Questo lavoro è stato sostenuto dal Centro per la Ricerca Florida Tumore al cervello, il Preston A. Wells Jr. Centro per la Terapia Tumore al cervello e il National Institutes of Health (1R21CA141020-01).

1. Preparazione sospensione singola cella Glioblastoma <ol><li> Cultura Gliomasphere è stabilito e mantenuto con il dosaggio neurosfere (NSA), come descritto in precedenza 2,11,12. </li><li> Al momento opportuno per passaging le gliomaspheres, il terreno contenente le sfere viene rimosso, collocato in un apposito tubo di tessuto sterile cultura dimensioni, e centrifugati a 800 rpm (110 g) per 5 min a temperatura ambiente. </li><li> Il supernatante viene scartato e il pellet di sfere viene risospeso in 1 ml di 0,05% tripsina-EDTA e incubate a 37 ° C in un bagno di acqua per 3-5 min. </li><li> Un volume uguale di inibitore della tripsina di soia viene poi utilizzato per interrompere l&#39;attività tripsina. </li><li> La sospensione cellulare viene delicatamente pipettato su e giù per assicurare omogeneità e neutralizzazione completa dell&#39;attività tripsina. </li><li> La sospensione cellulare viene nuovamente centrifugata a 800 rpm per 5 min. Il supernatante viene rimosso e le cellule sono risospese in 1 ml di NeuroCultNSC medio basale. </li><li> 10μl della cella singola sospensione viene miscelata con 90μl di trypan blu per eseguire una conta cellulare. </li></ol> 2. La colorazione con cellulare Trace Carboxyfluorescein succinimidile Ester (CFSE) Dye Green Fluorescent <ol><li> Per ogni 1 milione di cellule da colorare, 1 ml di reagente di colorazione viene preparata aggiungendo 1 ml di colorante 5 Cella CFSE mM Trace (Molecular Probes, Invitrogen) a 1 ml di terreno NeuroCult NSC Basal per dare una concentrazione finale di 5 mM colorazione . </li><li> Il reagente di colorazione contenente le cellule è mescolata bene fino omogeneità e incubate a temperatura ambiente per 10 min. </li><li> Durante l&#39;incubazione, gelida NeuroCult Medio NSC Basal viene preparata come la soluzione quenching. </li><li> Per interrompere il processo di colorazione, circa 5-10 volumi di soluzione ghiacciata quenching viene aggiunto ai CFSE-caricati cellule. </li><li> La soluzione viene quindi centrifugata a 800 rpm per 5 minuti e il supernatante viene scartato. </li><li> A °è punto, dovrebbe apparire un pellet di cellule sul fondo del tubo con un verde brillante tinta del colore, che indica la colorazione era successo. </li><li> Le cellule vengono lavate risospendendo il pellet in 1-2 ml di fresca Medio NeuroCult NSC Basal, centrifugazione a 800 rpm per 5 min e scartando il supernatante. Questa procedura viene ripetuta per un totale di tre lavaggi. </li><li> Dopo il terzo lavaggio, le cellule sono risospese in 1 ml di terreno NeuroCult NSC Basal e una conta cellulare viene eseguita. </li><li> Le cellule vengono quindi colorate CFSE piastrate in palloni di coltura tissutale sterili alla densità adeguata [50.000 cellule / ml] e posto a 37 ° C / 5% CO2 incubatore umidificato. </li></ol> 3. CFSE Caricamento di verifica <ol><li> Le cellule in coltura placcati NSA può essere monitorato con un microscopio a fluorescenza per verificare la colorazione CFSE. In questa fase, tutte le cellule mostrano colore verde brillante (Figura 1). In alternativa, una goccia di sospensione cellulare daCellule colorate CFSE potrebbe essere posto su un vetrino e coperto con vetrino di visualizzare le cellule al microscopio a fluorescenza. </li><li> Etichettatura CFSE successo è ulteriormente confermato tramite citometria a flusso. Fino a 1-5 x 10 5 cellule da ciascun gruppo è sufficiente per la verifica CFSE colorazione. Il CFSE cellule caricate e scaricate cellule di controllo negativo sono rispettivamente risospese in 1 x PBS-o NeuroCult Medio NSC Basal per un volume totale di 500 microlitri e posta in tubi separati FACS. </li><li> La citometria a flusso è regolato in base alle istruzioni per l&#39;uso i manuali per i relativi