Preston A. Wells Jr. Center for Brain Tumor Therapy; Diese Arbeit wurde durch Zuschüsse aus dem Florida-Zentrum für Hirntumor Forschung gefördert.

1. Erhebung von Primärdaten GBM Tissue <ol><li> Ein Glioblastom mutliforme (GBM) Tumor ist von einem Patienten mit der Diagnose Krebs und Chirurgie erhalten. </li><li> Arrangements muss mit der Neurochirurgie Team vorgenommen werden. Der Neurochirurg wird der resezierten GBM Tumor in einer angemessenen Größe Röhrchen mit neuralen Stammzellen (NSC) Basalmedium mit 10-15% Antibiotika (Peniciline / Streptomycin) ergänzt Platz. Kalte Medium muss auf OP-Saal durch Labor zur Verfügung gestellt werden. Alternativ kalten HEPES-gepufferte Minimum Essential Medium (HEM) oder Phosphat-gepufferter Kochsalzlösung (PBS) mit einer hohen Konzentration von Antibiotika kann auch für diesen Zweck verwendet werden. </li><li> Die resezierten Tumorgewebes ist das Labor auf Eis angeliefert und unter der Haube. </li></ol> 2. Dissoziation des Primärtumors in Single-Cell Suspension <ol><li> Excess der ursprünglichen Medium / PBS wird aus der Falcon-Röhrchen abgenommen und die Probe wird 2-3 mal mit 5-10m gewaschenl PBS / NSC Basalmedium zu entfernen Blut und Schmutz. Die PBS / Medium wird entfernt und die GBM Tumorgewebe wird in einer Petrischale. </li><li> Das Gewebe wird in kleine Stücke geschnitten und gehackt mit einem Nr. 10 Skalpell in kleine Stücke, um die Oberfläche für Trypsinierung zu erhöhen. Wiegemesser kann 1-3 Minuten je nach Größe des Tumors. </li><li> Das zerkleinerte Gewebe wird in 3-5ml vorgewärmtes% 0,05 Trypsin-EDTA für 10-15 Minuten bei 37 ° C Wasserbad trypsiniert. Eine elektronische Pipette wird verwendet, um die winzigen Tumor Stücke und Trypsin in ein 15ml Falcon-Röhrchen übertragen. </li><li> Ein gleiches Volumen von Sojabohnen-Trypsin-Inhibitor zugesetzt wird, um die enzymatische Reaktion Trypsin nach der Inkubationszeit zu stoppen. </li><li> Trypsin Inaktivierung durch Pipettieren der Suspension nach oben und unten mehrfach gesichert. Dann wird die Suspension pelletiert unten durch Zentrifugation bei 800rpm (110g) für 5min. </li><li> Der Überstand wird verworfen und die Gewebestücke werden in 1 ml steriler NSC ba resuspendiertsal Medium. Die Klumpen sind durch vorsichtiges Auf-und Abpipettieren (3-7 mal), bis eine glatte milchig einzigen Zellsuspension erreicht dissoziiert. Die Zahl der Pipettierschritte direkt abhängig von der Größe der Partikel in der zerkleinerte Gewebe. Lange und kräftige mechanische Dissoziation sollte vermieden werden, da es in Zelltod und eine Reduktion in der Sphäre Bildung könnte Folge sein. </li><li> Um un-dissoziierte Teile und Trümmer zu entfernen, ist 10-15 ml Basismedium in das Röhrchen gegeben und die Zellsuspension durch ein 40 Mikron Zelle Sieb in eine 50ml Tube gefiltert. </li><li> Die filtrierte Suspension wird bei 800rpm (110g) für 5min zentrifugiert. Der Überstand wird anschließend verworfen. </li><li> Pelletiert Zellen werden dann in 1-2ml komplette NSC Medium zur Zellzählung resuspendiert. </li></ol> 3. Zellzahl und Plating <ol><li> 10 l der Zellsuspension auf 90 ul von 0,04% Trypanblau in einer 1ml Eppendorf-Röhrchen aufgenommen. Hinweis: andere geeignete Zelle Verdünnunggen können ebenfalls verwendet werden. </li><li> Pipette auf und ab, um die Aussetzung zu mischen. 10 ul der Zellen / Trypanblau Mischung auf Zählkammer überführt, um die Zelldichte zu zählen. </li><li> Die Zellen werden in kompletten NSC Medium (eine Mischung aus NSC Basalmedium und NSC Verbreitung Ergänzung zu einem 9:1-Verhältnis) mit 20ng/ml EGF, 10ng/ml bFGF und 1μl/ml von 0,2% Heparin (2μg/ml) ergänzt in verchromt geeigneten Zellkultur Gefäße. 5, 20 und 40 ml Medium für T25, T80 und T175-Kolben verwendet werden, bzw.. Antibiotika können das Medium bei einer Konzentration von 1:100 hinzugefügt werden, um Wahrscheinlichkeit einer Kontamination zu verringern. </li><li> Der Kolben wird in einem Inkubator eingestellt bei 37 ° C und 5% CO 2 platziert. </li></ol> 4. Passagieren und Ausbau der GBM abgeleitet Kugeln: <ol><li> Wenn die Neurosphären einer durchschnittlichen Größe von 150-200 &amp;mgr; m im Durchmesser erreicht, wird die Kultur bereit für Subkultur. Der Inhalt jeder Flasche entnommen und in eine geeignete Größe sterile tiss platziertue Kultur Rohr, und zentrifugiert bei 800 rpm (110 g) für 5 min bei Raumtemperatur. </li><li> Der Überstand wird entfernt und das Pellet wird in 1 ml% 0,05 Trypsin-EDTA resuspendiert. </li><li> Um eine optimale Trypsinierung, wird die Zellsuspension bei 37 ° C im Wasserbad für 2-3 min inkubiert. Zum Stoppen der Trypsin-Aktivität ein gleiches Volumen an Sojabohnen-Trypsin-Inhibitor zur Zellsuspension gegeben und die Zellsuspension vorsichtig und pipettiert nach unten. </li><li> Die Zellsuspension wird bei 800 rpm (110g) für 5 min zentrifugiert. Dann wird der Überstand abgenommen und die Zellen werden in 1 ml der NSC-Medium resuspendiert. </li><li> Eine Zellzahl erfolgt wie oben beschrieben. </li><li> Die Zellen werden in einer Konzentration von 5x10 4 Zellen / ml in kompletten NSC Medium mit Wachstumsfaktoren und vernickelt in entsprechender Größe Gewebekultur Schiffe wie in den vorangegangenen Teil beschrieben ausplattiert. </li><li> Sekundäre Neurosphären werden in ca. 7-10 Tagen entsteht, wenn bei 37 ° C in einer feuchtenified Inkubator mit 5% CO 2. </li></ol> 5. Repräsentative Ergebnisse: Nach dem Ausstreichen der einzelnen Zellen von GBM Tumorgewebe entnommen, vermehren sich Tumorstammzellen-ähnliche Zellen und erzeugen kleine Cluster von Zellen wenige Zellen in 3-4 Tagen zusammen (siehe Video und Bild 1). Wie diese Cluster wachsen, erhalten sie eine Kugelform, so dass von 7-8 Tagen, korrekten Phase helle Kugeln mit einem durchschnittlichen Durchmesser von 150-200 Mikrometern (Sehen Sie das Video und Abbildung 2). Bei höherer Vergrößerung, gesunde Bereiche in der Regel zeigen microspikes an ihrer Peripherie. Nachdem große Kugel wie Trauben in 1-2 Tagen nach Einleitung Kultur ist durch die Existenz von nicht-dissoziierten Klumpen an der binging der Kultur und sollte nicht als echte Kugeln verwechselt werden. Die Menge der Ablagerungen in Primärtumor Bereich Kultur hängt von der ursprünglichen Quelle des Gewebes abhängig und ob es umfasst alle umliegenden Hirngewebe. Proper Gewebe Präparationstechnikeneinschließlich enzymatischer und mechanischer Dissoziation und anschließender Filtration der Probe mit einer ausreichenden Menge Medium kann in weniger Geröll in der Kultur führen. <img src="/files/ftp_upload/3633/3633fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Primary GBM Bereich Kultur 4 Tage nach dem Ausplattieren. Tumorstammzellen-ähnlichen Zellen vermehren sich und erzeugen kleine Gruppen von Zellen in 3-4 Tagen. Original-Vergrößerung, 20x. <img src="/files/ftp_upload/3633/3633fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Gang sind GBM Bereich Kultur 8 Tage nach dem Ausplattieren. Original-Vergrößerung, 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>

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Um neuralen Stamm-und Vorläuferzellen aus normalen Erwachsenen und fetale Gehirn 1, 2, 3, 4 zu isolieren und auch Tumorstammzellen-ähnliche Zellen aus Krebsgewebe wie Lungen-5, Prostata-6, 7 und Brust Gehirn 8, 9 der Neurosphäre Assay wurde häufig als die Methode der Wahl eingesetzt. Mit diesem einfachen und reproduzierbaren Test kann man erzeugen, eine unbestimmte Anzahl von Zellen aus resezierten Tumorgewebes, die ähnliche Merkmale wie somatische Stammzellen zeigen, ex vivo Multipotenz, die Fähigkeit, neue Tumoren nach der Implantation zu schaffen und sich selbst zu erneuern. Diese Zellen könnten verwendet werden, um die grundlegenden Krebs Zellbiologie einschließlich Zell-Zell-Interaktionen, und die Differenzierung, Migration, Invasion und Zelltod zu untersuchen. Darüber hinaus bieten isoliert Tumorstammzellen-ähnlichen Zellen ein unschätzbares Werkzeug zu untersuchen, wie Tumore zu bilden, Fortschritt und Rückfall und auch auf die zugrunde liegenden zellulären Mechanismen ableiten, die schließlich Erkenntnisse zu therapeutischen könnte entwirrenOptionen.

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Typ </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> NeuroCult NSC Basal Medium (Human) </td><td> Medium </td><td> Stem Cell Technologies </td><td> 05750 </td><td></td></tr><tr><td> NeuroCult NSC Proliferation Supplements (Human) </td><td> Medium ergänzen </td><td> Stem Cell Technologies </td><td> 05753 </td><td></td></tr><tr><td> % 0,05 Trypsin-EDTA </td><td> Reagens </td><td> Gibco </td><td> 25300-062 </td><td></td></tr><tr><td> * MEM </td><td> Reagens </td><td> Gibco </td><td> 41500-018 </td><td> HEM-Komponente </td></tr><tr><td> * HEPES </td><td> Reagens </td><td> Sigma </td><td> H4034 </td><td> HEM-Komponente </td></tr><tr><td> * Destilliertes Wasser </td><td> Reagens </td><td&gt; Gibco </td><td> 15230-147 </td><td></td></tr><tr><td> ** DNase I </td><td> Reagens </td><td> Roche </td><td> 104159 </td><td></td></tr><tr><td> ** Sojabohnentrypsininhibitor </td><td> Reagens </td><td> Sigma </td><td> T6522 </td><td></td></tr><tr><td> Pen / Strep </td><td> Reagens </td><td> Gibco </td><td> 15140-122 </td><td></td></tr><tr><td> Nr. 10 Skalpell </td><td> Chirurgisches Werkzeug </td><td> BD </td><td> 371610 </td><td></td></tr><tr><td> Petrischale </td><td> Kultur ware </td><td> BD Falcon </td><td> 353003 </td><td></td></tr><tr><td> Kleine Pinzette </td><td> Chirurgische Instrumente </td><td> Feine Science Tools </td><td> 11050-10 </td><td></td></tr><tr><td> Zellsieb </td><td> Sieb </td><td> BD Falcon </td><td> 352340 </td><td></td></tr><tr><td> T25-Kolben </td><td> Kultur ware </td><td> Nalge Nunc internationalen </td><td> 136196 </td><td></td></tr><tr><td> T80-Kolben </td><td> Kultur ware </td><td> Nalge Nunc international </td><td> 178905 </td><td></td></tr><tr><td> 15 ml Röhrchen </td><td> Kultur ware </td><td> BD Falcon </td><td> 352096 </td><td></td></tr><tr><td> 50 ml Röhrchen </td><td> Kultur ware </td><td> BD Falcon </td><td> 352070 </td><td></td></tr><tr><td> EGF </td><td> Wachstumsfaktor </td><td> R &amp; D </td><td> 2028-EG </td><td></td></tr><tr><td> b-FGF </td><td> Wachstumsfaktor </td><td> R &amp; D </td><td> 3139-FB </td><td></td></tr><tr><td> Heparin </td><td> Wachstumsfaktor </td><td> Sigma </td><td> H4784 </td><td> Rekonstituierte in PBS </td></tr></tbody></table> * Um HEM machen, mischen 1x10L Paket MEM and160ml 1M HEPES und bringen das Volumen auf 8,75 L mit destilliertem Wasser. Setzen Sie den End-pH um 7.4 Eind lagern bei 4 ° C. ** Zur Vorbereitung Trypsin-Inhibitor-Lösung, stellen Sie zunächst 10 ml DNase I-Lösung (100 mg DNase in 100 ml HEM gelöst) und fügen Sie dann 0,14 g Trypsin-Inhibitor zur DNase-Lösung und endlich das Volumen bis zu 1 Liter mit HEM. Keep Aliquots der Endprodukte in -20 ° C Gefrierschrank.

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

Stem-like Zellen in Tumoren wie Brust-, Lungen-, Darm-, Prostata und Gehirn isoliert worden. Ein kritischer Punkt bei all diesen Tumoren, insbesondere in Glioblastom mutliforme (GBM), zu identifizieren und zu isolieren tumorauslösende Zellpopulation (s), um ihre Rolle in der Tumorentstehung, Progression, und ein erneutes Auftreten zu untersuchen. Understanding tumorauslösende Zellpopulationen werden Hinweise auf der Suche nach wirksamen Therapien für diese Tumoren liefern. Die Neurosphäre Assay (NSA) aufgrund seiner Einfachheit und Reproduzierbarkeit als die Methode der Wahl für die Isolierung und Vermehrung von vielen dieser Tumorzellen eingesetzt. Dieses Protokoll zeigt die Neurosphäre Kultur-Methode zu isolieren und zu erweitern Stamm-ähnliche Zellen in operativ entfernt menschlichen GBM Tumorgewebe. Die Verfahren beinhalten eine erste chemische Verdauung und mechanische Dissoziation von Tumorgewebe und anschließend plating die daraus resultierenden einzigen Zellsuspension in NSA Kultur. Nach 7-10 Tagen, primäre Neurosphären von 150-200 um im Durchmesser beobachtet werden und sind für die weitere Passage und Expansion bereit.

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

Dieses Video zeigt, Protokoll der Isolierung und Expansion von Stammzellen, wie Zellen, die aus operativ entfernt menschlichen Glioblastom mutliforme (GBM) Tumorgewebe mit dem Neurosphäre Assay Kultur-Methode.

Isolation und Expansion der menschlichen Glioblastoma multiforme Tumorzellen unter Verwendung der Neurosphäre Assay

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 Aufrufe.  University of Florida. Dieses Video zeigt, Protokoll der Isolierung und Expansion von Stammzellen, wie Zellen, die aus operativ entfernt menschlichen Glioblastom mutliforme (GBM) Tumorgewebe mit dem Neurosphäre Assay Kultur-Methode.

Serie: 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.

Isolation und Expansion der erwachsenen Maus Neural Stem Cells mit dem Neurosphäre 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 ...

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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.

Gründung Embryonale Maus Neural Stem Cell Culture Mit dem Neurosphäre 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 ...

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.

Isolierung und Kultivierung humaner Zellen pilzförmige Papillen Geschmack

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-Umprogrammierung der Perizyten-abgeleiteten Zellen des erwachsenen menschlichen Gehirn in Neuronen induziert

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.

Quantitative Messung der relativen Retinsäure Niveaus in E8.5 Embryonen und Neurosphäre Kulturen Mit der F9 RARE-Lacz Zell-basierte 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.

Analyse der Spliceosomalen snRNA-Lokalisierung in menschlichen Hela-Zellen mit Mikroinjektion

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.

Ableitung, Expansion, Kryokonservierung und Charakterisierung von mikrovaskulären Endothelzellen des Gehirns aus humaninduzierten Pluripotenten Stammzellen

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 als assoziatives Lernwerkzeug zur kognitiven Beurteilung in Lern- und Gedächtnisstudien mit erwachsenen Zebrafischen

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.

Isolierung, Kultur und Charakterisierung von primären Schwann-Zellen, Keratinozyten und Fibroblasten aus der menschlichen Vorhaut

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.

Humane Mikroglia-ähnliche Zellen: Differenzierung aus induzierten pluripotenten Stammzellen und In-vitro-Lebendzell-Phagozytose-Assay mit humanen Synaptosomen

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