Ce travail a été financé par la subvention de la Société royale URF UF051616, Royaume-Uni et l&#39;European Research Council StG subvention à la norme BS 243261. Le travail en laboratoire est MZ également soutenue par une California Institute of Regenerative Medicine subvention RB1-01417.

1. Neural isolement de cellules souches <ol><li> Disséquer cerveaux entiers de E14-16 chez la souris et les placer dans le froid du milieu DMEM/F12 basale. Retirez toutes les méninges sous un microscope anatomique et les cerveaux de transfert dans un plat de 35 mm de Petri. </li><li> Utilisez une pince fine à dissocier mécaniquement cerveaux en fragments de tissus et de les transférer à un tube de 15 ml, puis centrifuger les échantillons à 800 tours par minute pendant 3 min pour éliminer les débris. </li><li> Ajouter DMEM/F12 contenant bFGF et EGF et triturer avec une pipette de 1 ml. </li><li> Passer la suspension de cellules à travers un tamis de cellules pour obtenir une suspension cellulaire unique. </li><li> Cellules de la plaque dans des flacons à 2-5 x 10 4 cellules / ml, puis effectuer un plein milieu changer tous les 3 jours et le passage des cellules tous les 6 jours. </li><li> Après au moins 5 passages, digest neurosphères à des cellules individuelles à l&#39;aide de la trypsine et l&#39;EDTA et de croître sur les chambres electrotactic poly-D-lysine/laminin-coated (préparé comme décrit ci-dessous en 2). Utilisez le milieu de culture contenant N2-supplément,bFGF et EGF à tout moment pour maintenir les propriétés de PNJ. </li></ol> 2. Préparation de la chambre de electrotactic <ol><li> Préparer 22 x 11 mm bandes de verre en divisant autoclave 22 x 22 mm d&#39;épaisseur des lamelles no.1 dans la moitié à l&#39;aide d&#39;un stylo en diamant. </li><li> Créer un verre autoportant et de dimensions intérieures 22 x 10 mm en recollant quatre verticalement debout 22 x 11 mm bandes avec de la graisse silicone de haute-vide. Autoriser les puits complètement sécher et durcir pendant la nuit. </li><li> Le lendemain, attacher deux 22 x 11 mm bandes de verre, parallèlement les uns aux autres en laissant un écart de 10 mm, à la base d&#39;une boîte de culture de 100 mm à l&#39;aide de la graisse silicone. Sceller la région entre ces bandes en plaçant un 22 x 22 mm lamelle à chaque extrémité, fixé au fond du récipient avec de la graisse sur trois côtés, mais pas celle la plus proche du centre. </li><li> Placez le verre bien préparé à l&#39;étape 2.2 sur le couvercle glisse de sorte que les murs intérieurs de créer un espace confiné pour l&#39;ensemencementles cellules sur le fond de la plaque. Imperméables à l&#39;eau tous les joints avec de la graisse de silicone. Manteau cette région confinée de manière séquentielle avec la poly-D-lysine, puis la laminine: ajouter 1 ml de poly-D-lysine dans la chambre et sortir pendant 5 min à permettre l&#39;acide poly-D-lysine pour se lier à la partie inférieure de la plaque; laver la chambre avec du PBS stérile à deux reprises, puis diluer la laminine dans du PBS stérile pour obtenir 20 pg / ml et utiliser pour couvrir le fond de la plaque. Laisser à température ambiante pendant la nuit. </li><li> Le lendemain, les cellules de récolte et de préparation de 1 ml de suspension contenant 1 x 104 cellules. Retirez la laminine de la vitre du bien, lui permettant de sécher complètement, et les remplacer par 1 ml de suspension cellulaire. Placer le plat dans l&#39;incubateur à 37 ° C pendant un minimum de 4 heures pour permettre la fixation. </li><li> Une fois que les cellules sont suffisamment confluente retirer tout le milieu de la chambre. Retirez délicatement le verre bien. Former un toit au-dessus des cellules en prenant soin de fixation, avec de la graisse de silicone, un autoclave 22 x 22 mm une lamelle en verrepontage entre les deux 22 x 11 mm bandes. Couvrir les cellules avec quelques gouttes de milieu pour éviter le dessèchement. </li><li> Former un réservoir moyen isolé à chaque extrémité de la chambre en créant deux barrières étanches graisse de silicone qui s&#39;exécutent d&#39;un bord de la coupelle à l&#39;autre, sur le toit chambre. Remplir la chambre avec un milieu frais, assurant un flux-travers d&#39;un réservoir de médium à l&#39;autre. Retour le plat dans l&#39;incubateur pendant 12 heures pour permettre la récupération des cellules. </li></ol> 3. L&#39;application d&#39;un champ électrique à la chambre de electrotactic <ol><li> Préparer un couvercle pour couvrir le plat en perçant deux trous, l&#39;un placé au-dessus de chaque réservoir de la chambre de la migration. </li><li> Remplacer tout le milieu dans la chambre du milieu de culture contenant 25 mM de tampon HEPES et transférer la capsule dans le système d&#39;imagerie à température contrôlée. Mettre en place les paramètres expérimentaux pour la time-lapse et multi-position d&#39;enregistrement. Aligner le chambre de sorte que la cathode et l&#39;anode sontà gauche et à droite respectivement, pour que le vecteur EF s&#39;étend horizontalement comme vu par le microscope et enregistrée dans le système d&#39;imagerie. </li><li> Remplir deux béchers avec une solution de Steinberg (58 mM de NaCl, 0,67 mM de KCl, 0,44 mM de Ca (NO 3) 2 .4 H 2 0, 1,3 mM MgSO 4 .7 H 2 0 et 4,6 mM Trizma de base, pH 7,4). Connecter un bécher séparée à chaque réservoir de médium à l&#39;aide de ponts de verre préparées à l&#39;avance des tubes de verre (environ 13 cm de long et environ 3 mm de diamètre, et pliée en forme de U par chauffage dans une flamme Bunsen), remplis de 2% (p / v) Steinberg&#39;s-agar solution, en passant par les trous dans le couvercle. Remplissez le circuit électrique en plaçant Ag / AgCl électrodes reliées à une alimentation CC dans chaque bécher de solution