Questo lavoro è stato sostenuto dalla Royal Society URF concessione UF051616, Regno Unito e il Consiglio europeo della ricerca StG concessione 243.261 a BS. Il lavoro in MZ laboratorio è anche supportato da una California Institute of Regenerative Medicine concessione RB1-01.417.

1. Neural isolamento delle cellule progenitrici <ol><li> Dissect cervelli interi da E14-16 topi e posto a freddo DMEM/F12 medio basale. Rimuovere tutte le meningi con un microscopio anatomico e cervelli di trasferimento in una capsula Petri 35 millimetri. </li><li> Utilizzare pinze sottili a dissociarsi meccanicamente cervello in frammenti di tessuto e trasferirli in una provetta da 15 ml, quindi centrifugare i campioni a 800 rpm per 3 min per rimuovere i detriti. </li><li> Aggiungi DMEM/F12 contenenti bFGF e di EGF e triturare con una pipetta 1 ml. </li><li> Far passare la sospensione cellulare attraverso un filtro cella per ottenere una sospensione di cellule singole. </li><li> Cellule Plate in palloni a 2-5 x 10 4 cellule / ml, quindi eseguire un mezzo completo cambio ogni 3 giorni e il passaggio delle cellule ogni 6 giorni. </li><li> Dopo almeno 5 passaggi, digest neurosfere di singole cellule con tripsina e EDTA e crescere sulle camere electrotactic poly-D-lysine/laminin-coated (preparato come descritto di seguito in 2). Utilizzare il terreno di coltura contenente N2-supplemento,bFGF EGF e in ogni momento di mantenere le proprietà di NPC. </li></ol> 2. Preparazione della camera electrotactic <ol><li> Preparare 22 x 11 mm strisce di vetro dividendo in autoclave 22 x 22 mm spessore coprioggetto n.1 in mezzo utilizzando una penna di diamante. </li><li> Creare un free-standing in vetro e di dimensioni interne 22 x 10 mm incollando insieme quattro in verticale in piedi 22 x 11 mm con strisce di alto vuoto grasso al silicone. Lasciare asciugare i pozzetti completamente e indurire durante la notte. </li><li> Il giorno seguente, collegare due strisce 22 x 11 mm di vetro, parallele fra loro lasciando uno spazio di 10 mm, alla base di un piatto di coltura da 100 mm con grasso siliconico. Sigillare la regione tra queste strisce inserendo un x 22 22 mm copertura antiscivolo a ciascuna estremità, fissato al fondo del recipiente con il grasso su tre lati, ma non quella più vicina al centro. </li><li> Posizionare il vetro ben preparata nella fase 2,2 sul coperchio scivola in modo che le pareti interne creare uno spazio confinato per la seminale cellule sul fondo della piastra. Acqua a prova di tutti i giunti con grasso al silicone. Coat questa regione limitata sequenzialmente con poli-D-lisina quindi laminina: aggiungere 1 ml di poli-D-lisina nella camera e lasciare per 5 minuti per consentire il poli-D-lisina di legarsi al fondo della piastra, il lavaggio della camera con PBS sterile due volte, quindi diluire laminina in PBS sterile per dare 20 pg / ml e utilizzare per coprire il fondo della piastra. Lasciare a temperatura ambiente per tutta la notte. </li><li> Il giorno seguente, le cellule raccolto e preparare 1 ml di sospensione contenente 1 x 104 cellule. Rimuovere laminina dal vetro bene, permettendo di asciugare completamente e sostituirlo con 1 ml di sospensione cellulare. Porre la capsula in incubatore a 37 ° C per un minimo di 4 ore per consentire fissaggio. </li><li> Una volta che le cellule confluenti sono sufficientemente rimuovere tutto mezzo dalla camera. Rimuovere con cautela il vetro bene. Formare un tetto sopra le celle con attenzione collegando, con grasso al silicone, uno sterilizzato 22 x 22 mm vetro coprioggettoponte tra le due strisce di 22 x 11 mm. Coprire le cellule con qualche goccia di medium per evitare l&#39;essiccamento fuori. </li><li> Forma un serbatoio isolato mezzo a ciascuna estremità della camera creando due barriere stagne grasso al silicone che corrono da un bordo del piatto all&#39;altro, oltre il tetto della camera. Riempire la camera con mezzo fresco, garantendo un flusso continuo da un serbatoio mezzo all&#39;altro. Ritorna il piatto per l&#39;incubatore per 12 ore per consentire il recupero delle cellule. </li></ol> 3. Applicazione di un campo elettrico alla camera electrotactic <ol><li> Preparare un coperchio per coprire il piatto forando due fori, uno posizionato su ciascun serbatoio della camera di migrazione. </li><li> Sostituire tutto medium nella camera con terreno di coltura contenente 25 mM di tampone HEPES e trasferire il piatto alla temperatura controllata sistema di imaging. Impostare i parametri sperimentali per il time-lapse e multi-posizione di registrazione. Allineare camera in modo che il catodo e l&#39;anodo sonosulla sinistra e destra rispettivamente, per assicurare che il vettore EF corre orizzontalmente come visto al microscopio e registrata nel sistema di imaging. </li><li> Riempire due bicchieri con la soluzione di Steinberg (58 mM NaCl, 0,67 mM KCl, 0,44 mM Ca (NO 3) 2 .4 H 2 0, 1,3 mM MgSO 4 .7 H 2 0 e 4,6 mM Trizma base, pH 7,4). Collegare un becher separato per ciascun serbatoio medio usando pre-preparati ponti di vetro (tubi di vetro ~ 13 cm di lunghezza e ~ 3 mm di diametro, e piegato a U da riscaldamento in una fiamma Bunsen), riempite con 2% (w / v) Steinberg&#39;s-agar soluzione, passando attraverso i fori del coperchio. Completare il circuito elettrico inserendo Ag / AgCl elettrodi collegati ad un alimentatore DC in ogni bicchiere di soluzione di Steinberg. </li><li> Impostare il selettore di tensione di alimentazione a 0 e accendere. Misurare la tensione attraverso la camera electrotactic mentre ruotando la manopola di tensione, usando un misuratore di tensione, e regolare a seconda delle esigenze sperimentali. </li><li> Start time-lapse. Eseguire una variazione media e regolare la tensione, come richiesto, ogni ora. Mezzo fresco, farmaci, agenti chimici o possono essere aggiunti ai serbatoi come richiesto. Quando esegue cambiamento mezzo, due opzioni può essere considerato come di seguito: <ol><li> Opzione n.1 - Per mettere in pausa il time-lapse temporaneamente, rimuovere con cautela ponti di vetro dalla camera per evitare perturbante coperchio coperchio, utilizzare un sterilizzato pipetta Pasteur gentilmente sostituire tutte di medie dimensioni con terreno fresco e mettere i ponti di vetro schiena, poi riprendere la registrazione. </li><li> Opzione No.2 - In alternativa, misura coperchio coperchio 4 con due fori (posizionato su ciascun serbatoio della camera di migrazione) può anche essere utilizzato per la variazione media. Due collegamento vetro ponti, gli altri due per cambiare mezzo. L&#39;opzione 2 consente di registrare in continuo, senza interferenze durante il cambio medio. </li></ol></li></ol> 4. Preparazione della fetta organotipica midollo spinale <ol><li> Dissect Lumbcorde ar spinale di due settimane vecchio C57 BL / 6 topi. </li><li> Tagliate midollo spinale in 500 sezioni micron di spessore con un elicottero del tessuto McIlwain. </li><li> Fette separati sotto un microscopio anatomico e selezionare fette con intatta sagittale / assiale struttura del midollo spinale. </li><li> Fette piastra in una capsula Petri 35 millimetri Matrigel contenente 30 microlitri e posizionare il più vicino al centro possibile e