendendo nuovamente il pellet in 1 mL/milza del cloruro di ammonio buffer (buffer di ACK, disponibili in commercio). Incubare le cellule per 1,5 min sul ghiaccio e successivamente lavare le cellule con 10 mL di PBS freddo mediante centrifugazione (come fatto in precedenza).</li>
		<li>Risospendere il pellet nello stesso tubo in 1 mL di PBS (pH 7,2) contenente 5% di siero bovino fetale per isolamento magnetico.</li>
	</ol>
</li>
	<li>Per eseguire l'isolamento magnetico, procedere alla selezione negativa di CD8+ T cellule utilizzando un kit di isolamento magnetico secondo le istruzioni del produttore o pubblicato protocolli15.</li>
	<li>Per eseguire la colorazione di CFSE, prima di contare il numero di CD8+ T cellule ottenute da OT-I topi utilizzando un contatore di cellule automatizzato (contatore di particelle). 1-5 di macchia x 107 cellule/mL in un volume di 1-5 mL con 5 µM CFSE in PBS per 7 min a 37 ° C, al riparo dalla luce in un tubo falcon di 15 ml.</li>
	<li>Placare la macchiatura CFSE aggiungendo lo stesso volume (1:1) di siero bovino fetale alle cellule e li Incubare per ulteriori 7 min a 37 ° C al riparo dalla luce. Lavare le cellule con 10 mL di PBS due volte.</li>
	<li>Contare le celle utilizzando un contatore di cellule automatizzato. Impostare il numero di cellulare da 3-5 x 107 cellule/mL di PBS al fine di iniettare 3-5 x 106 cellule/mouse in 100 µ l per via endovenosa (i.v.) tramite vena caudale.</li>
	<li>Per eseguire iniezioni della vena della coda, immobilizzare i topi in dispositivi appropriati. Scaldare la zona posteriore e la coda dei topi da iniettare utilizzando una lampada di luce rossa, posizionata tra 20 a 25 centimetri lontano i topi per 1-3 minuti consentire la vasodilatazione della vena della coda. Nota: Questo consente alla facilità della vena rilevamento e all'amministrazione della sospensione delle cellule.</li>
	<li>Garantire (con la mano appoggiata tra i topi e la lampada) che non è troppo caldo per i topi e quando le vene di coda sono chiaramente visibili, procedere all'iniezione. Iniettare le cellule nella vena caudale laterale o dorsale utilizzando una siringa da 1mL con un ago calibro 2516.</li>
</ol>2. immunizzazione (Endo-libero OVA + /-adiuvante)
<ol><li>Posizionare i topi trapiantati con OT-ho cellule (passaggio 1.7, Figura 1) in una camera di anestesia e isoflurano in ossigeno per amministrare un anestesia macchina14.</li>
	<li>Quando il mouse è completamente addormentato sotto anestesia, tirarla fuori dalla camera e radere il pelo del mouse sulla zona sopra gluteus superficialis (laterale bassa della schiena) utilizzando una macchina di taglio di capelli elettrico, al fine di eseguire un'iniezione pulito con una buona vista della zona di applicazione.</li>
	<li>Iniettare 50 µ l del vaccino, s.c. in zona rasata utilizzando un ago di calibro 25.</li>
</ol>3. isolamento dei linfociti e la macchiatura per analisi di citometria a flusso
<ol><li>Isolamento dei linfociti e splenocytes<ol>
<li>Eutanasia i topi vaccinati per inalazione di CO2 seguita da dislocazione cervicale.</li>
		<li>Estrarre i linfonodi drenanti e distanti e milza e metterli in piatti separati di Petri contenente 100 µm poro maglia tazze per rilascio di macinazione e delle cellule del tessuto. Seguire i passaggi 1.1.2 attraverso 1.1.3.</li>
		<li>Decantare il supernatante dopo centrifugazione e risospendere il pellet cellulare nello stesso volume del residuo PBS (circa 100 µ l).</li>
	</ol>
</li>
	<li>Macchiando per analisi di citometria a flusso<ol>
<li>In una provetta da 15 mL Centrifuga preparare un mix master contenenti gli anticorpi colorazione nelle concentrazioni illustrate nella tabella 1, producendo così un mix concentrato di colorazione di 2x. Preparare abbastanza volume di mix master di avere 100 µ l per campione. Mescolare le cellule e il mix master 1:1 e viene quindi incubato a 4° C per 30 min. Nota: Il volume finale di colorazione per campione dovrebbe essere 200 µ l (100 µ l di cella pellet + 100 µ l di mix master anticorpo).</li>
		<li>Lavare le cellule due volte mediante centrifugazione, come descritto in 1.1.3, aggiungere 10 mL di PBS, due volte.</li>
		<li>Risospendere le cellule marcate in 0,5-1 mL di PBS per acquisizione e trasferimento della sospensione per tubi cytometer di flusso. Sempre mantenere i campioni sul ghiaccio e al riparo dalla luce.</li>
	</ol>
</li>
</ol>4. flusso Cytometry
<ol><li>Sempre pre-filtrare i campioni di cellule utilizzando filtri di 70-100 µm.</li>
	<li>Preparare singola colorazione compensazione controlli per i fluorophores indicati nella tabella 1 (perline o celle). Nota: Compensazione dovrebbe essere eseguita nel citometro o analisi software17,18,19 e applicato a tutti i campioni.</li>
	<li>Seguire la strategia di gating rappresentata nella Figura 2. Brevemente, popolazioni di cancello in altezza di forward scatter vs zona di forward scatter (Figura 2A) e di nuovo a dispersione laterale ampia rispetto all'area di dispersione laterale (SSA, Figura 2B) al fine di escludere doppietti.</li>
	<li>Porta la popolazione da Figura 2B tracciando BV 650 (auto-fluorescenza) vs FITC (CFSE) al fine di discriminare il vero CFSE macchiato le cellule dalle cellule auto-fluorescente alte. Sono perline o cellule colorate con anticorpo coniugato a BV 650 nei controlli di compensazione. Questa trama (Figura 2) del BV 650 vs FITC è usata per cancello l'OT-ho CFSE macchiato le cellule.</li>
	<li>Al fine di distinguere le cellule derivate dal donatore vs topi destinatari del cancello la popolazione da Figura 2 C per Pe-Cy7 (Thy1.1 - CD90.1-) vs APC (CD8). Nota: Le cellule derivate da OT-I topi del donatore sono Thy1.1+ (CD90.1), mentre le cellule derivate da topi (C57BL/6, destinatario) WT sono Thy 1.2+ (CD90.2) (Figura 2D). Alcuni laboratori hanno OT-I topi in uno sfondo di Thy 1.2 e WT (C57BL/6) in un Thy1.1. In questo caso è necessario usare un anticorpo contro Thy 1.2 per OT-I cellulare gating.</li>
	<li>Ri-cancello CD8 cellule di+ da Thy1.1+ popolazione tracciando su un cancello composto da 450 BV (CD4) vs APC (CD8) (Figura 2E).</li>
	<li>Visualizzare la popolazione gated in Figura 2E utilizzando una visualizzazione dell'istogramma del CD8+ cellule che mostrano CFSE (FITC, 530/15 canale - laser blu-), cancello la popolazione proliferata includendo intensità da 102 fino al livello dove le popolazioni di controllo indivisa sono (intensità ~ 105, a seconda l'efficacia di CFSE colorazione e l'impostazione della tensione sul citofluorimetro) (Figura 2F).</li>
	<li>Fare un ulteriore cancello (non visibile in figura 2) tracciato marcatore FITC vs L/D (450/20 canale, UV laser) al fine di valutare le cellule morte in parallelo alla valutazione di proliferazione.</li>
	<li>Acquisire almeno 5000 eventi per i controlli di compensazione e 10,000 eventi (cancello E, Figura 2) dai campioni presso un citometro a flusso. Eseguire il citometro a flusso di basso o medio (non superare le portate di 20.000 eventi/s).</li>
</ol>

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Moderni vaccini sono idealmente ospita l'antigene purificato e adiuvanti, con l'eventuale aggiunta di un sistema di consegna come liposomi, particelle virus-like, nanoparticelle o vettori dal vivo. Un aspetto fondamentale durante la progettazione di un vaccino è quello di scegliere il giusto adiuvante secondo le necessità cliniche. Parte dell'ambito potrebbe coinvolgere favorendo un umorale vs risposta immunitaria cellulare (o entrambi), l'elezione di un locale vs una risposta immunitaria sistemica (o entrambi) e il ti...

Linfociti T citotossici (CTL) che inducono i vaccini sono fondamentali interventi terapeutici che sono stati sviluppati per combattere alcuni tipi di cancro1. CTL sono anche importanti per vaccini profilattici contro patogeni intracellulari2. Inoltre, CTL sono uno dei meccanismi di difesa immunitario pochi funzionalmente attivi in popolazioni a rischio come neonati3,4 quali dipendono anche CTL per combattere infezioni di vita iniziali5. A questo proposito, i vaccini contro il Virus respiratorio sinciziale (RSV) che sono stati sviluppati con un adiuvante che non suscitare le risposte CTL (allume) ha provocato un completo fallimento del vaccino che porta a gravi complicazioni su infezioni in infanti6. Questi effetti negativi della vaccinazione possono essere invertiti da un CD8+ di risposta delle cellule T7. Precedentemente abbiamo dimostrato che le principali citochine (interferoni di tipo I) suscitate da alcuni stimolatore di agonisti di geni (STING) di interferone sono essenziali per le risposte CTL generate da questi adiuvanti8, in parte misurando la proliferazione delle OT-I T cellule dopo vaccinazione e utilizzando questi risultati come una misura di CTL inducendo capacità osservata in esteso vaccinazione orari9. La misurazione della proliferazione di OT-ho CD8+ T cells in un mouse di destinatario C57BL/6 wild type (WT) di tintura di carboxyfluorescein succinimidyl ester (CFSE) diluizione è una stima robusta della capacità dell'adiuvante di un vaccino per generare cross-priming di SIINFEKL, (il peptide di immuno-dominante di ovoalbumina, uova). Variazioni di questa tecnica sono ampiamente utilizzati per la valutazione della proliferazione di OT-ho CD8+ e OT-II CD4+ T cellule. Ad esempio, esso è stato utilizzato in assenza delle citochine selezionate (topi KO) o per misurare l'efficacia del vaccino dopo il richiamo dell'antigene negli animali WT. Abbiamo messo a punto un protocollo breve (4 giorni esperimento) in cui dopo il trasferimento passivo di CFSE-macchiato OT-ho CD8+ T cells, una sottocutanea immunizzazione (s.c.) che consiste di una dose di 50 µ g di ovuli privo di endotossina completati con adiuvanti di prova viene somministrata (Figura 1). Il follow-up dei risultati 48 h dopo la vaccinazione fornisce una prova affidabile della capacità dell'adiuvante per generare risposte CTL. Da questa strategia, è possibile valutare la potenza della risposta immunitaria locale nel linfonodo drenante dopo immunizzazione, nonché l'entità della risposta misurando l'attività CTL nella milza (o linfonodi a distanza).

