Note tecniche: <ul><li> Gelificazione è iniziata con l&#39;aggiunta di sali. Pertanto, è fondamentale che i sali non vengono a contatto con PuraMatrix fino a gelificazione è desiderato, l&#39;esposizione a sali di molto bassa forza ionica (ad esempio, &lt;0,05 M) causerà gelificazione irreversibile. </li><li> Soluzioni rimanenze che vengono preparati possono essere usati in un secondo momento a condizione che non sono venuto in contatto con i sali. Non è necessario preoccuparsi di sonicazione ripetere. </li><li> Le bolle d&#39;aria può essere rimosso aliquoting PuraMatrix in provette sterili seguita da centrifugazione. </li><li> Prestate molta attenzione durante i cambi di mezzi di comunicazione come l&#39;aspirazione può facilmente interrompere il letto PuraMatrix. </li></ul> Materiali: BD PuraMatrix Peptide RAD16 Hydrogel-1% (Cat # 354250): la concentrazione del peptide totale della miscela non diluito è di 10 mg / ml. <ul><li> Acqua Sonicator Vasca o Vortex </li><li> 96-bene delle cellule piatto cultura </li><li> Colture Cellulari media </li><li> Sterile filtrata H 2 O </li><li> Saccarosio sterile filtrata 20% </li><li> Cellule (il tutto a 5.000 cellule / pozzetto): OVCAR-5 </li></ul> Tutte le fasi devono essere eseguite in condizioni asettiche. Procedimento: <ol><li> Sonicare PuraMatrix l&#39;1% (RAD16) in un sonicatore acqua del bagno per 30 minuti per ridurre la viscosità prima dell&#39;uso. </li><li> La soluzione PuraMatrix 1% può essere aliquotati in provette sterili per semplificare futuri esperimenti. <ol class="lalpha"><li> Evitare la formazione di bolle d&#39;aria, se si tratta di semplicemente rallenta il provette sterili contenenti PuraMatrix a 1000 rpm (300 xg) per 5 minuti. </li><li> La concentrazione del peptide totale della miscela non diluito è di 10 mg / ml. </li></ol></li><li> Due set di 4 pozzi saranno placcato a 2,5 mg / ml e 1,25 mg / ml di concentrazione del peptide totale, rispettivamente. </li><li> Diluire il PuraMatrix in ciascun pozzetto insieme con 0,2 micron sterile filtrata saccarosio 20% e il 13,3% di saccarosio al totale delle concentrazioni del peptide di 5 mg / ml e 2,5 mg / ml, rispettivamente, per ottenere una concentrazione di 2x BD PuraMatrix nel 10% di saccarosio. <ol class="lalpha"><li> Fare il 20% di saccarosio, diluire 10 g di saccarosio, poi volume di 50 ml di acqua. </li><li> 13% e il 10% di saccarosio può essere preparato diluendo la soluzione madre al 20%. <ol class="lroman"><li> 13% di saccarosio (per ogni ml 10): 6,5 ml di saccarosio al 20% + 3,5 ml sterili H 2 O. </li><li> 10% di saccarosio (per ogni ml 10): 5,0 ml di saccarosio al 20% + 5.0 ml sterile H 2 O. </li></ol></li></ol></li><li> Preparare una master mix di peptidi PuraMatrix in base al numero di pozzi che si intende piatto. (Vedi esempio sotto). Poi aliquota in singoli tubi contenenti 25 ml della master mix. <ol class="lalpha"><li> Il master mix dovrebbe essere preparato a due volte la concentrazione finale desiderata di peptide totale in quanto sarà diluito con un uguale volume di cellule durante la placcatura. <img src="/files/ftp_upload/1692/1692_fig11.jpg" alt="figura 1" /></li></ol></li><li> Preparare sospensioni cellulari da trypsinizing cellula colture cellulari monostrato. <ol class="lalpha"><li> 2 ml di 0,05% tripsina EDTA può essere utilizzato per ogni T-75 Flask. </li></ol></li><li> Neutralizzare la tripsina con l&#39;aggiunta dei terreni di coltura cellulare contenente