de Steinberg. </li><li> Réglez la molette de tension sur l&#39;alimentation à 0 et allumez-le. Mesurer la tension aux bornes de la chambre electrotactic tout en tournant le cadran de tension, à l&#39;aide d&#39;un voltmètre, et ajuster en fonction des besoins expérimentaux. </li><li> Début d&#39;enregistrement time-lapse. Effectuer une variation moyenne et réajuster la tension, selon les besoins, toutes les heures. Un milieu frais, les médicaments, les agents chimiques peuvent être ajoutées aux réservoirs, au besoin. Lorsque effectuer changement de milieu, deux options peuvent être considérées comme ci-dessous: <ol><li> Option n ° 1 - Pour mettre en pause l&#39;enregistrement time-lapse temporairement, retirer soigneusement les ponts en verre de la chambre à éviter de perturber Couvercle, utiliser un stérilisés pipette Pasteur doucement remplacer tout le milieu par du milieu frais et de mettre les ponts de verre arrière, puis reprendre l&#39;enregistrement. </li><li> Option n ° 2 - Par ailleurs, fait sur mesure Couvercle avec 4 trous (deux positionné au-dessus de chaque réservoir de la chambre de la migration) peut également être utilisé à des fins de changement de milieu. Deux ponts pour le verre de connexion, les deux autres pour changer à moyen terme. Option 2 permet l&#39;enregistrement en continu, sans ingérence lors du changement de support. </li></ol></li></ol> 4. Préparation de la tranche la moelle épinière organotypique <ol><li> Disséquer lumbcordons ar la colonne vertébrale de 2 semaines vieille BL C57 / 6 souris. </li><li> Trancher la moelle épinière en sections um d&#39;épaisseur 500 avec un hachoir de tissus McIlwain. </li><li> Tranches distinctes sous un microscope anatomique et sélectionner des tranches avec intacte sagittale / axiale structure de la moelle épinière. </li><li> Plate tranches dans un plat de 35 mm de Pétri contenant 30 ul Matrigel et les placer aussi près que possible du centre et de les maintenir dans un 5% de CO 2 incubateur à 37 ° C pendant au moins 30 min jusqu&#39;à ce que le Matrigel protéines s&#39;auto-assemblent pour produire un film mince qui recouvre la surface de la tranche la moelle épinière. Il est très important de s&#39;assurer que Matrigel a été entièrement assemblé, incuber la boîte de Pétri à 37 ° C pendant 30 min si nécessaire. </li><li> Ajouter 4-6 ml DMEM/F-12 milieu contenant 25 mM de tampon HEPES et 15-20% de sérum de veau foetal doucement de manière à éviter un écoulement moyen directement sur la tranche. Veiller à ce que la tranche n&#39;est pas entièrement submergée dans le milieu, laissant la surface des explants bien exposée aul&#39;air. Changer le milieu de deux fois par semaine. </li></ol> 5. L&#39;injection de Hoechst 33342 PNJ marquées dans la tranche la moelle épinière organotypique <ol><li> Préparer la suspension NPC à 1 × 10 6 cellules / ul. </li><li> Pré-incuber la suspension de cellules dans un milieu avec 5 uM Hoechst 33342 pour 30 min. </li><li> Utilisez tube capillaire en verre de micro-injection 2 pi de suspension dans la tranche la moelle épinière lentement sous un microscope. Assurez-vous que le tube capillaire en verre passe à travers le Matrigel (partie rose sous le microscope) et d&#39;atteindre à l&#39;intérieur de la tranche la moelle épinière (tissu gris sous le microscope), restent à l&#39;intérieur de la tranche la moelle épinière pendant au moins 30 secondes pour éviter la suspension de cellules de fonctionner plus. Mettez la boîte de Pétri contenant tranche la moelle épinière en incubateur (37 ° C 5% CO 2) et laissez-le pendant la nuit. </li><li> Le lendemain, appliquer une EF de 500 mV / mm à la tranche la moelle épinière contenant Hoechst 33342-labellisés PNJ dans la chambre electrotactic (en utilisant les méthodes décrites dans3). </li></ol> 6. Les résultats représentatifs Quand les PNJ ont été exposés à une gamme de FE physiologiques qu&#39;ils ont montré la migration des cellules fortement dirigée vers la cathode (Figure 1). La même observation a également été faite au niveau de la cellule unique sur le modèle organotypique tranche la moelle épinière ex vivo, une imitation environnement 3D dans des conditions in vivo (figure 2). <img alt="Figure 1" src="/files/ftp_upload/3453/3453fig1.jpg" /> Figure 1. PNJ montrent la migration dirigée dans FE. PC a montré la migration hautement orienté vers la cathode lors de l&#39;exposition pour les CE, les lignes rouges et les flèches bleues représentent les trajectoires et la direction de mouvement de la cellule (A). B montre les voies de migration des PNJ. Bar: 50 pm. <img alt="Figure 2" src="/files/ftp_upload/3453/3453fig2.jpg" /> Figure 2. PNJ transplantés afficher migration dirigée vers la cathode dans la tranche la moelle épinière organotypique </strong&gt;. (A) PNJ marqués avec Hoechst 33342 ont été transplanté dans la tranche la moelle épinière organotypique au point de départ du traitement EF. PNJ migré directionnelle vers la cathode pendant 2,5 heures, à quel point la polarité a été inversée EF (B). Modification de polarité EF déclenché un brusque retournement de électrotaxie vers la cathode nouvelle (C). (D) Image de PNJ transplantés dans la tranche la moelle épinière à la fin de l&#39;enregistrement time-lapse. (E) Une reconstruction 3D des PNJ transplantés dans la tranche la moelle épinière. 3D sections de balayage étaient 300 um d&#39;épaisseur, à partir du milieu et se terminant à la partie inférieure de la tranche. Les lignes pointillées indiquent la position relative de la même population de cellules transplantées au début, inversion, et les points d&#39;extrémité du traitement EF (A - C, respectivement). Têtes de flèches indiquent la même population de Hoechst 33342 PNJ labellisés. Bar: 50 pm.

<ol>
	<li>Huttenlocher, A., Horwitz, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing+with+electric+potential.">Wound healing with electric potential.</a> N. Engl. J. Med. 356, 303-303 (2007).</li><li>McCaig, C. D., Rajnicek, A. M., Song, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+cell+behavior+electrically:+current+views+and+future+potential.">Controlling cell behavior electrically: current views and future potential.</a> Physiol. Rev. 85, 943-943 (2005).</li><li>Borgens, R. B., Jaffe, L. F., Cohen, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+and+persistent+electrical+currents+enter+the+transected+lamprey+spinal+cord.">Large and persistent electrical currents enter the transected lamprey spinal cord.</a> PNAS. 77, 1209-1209 (1980).</li><li>Shapiro, S., Borgens, R., Pascuzzi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oscillating+field+stimulation+for+complete+spinal+cord+injury+in+humans:+a+phase+1+trial.">Oscillating field stimulation for complete spinal cord injury in humans: a phase 1 trial.</a> J. Neurosurg. Spine. 2, 3-3 (2005).</li><li>Zhao, M., Song, B., Pu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+signals+control+wound+healing+through+phosphatidylinositol-3-OH+kinase-gamma+and+PTEN.">Electrical signals control wound healing through phosphatidylinositol-3-OH kinase-gamma and PTEN.</a> Nature. 442, 457-457 (2006).</li><li>Yao, L., Shanley, L., McCaig, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+applied+electric+fields+guide+migration+of+hippocampal+neurons.">Small applied electric fields guide migration of hippocampal neurons.</a> J. Cell. Physiol. 216, 527-527 (2008).</li><li>Li, L., El-Hayek, Y. H., Liu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct-current+electrical+field+guides+neuronal+stem&#47;progenitor+cell+migration.">Direct-current electrical field guides neuronal stem&#47;progenitor cell migration.</a> Stem Cells. 26, 2193-2193 (2008).</li><li>Meng, X., Arocena, M., Penninger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI3K+mediated+electrotaxis+of+embryonic+and+adult+neural+progenitor+cells+in+the+presence+of+growth+factors.">PI3K mediated electrotaxis of embryonic and adult neural progenitor cells in the presence of growth factors.</a> Exp. Neurol. 227, 210-210 (2011).</li><li>Bonnici, B., Kapfhammer, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+stromal+cells+promote+neurite+extension+in+organotypic+spinal+cord+slice:+significance+for+cell+transplantation+therapy.">Bone marrow stromal cells promote neurite extension in organotypic spinal cord slice: significance for cell transplantation therapy.</a> Neurorehabil. Neural Repair. 22, 447-447 (2008).</li><li>Shichinohe, H., Kuroda, S., Tsuji, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+stromal+cells+promote+neurite+extension+in+organotypic+spinal+cord+slice:+significance+for+cell+transplantation+therapy.">Bone marrow stromal cells promote neurite extension in organotypic spinal cord slice: significance for cell transplantation therapy.</a> Neurorehabil. Neural Repair. 22, 447-447 (2008).</li><li>Song, B., Gu, Y., Pu, j. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+direct+current+electric+fields+to+cells+and+tissues+in+vitro+and+modulation+of+wound+electric+field+in+vivo.">Application of direct current electric fields to cells and tissues in vitro and modulation of wound electric field in vivo.</a> Nature Protocol. 2, 1479-1479 (2007).</li></ol>

Nous n&#39;avons rien à communiquer.

Les protocoles que nous utilisons sont basés sur des études antérieures 5,11. L&#39;utilisation de ces méthodes, la culture stable et électrique conditions actuelles peut être maintenue tout en appliquant un EF par des ponts d&#39;agar, la solution de Steinberg, et Ag / AgCl électrodes, à des cellules cultivées ou en tranches dans des chambres electrotactic conçus sur mesure de dimensions normalisées et précis. La profondeur de chambres peut être ajusté pour tenir compte d&#39;échantillon diff?...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> FGF-base humaine recombinante </td><td> Invitrogen </td><td> PHG0026 </td><td> 20 ng / ml </td></tr><tr><td> EGF humain recombinant </td><td> Invitrogen </td><td> PHG0311 </td><td> 20 ng / ml </td></tr><tr><td> N2-Supplément (100X) de liquide </td><td> Invitrogen </td><td> 02048 </td><td></td></tr><tr><td> DMEM/F12 (taux élevé de glucose) </td><td> Invitrogen </td><td> 31330-095 </td><td></td></tr><tr><td> Poly-D-Lysine </td><td> Millipore </td><td> A-003-E </td><td></td></tr><tr><td> Naturelle laminine de souris </td><td> Invitrogen </td><td> 23017-015 </td><td></td></tr><tr><td> Facteur de croissance réduit la membrane basale Matrix (Matrigel) </td><td> BD Biosciences </td&gt; <td> 354230 </td><td></td></tr><tr><td> Tampon HEPES </td><td> Gibco </td><td> 15630 </td><td></td></tr><tr><td> Tissue chopper McIlwain </td><td> Le Laboratoire Mickle Engineering Co Ltd </td><td> TC752-PD </td><td></td></tr><tr><td> Dow Corning graisse silicone de haute-vide </td><td> Sigma-Aldrich </td><td> Z273554 </td><td></td></tr></tbody></table>

electric field-controlled directed migration of neural progenitor cells in 2d and 3d environments

Endogènes des champs électriques (FE) sont naturellement présents in vivo et jouent un rôle crucial lors de tissus / organes de développement et de la régénération, y compris celle de la 1,2 du système nerveux central. Ces FE endogènes sont générées par la régulation cellulaire de transport ionique combinés à la résistance électrique de cellules et de tissus. Il a été rapporté que le traitement appliqué EF peut favoriser la réparation fonctionnelle des lésions de la moelle épinière chez les animaux et les humains 3,4. En particulier, la migration des cellules EF-dirigée a été démontrée dans une grande variété de types cellulaires 5,6, y compris les cellules progénitrices neurales (PNJ) 7,8. Demande de courant continu (DC) FE n&#39;est pas une technique couramment disponible dans la plupart des laboratoires. Nous avons décrit des protocoles détaillés pour l&#39;application de la FE DC à cultures cellulaires et tissulaires préalablement 5,11. Nous présentons ici une vidéo de démonstration de méthodes standards basées sur une intensité de champ calculée à mettre en place une 2Dd environnements 3D pour les PNJ, et d&#39;enquêter sur les réponses cellulaires à la stimulation EF dans les deux conditions de croissance des cellules simples en 2D, et la tranche cordon organotypique épinière en 3D. Le cordslice épinière est un tissu receveur idéal pour étudier les comportements des PNJ ex vivo, post-transplantation, parce que l&#39;organisation des tissus cytoarchitectoniques est bien conservé au sein de ces cultures 9,10. En outre, ce modèle ex vivo permet également des procédures qui ne sont pas techniquement possible de suivre des cellules in vivo en utilisant time-lapse d&#39;enregistrement au niveau cellule unique. Il est essentiel d&#39;évaluer de façon critique les comportements cellulaires, non seulement dans un environnement 2D, mais aussi dans un état ​​3D organotypique qui imite l&#39;environnement in vivo. Ce système permettra l&#39;imagerie haute résolution en utilisant la couverture à base de verre plats dans les tissus ou organes de la culture avec un suivi en 3D de la migration cellulaire unique in vitro et ex vivo et peut constituer une étape intermédiaire avant de passer à en viparadigmes VO.

Watch this Scientific Journal Video about Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments at JoVE.com

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Ce protocole illustre des méthodes utilisées pour établir des environnements 2D et 3D dans les chambres electrotactic conçus sur mesure, qui permettent de suivre les cellules<em&gt; In vivo / ex vivo</em&gt; En utilisant time-lapse d&#39;enregistrement au niveau cellule unique, afin d&#39;enquêter sur galvanotaxie ou électrotaxie et d&#39;autres réponses cellulaires à diriger à courant continu) des champs électriques (EFS).

Champ électrique contrôlé par les migrations des Réalisé cellules progénitrices neurales dans des environnements 2D et 3D

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

electric-field-controlled-directed-migration-neural-progenitor-cells

Research

JoVE Journal

Bioengineering

11.6K vues.  Cardiff University. Ce protocole illustre des méthodes utilisées pour établir des environnements 2D et 3D dans les chambres electrotactic conçus sur mesure, qui permettent de suivre les cellules In vivo / ex vivo En utilisant time-lapse d'enregistrement au niveau cellule unique, afin d'enquêter sur galvanotaxie ou électrotaxie et d'autres réponses cellulaires à diriger à courant continu) des champs électriques (EFS).

Vidéo: Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

La culture de cellules progénitrices neurales humaines dans un hydrogel en 3 dimensions Peptide auto-assemblage

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Recherche

Bio-ingénierie

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

Commande de cellules simples et simple Embryons en 3D Confinement: un nouveau dispositif de criblage à haut débit

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

Électrotaxie l&#39;étude des cellules du cancer du poumon à l&#39;aide d&#39;un double-Multichannel champ électrique microfluidique Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

Améliorer 2D et 3D Skin<em&gt; In vitro</em&gt; Modèles utilisant macromoléculaire Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

2D and 3D Matrices to Study Linear Invadosome Formation and Activity

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor cells have to cross anatomical barriers to invade and migrate through the surrounding tissue in order to reach blood or lymphatic vessels. This requires the interaction between cells and the extracellular matrix (ECM). At the cellular level, many cells, including the majority of cancer cells, are able to form invadosomes, which are F-actin-based structures capable of degrading ECM. Invadosomes are protrusive actin structures that recruit and activate matrix metalloproteinases (MMPs). The molecular composition, density, organization, and stiffness of the ECM are crucial in regulating invadosome formation and activation. In vitro, a gelatin assay is the standard assay used to observe and quantify invadosome degradation activity. However, gelatin, which is denatured collagen I, is not a physiological matrix element. A novel assay using type I collagen fibrils was developed and used to demonstrate that this physiological matrix is a potent inducer of invadosomes. Invadosomes that form along the collagen fibrils are known as linear invadosomes due to their linear organization on the fibers. Moreover, molecular analysis of linear invadosomes showed that the discoidin domain receptor 1 (DDR1) is the receptor involved in their formation. These data clearly demonstrate the importance of using a physiologically relevant matrix in order to understand the complex interactions between cells and the ECM.

This protocol describes how to prepare a 2D mixed matrix, consisting of gelatin and collagen I, and a 3D collagen I plug to study linear invadosomes. These protocols allow for the study of linear invadosome formation, matrix degradation activity, and the invasion capabilities of primary cells and cancer cell lines.

Matrices 2D et 3D pour étudier la formation et l&#39;activité invadosome linéaire

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

Caractérisation de la Migration cellulaire dans les trois dimensions In Vitro plaie environnements

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

Amélioré l’Hydrogel 3D des Cultures de cellules gliales primaires pour In Vitro la modélisation du neuro-inflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

Manipulation of Single Neural Stem Cells and Neurons in Brain Slices using Robotic Microinjection

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, manipulate, and follow single cells in the brain tissue with high resolution over time. This task is extremely challenging due to the complexity of tissues in the brain. We have recently developed a robot, that guide a microinjection needle into brain tissue upon utilizing images acquired from a microscope to deliver femtoliter volumes of solution into single cells. The robotic operation increases resulting an overall yield that is an order of magnitude greater than manual microinjection and allows for precise labeling and flexible manipulation of single cells in living tissue. With this, one can microinject hundreds of cells within a single organotypic slice. This article demonstrates the use of the microinjection robot for automated microinjection of neural progenitor cells and neurons in the brain tissue slices. More broadly, it can be used on any epithelial tissue featuring a surface that can be reached by the pipette. Once set up, the microinjection robot can execute 15 or more microinjections per minute. The microinjection robot because of its throughput and versality will make microinjection a broadly straightforward high-performance cell manipulation technique to be used in bioengineering, biotechnology, and biophysics for performing single-cell analyses in organotypic brain slices.

This protocol demonstrates the use of a robotic platform for microinjection into single neural stem cells and neurons in brain slices. This technique is versatile and offers a method of tracking cells in tissue with high spatial resolution.

Manipulation de cellules souches neurales et de neurones dans les tranches de cerveau à l’aide de microinjection robotique

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, ...

Electric-Field-Induced Neural Precursor Cell Differentiation in Microfluidic Devices

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell culture system that was used for a long-term cell differentiation study using microscopy. The microfluidic system consists of the following major components: an optically transparent electrotactic chip, a transparent indium-tin-oxide (ITO) heater, a culture media-filling pump, an electrical power supply, a high-frequency power amplifier, an EF multiplexer, a programmable X-Y-Z motorized stage, and an inverted phase-contrast microscope equipped with a digital camera. The microfluidic system is beneficial in simplifying the overall experimental setup and, in turn, the reagent and sample consumption. This work involves the differentiation of neural stem and progenitor cells (NPCs) induced by direct current (DC) pulse stimulation. In the stem cell maintenance medium, the mouse NPCs (mNPCs) differentiated into neurons, astrocytes, and oligodendrocytes after the DC pulse stimulation. The results suggest that simple DC pulse treatment could control the fate of mNPCs and could be used to develop therapeutic strategies for nervous system disorders. The system can be used for cell culture in multiple channels, for long-term EF stimulation, for cell morphological observation, and for automatic time-lapse image acquisition. This microfluidic system not only shortens the required experimental time, but also increases the accuracy of control on the microenvironment.

In this study, we present a protocol for the differentiation of neural stem and progenitor cells (NPCs) solely induced by direct current (DC) pulse stimulation in a microfluidic system.

Différenciation des cellules précurseurs neuronales induites par le champ électrique dans les appareils microfluidiques

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell ...

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

Dispositifs électriques et magnétiques de champ pour la stimulation des tissus biologiques

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...