<table>
	<tbody>
		<tr>
			<td>BD LSR Fortessa Cell Analyzer</td>
			<td>BD</td>
			<td>Special Order</td>
			<td>Flow Cytometer</td>
		</tr>
		<tr>
			<td>CFSE</td>
			<td>Molecular Probes</td>
			<td>C34554</td>
			<td>Proliferation Dye</td>
		</tr>
		<tr>
			<td>MojoSort Mouse CD8 T Cell Isolation Kit</td>
			<td>Biolegend</td>
			<td>480007</td>
			<td>Magnetic Isolation Beads and antibodies for negative selection of untouched CD8 T cells.</td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Blue Dead Cell Stain Kit, for UV excitation</td>
			<td>Molecular Probes</td>
			<td>L23105</td>
			<td>Dead Cell Marker</td>
		</tr>
		<tr>
			<td>CD90.1 (Thy-1.1) Monoclonal Antibody (HIS51), PE-Cyanine7</td>
			<td>eBioscience</td>
			<td>25-0900-82</td>
			<td>antibody</td>
		</tr>
		<tr>
			<td>APC anti-mouse CD8a Antibody</td>
			<td>BioLegend</td>
			<td>100712</td>
			<td>antibody</td>
		</tr>
		<tr>
			<td>BV421 Rat Anti-Mouse CD4</td>
			<td>BD</td>
			<td>740007</td>
			<td>antibody</td>
		</tr>
		<tr>
			<td>Z2 coulter Particle count and Size Analyzer</td>
			<td>Beckman Coulter</td>
			<td>9914591DA</td>
			<td>Cell counter. Z2 Automated particle/cell counter</td>
		</tr>
		<tr>
			<td>EndoGrade Ovalbumin (10 mg)</td>
			<td>Hyglos(Germany)</td>
			<td>321000</td>
			<td>Ovalbumin endotoxin free tested.</td>
		</tr>
		<tr>
			<td>Cell Strainer 100&#181;m nylon</td>
			<td>Corning</td>
			<td>352360</td>
			<td>Cell strainer (100 &#181;m pore mesh cups).</td>
		</tr>
		<tr>
			<td>Sample Vials</td>
			<td>Beckman Coulter</td>
			<td>899366014</td>
			<td>Sample vials for Z2 automated counter</td>
		</tr>
		<tr>
			<td>C57BL/6 mice (CD90.2)</td>
			<td>Harlan (Rossdorf, Germany)</td>
			<td></td>
			<td>Company is now Envigo</td>
		</tr>
		<tr>
			<td>OT-I (C57BL/6 background, CD90.1)</td>
			<td>Harlan (Rossdorf, Germany)</td>
			<td></td>
			<td>Inbreed at our animal facility. Company from where adquired is now Envigo</td>
		</tr>
		<tr>
			<td>FACS tubes</td>
			<td>Fischer (Corning)</td>
			<td>14-959-5</td>
			<td>Corning Falcon Round-Bottom Polystyrene Tubes</td>
		</tr>
		<tr>
			<td>Falcon 15 mL tubes</td>
			<td>Fischer (Corning)</td>
			<td>05-527-90</td>
			<td>Falcon 15mL Conical Centrifuge Tubes</td>
		</tr>
		<tr>
			<td>PBS (500 mL)</td>
			<td>Fischer (Gibco)</td>
			<td>20-012-027</td>
			<td>Gibco PBS (Phosphate Buffered Saline), pH 7.2</td>
		</tr>
		<tr>
			<td>Red lamp (heating lamp)</td>
			<td>Dirk Rossmann GmbH (Germany)</td>
			<td>405096</td>
			<td>Heating infrred lamp (100 wats)</td>
		</tr>
		<tr>
			<td>IsoFlo (Isoflurane)</td>
			<td>Abbott Laboratories (USA)</td>
			<td>5260.04-05.</td>
			<td>Isoflurane anesthesic (250 mL flask).</td>
		</tr>
		<tr>
			<td>Tabletop Anesthesia Machine/Mobile Anesthesia Machine with CO2 Absorber</td>
			<td>Parkland Scientific</td>
			<td>V3000PK</td>
			<td>Isoflurane anesthesia machine.</td>
		</tr>
		<tr>
			<td>RPMI 1640 medium</td>
			<td>Gibco (distributed by ThermoFischer)</td>
			<td>11-875-093</td>
			<td>Base medium with Glutamine (500 mL)</td>
		</tr>
		<tr>
			<td>Pen-Strept antibiotic solution (Gibco)</td>
			<td>Gibco (distributed by ThermoFischer)</td>
			<td>15-140-148</td>
			<td>Gibco Penicillin-Streptomycin (10,000 U/mL)</td>
		</tr>
		<tr>
			<td>Fetal Bobine Serum (Gibco)</td>
			<td>Gibco (distributed by ThermoFischer)</td>
			<td>10082147</td>
			<td>Fetal Bovine Serum, certified, heat inactivated, US origin</td>
		</tr>
		<tr>
			<td>ACK Lysing Buffer (100 ml)</td>
			<td>Gibco (distributed by ThermoFischer)</td>
			<td>A1049201</td>
			<td>Amonium Chloride Potasium (ACK) Whole Blood Lysis Buffer, suitable for erytrocyte lysis in spleen suspensions also</td>
		</tr>
		<tr>
			<td>Plastic Petri Dishes</td>
			<td>Nunc (distributed by ThermoFischer)</td>
			<td>150340</td>
			<td>60 x 15mm Plastic Petri Dish, Non-treated</td>
		</tr>
		<tr>
			<td>Cell Clump Filter</td>
			<td>CellTrics (Sysmex)</td>
			<td>04-004-2317</td>
			<td>CellTrics&#174; 50 &#956;m, sterile</td>
		</tr>
	</tbody>
</table>

rapid in vivo assessment of adjuvant's cytotoxic t lymphocytes generation capabilities for vaccine development

La valutazione dei vaccini moderni sottounità rivela che la generazione di anticorpi neutralizzanti è importante ma non sufficiente per la selezione di adiuvante. Di conseguenza, adiuvanti con capacità immuno-stimolante sia umorali e cellulari che sono in grado di promuovere le risposte citotossiche (CTL) di linfociti di T sono urgentemente necessari. Quindi, monitoraggio fedele dei candidati adiuvante che inducono cross-priming e successivamente migliorano CTL generazione rappresenta un passo cruciale nello sviluppo del vaccino. Qui vi presentiamo un'applicazione per un metodo che utilizza SIINFEKL specifici (OT-ho) cellule di T per monitorare la cross-presentazione del modello antigene ovoalbumina (uova) in vivo in presenza di candidati diversi adiuvante. Questo metodo rappresenta un test rapido per selezionare adiuvanti con le migliori funzionalità di cross-priming. La proliferazione di CD8+ T cellule è l'indicazione più preziosa di cross-priming ed è anche considerato come un correlato di indotta da adiuvante cross-presentazione. Questa funzionalità può essere valutata in differenti organi immuni come i linfonodi e la milza. Nella misura della generazione CTL possa anche essere monitorata, dando intuizioni sulla natura di un locale (svuotamento linfonodale principalmente) o una risposta sistemica (linfonodi a distanza e/o milza). Ulteriormente questa tecnica permette modifiche multiple per testare i farmaci che possono inibire specifiche vie di cross-presentazione e offre anche la possibilità di essere utilizzato in diversi ceppi di topi convenzionali e geneticamente modificati. In sintesi, l'applicazione che vi presentiamo qui sarà utile per i laboratori di vaccino nell'industria o academia che sviluppano o modificare coadiuvanti chimici per vaccino ricerca e sviluppo.

Al fine di testare i trattamenti utilizzando una diversa combinazione di adiuvanti (ADJ1 e ADJ2), abbiamo valutato la capacità di generazione di CTL misurando la proliferazione di passivamente trasferiti OT-ho CD8+ T cellule mediante citometria a flusso (Figura 2). Per questo, abbiamo precedentemente macchiato le cellule isolate dal drenante nei linfonodi e milza (tabella 1). Misurando la proliferazione di CD8+ T cellul...

Watch this Scientific Journal Video about Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development at JoVE.com

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

Qui presentiamo un'applicazione per una tecnica immunologica standard (CFSE macchiato OT-ho proliferazione) destinato a monitorare rapidamente adiuvante-mediata di linfociti T citotossici (CTL) generazione in vivo. Questa veloce stima delle capacità CTL è utile per lo sviluppo di vaccini profilattici contro patogeni intracellulari, come pure i vaccini terapeutici del cancro.