il 10% inattivato con il calore di siero fetale bovino (uso almeno 2 ml di mezzi per ml di tripsina utilizzato nel passaggio precedente). </li><li> Centrifugare le cellule a 1000 giri (~ 300 xg) per 5 minuti per creare un pellet di cellule. <ol><li> Risospendere le cellule nel 10% di saccarosio sterile filtrata. </li><li> Contare le celle e girare di nuovo a 1000 giri (~ 300 xg) per 5 minuti per rimuovere tracce di sale trovato nei media. </li></ol></li><li> Risospendere il OVCAR-5 cellule in 10% di saccarosio a 2,0 x 10 5 cellule / ml, in modo che il conteggio finale delle cellule per pozzetto saranno 5.000 cellule in ciascun pozzetto. Le seguenti operazioni devono essere eseguite rapidamente. </li><li> Aggiungere 25 microlitri di 2.0 x 10 5 cellule / ml di sospensione per il primo tubo singolo contenente 25 microlitri della master mix di PuraMatrix come indicato nello schema di cui sopra. <ol class="lalpha"><li> Mescolare rapidamente pipettando (~ 5 volte su e giù) nel tubo senza introdurre bolle d&#39;aria. </li><li> Pipettare quindi i 50 microlitri di cellule sospensione / PuraMatrix miscela nel pozzo appropriato di un piatto ben 96. </li><li> Avviare gelificazione del PuraMatrix BD pipettando gentilmente 150 microlitri terreni di coltura cellulare (RPMI + 10% FBS) al lato del bene <img src="/files/ftp_upload/1692/1692_fig12.jpg" alt="figura 2" /></li></ol></li><li> Ripetere la procedura descritta in 8 fino a quando tutti i pozzetti sono stati placcati. </li><li> Dopo 30 minuti molto delicatamente rimuovere ~ 2 / 3 della media con una pipetta 200 microlitri per equilibrare il pH del idrogel. <ol class="lalpha"><li> NON UTILIZZARE UN VUOTO IN QUESTO ASPIRATORE ostacolerà la MATRIX </li><li> Posizionare la punta della pipetta nel pozzetto in modo chenon toccare il letto PuraMatrix e delicatamente rimuovere il supporto dal pozzo. </li><li> Si deve prestare attenzione a non disturbare la matrice durante la coltura di cellule in PuraMatrix. </li></ol></li><li> Molto delicatamente aggiungere 150 ml di mezzi freschi colture cellulari al lato del pozzetto contenente cellule incapsulate in PuraMatrix. </li><li> Cambiare il terreni di coltura di cellule in ciascun pozzetto ogni 48 ore con terreni di coltura fresco con attenzione ripetendo i punti 10 e 11. </li></ol> Rappresentante dei risultati: <img src="/files/ftp_upload/1692/1692_fig1tmb.jpg" alt="figura 1" border="0" /> Figura 1. OVCAR-5 cellule sono state incapsulate, come descritto nel protocollo di cui sopra. Immagini sono state acquisite utilizzando un microscopio rovesciato Olympus FV1000 dotato di Spectra-Physics DeepSee Ti: zaffiro laser sintonizzati a 750 nm. Sessioni di imaging sono state condotte nei giorni 1, 3, 5 e 7 post-placcatura. A distanza di lavoro, l&#39;acqua immergendo obiettivo 40x (Olympus LUMPLFLN 0,8 NA) è stato utilizzato per l&#39;immagine attraverso la PuraMatrix. Volumi tridimensionali sono stati raccolti attraverso l&#39;acquisizione di immagini in stack 1,47 micron passi assiale. Due non-descanned rivelatori di raccolti tramite l&#39;emissione di autofluorescenza violetto (440-490 nm) e verde (510-550 nm) Filtri a banda passante. Le immagini finali e filmati stack di profondità sono stati elaborati utilizzando ImageJ unendo entrambi i canali di rilevazione. Si prega di <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/1692/1692_fig1.jpg">cliccare qui</a> per vedere una versione più grande della figura 1.

<ol>
	<li>Gelain, F., Bottai, D., Vescovi, A., Zhang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designer+self-assembling+Peptide+nanofiber+scaffolds+for+adult+mouse+neural+stem+cell+3-dimensional+cultures.">Designer self-assembling Peptide nanofiber scaffolds for adult mouse neural stem cell 3-dimensional cultures.</a> PLoS One. 1, e119-e119 (2006).</li></ol>

puramatrix encapsulation of cancer cells

Crescente evidenza suggerisce che le cellule tumorali coltura in tre dimensioni con maggiore precisione ricapitola la complessità della biologia del tumore. Molti di questi modelli utilizzano membrane ricostituito scantinato derivati ​​da animali che contengono una quantità variabile di fattori di crescita e di citochine che possono influenzare la crescita dei modelli di cultura queste cellule. Qui, descriviamo in dettaglio la preparazione e l&#39;uso di PuraMatrix, disponibile in commercio gel peptide auto assemblaggio che è privo di materiale di origine animale e agenti patogeni a integrare e propagare il cancro ovarico linea cellulare, OVCAR-5. Iniziamo descrivendo come preparare il PuraMatrix prima dell&#39;uso. Successivamente, dimostriamo come mixare correttamente il PuraMatrix e sospensione cellulare per incapsulare le cellule in idrogel. Al momento l&#39;aggiunta di terreni di coltura cellulare o iniezione in un ambiente fisiologico, il componente del peptide PuraMatrix rapidamente auto assembla in un idrogel 3D che mostra una scala nanometrica struttura fibrosa con una dimensione dei pori media di 500-200 nm<sup&gt; 1</sup&gt;. Inoltre, ci mostrano come si propagano le culture cresciute in PuraMatrix incapsulato. Quando incapsulato in PuraMatrix, OVCAR-5 cellule assemblano in tre strutture acinose dimensionale che si avvicinano maggiormente la morfologia dei noduli micrometastatica osservati nella clinica di monostrato in modelli in vitro. Usando la microscopia confocale ci illustrano l&#39;aspetto di rappresentante OVCAR-5 cellule incapsulate in PuraMatrix il giorno 1, 3, 5, e 7 placcatura post. L&#39;uso di PuraMatrix alle cellule tumorali cultura dovrebbe migliorare la nostra comprensione della malattia e ci permettono di valutare la risposta al trattamento in sistemi modello più clinicamente predittiva.

Watch this Scientific Journal Video about PuraMatrix Encapsulation of Cancer Cells at JoVE.com

PuraMatrix Encapsulation of Cancer Cells

Questo video dimostra come incapsulare le cellule tumorali e cultura in PuraMatrix, un assemblaggio di auto in commercio gel peptide.

Incapsulamento PuraMatrix delle cellule tumorali

Increasing evidence suggests that culturing cancer cells in three dimensions more accurately recapitulates the complexity of tumor biology. Many of these ...

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

16.6K Views.  Harvard Medical School. Questo video dimostra come incapsulare le cellule tumorali e cultura in PuraMatrix, un assemblaggio di auto in commercio gel peptide.

Video: PuraMatrix Encapsulation of Cancer Cells

A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events leading to stromal activation and establishment of the tumor microenvironment (TME). Several in vitro and in vivo models of the TME have been developed; however, in general these models do not readily permit isolation of individual cell populations, under non-perturbing conditions, for further study. To circumvent this difficulty, we have employed an in vitro TME model using a cell growth substrate consisting of a permeable microporous membrane insert that permits simple generation of highly enriched cell populations grown intimately, yet separately, on either side of the insert&#39;s membrane for extended co-culture times. Through use of this model, we are capable of generating greatly enriched cancer-associated fibroblast (CAF) populations from normal diploid human fibroblasts following co-culture (120 hr) with highly metastatic human breast carcinoma cells, without the use of fluorescent tagging and/or cell sorting. Additionally, by modulating the pore-size of the insert, we can control for the mode of intercellular communication (e.g., gap-junction communication, secreted factors) between the two heterotypic cell populations, which permits investigation of the mechanisms underlying the development of the TME, including the role of gap-junction permeability. This model serves as a valuable tool in enhancing our understanding of the initial events leading to cancer-stroma initiation, the early evolution of the TME, and the modulating effect of the stroma on the responses of cancer cells to therapeutic agents.

We adapted a permeable microporous membrane insert to mimic the tumor microenvironment (TME). The model consists of a mixed cell culture, allows simplified generation of highly enriched individual cell populations without using fluorescent tagging or cell sorting, and permits studying intercellular communication within the TME under normal or stress conditions.

Un Mimic del microambiente tumorale: Un metodo semplice per la generazione di popolazioni di cellule arricchito e Investigating intercellulare Comunicazione

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events ...

Ricerca

Ricerca sul cancro

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, ...

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

In vitro test Clearance mesoteliali che modella i primi passi di metastasi del cancro ovarico

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

Medicina

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

Ingegneria dei tessuti del tumore stromale Microenvironment con l&#39;applicazione di Invasion Cancer Cell

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

Bioingegneria

Isolation of Proximal Fluids to Investigate the Tumor Microenvironment of Pancreatic Adenocarcinoma

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables associated to specific pancreatic pathologies to help preoperative differential diagnosis and patient profiling. Pancreatic juice is a relatively unexplored body fluid, which, due to its close proximity to the tumor site, reflects changes in the surrounding tissue. Here we describe in detail the intraoperative collection procedure. Unfortunately, translating pancreatic juice collection to murine models of PDAC, to perform mechanistic studies, is technically very challenging. Tumor interstitial fluid (TIF) is the extracellular fluid, outside blood and plasma, which bathes tumor and stromal cells. Similarly to pancreatic juice, for its property to collect and concentrate molecules that are found diluted in plasma, TIF can be exploited as an indicator of microenvironmental alterations and as a valuable source of disease-associated biomarkers. Since TIF is not readily accessible, various techniques have been proposed for its isolation. We describe here two simple and technically undemanding methods for its isolation: tissue centrifugation and tissue elution.

Pancreatic juice is a precious source of biomarkers for human pancreatic cancer. We describe here a method for intraoperative collection procedure. To overcome the challenge of adopting this procedure in murine models, we suggest an alternative sample, tumor interstitial fluid, and describe here two protocols for its isolation.

Isolamento di fluidi prossimali per studiare il microambiente tumorale dell'adenocarcinoma pancreatico

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables ...

Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human cancer cells in mitosis. The phenanthridine PJ-34 was the most potent molecule. Declustering of extra-centrosomes causes mitotic failure and cell death in multi-centrosomal cells. Most solid human cancers have high occurrence of extra-centrosomes. The activity of PJ-34 was documented in real-time by confocal imaging of live human breast cancer MDA-MB-231 cells transfected with vectors encoding for fluorescent &gamma;-tubulin, which is highly abundant in the centrosomes and for fluorescent histone H2b present in the chromosomes. Aberrant chromosomes arrangements and de-clustered &gamma;-tubulin foci representing declustered centrosomes were detected in the transfected MDA-MB-231 cells after treatment with PJ-34. Un-clustered extra-centrosomes in the two spindle poles preceded their cell death. These results linked for the first time the recently detected exclusive cytotoxic activity of PJ-34 in human cancer cells with extra-centrosomes de-clustering in mitosis, and mitotic failure leading to cell death. According to previous findings observed by confocal imaging of fixed cells, PJ-34 exclusively eradicated cancer cells with multi-centrosomes without impairing normal cells undergoing mitosis with two centrosomes and bi-focal spindles. This cytotoxic activity of PJ-34 was not shared by other potent PARP1 inhibitors, and was observed in PARP1 deficient MEF harboring extracentrosomes, suggesting its independency of PARP1 inhibition. Live confocal imaging offered a useful tool for identifying new molecules eradicating cells during mitosis.

The cytotoxic activity of the phenanthridine PJ-34 in cancer cells undergoing mitosis was documented in real time by live confocal imaging. PJ-34 eradicated human breast cancer MDA-MB-231 cells harboring extra-centrosomes in mitosis. Unlike normal bi-focal mitosis, the extra-centrosomes were not clustered in the two spindle poles in the presence of PJ-34.

Cella della morte associata a mitosi anomalo osservato da Imaging confocale in cellule vive di cancro

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human ...

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 ...

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular weight metabolites in tissues of the treated host to achieve long-term survival. The semipermeable membrane allows engrafted encapsulated cells to avoid rejection by the immune system. The encapsulation procedure was designed to enable a controlled release of bioactive compounds, such as insulin, other hormones, and cytokines. Here we describe a method for encapsulation of catabolic cells, which consume lipids for heat production and energy dissipation (thermogenesis) in the intra-abdominal adipose tissue of obese mice. Encapsulation of thermogenic catabolic cells may be potentially applicable to the prevention and treatment of obesity and type 2 diabetes. Another potential application of catabolic cells may include detoxification from alcohols or other toxic metabolites and environmental pollutants.

Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

Incapsulamento termogenico preadipocytes al trapianto nel tessuto adiposo Depositi tessuti

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

Un Cancer Cell Saggio sferoide valutare Invasion in un Ambiente 3D

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential.  Most traditional assays, however, do not examine these ...

Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing factor in the overall outcome. Immunoblotting, flow cytometry and fixed cell experiments are often used to study how cells respond to anti-cancer drugs. These methods are important, but they have several shortcomings. Variability in drug responses between cancer and normal cells, and between cells of different cancer origin, and transient and rare responses are difficult to understand using population averaging assays and without being able to directly track and analyze them longitudinally. The microscope is particularly well suited to image live cells. Advancements in technology enable us to routinely image cells at a resolution that enables not only cell tracking, but also the observation of a variety of cellular responses. We describe an approach in detail that allows for the continuous time-lapse imaging of cells during the drug response for essentially as long as desired, typically up to 96 hr. Using variations of the approach, cells can be monitored for weeks. With the employment of genetically encoded fluorescent biosensors numerous processes, pathways and responses can be followed. We show examples that include tracking and quantification of cell growth and cell cycle progression, chromosome dynamics, DNA damage, and cell death. We also discuss variations of the technique and its flexibility, and highlight some common pitfalls.

Here, we describe a method of long-term time-lapse microscopy to longitudinally track single cells in response to anti-cancer therapeutics.

Attraverso lo specchio: Time-lapse Microscopia e longitudinale monitoraggio di singole cellule allo Studio Therapeutics anti-cancro

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing ...