Ce travail a été soutenu par des subventions du Centre de Recherche en Floride tumeur cérébrale; Preston A. Wells Jr. Center for Therapy tumeur au cerveau.

1. Prélèvement de tissus GBM primaire <ol><li> Un glioblastome mutliforme (GBM) tumeur est obtenue à partir d&#39;un patient diagnostiqué avec le cancer et subissant une chirurgie. </li><li> Dispositions doivent être prises avec l&#39;équipe de neurochirurgie. Le neurochirurgien aura lieu la tumeur réséquée GBM dans un tube de taille appropriée contenant des cellules souches neurales (NSC) milieu de base additionné d&#39;antibiotiques 10-15% (Peniciline / streptomycine). Milieu froid doit être fournie à la salle d&#39;opération par le laboratoire. Alternativement, le froid tamponné HEPES milieu essentiel minimum (HEM) ou de tampon phosphate salin (PBS) à forte concentration d&#39;antibiotiques peuvent également être utilisés à cette fin. </li><li> Le tissu tumoral réséqué est livré au laboratoire sur la glace et placé sous le capot. </li></ol> 2. La dissociation de la tumeur primitive en une seule cellule de suspension <ol><li> L&#39;excès du support original / PBS est éliminé du tube Falcon et l&#39;échantillon est lavé 2-3 fois avec le 5-10ml de PBS / NSC milieu basal pour enlever le sang et les débris. Le PBS / milieu est éliminé et le tissu tumoral GBM est placé dans une boîte de Pétri. </li><li> Le tissu est coupé en petits morceaux et émincé avec une lame de bistouri n ° 10 en petits morceaux pour augmenter la surface pour le processus de traitement à la trypsine. Mincing peut prendre 1-3 minutes selon la taille de la tumeur. </li><li> Le tissu émincé est trypsinisées en 3-5ml de pré-chauffé à 0,05% de trypsine-EDTA pendant 10-15 minutes dans un bain d&#39;eau à 37 ° C. Une pipette électronique est utilisé pour transférer les morceaux minuscules tumeurs et de la trypsine dans un tube de 15ml Falcon. </li><li> Un volume égal de l&#39;inhibiteur de trypsine de soja est ajouté pour arrêter la réaction enzymatique trypsine après la période d&#39;incubation. </li><li> Inactivation trypsine est assurée par la suspension de pipetage de haut en bas plusieurs fois. Ensuite, la suspension est granulé par centrifugation à 800rpm (110g) pendant 5 min. </li><li> Le surnageant est éliminé et les morceaux de tissus sont remis en suspension dans 1ml de solution stérile NSC bamoyennes sal. Les bouquets sont dissociés par un léger pipetage haut et en bas (3-7 fois) jusqu&#39;à ce qu&#39;une bonne suspension laiteuse seule cellule est atteint. Le nombre d&#39;étapes de pipetage dépend directement de la taille des particules dans les tissus hachés. De longs et vigoureux dissociation mécanique devrait être évitée car elle pourrait entraîner la mort cellulaire et une réduction de la formation sphère. </li><li> Pour supprimer l&#39;ONU dissocié des pièces et des débris, 10-15 ml de milieu de base est ajoutée dans le tube et la suspension cellulaire est filtrée à travers une passoire de 40 microns de cellules dans un tube de 50ml. </li><li> La suspension filtrée est centrifugé à 800rpm (110g) pendant 5 min. Le surnageant est éliminé par la suite. </li><li> Cellules Pelletted sont ensuite resuspendues dans 1-2ml de NSC milieu complet pour le comptage cellulaire. </li></ol> 3. Numération cellulaire et placage <ol><li> 10 pl de la suspension cellulaire sont ajoutés à 90 uL de 0,04% bleu trypan dans un tube eppendorf 1ml. Remarque: d&#39;autres cellules appropriées dilutiontions peuvent également être utilisés. </li><li> Pipeter haut et en bas pour mélanger la suspension. 10 uL des cellules / bleu trypan mélange est transféré à hémocytomètre pour compter la densité cellulaire. </li><li> Les cellules sont étalées au NSC milieu complet (un mélange de NSC milieu basal et NSC prolifération supplément à un ratio de 9:1) complété avec 20ng/ml EGF, bFGF et 10ng/ml 1μl/ml de l&#39;héparine de 0,2% (2μg/ml) dans appropriée des récipients de culture tissulaire. 5 ml, 20 et 40 du milieu est utilisé pour des flacons T25, T80 et T175, respectivement. Des antibiotiques peuvent être ajoutés dans le milieu à une concentration de 1:100 pour diminuer risques de contamination. </li><li> Le ballon est placé dans un incubateur réglé à 37 ° C et 5% de CO 2. </li></ol> 4. Le repiquage et l&#39;expansion du GBM sphères dérivés: <ol><li> Lorsque les neurosphères atteint une taille moyenne de 150-200 m de diamètre, la culture est prêt pour la sous-culture. Le contenu de chaque flacon est retiré et placé dans une taille appropriée TISS stérilestube de culture ue, et centrifugé à 800 rpm (110 g) pendant 5 min à température ambiante. </li><li> Le surnageant est éliminé et le culot est resuspendu dans 1 ml de 0,05% de trypsine-EDTA. </li><li> Pour parvenir à une trypsinisation optimale, la suspension cellulaire est incubée à 37 ° C dans un bain d&#39;eau pendant 2-3 min. Pour arrêter l&#39;activité de la trypsine un volume égal de l&#39;inhibiteur de trypsine de soja est ajoutée à la suspension cellulaire et la suspension cellulaire est doucement introduit à la pipette de haut en bas. </li><li> La suspension cellulaire est centrifugée à 800 tours par minute (110g) pendant 5 min. Ensuite, le surnageant est éliminé et les cellules sont remises en suspension dans 1 ml de milieu NSC. </li><li> Un comptage des cellules est effectué comme décrit précédemment. </li><li> Les cellules sont étalées à une concentration de 5x10 4 cellules / ml dans NSC milieu complet additionné de facteurs de croissance et plaquées dans des navires de taille appropriée la culture de tissus tels que décrits dans la partie précédente. </li><li> Neurosphères secondaires se forment dans les 7-10 jours lorsqu&#39;elle est incubée à 37 ° C dans un environnement humideified incubateur avec 5% de CO 2. </li></ol> 5. Les résultats représentatifs: Après étalement de la récolte des cellules individuelles à partir de tissus tumoraux GBM, une tumeur proche des cellules souches prolifèrent et génèrent de petits amas de cellules composée de quelques cellules dans 3-4 jours (voir la vidéo et graphique 1). Comme ces grappes de croître, ils acquièrent une forme plus sphérique afin que par 7-8 jours, des sphères lumineuses bonne phase avec un diamètre moyen de 150-200 microns forme (voir la vidéo et graphique 2). A plus fort grossissement, de sphères saines habituellement démontrer microspikes à leur périphérie. Ayant grande sphère comme les grappes de 1-2 jours après l&#39;initiation de la culture est due à l&#39;existence de non-dissociée des touffes à l&#39;binging de la culture et ne doivent pas être confondus avec les sphères vrai. La quantité de débris dans la culture sphère tumeur primitive varie en fonction de la source initiale de tissu et si oui ou non il comprend tout tissu cérébral environnant. Techniques appropriées de préparation des tissusnotamment la dissociation enzymatique et mécanique et la filtration ultérieure de l&#39;échantillon avec une quantité suffisante de milieu peut aboutir à moins de débris dans la culture. <img src="/files/ftp_upload/3633/3633fig1.jpg" alt="Figure 1" /> Figure 1. Primaires de la culture sphère GBM 4 jours après placage. Tumeur proche des cellules souches prolifèrent et génèrent de petits amas de cellules dans les 3-4 jours. Grossissement original; 20x. <img src="/files/ftp_upload/3633/3633fig2.jpg" alt="Figure 2" /> Figure 2. Passage d&#39;une culture sphère GBM 8 jours après placage. Grossissement original; 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>

Pas de conflits d&#39;intérêt déclarés.

