Questo lavoro è stato supportato da sovvenzioni del Centro per la ricerca della Florida Brain Tumor; Preston A. Wells Jr. Center for Brain Tumor Therapy.

1. Raccolta di tessuto GBM primario <ol><li> Un mutliforme glioblastoma (GBM) tumore è ottenuto da un paziente diagnosticato con il cancro e sottoposti a intervento chirurgico. </li><li> Accordi deve essere fatta con il team di neurochirurgia. Il neurochirurgo metterà GBM il tumore asportato in un tubo di dimensioni adeguate contenente cellule staminali neurali (NSC) medio basale completato con 10-15% di antibiotici (Peniciline / streptomicina). Medium freddo devono essere fornite alla sala operatoria da laboratorio. In alternativa, freddo HEPES-buffered medio minimo indispensabile (HEM) o tampone fosfato (PBS) con elevata concentrazione di antibiotici può essere utilizzato anche per questo scopo. </li><li> Il tessuto tumorale asportato viene consegnato al laboratorio su ghiaccio e posto sotto il cofano. </li></ol> 2. Dissociazione del tumore primario in cella singola Sospensione <ol><li> Eccesso del mezzo originale / PBS è stato rimosso dal tubo falco e il campione viene lavato 2-3 volte con 5-10ml di PBS / NSC medio basale per rimuovere sangue e detriti. Il PBS / Medium è stato rimosso e il tessuto tumorale GBM è posto in una capsula di Petri. </li><li> Il tessuto viene tagliato in piccoli pezzi e macinata con una lama di 10 N. bisturi in piccoli pezzi per aumentare la superficie per il processo tripsinizzazione. Macinazione può richiedere 1-3 minuti a seconda delle dimensioni del tumore. </li><li> Il tessuto è trypsinized tritata in 3-5ml di pre-riscaldato 0,05% tripsina-EDTA per 10-15 minuti a bagnomaria a 37 ° C. Una pipetta elettronica è utilizzato per trasferire i pezzi tumore piccolo e tripsina in un tubo 15ml Falcon. </li><li> Un uguale volume di inibitore della tripsina di soia viene aggiunta per bloccare la reazione enzimatica tripsina dopo il periodo di incubazione. </li><li> Inattivazione della tripsina è assicurata pipettando la sospensione su e giù diverse volte. Poi, la sospensione è pellet dalla centrifugazione a 800 giri (110 g) per 5min. </li><li> Il supernatante viene scartato ed i pezzi di tessuto sono risospesi in 1 ml di soluzione sterile NSC basal media. Le macchie sono dissociate pipettando gentilmente su e giù (3-7 volte) fino a una superficie liscia sospensione lattiginosa singola cella è raggiunto. Il numero di passi pipettamento dipende direttamente dalla dimensione delle particelle nel tessuto tritato. Lungo e vigoroso dissociazione meccanica dovrebbe essere evitata in quanto potrebbe provocare la morte cellulare e una riduzione della formazione di sfera. </li><li> Per rimuovere un-dissociato pezzi e detriti, 10-15 ml di terreno di base viene aggiunto il tubo e la sospensione cellulare viene filtrata attraverso un filtro a 40 micron delle cellule in una provetta da 50 ml. </li><li> La sospensione è filtrata centrifugato a 800 giri (110 g) per 5min. Il supernatante viene scartato in seguito. </li><li> Pellettati cellule vengono poi risospeso in 1-2ml di completo supporto NSC per il conteggio delle cellule. </li></ol> 3. Conteggio delle cellule e placcatura <ol><li> 10 ml di sospensione cellulare viene aggiunto a 90 ml di 0,04% tripan blu in un tubo di 1 ml Eppendorf. Nota: altre appropriato delle cellule diluizionezioni possono anche essere utilizzati. </li><li> Pipetta su e giù per miscelare la sospensione. 