mantenuti in 5% CO 2 incubatore a 37 ° C per almeno 30 min fino proteine ​​Matrigel auto-assemblano per produrre un film sottile che copre la superficie della fetta midollo spinale. È molto importante assicurarsi che Matrigel è stato completamente assemblato, incubare la piastra di Petri a 37 ° C per altri 30 minuti, se necessario. </li><li> Aggiungere 4-6 ml DMEM/F-12 mezzo contenente 25 mM di tampone HEPES e 15-20% siero di vitello fetale delicatamente in modo da evitare flusso medio direttamente sulla fetta. Assicurarsi che la sezione non è completamente sommerso nel mezzo, lasciando la superficie di espianti ben espostol&#39;aria. Modificare media due volte a settimana. </li></ol> 5. L&#39;iniezione di Hoechst 33342 NPC etichettati nella fetta organotipica midollo spinale <ol><li> Preparare la sospensione NPC a 1 × 10 6 cellule / microlitro. </li><li> Pre-incubare la sospensione cellulare in terreno con 5 pM Hoechst 33342 per 30 min. </li><li> Utilizzare tubo capillare di vetro a microinject 2 microlitri di sospensione nella sezione midollo spinale lentamente sotto un microscopio. Assicurarsi capillare di vetro passa attraverso il matrigel (parte rosa sotto microscopio) e raggiungere all&#39;interno della fetta midollo spinale (tessuto grigio sotto microscopio), rimangono all&#39;interno della fetta midollo spinale per almeno 30 secondi per evitare la sospensione cellulare investito. Mettere la piastra di Petri contenente fetta del midollo spinale in incubatore (37 ° C 5% CO 2) e lasciare tutta la notte. </li><li> Il giorno seguente, applicare un EF di 500 mV / mm alla fetta midollo spinale contenente Hoechst 33342-marcati NPC nella camera electrotactic (utilizzando i metodi descritti nella3). </li></ol> 6. Risultati rappresentativi Quando NPC sono stati esposti ad una serie di EF fisiologiche, hanno mostrato la migrazione cellulare altamente orientata verso il catodo (Figura 1). La stessa osservazione è stata fatta a livello di singola cellula sulla organotipica spinale modello fetta cavo ex vivo, uno mimando ambiente 3D in condizioni in vivo (Figura 2). <img alt="Figura 1" src="/files/ftp_upload/3453/3453fig1.jpg" /> Figura 1. NPCs mostrano la migrazione diretta in EF. PC ha mostrato grande migrazione diretta verso il catodo, quando esposti ad EF, linee rosse e frecce blu rappresentano le traiettorie e la direzione di movimento delle cellule (A). B mostra i percorsi di migrazione degli NPC. Bar: 50 micron. <img alt="Figura 2" src="/files/ftp_upload/3453/3453fig2.jpg" /> Figura 2. NPC trapiantati mostrano la migrazione diretta verso il catodo nella fetta organotipica midollo spinale </strong&gt;. (A) NPC marcati con Hoechst 33342 sono stati trapiantati in sezione organotipica midollo spinale al punto di partenza del trattamento EF. NPC migrazione direzionale verso il catodo per 2,5 ore, a questo punto la polarità EF è stata invertita (B). Alterare la polarità EF ha innescato una brusca inversione di electrotaxis nuovo verso il catodo (C). (D) Immagine di NPC trapiantate all&#39;interno della fetta di midollo spinale, alla fine del time-lapse. (E) Una ricostruzione 3D di NPC trapiantate all&#39;interno della fetta di midollo spinale. 3D sezioni scansione erano di 300 pm di spessore, a partire dalla metà e termina nella parte inferiore della fetta. Le linee tratteggiate indicano le posizioni relative della stessa popolazione di cellule trapiantate all&#39;inizio, inversione, e punti finali del trattamento EF (A - C, rispettivamente). Punte di freccia indicano la stessa popolazione di Hoechst 33342 NPC etichettati. Bar: 50 micron.

