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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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><tbody><tr><td>Growth Factor Reduced Matrigel</td><td>BD Biosciences</td><td>354230</td><td>Avoid repeat freeze thaw&amp;nbsp;</td></tr><tr><td>Lab-Tek glass hamber slides 8-well</td><td>Thermo Fisher Scientific</td><td>177402</td><td>Precool on ice&amp;nbsp;</td></tr><tr><td>Lab-Tek glass chamber slides 2-well&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>177380</td><td>Precool on ice&amp;nbsp;</td></tr><tr><td>Goat anti mouse F(ab&amp;acute;)&lt;sub&gt;2 &lt;/sub&gt;fragment</td><td>Jackson Immunoresearch</td><td>115-006-006</td><td></td></tr><tr><td>Normal goat serum</td><td>Jackson Immunoresearch</td><td>005-000-121</td><td></td></tr><tr><td>Fluorescent conjugated Alexa antibodies</td><td>Molecular Probes</td><td></td><td>Please look at molecular probes website</td></tr><tr><td>Prolong gold antifade reagent with DAPI</td><td>Molecular Probes</td><td>P36935</td><td></td></tr><tr><td>3D Culture Cell Harvesting kit</td><td>Trevigen</td><td>3448-020-k</td><td></td></tr><tr><td>Anti-Apha-6-integrin</td><td>Fisher Scientific</td><td>MAB1378</td><td>1:100 dilution for IF</td></tr><tr><td>Anti-GM130</td><td>BD Biosciences</td><td>610822</td><td>1:100 dilution for IF</td></tr><tr><td>Anti-phospho-histone 3 (Ser10)FITC Conjugated</td><td>Fisher Scientific</td><td>16-222</td><td>1:100 dilution for IF</td></tr><tr><td>Anti-Cleaved caspase 3 (Asp 175)</td><td>Cell Signaling</td><td>9661s</td><td>1:50 dilution for IF</td></tr><tr><td>Anti-E-cadherin</td><td>BD Biosciences</td><td>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

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract (ECME)-based Three-dimensional System

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, thus greatly influencing normal tissue homeostasis and disease outcome. In breast cancer, tumor-associated macrophages (TAMs) play a critical role in disease progression, metastasis, and recurrence; therefore, understanding the mechanisms of monocyte chemoattraction to the tumor microenvironment and their interactions with tumor cells is important to control the disease. Here, we provide a detailed description of a three-dimensional (3D) co-culture system of human breast cancer (BrC) cells and human monocytes. BrC cells produced high basal levels of regulated on-activation, normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), and granulocyte-macrophage colony-stimulating factor (G-CSF), while in co-culture with monocytes, pro-inflammatory cytokines Interleukin (IL)-1 beta (IL-1&#946;) and IL-8 were enriched together with matrix metalloproteinases (MMP)-1, MMP-2, and MMP-10. This tumor stroma microenvironment promoted resistance to anoikis in MCF-10A 3D acini-like structures, chemoattraction of monocytes, and invasion of aggressive BrC cells. The protocols presented here provide an affordable alternative to study intra-tumor communication and are an example of the great potential that in vitro 3D cell systems provide to interrogate specific features of tumor biology related to tumor aggression.

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System

Here, we describe a three-dimensional culture method to analyze the morphology of primary breast cancer cells, as well as to study their direct/indirect interactions with monocytes and the outcomes such as collagen degradation, immune cell recruitment, cell invasion, and promotion of cancer-related inflammation.

Analizzando la comunicazione tra i monociti e le cellule di cancro al seno primario in una tabella extracellulare estrarre (ECME)-sistema tridimensionale basato su

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, ...

Ricerca

Ricerca sul cancro

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

Saggio sferoide Misura TGF-β-indotta Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

Medicina

Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture

The differentiation of acinar cells to ductal cells during pancreatitis and in the early development of pancreatic cancer is a key process that requires further study. To understand the mechanisms regulating acinar-to-ductal metaplasia (ADM), ex vivo 3D culture and differentiation of primary acinar cells to ductal cells offers many advantages over other systems. With the technique herein, modulation of protein expression is simple and quick, requiring only one day to isolate, stimulate or virally infect, and begin culturing primary acinar cells to investigate the ADM process. In contrast to using basement membrane matrix, the seeding of acinar cell clusters in collagen I extracellular matrix, allows acinar cells to retain their acinar identity before manipulation. This is vital when testing the contribution of various components to the induction of ADM. Not only are the effects of cytokines or other ectopically administered factors testable through this technique, but the contribution of common mutations, increased protein expression, or knockdown of protein expression is testable via viral infection of primary acinar cells, using adenoviral or lentiviral vectors. Moreover, cells can be re-isolated from collagen or basement membrane matrix at the endpoint and analyzed for protein expression.