10 L delle cellule / trypan miscela blu è trasferito a emocitometro per contare la densità delle cellule. </li><li> Le cellule sono placcati in completo medio NSC (una miscela di media NSC basale e NSC supplemento di proliferazione nucleare in un rapporto 9:1) integrata con 20ng/ml EGF, bFGF 10ng/ml e 1μl/ml dello 0,2% eparina (2μg/ml) in appropriato tessuto cultura navi. Ml 5, 20 e 40 di media è utilizzato per fiaschi T25, T80 e T175, rispettivamente. Gli antibiotici possono essere aggiunti al mezzo ad una concentrazione di 1:100 per diminuire la possibilità di contaminazione. </li><li> Il pallone viene posto in un incubatore a 37 ° C e 5% di CO 2. </li></ol> 4. Passaging e l&#39;espansione del GBM sfere derivati: <ol><li> Quando il neurosfere raggiunto una dimensione media di 150-200 micron di diametro, la cultura è pronto per sottocultura. Il contenuto di ciascun pallone viene rimosso e collocato in una dimensione appropriata TISS steriletubo cultura ue, e centrifugati a 800 giri (110 g) per 5 minuti a temperatura ambiente. </li><li> Il surnatante viene rimosso e il pellet viene risospeso in 1 ml di 0,05% tripsina-EDTA. </li><li> Per ottenere un tripsinizzazione ottimale, la sospensione cellulare viene incubato a 37 ° C a bagnomaria per 2-3 min. Per interrompere l&#39;attività tripsina un uguale volume di inibitore della tripsina di soia viene aggiunta alla sospensione cellulare e la sospensione cellulare viene delicatamente pipettato su e giù. </li><li> La sospensione cellulare viene centrifugata a 800 rpm (110g) per 5 min. Poi, il surnatante viene rimosso e le cellule sono risospese in 1 ml di terreno NSC. </li><li> Un conteggio delle cellule viene eseguita come descritto in precedenza. </li><li> Le cellule sono placcati in una concentrazione di 5x10 4 cellule / ml in totale medio NSC completato con fattori di crescita e placcato in vasi di dimensioni adeguate alla coltura di tessuti come descritto nella parte precedente. </li><li> Neurosfere secondarie si formano in 7-10 giorni, quando incubato a 37 ° C in ambienti umidiified incubatore con il 5% di CO 2. </li></ol> 5. Rappresentante dei risultati: Dopo la placcatura singole cellule raccolte dal tessuto tumorale GBM, tumore-come le cellule staminali proliferare e generare piccoli gruppi di cellule composto da poche cellule in 3-4 giorni (vedi il video e la Figura 1). Come questi gruppi crescono, acquisiscono una forma più sferica in modo che entro 7-8 giorni, sfere in fase corretta luminoso con un diametro medio di 150-200 micron forma (Guarda il video e la Figura 2). A maggiore ingrandimento, sfere sano di solito dimostrare microspikes alla loro periferia. Avendo grande sfera come grappoli in 1-2 giorni dopo l&#39;inizio della cultura è dovuta alla presenza di non dissociati aggregati al binging della cultura e non deve essere scambiato come sfere vero. La quantità di detriti nel campo del tumore primario cultura varia a seconda della fonte iniziale del tessuto e se non si include qualsiasi tessuto cerebrale circostante. La corretta tessuto tecniche di preparazionecompresi dissociazione enzimatica e meccanica, e successive di filtrazione del campione con una quantità sufficiente di media può portare a meno detriti nella cultura. <img src="/files/ftp_upload/3633/3633fig1.jpg" alt="Figura 1" /> Figura 1. Primaria cultura sfera GBM 4 giorni dopo la placcatura. Tumore-come le cellule staminali proliferare e generare piccoli gruppi di cellule in 3-4 giorni. Ingrandimento originale; 20x. <img src="/files/ftp_upload/3633/3633fig2.jpg" alt="Figura 2" /> Figura 2. Passage una cultura sfera GBM 8 giorni dopo placcatura. Ingrandimento originale; 20x.

<ol>
	<li>Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+neurons+and+astrocytes+from+isolated+cells+of+the+adult+mammalian+central+nervous+system.">Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.</a> Science. 255, 1707-1710 (1992).</li><li>Siebzehnrubl, F. A., Vedam-Mai, V., Azari, H., Reynolds, B. A., Deleyrolle, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Characterization+of+Adult+Neural+Stem+Cells.">Isolation and Characterization of Adult Neural Stem Cells.</a> Methods Mol Biol. 750, 61-77 (2011).</li><li>Azari, H., Rahman, M., Sharififar, S., Reynolds, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Expansion+of+the+Adult+Mouse+Neural+Stem+Cells+Using+the+Neurosphere+Assay.">Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay.</a> J. Vis. Exp. (45), e2393-e2393 (2010).</li><li>Azari, H., Sharififar, S., Rahman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+Embryonic+Mouse+Neural+Stem+Cell+Culture+Using+the+Neurosphere+Assay.">Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay.</a> J. Vis. Exp. (47), e2457-e2457 (2011).</li><li>Eramo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+expansion+of+the+tumorigenic+lung+cancer+stem+cell+population.">Identification and expansion of the tumorigenic lung cancer stem cell population.</a> Cell Death Differ. 15, 504-514 (2008).</li><li>Patrawala, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+purified+CD44++prostate+cancer+cells+from+xenograft+human+tumors+are+enriched+in+tumorigenic+and+metastatic+progenitor+cells.">Highly purified CD44+ prostate cancer cells from xenograft human tumors are enriched in tumorigenic and metastatic progenitor cells.</a> Oncogene. 25, 1696-1708 (2006).</li><li>Li, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+resistance+of+tumorigenic+breast+cancer+cells+to+chemotherapy.">Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy.</a> J Natl. Cancer. Inst. 100, 672-679 (2008).</li><li>Galli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+tumorigenic,+stem-like+neural+precursors+from+human+glioblastoma.">Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma.</a> Cancer Res. 64, 7011-7021 (2004).</li><li>Deleyrolle, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+label-retaining+tumour-initiating+cells+in+human+glioblastoma.">Evidence for label-retaining tumour-initiating cells in human glioblastoma.</a> Brain. 134, 1331-1343 (2011).</li></ol>

Nessun conflitto di interessi dichiarati.

Per isolare staminali neurali e cellule progenitrici da adulto normale e il cervello del feto 1, 2, 3, 4 e anche tumore-come le cellule staminali dai tessuti tumorali come il polmone 5, 6 della prostata, della mammella e del cervello 7 8, 9 il test neurosfere è stato frequentemente utilizzati come metodo di scelta. Da questo test semplice e riproducibile, si può generare un numero indefinito di cellule dal tessuto tumorale asportato che mostrano caratteristiche simili a quelle delle cellule staminali somatiche; multipotenza ex vivo, la possibilità di creare nuovi tumori al momento dell&#39;impianto, e di auto-rinnovamento. Queste cellule potrebbero essere usate per studiare la biologia di base delle cellule tumorali tra cui cellula-cellula interazioni, e la differenziazione, migrazione, invasione e morte cellulare. Inoltre, isolato tumore-come le cellule staminali di fornire uno strumento prezioso per studiare come si formano i tumori, il progresso, e la ricaduta e anche per svelare i meccanismi alla base derivanti cellulari che alla fine potrebbe fornire spunti per terapeuticoopzioni.

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Tipo </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> NeuroCult NSC medio basale (Human) </td><td> Medio </td><td> Stem Cell Technologies </td><td> 05750 </td><td></td></tr><tr><td> NeuroCult NSC Supplementi proliferazione (Human) </td><td> Supplemento media </td><td> Stem Cell Technologies </td><td> 05753 </td><td></td></tr><tr><td> 0,05% tripsina-EDTA </td><td> Reagente </td><td> Gibco </td><td> 25300-062 </td><td></td></tr><tr><td> * MEM </td><td> Reagente </td><td> Gibco </td><td> 41500-018 </td><td> HEM componente </td></tr><tr><td> * HEPES </td><td> Reagente </td><td> Sigma </td><td> H4034 </td><td> HEM componente </td></tr><tr><td> * Acqua distillata </td><td> Reagente </td><td&gt; Gibco </td><td> 15230-147 </td><td></td></tr><tr><td> ** DNasi I </td><td> Reagente </td><td> Roche </td><td> 104159 </td><td></td></tr><tr><td> ** Soia inibitore della tripsina </td><td> Reagente </td><td> Sigma </td><td> T6522 </td><td></td></tr><tr><td> Pen / Strep </td><td> Reagente </td><td> Gibco </td><td> 15140-122 </td><td></td></tr><tr><td> N. 10 lame bisturi </td><td> Strumento chirurgico </td><td> BD </td><td> 371610 </td><td></td></tr><tr><td> Petri Dish </td><td> Cultura software </td><td> BD Falcon </td><td> 353003 </td><td></td></tr><tr><td> Pinzetta </td><td> Strumenti chirurgici </td><td> Strumenti Scienza multa </td><td> 11050-10 </td><td></td></tr><tr><td> Cellulare filtro </td><td> Setaccio </td><td> BD Falcon </td><td> 352340 </td><td></td></tr><tr><td> T25 fiasco </td><td> Cultura software </td><td> Nalge Nunc internazionale </td><td> 136196 </td><td></td></tr><tr><td> T80 fiasco </td><td> Cultura software </td><td> Nalge Nunc internazionale </td><td> 178905 </td><td></td></tr><tr><td> Provette da 15 ml </td><td> Cultura software </td><td> BD Falcon </td><td> 352096 </td><td></td></tr><tr><td> 50 ml provette </td><td> Cultura software </td><td> BD Falcon </td><td> 352070 </td><td></td></tr><tr><td> FEG </td><td> Fattore di crescita </td><td> R &amp; S </td><td> 2028-EG </td><td></td></tr><tr><td> b-FGF </td><td> Fattore di crescita </td><td> R &amp; S </td><td> 3139-FB </td><td></td></tr><tr><td> Eparina </td><td> Fattore di crescita </td><td> Sigma </td><td> H4784 </td><td> Ricostituita in PBS </td></tr></tbody></table> * Per fare HEM, mescolare pacchetto 1x10L di MEM and160ml di HEPES 1M e portare il volume a 8,75 L con acqua distillata. Impostare il PH finale a 7,4 unod conservarlo a 4 ° C. ** Per preparare soluzione inibitore della tripsina, per prima cosa 10 ml di soluzione di DNasi I (100 mg DNasi sciolto in 100 ml di HEM) e aggiungere 0,14 g di inibitore della tripsina alla soluzione di DNasi e, infine, portare al volume di 1 litro con HEM. Mantenere aliquote dei prodotti finali in congelatore -20 ° C.

isolation and expansion of human glioblastoma multiforme tumor cells using the neurosphere assay

-Come le cellule staminali sono state isolate nei tumori come mammella, polmone, colon, prostata e cervello. Un problema critico in tutti questi tumori, soprattutto in mutliforme glioblastoma (GBM), è quello di identificare e isolare popolazione di cellule tumorali di iniziare (s) per studiare il loro ruolo nella formazione del tumore, la progressione e le recidive. Tumore comprensione iniziare popolazioni di cellule fornirà gli indizi per trovare approcci terapeutici efficaci per questi tumori. Il test neurosfere (NSA) per la sua semplicità e la riproducibilità è stata utilizzata come metodo di scelta per l&#39;isolamento e la propagazione di molte di queste cellule tumorali. Questo protocollo viene illustrato il metodo neurosfere cultura per isolare ed espandere cellule staminali, come in chirurgicamente asportato tessuto tumorale umano GBM. Le procedure comprendono una digestione iniziale dissociazione chimica e meccanica del tessuto tumorale e, successivamente, placcatura la sospensione risultante singola cellula in coltura NSA. Dopo 7-10 giorni, neurosfere primarie del 150-200 micron di diametro può essere osservato e sono pronti per un ulteriore passaging ed espansione.

Watch this Scientific Journal Video about Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay at JoVE.com

Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

Questo protocollo video dimostra l&#39;isolamento e l&#39;espansione delle cellule staminali come da chirurgicamente asportato mutliforme glioblastoma umano (GBM) tessuti tumorali utilizzando il metodo neurosfere cultura test.

Isolamento ed espansione di cellule umane Glioblastoma Multiforme Tumore del saggio di neurosfere

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in ...

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Neuroscience

29.6K Views.  University of Florida. Questo protocollo video dimostra l'isolamento e l'espansione delle cellule staminali come da chirurgicamente asportato mutliforme glioblastoma umano (GBM) tessuti tumorali utilizzando il metodo neurosfere cultura test.

Video: Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

Isolamento ed espansione della cellule staminali neurali del mouse utilizzando il saggio neurosfere

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

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Neuroscienze

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

Stabilire embrionali di topo Neural Stem Cell Culture del saggio di neurosfere

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Isolation and Culture of Human Fungiform Taste Papillae Cells

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices1,2,3,4. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d5,6,7,8. We recently reported on a successful method for the extended culture of rodent taste cells9. We here report for the first time the establishment of an in vitro culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C &beta;2, (PLC-&beta;2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-&beta;2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro preparation.
Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al. 201110.

We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.

Isolamento e la coltura di cellule umane Gusto papille fungiformi

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple ...

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis and enable new strategies for in vitro modeling or repairing the diseased brain. Identifying brain-resident non-neuronal cell types amenable to direct conversion into iNs might allow for launching such an approach in situ, i.e. within the damaged brain tissue. Here we describe a protocol developed in the attempt of identifying cells derived from the adult human brain that fulfill this premise. This protocol involves: (1) the culturing of human cells from the cerebral cortex obtained from adult human brain biopsies; (2) the in vitro expansion (approximately requiring 2-4 weeks) and characterization of the culture by immunocytochemistry and flow cytometry; (3) the enrichment by fluorescence-activated cell sorting (FACS) using anti-PDGF receptor-&#946; and anti-CD146 antibodies; (4) the retrovirus-mediated transduction with the neurogenic transcription factors sox2 and ascl1; (5) and finally the characterization of the resultant pericyte-derived induced neurons (PdiNs) by immunocytochemistry (14 days to 8 weeks following retroviral transduction). At this stage, iNs can be probed for their electrical properties by patch-clamp recording. This protocol provides a highly reproducible procedure for the in vitro lineage conversion of brain-resident pericytes into functional human iNs.

Targeting brain-resident cells for direct lineage-reprogramming offers new perspectives for brain repair. Here we describe a protocol of how to prepare cultures enriched for brain-resident pericytes from the adult human cerebral cortex and convert these into induced neurons by retrovirus-mediated expression of the transcription factors Sox2 and Ascl1.

Lineage-riprogrammazione di cellule pericyte-derivate del cervello umano adulto in neuroni indotti

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, neurogenesis, patterning of the hindbrain and spinal cord, and the development of multiple organ systems. Due to its chemical nature as a small amphipathic lipid, direct detection and visualization of RA histologically remains technically impossible. Currently, methods used to infer the presence and localization of RA make use of reporter systems that detect the biological activity of RA. Most established reporter systems, both transgenic mice and cell lines, make use of the highly potent RA response element (RARE) upstream of the RAR-beta gene to drive RA-inducible expression of reporter genes, such as beta-galactosidase or luciferase. The transgenic RARE-LacZ mouse is useful in visualizing spatiotemporal changes in RA signaling especially during embryonic development. However, it does not directly measure overall RA levels. As a reporter system, the F9 RARE-LacZ cell line can be used in a variety of ways, from simple detection of RA to quantitative measurements of RA levels in tissue explants. Here we describe the quantitative determination of relative RA levels generated in embryos and neurosphere cultures using the F9 RARE-LacZ reporter cell line.

Methods to accurately measure retinoic acid (RA) levels in small amounts of tissue do not exist. This protocol describes the easy, quantitative measurement of relative RA levels in E8.5 embryos and neurospheres using an RA reporter cell line.