<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>

Non abbiamo nulla da rivelare.

I protocolli che usiamo sono basati su studi precedenti 5,11. L&#39;utilizzo di questi metodi, cultura stabile ed elettrico condizioni attuali può essere mantenuta, mentre l&#39;applicazione di un EF via ponti agar, soluzione Steinberg, e Ag / AgCl elettrodi, alle cellule o fette coltivate in camere electrotactic custom-progettati di dimensioni standardizzate e precise. La profondità delle camere può essere regolata per accogliere campione per diversi spessori 11, e nel caso di cellule, la dimen...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> FGF-base ricombinante umano </td><td> Invitrogen </td><td> PHG0026 </td><td> 20 ng / mL </td></tr><tr><td> EGF umano ricombinante </td><td> Invitrogen </td><td> PHG0311 </td><td> 20 ng / mL </td></tr><tr><td> N2-Supplement (100X) di liquido </td><td> Invitrogen </td><td> 02048 </td><td></td></tr><tr><td> DMEM/F12 medio (glucosio) </td><td> Invitrogen </td><td> 31330-095 </td><td></td></tr><tr><td> Poly-D-lisina </td><td> Millipore </td><td> A-003-E </td><td></td></tr><tr><td> Naturale del mouse laminina </td><td> Invitrogen </td><td> 23017-015 </td><td></td></tr><tr><td> Fattore di crescita ridotta Basement Membrane Matrix (Matrigel) </td><td> BD Biosciences </td&gt; <td> 354230 </td><td></td></tr><tr><td> Tampone HEPES </td><td> Gibco </td><td> 15630 </td><td></td></tr><tr><td> McIlwain tessuto chopper </td><td> Il Laboratorio Mickle Engineering Co Ltd </td><td> TC752-PD </td><td></td></tr><tr><td> Dow Corning alto vuoto grasso al silicone </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

Endogeni campi elettrici (EFS) si trovano naturalmente nel vivo e giocare un ruolo critico durante tessuto / organo di sviluppo e rigenerazione, compresa quella del 1,2 del sistema nervoso centrale. Questi EF endogeni sono generati da regolazione cellulare di trasporto ionico combinata con la resistenza elettrica di cellule e tessuti. È stato riportato che applicata EF trattamento può promuovere la riparazione funzionale di lesioni del midollo spinale negli animali e nell&#39;uomo 3,4. In particolare, EF-diretta migrazione delle cellule è stata dimostrata in un&#39;ampia varietà di tipi cellulari 5,6, incluse le cellule progenitrici neurali (NPC) 7,8. Applicazione di corrente continua (DC) EFS non è una tecnica comunemente disponibili nella maggior parte dei laboratori. Abbiamo descritto i protocolli modalità di applicazione di EFS DC per colture cellulari e tissutali in precedenza 5,11. Qui vi presentiamo un video dimostrativo dei metodi standard basato su una intensità di campo calcolato per istituire uno 2Dd ambienti 3D per i PNG, e per indagare le risposte cellulari alla stimolazione EF in entrambe le singole condizioni di crescita delle cellule in 2D, e la fetta organotipica midollo spinale in 3D. Il cordslice spinale è un tessuto destinatario ideale per studiare i comportamenti degli NPC ex vivo, post-trapianto, perché l&#39;organizzazione del tessuto citoarchitettonica è ben conservato all&#39;interno di queste culture 9,10. Inoltre, questo modello ex vivo permette inoltre procedure che non sono tecnicamente realizzabile per monitorare le cellule in vivo usando time-lapse a livello di singola cellula. È essenziale valutare criticamente comportamenti delle cellule non solo in un ambiente 2D, ma anche in una condizione 3D organotipica che imita l&#39;ambiente in vivo. Questo sistema consentirà di imaging ad alta risoluzione con copertura in vetro a base di pesce in tessuto o cultura organistica con tracking 3D della migrazione singola cellula in vitro ed ex vivo e può essere una tappa intermedia prima di passare in vivo paradigmi.

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

Questo protocollo vengono utilizzati dei metodi utilizzati per stabilire gli ambienti 2D e 3D in custom-designed camere electrotactic, che permette di monitorare le celle<em&gt; In vivo / ex vivo</em&gt; Con time-lapse a livello di singola cellula, al fine di indagare galvanotaxis / electrotaxis e le altre risposte cellulari a corrente continua (DC) campi elettrici (EFS).

Di campo elettrico controllato migrazione diretta di cellule progenitrici neurali in ambienti 2D e 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 Views.  Cardiff University. Questo protocollo vengono utilizzati dei metodi utilizzati per stabilire gli ambienti 2D e 3D in custom-designed camere electrotactic, che permette di monitorare le celle In vivo / ex vivo Con time-lapse a livello di singola cellula, al fine di indagare galvanotaxis / electrotaxis e le altre risposte cellulari a corrente continua (DC) campi elettrici (EFS).

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

Adaptation of a Haptic Robot in a 3T fMRI

Functional magnetic resonance imaging (fMRI) provides excellent functional brain imaging via the BOLD signal 1 with advantages including non-ionizing radiation, millimeter spatial accuracy of anatomical and functional data 2, and nearly real-time analyses 3. Haptic robots provide precise measurement and control of position and force of a cursor in a reasonably confined space. Here we combine these two technologies to allow precision experiments involving motor control with haptic/tactile environment interaction such as reaching or grasping. The basic idea is to attach an 8 foot end effecter supported in the center to the robot 4 allowing the subject to use the robot, but shielding it and keeping it out of the most extreme part of the magnetic field from the fMRI machine (Figure 1).
The Phantom Premium 3.0, 6DoF, high-force robot (SensAble Technologies, Inc.) is an excellent choice for providing force-feedback in virtual reality experiments 5, 6, but it is inherently non-MR safe, introduces significant noise to the sensitive fMRI equipment, and its electric motors may be affected by the fMRI's strongly varying magnetic field. We have constructed a table and shielding system that allows the robot to be safely introduced into the fMRI environment and limits both the degradation of the fMRI signal by the electrically noisy motors and the degradation of the electric motor performance by the strongly varying magnetic field of the fMRI. With the shield, the signal to noise ratio (SNR: mean signal/noise standard deviation) of the fMRI goes from a baseline of &tilde;380 to &tilde;330, and &tilde;250 without the shielding. The remaining noise appears to be uncorrelated and does not add artifacts to the fMRI of a test sphere (Figure 2). The long, stiff handle allows placement of the robot out of range of the most strongly varying parts of the magnetic field so there is no significant effect of the fMRI on the robot. The effect of the handle on the robot's kinematics is minimal since it is lightweight (&tilde;2.6 lbs) but extremely stiff 3/4" graphite and well balanced on the 3DoF joint in the middle. The end result is an fMRI compatible, haptic system with about 1 cubic foot of working space, and, when combined with virtual reality, it allows for a new set of experiments to be performed in the fMRI environment including naturalistic reaching, passive displacement of the limb and haptic perception, adaptation learning in varying force fields, or texture identification 5, 6.

The adaptation and use of a haptic robot in a 3T fMRI is described.

Adattamento di un robot tattile in un fMRI 3T

Functional magnetic resonance imaging (fMRI) provides excellent functional brain imaging via the BOLD signal 1 with advantages including non-ionizing ...

Ricerca

Bioingegneria

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 coltivazione di cellule progenitrici neurali umane in un 3-dimensionale di auto-assemblaggio 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 ...

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.

Ordinazione di singole cellule e singola embrioni in 3D confinamento: Un nuovo dispositivo per l&#39;alta Screening contenuti

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

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

A base di seta-collagene Engineered Modello 3D del tessuto neurale polarizzata

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

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.

Electrotaxis Studi di polmone cellule tumorali usando un Multicanale Dual-campo elettrico Microfluidic 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.

Migliorare 2D e 3D della pelle<em&gt; In Vitro</em&gt; modelli con Macromolecole affollamento

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.

Matrici 2D e 3D per studiare formazione e attività lineare invazienti

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.

Che caratterizzano la migrazione delle cellule all'interno del tridimensionale In Vitro ferita ambienti

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.

Migliorata 3D idrogel culture di cellule gliali primari In Vitro modellazione del Neuroinflammation

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.

Manipolazione di singole cellule staminali neurali e neuroni nelle fette cerebrali utilizzando la microiniezione robotica

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