Rapida In Vivo valutazione della funzionalità di generazione i linfociti T citotossici di adiuvante per lo sviluppo di vaccini

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. ...

rapid-vivo-assessment-adjuvant-s-cytotoxic-t-lymphocytes-generation

Research

JoVE Journal

Medicine

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

8.6K Views.  Helmholtz Centre for Infection Research. Qui presentiamo un'applicazione per una tecnica immunologica standard (CFSE macchiato OT-ho proliferazione) destinato a monitorare rapidamente adiuvante-mediata di linfociti T citotossici (CTL) generazione in vivo. Questa veloce stima delle capacità CTL è utile per lo sviluppo di vaccini profilattici contro patogeni intracellulari, come pure i vaccini terapeutici del cancro.

Video: Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

Teratoma Generation in the Testis Capsule

Pluripotent stem cells (PSCs) have the unique characteristic that they can differentiate into cells from all three germ layers. This makes them a potentially valuable tool for the treatment of many different diseases. With the advent of induced pluripotent stem cells (iPSCs) and continuing research with human embryonic stem cells (hESCs) there is a need for assays that can demonstrate that a particular cell line is pluripotent. Germline transmission has been the gold standard for demonstrating the pluripotence of mouse embryonic stem cell (mESC) lines1,2,3. Using this assay, researchers can show that a mESC line can make all cell types in the embryo including germ cells4. With the generation of human ESC lines5,6, the appropriate assay to prove pluripotence of these cells was unclear since human ESCs cannot be tested for germline transmission. As a surrogate, the teratoma assay is currently used to demonstrate the pluripotency of human pluripotent stem cells (hPSCs)7,8,9. Though this assay has recently come under scrutiny and new technologies are being actively explored, the teratoma assay is the current gold standard7. In this assay, the cells in question are injected into an immune compromised mouse. If the cells are pluripotent, a teratoma will eventually develop and sections of the tumor will show tissues from all 3 germ layers10. In the teratoma assay, hPSCs can be injected into different areas of the mouse. The most common injection sites include the testis capsule, the kidney capsule, the liver; or into the leg either subcutaneously or intramuscularly11. Here we describe a robust protocol for the generation of teratomas from hPSCs using the testis capsule as the site for tumor growth.

Human pluripotent stem cells (hPSCs) have the potential to treat a myriad of different diseases. The utility of these cells lies in the fact that they can differentiate into any cell type in the body. Here we describe the teratoma assay, which is used to demonstrate the pluripotence of hPSCs.

Generation teratoma nella Capsule Testicolo

Pluripotent stem cells (PSCs) have the unique characteristic that they can differentiate into cells from all three germ layers.  This makes them a ...

Ricerca

Medicina

Rapid Point-of-Care Assay of Enoxaparin Anticoagulant Efficacy in Whole Blood

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the low-molecular-weight-heparin (LMWH), enoxaparin. There are also urgent clinical situations where it would be important if this could be determined rapidly. The present assay is designed to accomplish this. We only assayed human blood samples that were spiked with known concentrations of enoxaparin. The essential feature of the present assay is the quantification of the efficacy of enoxaparin in a patient's blood sample by degrading it to complete inactivity with heparinase. Two blood samples were drawn into Vacutainer tubes (Becton-Dickenson; Franklin Lakes, NJ) that were spiked with enoxaparin; one sample was digested with heparinase for 5 min at 37 &deg;C, the other sample represented the patient's baseline anticoagulated status. The percent shortening of clotting time in the heparinase-treated sample, as compared to the baseline state, yielded the anticoagulant contribution of enoxaparin. We used the portable, battery operated Hemochron 801 apparatus for measurements of clotting times (International Technidyne Corp., Edison, NJ). The apparatus has 2 thermostatically controlled (37 &deg;C) assay tube wells. We conducted the assays in two types of assay cartridges that are available from the manufacturer of the instrument. One cartridge was modified to increase its sensitivity. We removed the kaolin from the FTK-ACT cartridge by extensive rinsing with distilled water, leaving only the glass surface of the tube, and perhaps the detection magnet, as activators. We called this our minimally activated assay (MAA). The use of a minimally activated assay has been studied by us and others. 2-4 The second cartridge that was studied was an activated partial thromboplastin time (aPTT) assay (A104). This was used as supplied from the manufacturer. The thermostated wells of the instrument were used for both the heparinase digestion and coagulation assays. The assay can be completed within 10 min. The MAA assay showed robust changes in clotting time after heparinase digestion of enoxaparin over a typical clinical concentration range. At 0.2 anti-Xa I.U. of enoxaparin per ml of blood sample, heparinase digestion caused an average decrease of 9.8% (20.4 sec) in clotting time; at 1.0 I.U. per ml of enoxaparin there was a 41.4% decrease (148.8 sec). This report only presents the experimental application of the assay; its value in a clinical setting must still be established.

Demonstration of a rapid quantitative assay of the inhibition of blood coagulation by the low-molecular-weight-heparin, enoxaparin. The contribution of enoxaparin is assessed by removing its influence through digestion with heparinase. A fuller description of the assay is detailed in our published paper.1 The assay still requires clinical confirmation.

Rapid Point-of-Care test di efficacia anticoagulante Enoxaparina nel sangue intero

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19 F MRI per il tracciamento cellulare

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic Therapy is a novel approach for a more tumor selective treatment.
Photodynamic Therapy (PDT) that makes use of a nontoxic photosensitizer (PS), which, upon activation with light of a specific wavelength in the presence of oxygen, generates oxygen radicals that elicit a cytotoxic response1. Despite its approval almost twenty years ago by the FDA, PDT is nowadays only used to treat a limited number of cancer types (skin, bladder) and nononcological diseases (psoriasis, actinic keratosis)2.
The major advantage of the use of PDT is the ability to perform a local treatment, which prevents systemic side effects. Moreover, it allows the treatment of tumors at delicate sites (e.g. around nerves or blood vessels). Here, an intraoperative application of PDT is considered in osteosarcoma (OS), a tumor of the bone, to target primary tumor satellites left behind in tumor surrounding tissue after surgical tumor resection. The treatment aims at decreasing the number of recurrences and at reducing the risk for (postoperative) metastasis.
In the present study, we present in vitro PDT procedures to establish the optimal PDT settings for effective treatment of widely used OS cell lines that are used to reproduce the human disease in well established intratibial OS mouse models. The uptake of the PS mTHPC was examined with a spectrophotometer and phototoxicity was provoked with laser light excitation of mTHPC at 652 nm to induce cell death assessed with a WST-1 assay and by the counting of surviving cells. The established techniques enable us to define the optimal PDT settings for future studies in animal models. They are an easy and quick tool for the evaluation of the efficacy of PDT in vitro before an application in vivo.

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

The protocol reported here allows the evaluation of the efficacy of photodynamic therapy (PDT) in cell lines and the optimization of the PDT settings before applying the therapy in animal models.

Citotossico Efficacia della terapia fotodinamica in cellule di osteosarcoma<em&gt; In Vitro</em

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic ...

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

Sequenziale<em&gt; In vivo</em&gt; Imaging di osteogeniche cellule staminali / progenitrici durante la frattura di riparazione

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

Valutazione del cancro ovarico Spheroid attaccamento e l&#39;invasione delle cellule mesoteliali in tempo reale

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

Raccolta ed estrazione di Saliva DNA per il sequenziamento di nuova generazione

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.1 However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. 1-3
To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 &#181;l&#160;of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes.
Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival. Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and delivery.

Valutazione della solidità del grasso umano iniezione in topi nudi con Micro-Tomografia Computerizzata

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

Generazione di Microtumors Uso 3D Biogel Umano Sistema Cultura e cellule di glioblastoma paziente-derivato per Kinomic Profiling and Drug Testing risposta

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

L&#39;esaurimento delle cellule di topo da xenotrapianti tumorali umani migliora in modo significativo Analisi A valle delle cellule bersaglio

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...