Primary acinar cell isolation, protein expression or activity modulation, culture, and down-stream applications are abundantly useful in the ex vivo study of acinar-to-ductal metaplasia (ADM), an early event in the development of pancreatic cancer.

Che imita e manipolare Metaplasia acinoso--Ductal pancreatico nella coltura cellulare 3-dimensionale

The differentiation of acinar cells to ductal cells during pancreatitis and in the early development of pancreatic cancer is a key process that requires ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

Valutazione in vitro della trasformazione oncogenica nelle cellule epiteliali mammarie umane

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

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 ...

Phospholipid Mediator Induced Transformation in Three-Dimensional Cultures

Several models have been developed to study cancer, such as rodent models and established cell lines. Valuable insights into carcinogenesis have been provided by studies using these models. Cell lines have provided an understanding of the deregulation of molecular signaling associated with breast tumorigenesis, while rodent models are widely used to study cellular and molecular characteristics of breast cancer in vivo. The establishment of 3D cultures of breast epithelial and cancerous cells aids in bridging the gap between in vivo and in vitro models by mimicking the in vivo conditions in vitro. This model can be used to understand the deregulation of complex molecular signaling events and the cellular characteristics during breast carcinogenesis. Here, a 3D culture system is modified to study a phospholipid mediator-induced (Platelet Activating Factor, PAF) transformation. Immunomodulators and other secreted molecules play a major role in tumor initiation and progression in the breast. In the present study, 3D acinar cultures of breast epithelial cells are exposed to PAF exhibited transformation characteristics such as loss of polarity and altered cellular characteristics. This 3D culture system will assist in shedding light on genetic and/or epigenetic perturbations induced by various small molecule entities in the tumor microenvironment. Additionally, this system will also provide a platform for the identification of novel as well as known genes that may be involved in the process of transformation.

The present protocol describes the setting up of 3D 'on top' cultures of a non-transformed breast epithelial cell line, MCF10A, that has been modified to study Platelet Activating Factor (PAF) induced transformation. Immuno-fluorescence has been used to assess the transformation and is discussed in detail.

Trasformazione indotta dal mediatore dei fosfolipidi in colture tridimensionali

Several models have been developed to study cancer, such as rodent models and established cell lines. Valuable insights into carcinogenesis have been ...

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 ...

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, including breast carcinomas, are complex 3D tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix (ECM). Yet most in vitro models of breast carcinoma consist only of cancer epithelial cells, omitting the stroma and, therefore, the 3D architecture of a tumor in vivo. Appropriate 3D modeling of carcinoma is important for accurate understanding of tumor biology, behavior, and response to therapy. However, the duration of culture and volume of 3D models is limited by the availability of oxygen and nutrients within the culture. Herein, we demonstrate a method in which breast carcinoma epithelial cells and stromal fibroblasts are incorporated into ECM to generate a 3D breast cancer surrogate that includes stroma and can be cultured as a solid 3D structure or by using a perfusion bioreactor system to deliver oxygen and nutrients. Following setup and an initial growth period, surrogates can be used for preclinical drug testing. Alternatively, the cellular and matrix components of the surrogate can be modified to address a variety of biological questions. After culture, surrogates are fixed and processed to paraffin, in a manner similar to the handling of clinical breast carcinoma specimens, for evaluation of parameters of interest. The evaluation of one such parameter, the density of cells present, is explained, where ImageJ and CellProfiler image analysis software systems are applied to photomicrographs of histologic sections of surrogates to quantify the number of nucleated cells per area. This can be used as an indicator of the change in cell number over time or the change in cell number resulting from varying growth conditions and treatments.

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.

Preparazione e analisi di<em&gt; In Vitro</em&gt; Tridimensionale carcinoma mammario mondo dei replicanti

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, ...

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

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

Isolamento di cellule epiteliali mammarie da tridimensionale Mixed-cell Spheroid Co-cultura

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...