parametri. CFSE ha un massimo di eccitazione lunghezza d&#39;onda di 492 nm e un massimo di emissione di 517 nm. </li><li> Primo, le cellule di controllo scarichi vengono eseguiti e gli eventi acquisiti vengono registrati in scatter in avanti e laterale (FSC e SSC) e trame istogramma, e la popolazione cellulare principale è gated (Figura 2A). </li><li> Analogamente, le cellule CFSE caricati vengono analizzati utilizzando lo stesso parametros usato per cellule di controllo (Figura 2B). </li></ol> 4. Isolamento del Slow-dividendo [CFSE cioè di fissaggio] popolazione di cellule Uso fluorescente Ordinamento attivato cellulare (FACS) <ol><li> Sulla divisione, CFSE intensità diminuisce e cellule hGBM formare gliomaspheres che sono composti da cellule che presentano diversi livelli di intensità di fluorescenza verde (Figura 3). Quando i gliomaspheres hanno raggiunto una dimensione media di circa 150-200 micron di diametro (circa 5 a 10 giorni dopo carico CFSE seconda del tasso di crescita della linea cellulare), il terreno contenente le sfere viene rimosso, collocato in una dimensione appropriata coltura tissutale sterili tubo, e centrifugati a 800 rpm per 5 min a temperatura ambiente. </li><li> Una cella singola sospensione è preparata come descritto nei passaggi precedenti e le cellule sono risospese in PBS per una conta cellulare al fine di preparare una sospensione cellulare con una densità fino a 10 7 cellule per millilitro. </li><li> A poppaer risospendere le cellule in un volume appropriato di PBS, ioduro di propidio (PI, Sigma, 500 mg / ml) viene aggiunto ad una concentrazione di 1 pl / ml di sospensione cellulare per escludere cellule morte quando ordinati / analizzate al citofluorimetro. </li><li> Due 15 ml provette sterili di coltura di tessuti contenenti ciascuno 1 ml di completo Medio NeuroCult NSC viene utilizzato per raccogliere le cellule selezionate lento divisione (cellule CFSE di sostegno) e le cellule veloci divisione (cellule CFSE diluizione). </li><li> In primo luogo, la sospensione di cellule dal gruppo di cellule scarico viene fatto passare attraverso il citofluorimetro. </li><li> Le cellule (eventi) vengono riportati sulla base forward scatter (FSC) rispetto side scatter (SSC) e parametri di tensione per entrambi i parametri vengono regolati in modo che la maggior parte delle cellule sono incluse nel diagramma di flusso. </li><li> Per escludere le cellule morte o danneggiate, le cellule vengono tracciati sulla base di reattività SSC rispetto PI e la singola popolazione cellulare in tempo reale (PI negativo) è sorvegliato. </li><li> Poi, una popolazione cellulare omogenea viene selezionato in base forward scatter (FSC) rispetto side scatter (SSC). </li><li> La popolazione omogenea di cellule singole vivo viene tracciata in un istogramma basata sulla frequenza cella rispetto intensità CFSE impostato su una scala logaritmica. L&#39;intensità del laser CFSE viene regolata in modo da mettere il segnale negativo tra 0 e 100. </li><li> Usando gli stessi parametri e di una strategia di gating simile, le cellule CFSE etichettati vengono eseguiti attraverso il citofluorimetro e analizzati (Figura 4C). </li><li> Successivamente, una popolazione lenta divisione di cellule e una popolazione complessiva (più veloci le cellule in divisione) sono identificati in base alla loro capacità di trattenere CFSE (CFSE high-top 5% e CFSE basso bottom 85%, rispettivamente). </li><li> Queste diverse popolazioni cellulari sono poi suddivisi in separati 15 provette sterili di coltura dei tessuti ml contenenti 1 ml di completo supporto NSC. </li><li> Cellule filtrate vengono quindi centrifugati a 800 rpm per 5 min. </li><li> Il supernatante viene scartato e il pellet viene risospeso in un idoneotuno volumi di mezzo (a seconda del numero di eventi isolati o dimensioni pellet) e una conta cellulare viene eseguita. </li><li> Cellule da diverse popolazioni filtrate vengono piastrate a 50.000 