Questo lavoro è stato sostenuto da sovvenzioni NIH R01 CA107469, R01 CA125577, e U01CA154224 di CG Kleer, la University of Cancer Support Center del Michigan.

1. Preparazione dei Materiali e cultura dei media <ol><li> Fattore di crescita Thaw ridotta (GFR) Matrigel a 4 ° CO / N. </li><li> Warm up supporti completi per la linea cellulare richiesto a 37 ° C. MCF10A-ATCC specificato media; F-12 medio di HME-Ham supplementato con 5% di siero fetale bovino, 1 mg / ml di idrocortisone, 5 pg / ml di insulina, 10 ng / ml di fattore di crescita epidermico, e 100 ng / ml di tossina del colera; F-12 supporti di SUM149-Ham, completata con il 5% FBS, 2 mg / ml di idrocortisone e 5 mg / ml di insulina; e MDA-MB-231-10% FBS-DMEM </li><li> Preraffreddare uno scivolo camera 8 pozzetti su ghiaccio. </li><li> Preparare cappa coltura tissutale e forniture. </li></ol> 2. Tre Culture dimensionale in 8 pozzetti diapositive Camera <ol><li> Coat ciascun pozzetto della slitta 8 pozzetti con 40 ml di GFR Matrigel. Aggiungere il Matrigel nel centro della diapositiva e poi diffusa utilizzando una punta di pipetta P200. Cercare di diffondere il più uniformemente possibile, evitando le bolle e invadendo che può portare aformazione di menisco. </li><li> Incubare i vetrini a 37 ° C in 5% CO 2. Mentre il Matrigel viene consolidando, trypsinize le cellule, raccogliere e girare a 150 xg nel tessuto della cultura centrifugare per 4 min. </li><li> Contare le cellule e diluire a 12.500 cellule / ml in mezzo completo della rispettiva linea cellulare. Aggiungere GFR Matrigel al 2% e aggiungere 400 microlitri di ciascun pozzetto di una diapositiva camera Matrigel prerivestita. </li><li> Incubare i vetrini in CO 2 incubatore. Dare freschi cambiamenti multimediali completi ogni 4 ° giorno fino a 15 giorni. </li><li> Il trattamento con inibitori farmacologici: Per gli studi inibitori aggiungono piccole molecole inibitrici ai media completo al momento della placcatura seguita da cambiamenti dei media freschi integrati con gli inibitori ogni tre giorni durante la crescita delle cellule di GFR Matrigel. </li><li> Il trattamento con proteina purificata: <ol><li> Piastra le cellule seguenti passaggi 2,1-2,3. Lasciare le culture di crescere per 4 giorni, seguiti da starvati sieroavanti per 16 ore. </li><li> Al giorno 5, trattare le cellule con una concentrazione adeguata di proteine ​​purificate in 0.1% FBS completato media per le cellule affamate di siero. Dare cambiamento fresca media con proteina purificata dopo 2 giorni. </li><li> Al giorno 10, sostituire il supporto condizionata e supporti completi HME. Lasciare le colture a crescere per altri 5 giorni. </li></ol></li><li> Immunofluorescenza delle strutture acinose coltivate su Matrigel: <ol><li> Preparare 2% paraformaldeide, 0,5% Triton X-100 e glicina 100 mM in 1x PBS, pH 7,4. Preparare tampone immunofluorescenza (IF buffer) che comprende 1x PBS contenente 0,1% di albumina di siero bovino, 0,2% Triton X-100, 0,05% Tween-20. </li><li> Utilizzare un puntale P200 per aspirare accuratamente il supporto condizionato. </li><li> Fissare le strutture acinose con 2% paraformaldeide preparata a temperatura ambiente (RT) per 20 min. </li><li> Permeabilize le cellule con 0,5% Triton X-100 per 10 minuti a 4 ° C. </li><li> Lavare la culturas tre volte con glicina 100 mM, 10-15 min per ogni lavaggio. </li><li> Bloccare le cellule con 10% di siero di capra in tampone IF, chiamato blocco primario, per 1 ora a RT. </li><li> Incubare le colture con 20 ug / ml di capra anti F (ab &#39;) 2 frammento nel buffer di blocco primario per 30 min mouse. Questo passaggio è importante per bloccare il mouse specie IgG immunoreattivi in ​​Matrigel. </li><li> Incubare con anticorpo primario nella seconda soluzione di blocco (blocco Primario +20 mcg / ml di capra anti-topo F (ab &#39;) 2 frammento) O / N a 4 ° C. </li><li> Lavare tre volte con tampone IF per 10-15 minuti ciascuno con dolce dondolio. </li><li> Incubare con fluorescenti anticorpi secondari coniugati nel tampone IF per 45 min. </li><li> Lavare 3x per 10 minuti ciascuno con tampone IF con dolce dondolio. </li><li> Montare i vetrini con il reagente anti-sbiadimento contenente DAPI per visualizzare i nuclei. </li><li> Lasciare i vetrini si asciughino O / N a RT. </li><li> Acquisizione Image: Acquisire le immagini confocale ai mi coloniadsection di cellule cresciute in cima Matrigel. Per ulteriori informazioni acquisire una serie di sezioni ottiche per tutta la lunghezza della struttura acinose da Z impilamento. </li></ol></li></ol> 3. Three Dimensional Cultura in 2-bene Slides Camera per Immunoblotting <ol><li> Preraffreddare 2 ben vetrino camera su ghiaccio. Seguire i passaggi 2,1-2,4 scaling up i reagenti per la camera 2 e scivolo ad esempio il cappotto con 160 ml di Matrigel e aggiungere 1,6 ml di cellula / Matrigel mescolare in ciascun pozzetto della diapositiva 2-bene. </li><li> Raccogliere le strutture acinose dal Matrigel utilizzando il kit di raccolta di cellule coltura 3D disponibili in commercio seguendo le istruzioni del produttore. </li><li> Diluire il lavaggio delle cellule e di raccolta delle cellule buffer a 1x e preraffreddare a 4 ° C. Lavaggio e stacchi di Matrigel devono essere eseguite su ghiaccio. </li><li> Lavare le strutture acinose 3x utilizzando il tampone di lavaggio delle cellule. </li><li> Raccogliere la acini in 15 ml provette per centrifuga con bu raccolta di celluleffer seguita da 30 minuti di incubazione su un agitatore a 4 ° C. Mescolare bene la sospensione con una pipetta 1 ml. Questo romperà il Matrigel in piccoli pezzi e rilasciare la maggior parte delle cellule nel buffer raccolta di cellule. </li><li> Agglomerare le cellule a 200 xg in una centrifuga cultura. Rimuovere lo strato galleggiante idrogel dalle cellule pellet con una pipetta P200. </li><li> Risospendere lisati in tampone campione SDS forniti con il kit. Tampone di lisi RIPA può anche essere usato. </li></ol> 4. Quantificazione dei vs maligna benigna fenotipo seno <ol><li> Crescere cellule in triplicato per ogni set di trattamento seguendo i passaggi 2,1-2,4. </li><li> Prendere immagini a contrasto di fase di strutture acinose da ciascun pozzetto a 5X o 10X risoluzione usando un microscopio a contrasto di fase collegato a un cambio diapositiva e un computer. Prendete le immagini spostando il vetrino da un fotogramma all&#39;altro e quindi copre l&#39;intero bene della diapositiva incamerata. </li><li> Contare il numero totale di strutture acinose,il numero di acini singolo piatto rotondo e il numero di strutture multiacinar casaccio o crescenti strutture prive organizzazione e con processi di ramificazione provenienti da esso. </li><li> Calcolare la percentuale di strutture acinose piatto rotondo singoli vs strutture maligni e la trama come grafico a colonne. Calcolare la significatività con il test t di Student. </li></ol>

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Gli autori dichiarano interessi finanziari concorrenti.

Abbiamo descritto qui la coltura tridimensionale di cellule epiteliali mammarie su una membrana basale ricostituita e l&#39;utilità di questo sistema di distinguere tra vs maligno benigno fenotipo seno. Questo sistema può essere utilizzato anche per coltura diversi tipi di cellule di altri tessuti ghiandolari ed è stato riportato che sono stati utilizzati con successo per cultura prostata epiteliale, epiteliale bronchiale, e cellule epiteliali tiroidee, per citarne alcuni 12-14. L&#39;importanza della cult...

Tre coltura tridimensionale (3D) di cellule epiteliali mammarie sulla membrana basale ricostituita è un importante sistema modello per studiare il complesso fenotipo e segnalazione associato di seno normale e cancro al seno 2,3. L&#39;unità funzionale del seno è il terminale condotto unità lobulare (TDLU) che consiste in un piccolo dotto che si dirama in acini. Gli acini sono strutture altamente organizzate, composto da due strati di cellule, cellule epiteliali e mioepiteliali, che sono circondate da una membrana basale 5. Le caratteristiche più distintive di acini normali sono polarizzazione cellulare, attacco alla membrana basale sottostante e comunicazione varie cellule specializzate. Questa organizzazione intricato è interrotto nei carcinomi invasivi. Le culture 2D ampiamente utilizzato, dove le cellule sono coltivate in monostrato, non consentono la formazione di acini. Così, la coltura monostrato manca nel fornire un sistema per catturare l&#39;intricata relazione tra le cellule epitelialiin seno normale e la loro deregolamentazione nel cancro al seno. Culture epiteliali monotipica funzionali delle cellule mammarie sono state sviluppate in diversi laboratori 2,3. Cultura 3D coinvolge la crescita delle cellule del seno su Matrigel, che è un estratto solubilizzato derivato da cellule di topo sarcoma Engelbreth-Holm-Swarm ed è disponibile in commercio. Altri substrati 3D come il collagene che vengono utilizzati anche. Le cellule del seno possono essere coltivate in 3D da 3D saggio incorporato, in cui le cellule sono coltivate incorporato in Matrigel 2. In alternativa, le cellule possono essere coltivate da 3D sul dosaggio superiore, chiamato anche saggio overlay 3D, che è conveniente in quanto richiede minor volume di Matrigel, e facilita il controllo di lasso di tempo di formazione della colonia mediante imaging a contrasto di fase ed è ideale per l&#39;imaging in situ 2 -3. Il 3D sulla cima saggio è stato usato per definire la correlazione tra il fenotipo acinose e profilo di espressione genica 7. Cultura 3D può essere effectively utilizzato per distinguere le cellule normali e benigne da carcinoma invasivo. Cellule del seno normale e benigni formano la crescita arrestato strutture polarizzate con un lume ben definite nel centro di Matrigel mentre le cellule di carcinoma invasivo crescono prolifico e casaccio, senza compensazione del lume. E6/E7 immortalato cellule epiteliali mammarie umane e linea cellulare MCF10A, che è una linea cellulare spontaneamente immortalato isolato da un paziente di 36 anni con i cambiamenti fibrocistica, sono stati utilizzati con successo per riconquistare il fenotipo benigna del seno in 3D 3,8 e hanno servito come un sistema modello per studiare la funzione di soppressore tumorale oncogenica e di diverse molecole 8,9. Queste cellule benigne può essere progettato per manipolare gene (s) di interesse con l&#39;introduzione di lentivirus / retrovirus. Se il gene (s) di interesse è un soppressore del tumore, l&#39;abbattimento shRNA mediata porterà ad una trasformazione maligna mentre se il gene di interesse è una sovraespressione oncogene in cellule benignedovrebbe mostrare un fenotipo maligno. In entrambi i casi le strutture acinose formate in 3D possono catturare la trasformazione maligna. Benigni singole cellule placcato in 3D iniziano a proliferare e formare rotondo strutture sferiche. Di giorno 5-8 questo aggregato sferico delle cellule sarà differenziarsi in uno strato circostante polarizzata di celle che è in contatto con la Matrigel, e una massa interna di cellule meno polarizzate, che mancano contatto con la Matrigel. Nei prossimi 10-15 giorni le cellule interne inizieranno a morire per apoptosi formando un chiaro lume. Le cellule maligne cresceranno a casaccio perdere la loro polarità. Cellule altamente maligne di solito formano ramificazione delle strutture. Queste differenze nel fenotipo possono essere catturati da tempo di imaging a contrasto di fase, periodo e in situ immunoistochimica con marcatori molecolari rilevanti. I marcatori più comunemente utilizzati sono alfa 6-integrina, un marcatore polarità basale, in combinazione con il marcatore di proliferazione fosfo-istone 3 e il apoptotic marcatore spaccati caspasi-3. Quest&#39;ultimo è importante determinare se vi è formazione di lumen nel centro degli acini, una caratteristica delle cellule normali del seno e benigne. Altri marcatori di polarità e di contatto cellula-cellula comprendono GM130, laminina, ERM, E-caderina. Alternativamente le linee di cellule di cancro al seno può essere progettato per manipolare il gene di interesse. In questo caso reversione ad una maggiore crescita arrestata fenotipo benigno può essere utilizzato come lettura per distinguere dal fenotipo cancro. Cultura 3D può essere utilizzato anche con attenzione a delineare le vie di segnalazione coinvolte nella trasformazione maligna 10-11. Utilizzando il sopra citato le letture, inibitori farmacologici, anticorpi bloccanti o proteine ​​ricombinanti può essere utilizzato da individuare nella segnalazione molecolare che porta alla trasformazione maligna. Con un impegno costante da vari laboratori è possibile estrarre le cellule dal 3D per isolare l&#39;RNA e anche preparare lisati per immunoblotting e immunitànoprecipitation. Queste strategie sono descritte in un graduale e modo dettagliato nel protocollo.

<table border="0" cellpadding="0" cellspacing="0" width="585">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Growth Factor Reduced matrigel</td>
			<td style="width:83px;">BD biosciences</td>
			<td style="width:119px;">354230</td>
			<td style="width:167px;">Avoid repeat freeze thaw&#160;</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Lab-Tek glass hamber slides 8-well</td>
			<td style="width:83px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">177402</td>
			<td>Precool on ice&#160;</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Lab-Tek glass chamber slides 2- well&#160;</td>
			<td style="width:83px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">177380</td>
			<td>Precool on ice&#160;</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">goat anti mouse F(ab&#180;)2 fragment</td>
			<td style="width:83px;">Jackson Immunoresearch</td>
			<td style="width:119px;">115-006-006</td>
			<td></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Normal goat serum</td>
			<td style="width:83px;">Jackson Immunoresearch</td>
			<td style="width:119px;">005-000-121</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Fluorescent conjugated Alexa antibodies</td>
			<td style="width:83px;">Molecular probes</td>
			<td style="width:119px;"></td>
			<td>Please look at molecular probes website</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Prolong gold antifade reagent with DAPI</td>
			<td style="width:83px;">Molecular Probes</td>
			<td style="width:119px;">P36935</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">3D Culture Cell Harvesting kit</td>
			<td style="width:83px;">Trevigen</td>
			<td style="width:119px;">3448-020-k</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-Apha-6-integrin</td>
			<td style="width:83px;">Fisher Scientifics&#160;</td>
			<td style="width:119px;">MAB1378</td>
			<td>1:00 dilution for IF</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-GM130</td>
			<td style="width:83px;">BD Biosciences</td>
			<td style="width:119px;">610822</td>
			<td>1:00 dilution for IF</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-phospho-histone 3 (Ser10)FITC Conjugated</td>
			<td style="width:83px;">Fisher Scientifics&#160;</td>
			<td style="width:119px;">16-222</td>
			<td>1:100 dilution for IF</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-Cleaved caspase 3 (Asp 175)</td>
			<td style="width:83px;">Cell Signaling</td>
			<td style="width:119px;">9661s</td>
			<td>1:50 dilution for IF</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-E-cadherin</td>
			<td style="width:83px;">BD Biosciences</td>
			<td style="width:119px;">610181</td>
			<td>1:200 dilution for IF</td>
		</tr>
	</tbody>
</table>

three dimensional cultures: a tool to study normal acinar architecture vs. malignant transformation of breast cells

Carcinomi mammari invasivi sono un gruppo di tumori epiteliali maligni caratterizzato dall&#39;invasione dei tessuti adiacenti e la propensione a metastatizzare. L&#39;interazione dei segnali tra le cellule tumorali e il loro microambiente esercita una forte influenza sulla crescita del cancro al seno e comportamento biologico 1. Tuttavia, la maggior parte di questi segnali dalla matrice extracellulare sono persi o loro rilevanza è understudied quando le cellule vengono coltivate in coltura due dimensioni (2D) come un monostrato. Negli ultimi anni, tridimensionale coltura su una membrana basale ricostituita (3D) è emerso come un metodo di scelta per ricapitolare dell&#39;architettura tissutale di cellule mammarie benigne e maligne. Le cellule coltivate in 3D conservano gli spunti importanti della matrice extracellulare e forniscono un fisiologicamente rilevanti ex vivo di sistema 2,3. Di nota, vi è una crescente evidenza che suggerisce che le cellule si comportano diversamente quando coltivate in 3D rispetto al 2D 4. Cultura 3Dpuò essere usato efficacemente come mezzo per differenziare il fenotipo maligno dal fenotipo seno benigna e per consolidare la segnalazione cellulare e molecolare coinvolto 3. Una delle caratteristiche distintive di cellule epiteliali benigne è che essi sono polarizzati in modo che il citoplasma apicale è verso il lume e il citoplasma basale poggia sulla membrana basale. Questa polarità apico-basale si perde in carcinomi mammari invasivi, che sono caratterizzate da disorganizzazione e la formazione di anastomosi e la ramificazione tubuli che si infiltra a casaccio lo stroma circostante cellulare. Queste differenze istopatologici tra ghiandola benigna e il carcinoma invasivo possono essere riprodotti in 3D 6,7. Utilizzando le opportune read-out come il quantificazione di singole strutture acinose rotonde, o differenziale espressione di marcatori molecolari validati per la proliferazione cellulare, la polarità e l&#39;apoptosi in combinazione con altre tecniche di biologia molecolare e cellulare, cultur 3De in grado di fornire uno strumento importante per comprendere meglio i cambiamenti cellulari durante la trasformazione maligna e per delineare la segnalazione responsabile.

Cultura 3D può essere efficacemente utilizzata per differenziare fenotipo maligno delle cellule epiteliali mammarie umane dal seno benigno. Singole cellule benigne placcato in Matrigel crescere come strutture sferiche organizzate che possono essere facilmente imaged singola acini piatto rotondo in un piano utilizzando l&#39;imaging a contrasto di fase. Le singole cellule maligne piastrate su Matrigel crescono a casaccio, mostrano molto alto indice di rifrazione e sono difficili da immagine in un piano. Come mostrato in...

Watch this Scientific Journal Video about Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells at JoVE.com

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Tre coltura tridimensionale di cellule epiteliali mammarie su una membrana basale ricostituita è un metodo utile ricordare l&#39;architettura in vivo del seno benigno, e per differenziare il fenotipo maligno dal fenotipo seno benigno. È importante sottolineare che questo sistema può essere applicato a studiare carcinomi invasivi in ​​altri tessuti.

Tre Culture dimensionali: strumento per studiare normale acinose Architettura vs trasformazione maligna delle cellule mammarie

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

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JoVE Journal

Medicine

19.8K Views.  University of Michigan Comprehensive Cancer Center.  Tre coltura tridimensionale di cellule epiteliali mammarie su una membrana basale ricostituita è un metodo utile ricordare l'architettura in vivo del seno benigno, e per differenziare il fenotipo maligno dal fenotipo seno benigno. È importante sottolineare che questo sistema può essere applicato a studiare carcinomi invasivi in ​​altri tessuti. 

Video: Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Neuroendocrini umani cella linee tumorali come un modello tridimensionale per lo studio dei diritti dell&#39;uomo e terapia dei tumori neuroendocrini

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

Ricerca

Medicina

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

A Three-dimensional Tissue Culture modello per studiare Primario Osso Osseo e le sue Malignità

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

Quantificazione del cancro al seno Cellula Invasività Utilizzando una tridimensionale (3D) Modello

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

Iniezione ortotopico di cellule di carcinoma mammario nel mammaria Fat Pad di Mice per studiare la crescita del tumore.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

Utilizzando virus adeno-associati come strumento per studiare le barriere della retina nella malattia

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Tridimensionale (3D) Tumore Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

Utilizzo del saggio soft Agar Colony Formazione per identificare inibitori di tumorigenicità in cellule di carcinoma mammario

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

Tridimensionale alginato-tallone coltura di cellule umane adenoma ipofisario

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

Utilizzando Saccadometry con stimolazione cerebrale profonda per studiare normali e patologiche funzione cerebrale

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...

A Clinical Trial Assessing the Safety, Efficacy, and Delivery of Olive-Oil-Based Three-Chamber Bags for Parenteral Nutrition

Limited evidence exists to precisely estimate efficacy and safety differences between parenteral nutrition (PN) prepared using olive-oil-based three-chamber bags (3CBs) and soybean-oil-based compounded bags (CoBs) in hospitalized adult patients. We designed a multicenter, randomized, prospective, open-label, noninferiority protocol to compare the efficacy, safety, and distribution of olive-oil-based 3CBs and soybean-oil-based CoB formulations in adult Chinese patients requiring PN during surgical intervention. Subjects were randomized to receive either one of the study treatments using an interactive voice or web-based recognition system in accordance with the randomization code. Randomization was further stratified based on the study site and surgical category. Both treatment groups received similar amounts of calories and protein. In addition, the two study treatments contained a similar composition of the amino-acid component. The only difference between the two PN formulations was the lipid constitution. The duration of administration of study treatments was a minimum of 5 days up to a maximum of 14 days after the surgical procedure. The primary efficacy endpoint was serum prealbumin levels on day 5 of the study. Noninferiority was proved if the anti-log of the lower bound of the 95% confidence interval (CI) of the treatment difference was at least 0.80. Other efficacy measures included treatment preparation time; duration to achieve tolerability of oral nutrition; associated infectious complications; length of hospitalization; and laboratory assessment of markers of nutrition, inflammation, metabolism, and oxidative stress. A total of 458 patients were enrolled in the study. The results showed that olive-oil-based 3CBs were non-inferior to soybean-based CoBs, besides being well tolerated. The infection rate was found to be significantly lower in the olive-oil-based 3CB group. Thus, this study may be used as a reference for future research on lipid emulsion and 3CBs.

Here, we present a protocol for a study comparing the efficacy, safety, and delivery of olive-oil-based 3CB and soybean-oil-based CoB formulations in adults requiring parenteral nutrition. The results revealed that olive-oil-based 3CBs is non-inferior and well tolerated compared to soybean formulations.