Pour isoler souches neurales et de cellules progénitrices d&#39;adulte normal et le cerveau du fœtus 1, 2, 3, 4 et aussi une tumeur proche des cellules souches provenant de tissus de cancer comme le poumon 5, de la prostate 6, 7 et le cerveau maternel 8, 9 le dosage a été Neurosphère fréquemment utilisées comme la méthode de choix. L&#39;utilisation de ce test simple et reproductible, on peut générer un nombre indéfini de cellules de tissu tumoral réséqué qui montrent les mêmes caractéristiques que les cellules souches somatiques; multipotence ex vivo, la capacité de créer de nouvelles tumeurs lors de l&#39;implantation, et l&#39;auto-renouvellement. Ces cellules pourraient être utilisées pour étudier la biologie fondamentale sur le cancer des cellules dont la cellule à cellule des interactions, et de la différenciation, la migration, l&#39;invasion et la mort cellulaire. En outre, les tumeurs isolées proche des cellules souches de fournir un outil précieux pour étudier comment se forment les tumeurs, les progrès et rechutes et aussi de démêler les sous-tendent les mécanismes cellulaires dérivant qui pourraient éventuellement fournir des indications aux thérapeutiquesoptions.

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Tapez </td><td> Société </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> NeuroCult NSC milieu de base (l&#39;homme) </td><td> Medium </td><td> Stem Cell Technologies </td><td> 05750 </td><td></td></tr><tr><td> NeuroCult NSC Suppléments prolifération (Human) </td><td> Supplément de milieu </td><td> Stem Cell Technologies </td><td> 05753 </td><td></td></tr><tr><td> 0,05% de trypsine-EDTA </td><td> Réactif </td><td> Gibco </td><td> 25300-062 </td><td></td></tr><tr><td> * MEM </td><td> Réactif </td><td> Gibco </td><td> 41500-018 </td><td> Composante HEM </td></tr><tr><td> * HEPES </td><td> Réactif </td><td> Sigma </td><td> H4034 </td><td> Composante HEM </td></tr><tr><td> * L&#39;eau distillée </td><td> Réactif </td><td&gt; Gibco </td><td> 15230-147 </td><td></td></tr><tr><td> ** DNase I </td><td> Réactif </td><td> Roche </td><td> 104159 </td><td></td></tr><tr><td> ** Inhibiteur de la trypsine de soja </td><td> Réactif </td><td> Sigma </td><td> T6522 </td><td></td></tr><tr><td> Pen / Strep </td><td> Réactif </td><td> Gibco </td><td> 15140-122 </td><td></td></tr><tr><td> N ° 10 lame de bistouri </td><td> Outil chirurgical </td><td> BD </td><td> 371610 </td><td></td></tr><tr><td> Boîte de Pétri </td><td> Culture de la céramique </td><td> BD Falcon </td><td> 353003 </td><td></td></tr><tr><td> Une petite pince </td><td> Outils chirurgicaux </td><td> Outils Fine Science </td><td> 11050-10 </td><td></td></tr><tr><td> Tamis cellulaire </td><td> Tamis </td><td> BD Falcon </td><td> 352340 </td><td></td></tr><tr><td> T25 flacon </td><td> Culture de la céramique </td><td> Nalge Nunc internationale </td><td> 136196 </td><td></td></tr><tr><td> T80 flacon </td><td> Culture de la céramique </td><td> Nalge Nunc International </td><td> 178905 </td><td></td></tr><tr><td> Tubes de 15 ml </td><td> Culture de la céramique </td><td> BD Falcon </td><td> 352096 </td><td></td></tr><tr><td> Tubes de 50 ml </td><td> Culture de la céramique </td><td> BD Falcon </td><td> 352070 </td><td></td></tr><tr><td> EGF </td><td> Facteur de croissance </td><td> R &amp; D </td><td> 2028-EG </td><td></td></tr><tr><td> b-FGF </td><td> Facteur de croissance </td><td> R &amp; D </td><td> 3139-FB </td><td></td></tr><tr><td> Héparine </td><td> Facteur de croissance </td><td> Sigma </td><td> H4784 </td><td> Reconstitué en PBS </td></tr></tbody></table> * Pour faire HEM, mélanger des paquets 1x10L de MEM and160ml d&#39;HEPES 1M et porter le volume à 8,75 L en utilisant de l&#39;eau distillée. Réglez le pH final à 7,4 uned stocker à 4 ° C. ** Pour préparer la solution d&#39;inhibiteur de trypsine, faites d&#39;abord 10 ml de solution de DNase I (100 mg DNase dissous dans 100 ml de HEM), puis ajouter 0,14 g d&#39;inhibiteur de la trypsine à la solution de DNase et enfin faire le volume à 1 litre avec de HEM. Gardez aliquotes des produits finaux de -20 ° C au congélateur.

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

Proche des cellules souches ont été isolées dans les tumeurs comme le sein, poumon, côlon, prostate et du cerveau. Une question essentielle dans toutes ces tumeurs, en particulier dans mutliforme glioblastome (GBM), est d&#39;identifier et d&#39;isoler la tumeur population cellulaire initiateur (s) pour enquêter sur leur rôle dans la formation tumorale, la progression, et les récidives. Comprendre la tumeur initier les populations de cellules fournira des indices pour trouver des approches thérapeutiques efficaces pour ces tumeurs. Le dosage de Neurosphère (NSA) en raison de sa simplicité et sa reproductibilité a été utilisée comme méthode de choix pour l&#39;isolement et la propagation de nombreuses de cette cellules tumorales. Ce protocole illustre la méthode de culture Neurosphère à isoler et à étendre aux cellules souches dans la résection chirurgicale du tissu tumoral humain GBM. Les procédures comprennent une digestion chimique initial et de dissociation mécanique du tissu tumoral, et par la suite plaquage de la suspension cellulaire unique résultant de la culture NSA. Après 7-10 jours, primaire neurosphères du 150-200 m de diamètre peuvent être observés et sont prêts pour d&#39;autres repiquage et d&#39;expansion.

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

Ce protocole vidéo démontre l&#39;isolement et l&#39;expansion des cellules souches comme des cellules de glioblastome mutliforme résection chirurgicale humaine (GBM) tissu tumoral en utilisant la méthode de dosage Neurosphère culture.

L&#39;isolement et l&#39;expansion des cellules humaines de glioblastome multiforme tumeur utilisant le test Neurosphère

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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29.6K vues.  University of Florida. Ce protocole vidéo démontre l'isolement et l'expansion des cellules souches comme des cellules de glioblastome mutliforme résection chirurgicale humaine (GBM) tissu tumoral en utilisant la méthode de dosage Neurosphère culture.

Vidéo: 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.

L&#39;isolement et expansion des cellules souches neurales de souris adultes utilisant le test Neurosphère

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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Neurosciences

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.

Établir embryonnaires de la souris culture de cellules souches neurales utilisant le test Neurosphère

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.

Isolement et culture de cellules humaines fongiformes papilles gustatives

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 reprogrammation de cellules pericyte dérivés du cerveau humain adulte en neurones induits

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.

Mesure quantitative des niveaux relatifs à l&#39;acide rétinoïque E8.5 Embryons et Neurosphère Cultures Utilisation du F9 RARE-Lacz base de cellules 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 de la localisation spliceosomale snRNA dans les cellules humaines d'Hela utilisant la microinjection

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.

Dérivation, expansion, cryopréservation et caractérisation des cellules endothéliales microvasculaires cérébrales à partir de cellules souches pluripotentes induites par l’homme

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 en tant qu’outil d’apprentissage associatif pour l’évaluation cognitive dans les études d’apprentissage et de mémoire à l’aide de poissons-zèbres adultes

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.

Isolement, culture et caractérisation des cellules de Schwann primaires, des kératinocytes et des fibroblastes du prépuce humain

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.

Cellules humaines de type microglie : différenciation à partir de cellules souches pluripotentes induites et dosage in vitro de la phagocytose sur cellules vivantes à l’aide de synaptosomes humains

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