cellule per ml in un matraccio di coltura tissutale appropriate sterile e posto a 37 ° C / 5% CO2 incubatore umidificato per 5-10 giorni prima di essere serialmente diversi passaggi o utilizzato per esperimenti a valle. </li></ol> 5. Risultati rappresentativi Dopo il caricamento CFSE, tutte le cellule presenti un&#39;intensa fluorescenza verde come visualizzato al microscopio (Figura 1). Questo verdastro colore può essere visto anche ad occhio nudo (vedi il video). Analizzare queste cellule con citometria di flusso mostra anche altamente fluorescenza verde intenso rispetto a cellule di controllo (Figura 2). Su cellule CFSE placcatura caricati nella cultura neurosfere, queste cellule proliferano e formano 150-200 micron gliomaspheres in 5-10 giorni. Come le cellule proliferano, t<html> colorante fluorescente che è equamente distribuito tra cellule figlie su divisioni, portando alla dimezzamento dell&#39;intensità di fluorescenza di ogni divisione cellulare. Glioblastomi sono funzionalmente eterogenei e sono composti da cellule proliferanti su diverse scale temporali. Il metodo descritto in questo protocollo permette di identificare e isolare cellule in divisione rapida e lenta in virtù della loro capacità di diluire o mantenere CFSE, rispettivamente (figura 3, anche il video). Citometria di flusso dell&#39;analisi gliomaspheres dissociate generati da cellule CFSE-caricati in confronto alle cellule scariche dimostra diverse proprietà di ritenzione CFSE (Figura 4). Veloce cellule in divisione (in basso 85%) o lento (cellule che si dividono i primi 5% - CFSE alto) gliomapshere mostra la formazione di abilità quando coltivate in condizioni di saggio neurosfere (Figura 5). gura 1 &quot;/&gt; Figura 1. Cellule tumorali dissociati subito dopo il caricamento con la tintura CFSE. Le cellule presentano colore verde brillante dimostrando etichettatura di successo. Originale ingrandimento, 10 x. <img src="/files/ftp_upload/3918/3918fig2.jpg" alt="Figura 2" /> Figura 2. Loading conferma CFSE in citometria a flusso. A) cellule scaricato, B) CFSE caricato cellule e C) Confronto tra intensità di fluorescenza CFSE in cellule cariche rispetto a vuoto. Si noti che vi è di diversi ordini di grandezza differenza nell&#39;intensità di fluorescenza tra le cellule marcate e non marcato. <img src="/files/ftp_upload/3918/3918fig3.jpg" alt="Figura 3" /> Figura 3. A gliomasphere rappresentante derivata da un CFSE caricato a cella 6 giorni dopo placcatura. Alcune cellule presentano una colorazione CFSE molto luminoso. Originale ingrandimento, 63 x. <img src = &quot;/ files/ftp_upload/3918/3918fig4.jpg&quot; alt = &quot;Figura 4&quot; /&gt; Figura 4. Rappresentante citometria a flusso trame / istogrammi per l&#39;isolamento di cellule in divisione veloci e lente 6 giorni dopo la colorazione CFSE. A) FSC e SSC per mostrare tutta la popolazione cellulare, B) reattività SSC vs PI di escludere le cellule morte o danneggiate, C) FSC e SSC a cancello una popolazione omogenea cellule vive, D) dimostra la presenza di cellule CFSE ritegno 6 giorni dopo etichettatura CFSE come evidenziato da una maggiore intensità di fluorescenza rispetto al controllo negativo. L&#39;istogramma mostra anche che l&#39;intensità CFSE decaduto nel tempo. E) Istogramma visualizzazione della strategia di gating per identificare cellule CFSE fissaggio (top 5%) e cellule CFSE diluenti (bottom 85%). <img src="/files/ftp_upload/3918/3918fig5.jpg" alt="Figura 5" /> Gliomaspheres rappresentativi Figura 5. Generati da <strong&gt; A) in rapida divisione delle cellule (CFSE basso) e delle cellule B) slow-divisori (CFSE alto). Originale ingrandimento:. 10x e 20x rispettivamente C) conta di cellule ottenute da Rappresentante veloce o lento dividendo derivato dalle cellule di coltura al momento il passaggio. P &lt;0.05, t-test. <a href="http://www.jove.com/files/ftp_upload/3918/3918fig5large.jpg" target="_blank">Clicca qui per ingrandire la figura</a> .

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Nessun conflitto di interessi dichiarati.

Un numero crescente di studi hanno dimostrato l&#39;importanza di una lenta separazione sub-compartimento di cellule nel cancro che contribuiscono alla formazione di tumori e di resistenza al trattamento 2-8. Pertanto è fondamentale per descrivere e standardizzare metodi sperimentali che consentano lo studio di questa sottopopolazione di cellule o più in generale per confrontare sottopopolazioni con differenti tassi di crescita. Utilizzando CFSE per identificare e isolare sotto-frazioni di cellule in base a...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Tipo </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> NeuroCult NSC media basale (umano) </td><td> Medio </td><td> Stem Cell Technologies </td><td> 05750 </td></tr><tr><td> NeuroCult NSC Supplementi proliferazione (umano) </td><td> Supplemento Media </td><td> Stem Cell Technologies </td><td> 05753 </td></tr><tr><td> 0,05% tripsina-EDTA </td><td> Reagente </td><td> Gibco </td><td> 25300-062 </td></tr><tr><td> Inibitore della tripsina di soia </td><td> Reagente </td><td> Sigma </td><td> T6522 </td></tr><tr><td> Penicillina / streptomicina </td><td> Reagente </td><td> Gibco </td><td> 15140-122 </td></tr><tr><td> T25 pallone </td><td> Cultura ware </td><td> Nalge Nunc internazionale </td><td> 136196 </td></tr><tr><td> T80 pallone </td><td> Cultura ware </td><td> Nalge Nunc internazionale </td><td> 178905 </td></tr><tr><td> 15 ml provette </td><td> Cultura ware </td><td> BD Falcon </td><td> 352096 </td></tr><tr><td> 50 ml provette </td><td> Cultura ware </td><td> BD Falcon </td><td> 352070 </td></tr><tr><td> FEG </td><td> Fattore di crescita </td><td> R &amp; S </td><td> 2028-EG </td></tr><tr><td> CellTrace CFSE cellulare Kit proliferazione </td><td> Cell Division fluorescente monitoraggio Dye </td><td> Molecular Probes, Invitrogen </td><td> C34554 </td></tr></tbody></table>

identification and isolation of slow-dividing cells in human glioblastoma using carboxy fluorescein succinimidyl ester (cfse)

Eterogeneità tumorale rappresenta una caratteristica fondamentale di supporto robustezza tumore e presenta un ostacolo centrale per lo sviluppo di strategie terapeutiche 1. Per superare il problema della eterogeneità tumorale, è essenziale sviluppare saggi e strumenti che consentono fenotipica, (epi) identificazione genetica e funzionale e caratterizzazione di sottopopolazioni tumorali che guidano patologie malattie specifiche e rappresentano obiettivi clinicamente rilevanti. È ormai noto che i tumori esporre distinti sotto-frazioni di cellule con differenti frequenze di divisione cellulare, e che i criteri funzionali essendo ciclo lento è correlata positivamente con capacità di formazione di tumori in diversi tumori inclusi quelli del cervello, la mammella, della pelle e pancreas così come la leucemia 2-8. Il colorante fluorescente carbossifluoresceina succinimmidil estere (CFSE) è stato utilizzato per il monitoraggio della frequenza di divisione delle cellule in vitro e in vivo in ematica tumors e tumori solidi come il glioblastoma 2,7,8. La cellula-permeant non fluorescente pro-farmaco di CFSE viene convertito da esterasi intracellulari in un composto fluorescente, che è trattenuta all&#39;interno delle cellule da covalentemente legame alle proteine ​​per reazione della sua porzione succinimidil con gruppi amminici intracellulari di formare legami ammidici stabili 9. Il colorante fluorescente è equamente distribuito tra cellule figlie upon divisioni, portando alla dimezzamento dell&#39;intensità di fluorescenza con ogni divisione cellulare. Ciò consente il monitoraggio della frequenza del ciclo cellulare fino a otto-dieci giri di divisione 10. Capacità di ritenzione CFSE è stato utilizzato con le cellule tumorali del cervello per identificare e isolare una sottopopolazione lento in bicicletta (top 5% dye-fissaggio celle) ha dimostrato di essere arricchito in attività delle cellule staminali del cancro 2. Questo protocollo descrive la tecnica di colorazione delle cellule con CFSE e l&#39;isolamento di singole popolazioni in una cultura umana glioblastoma (GBM) derivati ​​da cellule che posseggono differenti tassi di divisione in citometria a flusso 2. La tecnica è servito per identificare e isolare un tumore lento ciclismo popolazione di cellule cerebrali in virtù della loro capacità di trattenere l&#39;etichettatura CFSE.

Watch this Scientific Journal Video about Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE at JoVE.com

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE

Questo protocollo video dimostra l&#39;applicazione del colorante fluorescente carbossifluoresceina succinimmidil estere (CFSE) per l&#39;identificazione e la separazione di differenti sottopopolazioni di cellule di glioblastoma umano basati sulla frequenza di divisione cellulare.

Identificazione e isolamento di Slow-divisione delle cellule nel glioblastoma umano per mezzo di Carboxy fluoresceina succinimidile Ester (CFSE)

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic ...

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Research

JoVE Journal

Medicine

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)

14.0K Views.  The University of Florida. Questo protocollo video dimostra l'applicazione del colorante fluorescente carbossifluoresceina succinimmidil estere (CFSE) per l'identificazione e la separazione di differenti sottopopolazioni di cellule di glioblastoma umano basati sulla frequenza di divisione cellulare.

Video: Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Differenziazione Isolamento, caratterizzazione e comparata delle cellule umane Dental Pulp staminali derivate da denti permanenti mediante due diversi metodi

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Ricerca

Medicina

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

Isolamento di cancro cellule staminali da campioni umani di cancro alla prostata

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

Isolamento, Crioconservazione e Cultura umani Cellule epiteliali Amnion per le applicazioni cliniche

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

Isolamento di cellule umane endoteliali linfatico da Multi-parametro fluorescenza-attivato cell sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Studiare cancro del pancreas staminali caratteristiche cellulari per lo sviluppo di nuove strategie di trattamento

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

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

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

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

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

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

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

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

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

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

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

Intranasal Delivery of Therapeutic Stem Cells to Glioblastoma in a Mouse Model

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of therapeutic stem cells via the intranasal route is a recently discovered alternative strategy, with strong potential for clinical translation, due to its non-invasive nature compared to intracranial implantation or delivery via systemic routes. The lack of blood brain barrier further strengthens the therapeutic potential of stem cells undergoing intranasal brain entry. This article summarizes the essential techniques utilized in our studies and outlines the basic principles of intranasal strategy for stem cell delivery using a mouse model of intracranial glioma xenografts. We demonstrate the optimized procedures that generate consistent and reproducible results with specific predetermined experimental parameters and offer guidelines for streamlined work flow that ensure efficient execution and reliable experimental outcome. The article is designed to serve as a baseline for further experimental customization based on hypothesis, stem cell types, or tumor specifics.

Stem cells are promising therapeutic carriers to treat brain tumors due to their intrinsic tumor tropism. Non-invasive intranasal stem cell delivery bypasses the blood brain barrier and demonstrates strong potential for clinical translation. This article summarizes the basic principles of intranasal stem cell delivery in a mouse model of glioma.

Intranasale consegna delle cellule staminali terapeutiche a Glioblastoma in un modello di mouse

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of ...

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the development of obesity-associated complications such as type 2 diabetes. To quantitatively and qualitatively investigate the immune cell subsets in human AT deposits, we have developed a flow cytometry approach. The stromal vascular fraction (SVF), containing the immune cells, is isolated from subcutaneous and visceral AT biopsies by collagenase digestion. Adipocytes are removed after centrifugation. The SVF cells are stained for multiple membrane-bound markers selected to differentiate between immune cell subsets and analyzed using flow cytometry. As a result of this approach, pro- and anti-inflammatory macrophage subsets, dendritic cells (DCs), B-cells, CD4+ and CD8+ T-cells, and NK cells can be detected and quantified. This method gives detailed information about immune cells in AT and the amount of each specific subset. Since there are numerous fluorescent antibodies available, our flow cytometry approach can be adjusted to measure various other cellular and intracellular markers of interest.

This article describes a method to analyze immune cell content of adipose tissue by isolation of immune cells from adipose tissue and subsequent analysis using flow cytometry.

Caratterizzazione di cellule immunitarie nel tessuto adiposo umano mediante citometria a flusso

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the ...

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

Isolamento di cellule deciduali umane primarie dalle membrane fetali del termine intempestivo della placenta

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...