Misurazione quantitativa dei livelli relativi dell&#39;acido retinoico nelle E8.5 embrioni e neurosfere Culture Utilizzando il F9 RARE-LacZ cellulare a base di Reporter Assay

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, ...

Analysis of Spliceosomal snRNA Localization in Human Hela Cells Using Microinjection

Biogenesis of spliceosomal snRNAs is a complex process involving both nuclear and cytoplasmic phases and the last step occurs in a nuclear compartment called the Cajal body. However, sequences that direct snRNA localization into this subnuclear structure have not been known until recently. To determine sequences important for accumulation of snRNAs in Cajal bodies, we employed microinjection of fluorescently labelled snRNAs followed by their localization inside cells. First, we prepared snRNA deletion mutants, synthesized DNA templates for in vitro transcription and transcribed snRNAs in the presence of UTP coupled with Alexa488. Labelled snRNAs were mixed with 70 kDa-Dextran conjugated with TRITC, and microinjected to the nucleus or the cytoplasm of human HeLa cells. Cells were incubated for 1 h and fixed and the Cajal body marker coilin was visualized by indirect immunofluorescence, while snRNAs and dextran, which serves as a marker of nuclear or cytoplasmic injection, were observed directly using a fluorescence microscope. This method allows for efficient and rapid testing of how various sequences influence RNA localization inside cells. Here, we show the importance of the Sm-binding sequence for efficient localization of snRNAs into the Cajal body.

Biogenesis of spliceosomal snRNAs is a complex process involving various cellular compartments. Here, we employed microinjection of fluorescently labelled snRNAs in order to monitor their transport inside the cell.

Analisi della localizzazione snRNA Spliceosomic nelle cellule umane di Hela utilizzando la microiniezione

Biogenesis of spliceosomal snRNAs is a complex process involving both nuclear and cytoplasmic phases and the last step occurs in a nuclear compartment ...

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models for studying blood-brain barrier (BBB) function. This modified protocol provides detailed steps to derive, expand, and cryopreserve BMECs from human iPSCs using a different donor and reagents than those reported in previous protocols. iPSCs are treated with essential 6 medium for 4 days, followed by 2 days of human endothelial serum-free culture medium supplemented with basic fibroblast growth factor, retinoic acid, and B27 supplement. At day 6, cells are sub-cultured onto a collagen/fibronectin matrix for 2 days. Immunocytochemistry is performed at day 8 for BMEC marker analysis using CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. Western blotting is performed to confirm BMEC marker expression, and absence of SOX17, an endodermal marker. Angiogenic potential is demonstrated with a sprouting assay. Trans-endothelial electrical resistance (TEER) is measured using chopstick electrodes and voltohmmeter starting at day 7. Efflux transporter activity for ATP binding cassette subfamily B member 1 and ATP binding cassette subfamily C member 1 is measured using a multi-plate reader at day 8. Successful derivation of BMECs is confirmed by the presence of relevant cell markers, low levels of SOX17, angiogenic potential, transporter activity, and TEER values ~2000 &#937; x cm2. BMECs are expanded until day 10 before passaging onto freshly coated collagen/fibronectin plates or cryopreserved. This protocol demonstrates that iPSC-derived BMECs can be expanded and passaged at least once. However, lower TEER values and poorer localization of BMEC markers was observed after cryopreservation. BMECs can be utilized in co-culture experiments with other cell types (neurons, glia, pericytes), in three-dimensional brain models (organ-chip and hydrogel), for vascularization of brain organoids, and for studying BBB dysfunction in neuropsychiatric disorders.

This protocol details an adapted method to derive, expand, and cryopreserve brain microvascular endothelial cells obtained by differentiating human induced pluripotent stem cells, and to study blood brain barrier properties in an ex vivo model.

Derivazione, espansione, crioconservazione e caratterizzazione di cellule endoteliali microvascolari cerebrali da cellule staminali pluripotenti indotte dall'uomo

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...

Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and traumatic brain injury (TBI). Zebrafish are an important neuroscience model due to their transparency during development and robust regenerative capabilities following neurotrauma. While various cognitive tests exist in zebrafish, most of the cognitive assessments that are rapid examine non-associative learning. At the same time, associative-learning assays often require multiple days or weeks. Here, we describe a rapid associative-learning test that utilizes an adverse stimulus (electric shock) and requires minimal preparation time. The shuttle box assay, presented here, is simple, ideal for novice investigators, and requires minimal equipment. We demonstrate that, following TBI, this shuttle box test reproducibly assesses cognitive deficit and recovery from young to old zebrafish. Additionally, the assay is adaptable to examine either immediate or delayed memory. We demonstrate that both a single TBI and repeated TBI events negatively affect learning and immediate memory but not delayed memory. We, therefore, conclude that the shuttle box assay reproducibly tracks the progression and recovery of cognitive impairment.

Learning and memory are potent metrics in studying either developmental, disease-dependent, or environmentally induced cognitive impairments. Most cognitive assessments require specialized equipment and extensive time commitments. However, the shuttle box assay is an associative learning tool that utilizes a conventional gel box for rapid and reliable assessment of adult zebrafish cognition.

Shuttle Box Assay come strumento di apprendimento associativo per la valutazione cognitiva negli studi sull'apprendimento e sulla memoria utilizzando Zebrafish adulto

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and ...

Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid enzymatic dissociation of skin. Primary keratinocytes, fibroblasts, and Schwann cells are all harvested from the human newborn foreskin, which is available following standard of care procedures. The removed skin is disinfected, and the subcutaneous fat and muscle are removed using a scalpel. The method consists of enzymatic and mechanical separation of epidermal and dermal layers, followed by additional enzymatic digestion to obtain single-cell suspensions from each of these skin layers. Finally, single cells are grown in appropriate cell culture media following standard cell culture protocols to maintain growth and viability over weeks. Together, this simple protocol allows isolation, culturing, and characterization of all three cell types from a single piece of skin for in vitro evaluation of skin-nerve models. Additionally, these cells can be used together in co-cultures to gauge their effects on each other and their responses to in vitro trauma in the form of scratches performed robotically in the culture associated with wound healing.

The study of wound healing associated with musculoskeletal injury often requires the assessment of in vitro interactions between Schwann cells (SCs), keratinocytes, and fibroblasts. This protocol describes the isolation, culturing, and characterization of these primary cells from the human foreskin.

Isolamento, coltura e caratterizzazione di cellule primarie di Schwann, cheratinociti e fibroblasti dal prepuzio umano

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid ...

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple neurological diseases. Studying human microglia in health and disease represents a challenge due to the extremely limited supply of human cells. Induced pluripotent stem cells (iPSCs) derived from human individuals can be used to circumvent this barrier. Here, it is demonstrated how to differentiate human iPSCs into microglia-like cells (iMGs) for in vitro experimentation. These iMGs exhibit the expected and physiological properties of microglia, including microglia-like morphology, expression of proper markers, and active phagocytosis. Additionally, documentation for isolating and labeling synaptosome substrates derived from human iPSC-derived lower motor neurons (i3LMNs) is provided. A live-cell, longitudinal imaging assay is used to monitor engulfment of human synaptosomes labeled with a pH-sensitive dye, allowing for investigations of iMG's phagocytic capacity. The protocols described herein are broadly applicable to different fields that are investigating human microglia biology and the contribution of microglia to disease.

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

This protocol describes the differentiation process of human induced pluripotent stem cells (iPSCs) into microglia-like cells for in vitro experimentation. We also include a detailed procedure for generating human synaptosomes from iPSC-derived lower motor neurons that can be used as a substrate for in vitro phagocytosis assays using live-cell imaging systems.

Cellule simili alla microglia umana: differenziazione da cellule staminali pluripotenti indotte e saggio di fagocitosi di cellule vive in vitro utilizzando sinaptosomi umani

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple ...