Diese Arbeit wurde von der Royal Society URF Zuschuss UF051616, Großbritannien und den European Research Council StG Zuschuss 243261 BS unterstützt. Die Arbeit im Labor MZ wird auch durch eine California Institute of Regenerative Medicine Zuschuss RB1-01417 unterstützt.

1. Neurale Vorläuferzellen Zellisolation <ol><li> Sezieren ganze Gehirne von E14-16-Mäusen und in kaltem DMEM/F12 Basismedium. Entfernen Sie alle Meningen unter einem Mikroskop anatomischen und Transfer Gehirne in eine 35 mm Petrischale. </li><li> Verwenden feinen Pinzette mechanisch zu distanzieren Gehirne in Gewebefragmente und übertragen Sie sie auf einem 15 ml-Tube, dann zentrifugieren Proben bei 800 rpm für 3 Minuten, um Verschmutzungen zu entfernen. </li><li> Add DMEM/F12 mit bFGF und EGF und verreiben mit einer 1 ml Pipette. </li><li> Übergeben Sie die Zellsuspension durch ein Sieb, um Zelle eine einzelne Zelle Suspension zu erhalten. </li><li> Platte Zellen in Kolben bei 2-5 x 10 4 Zellen / ml, führen Sie eine vollständige Medium alle 3 Tage und den Durchgang Zellen alle 6 Tage. </li><li> Nach mindestens 5 Passagen, Digest Neurosphären, um einzelne Zellen unter Verwendung von Trypsin und EDTA und wachsen auf poly-D-lysine/laminin-coated electrotactic Kammern (hergestellt wie unter Punkt 2 beschrieben). Mit dem wachsenden Medium mit N2-Supplement,bFGF und EGF zu jeder Zeit zu pflegen Eigenschaften der NPCs. </li></ol> 2. Herstellung der Kammer electrotactic <ol><li> Planen 22 x 11 mm Glasstreifen durch Teilen autoklaviert 22 x 22 mm Dicke Nr.1 ​​Deckgläser in der Mitte mit einem Diamant-Stift. </li><li> Erstellen Sie eine frei stehende Glas auch von Innenmaßen 22 x 10 mm miteinander verklebt, vier vertikal stehend 22 x 11 mm Streifen mit Hochvakuum-Silikonfett. Lassen Sie die Wells vollständig trocknen und aushärten über Nacht. </li><li> Am folgenden Tag legen zwei 22 x 11 mm Glas-Streifen, die parallel zueinander, wobei ein Spalt von 10 mm, auf der Basis eines 100 mm Kulturschale mit Silikonfett. Abdichten den Bereich zwischen diesen Streifen, indem eine 22 x 22 mm Deckglas an jedem Ende, an der Unterseite der Schale mit Fett auf drei Seiten, aber nicht dem Zentrum am nächsten. </li><li> Das Glas gut in Schritt 2.2 auf den Deckel rutscht vorbereitet, so dass die Innenwände einen geschlossenen Raum zu schaffen für die Aussaatdie Zellen auf der Unterseite der Platte. Wasserdichte alle Fugen mit Silikon einfetten. Coat diese Region beschränkt nacheinander mit Poly-D-Lysin dann Laminin: 1 ml Poly-D-Lysin in die Kammer zu verlassen und für 5 Minuten, damit das Poly-D-Lysin an der Unterseite der Platte zu binden; waschen Sie die Kammer mit sterilem PBS zweimal, dann verdünnt Laminin in sterilem PBS auf 20 ug / ml zu erhalten und zu verwenden, um die Unterseite der Platte bestimmt. Lassen Sie bei Raumtemperatur über Nacht. </li><li> Am folgenden Tag, Ernte-Zellen und bereiten 1 ml Suspension mit 1 x 104 Zellen. Entfernen Sie Laminin aus dem Glas gut, so dass es an der Luft trocknen vollständig und ersetzen mit 1 ml Zellsuspension. Die Schale im Inkubator bei 37 ° C für mindestens 4 Stunden zur Befestigung zu ermöglichen. </li><li> Sobald die Zellen konfluent sind ausreichend entfernen Medium aus der Kammer. Entfernen Sie vorsichtig das Glas gut. Bilden ein Dach über den Zellen durch vorsichtiges Befestigung, mit Silikonfett, einer autoklavierten 22 x 22 mm DeckglasBrückenschlag zwischen den beiden 22 x 11 mm Streifen. Die Zellen mit ein paar Tropfen des Mediums zu vermeiden Austrocknen. </li><li> Bilden eine isolierte Mediumspeicher an jedem Ende der Kammer durch die Schaffung von zwei wasserdichte Silikonfett Barrieren, die von einem Rand der Schale zur anderen laufen, über die Kammer Dach. Füllen der Kammer mit frischem Medium, wodurch eine Durchströmung von einem Medium Reservoir zu dem anderen. Senden Sie das Gericht in den Inkubator für 12 Stunden auf die Erholung der Zellen zu ermöglichen. </li></ol> 3. Anwendung eines elektrischen Feldes an die Kammer electrotactic <ol><li> Bereiten Sie einen Deckel auf die Schüssel durch das Bohren Sie zwei Löcher, eins über jedem Behälter der Migration Kammer positioniert zu decken. </li><li> Ersetzen Sie alle Medium in der Kammer mit Kulturmedium mit 25 mM HEPES-Puffer und die Schale in der temperaturgeführten Imaging-System. Richten Sie die experimentellen Parameter für Zeitraffer-und Multi-Positions-Aufnahme. Richten der Kammer, so dass die Kathode und Anodeauf der linken und rechten festgelegt, um sicherzustellen, dass die EF-Vektor horizontal verläuft, wie sie das Mikroskop betrachtet und in das Abbildungssystem. </li><li> Füllen Sie zwei Bechergläser mit Steinberg-Lösung (58 mM NaCl, 0,67 mM KCl, 0,44 mM Ca (NO 3) 2 · 4H 2 0, 1,3 mM MgSO 4 · 7H 2 0 und 4,6 mm Trizma Base, pH 7,4). Eine separate Becher auf jedes Medium Behälter unter Verwendung vorgefertigter Glasbrücken (Glasröhren ~ 13 cm lang und ca. 3 mm im Durchmesser, und Biegen zu einer U-Form durch Erhitzen in einem Bunsenbrenner), mit 2% (w / gefüllt v) Steinberg&#39;s-Agar-Lösung, die durch die Löcher im Deckel. Füllen Sie den Stromkreis, indem Ag / AgCl-Elektroden verbunden mit einer DC-Stromversorgung in jedem Becher der Steinberg-Lösung. </li><li> Stellen Sie die Spannung Regler an der Stromversorgung auf 0 und schalten Sie ein. Messen der Spannung über dem electrotactic Kammer beim Einschalten die Spannung Zifferblatt, mit einem Voltmeter, und Anpassung an die experimentellen Anforderungen. </li><li> Start Zeitraffer-Aufnahme. Führen Sie ein Mediumwechsel und justieren Sie die Spannung, je nach Bedarf, jede Stunde. Frisches Medium, Drogen oder chemische Mittel können zu den Stauseen hinzugefügt werden, wie erforderlich. Wenn durchführen Medium ändern, können zwei Optionen, wie unten in Betracht gezogen werden: <ol><li> Option Nr.1 ​​- Zum Anhalten der Zeitraffer-Aufnahme vorübergehend, entfernen Sie vorsichtig Glas Brücken von Kammer zu vermeiden störende Deckelklappe, verwenden Sie einen sterilisierten Pasteurpipette vorsichtig ersetzen alle Medium durch frisches Medium und stellte das Glas wieder Brücken, dann die Aufnahme fortzusetzen. </li><li> Option Nr.2 - Alternativ, custom made Deckel Deckel mit 4 Löchern (zwei über jedem Reservoir der Migration Kammer positioniert) kann auch für mittlere Veränderung Zweck verwendet werden. Zwei für Glasbrücken Verbindung, die beiden anderen für Medienwechsel. Option 2 ermöglicht eine kontinuierliche Aufzeichnung ohne Störungen während Mediumwechsel. </li></ol></li></ol> 4. Herstellung des organotypischen Rückenmarks Slice <ol><li> Sezieren Lumbar Rückenmark von 2 Wochen alten C57 BL / 6 Mäuse. </li><li> In Scheiben schneiden Rückenmark in 500 pm dicke Schnitte mit einem McIlwain Gewebe Chopper. </li><li> Separate Scheiben unter einem Mikroskop anatomischen und wählen Scheiben mit intakten sagittal / axial Rückenmark Struktur. </li><li> Teller Scheiben in einer 35 mm Petrischale mit 30 ul Matrigel und legen Sie sie in der Mitte erscheint wie möglich zu schließen und pflegen sie in einem 5% CO 2-Inkubator bei 37 ° C für mindestens 30 min, bis das Matrigel Proteine ​​selbst zusammen zu produzieren ein dünner Film, der die Oberfläche des Rückenmarks Schicht umfasst. Es ist sehr wichtig, um sicherzustellen, dass Matrigel wurde komplett zusammengebaut, inkubieren Sie die Petrischale bei 37 ° C für weitere 30 min, wenn nötig. </li><li> Fügen Sie 4-6 ml DMEM/F-12-Medium mit 25 mM HEPES-Puffer und 15 bis 20% fötalem Kälberserum sehr schonend, so dass mittel fließen direkt auf die Scheibe zu vermeiden. Sicherstellen, dass die Scheibe nicht vollständig in Medium eingetaucht, so dass die Oberfläche der Explantate gut ausgesetztdie Luft. Ändern Medium zweimal wöchentlich. </li></ol> 5. Die Injektion von Hoechst 33342 beschriftet NPCs in den organotypischen Rückenmark Scheibe <ol><li> Bereiten Sie den NPC Suspension bei 1 × 10 6 Zellen / ul. </li><li> Pre-Inkubation der Zellsuspension in Medium mit 5 uM Hoechst 33342 für 30 min. </li><li> Verwenden Sie kapillaren Glasröhre bis 2 ul Suspension in das Rückenmark Scheibe langsam microinject unter dem Mikroskop. Stellen Sie sicher, Kapillar-Glasrohr geht durch den Matrigel (rosa Teil unter dem Mikroskop) und erreichen an der Innenseite der Scheibe des Rückenmarks (graue Gewebe unter dem Mikroskop), im Inneren des Rückenmarks Slice für mindestens 30 Sekunden bleiben, um Zellsuspension überfahren zu vermeiden. Legen Sie die Petrischale mit Rückenmark Scheibe in Inkubator (37 ° C 5% CO 2) und lassen Sie es über Nacht. </li><li> Am nächsten Tag, wenden eine EF von 500 mV / mm des Rückenmarks Schicht mit Hoechst 33342-markierten NPCs in der electrotactic Kammer (unter Verwendung der Verfahren beschrieben3). </li></ol> 6. Repräsentative Ergebnisse Wenn NPCs wurden auf einen Bereich von physiologischen EF zeigten sie stark gerichtete Wanderung in Richtung der Kathode (1) ausgesetzt ist. Die gleiche Beobachtung wurde auch bei einer einzelnen Zelle auf der organotypischen Rückenmarks Slice ex vivo-Modell, eine 3D-Umgebung in vivo Bedingungen nachahmen (Abbildung 2). <img alt="1" src="/files/ftp_upload/3453/3453fig1.jpg" /> Abbildung 1. NPCs zeigen gerichtete Wanderung in EFs. PCs zeigten hoch gerichtete Wanderung zur Kathode, wenn EFS ausgesetzt sind, repräsentieren roten Linien und blauen Pfeile Flugbahnen und die Richtung der Bewegung der Zellen (A). B zeigt die Migrationswege von NPCs. Bar: 50 um. <img alt="2" src="/files/ftp_upload/3453/3453fig2.jpg" /> Abbildung 2. Die transplantierten NPCs für gerichtete Wanderung in Richtung der Kathode in der organotypischen Rückenmarks Slice </strong&gt;. (A) NPCs mit Hoechst 33342 beschriftet wurden in den organotypischen Rückenmark Scheibe am Startpunkt des EF Behandlung transplantiert. NPCs migriert gerichtet in Richtung der Kathode für 2,5 Stunden, an welchem ​​Punkt die EF Polarität umgekehrt wurde (B). Ändern EF Polarität löste eine scharfe Umkehr der electrotaxis gegenüber der neuen Kathode (C). (D) Bild von transplantierten NPCs innerhalb des Rückenmarks Scheibe am Ende der Zeitraffer-Aufnahme. (E) Eine 3D-Rekonstruktion von transplantierten NPCs innerhalb des Rückenmarks in Scheiben schneiden. 3D-Scanning-Sektionen waren 300 um Dicke, ausgehend von der Mitte und endet an der Unterseite der Scheibe. Die gepunkteten Linien zeigen die relativen Positionen der gleichen Population von transplantierten Zellen am Anfang, Umkehr und Endpunkte der EF Behandlung (A - C, jeweils). Pfeilspitzen zeigen den gleichen Population von Hoechst 33342 markierten NPCs. Bar: 50 um.

<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>

Wir haben nichts zu offenbaren.

Die Protokolle verwenden wir auf der Basis früherer Studien 5,11. Mit Hilfe dieser Methoden stabilen Kultur und elektrischer Strom Bedingungen aufrecht erhalten, während ein EF-Agar über Brücken, Steinberg-Lösung und Ag / AgCl-Elektroden, um Zellen oder in Scheiben individuell gestaltet electrotactic Kammern von standardisierten und präzise Abmessungen kultiviert werden. Die Tiefe der Kammern kann eingestellt werden, um Platz für verschiedene Dicken Probe 11 zu, und im Falle von Zellen, Kamm...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> FGF-basic Rekombinantes humanes </td><td> Invitrogen </td><td> PHG0026 </td><td> 20 ng / ml </td></tr><tr><td> Rekombinantes humanes EGF </td><td> Invitrogen </td><td> PHG0311 </td><td> 20 ng / ml </td></tr><tr><td> N2-Supplement (100X) Flüssigkeit </td><td> Invitrogen </td><td> 02048 </td><td></td></tr><tr><td> DMEM/F12-Medium (high Glucose) </td><td> Invitrogen </td><td> 31330-095 </td><td></td></tr><tr><td> Poly-D-Lysin </td><td> Millipore </td><td> A-003-E </td><td></td></tr><tr><td> Natürliche Mauslaminin </td><td> Invitrogen </td><td> 23017-015 </td><td></td></tr><tr><td> Wachstumsfaktor reduziert Basalmembran-Matrix (Matrigel) </td><td> BD Biosciences </td&gt; <td> 354230 </td><td></td></tr><tr><td> HEPES-Puffer </td><td> Gibco </td><td> 15630 </td><td></td></tr><tr><td> McIlwain Gewebe Chopper </td><td> Die Mickle Laboratory Engineering Co Ltd </td><td> TC752-PD </td><td></td></tr><tr><td> Dow Corning Hochvakuum-Silikonfett </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

Endogene elektrische Felder (EF) kommen natürlicherweise in vivo und spielen eine entscheidende Rolle bei der Gewebe / Organ Entwicklung und Regeneration, einschließlich der des zentralen Nervensystems 1,2. Diese endogenen EF durch zellulären Regulation der Ionentransport mit dem elektrischen Widerstand von Zellen und Geweben kombiniert erzeugt. Es wurde berichtet, dass EF angewandte Behandlung kann der funktionellen Wiederherstellung von Verletzungen des Rückenmarks bei Tieren und Menschen 3,4 zu fördern. Insbesondere hat EF-gerichtete Wanderung in einer Vielzahl von Zelltypen 5,6, einschließlich neuralen Vorläuferzellen (NPC) 7,8 nachgewiesen. Anlegen eines Gleichstroms (DC) EF ist nicht eine allgemein verfügbare Technik in den meisten Laboratorien. Wir haben detaillierte Protokolle für die Anwendung von DC EFs beschrieben, um Zell-und Gewebekulturen zuvor 5,11. Hier präsentieren wir eine Video-Demonstration der Standard-Methoden auf einem berechneten Feldstärke einzurichten 2D-Basis eind 3D-Umgebungen für NPCs und zu zellulären Reaktionen auf EF Stimulation in beiden einzigen Zelle Wachstumsbedingungen in 2D, und der organotypischen Rückenmark Scheibe in 3D untersuchen. Die Wirbelsäule ist ein idealer cordslice Empfänger Gewebe zum Studium NPC ex vivo Verhalten, nach der Transplantation, weil die zytoarchitektonischen Gewebeorganisation wird auch in diesen Kulturen 9,10 erhalten. Darüber hinaus ist diese ex vivo-Modell ermöglicht auch Verfahren, die derzeit technisch nicht möglich, Zellen in vivo mittels Zeitraffer-Aufnahme auf der Ebene einzelner Zellen zu verfolgen. Es ist von entscheidender Zelle muss auf das Verhalten nicht nur in einer 2D-Umgebung zu bewerten, sondern auch in einer 3D-organotypischen Bedingung, die die in vivo-Umgebung imitiert. Dieses System ermöglicht hochauflösende Bildgebung mit Deckglas-basierte Gerichte im Gewebe oder Organ Kultur mit 3D-Tracking einzelner Zellmigration in vitro und ex vivo und kann ein Zwischenschritt, bevor sie auf in sein vivo Paradigmen.

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

Dieses Protokoll zeigt Methoden verwendet werden, um 2D-und 3D-Umgebungen in speziell angefertigten electrotactic Kammern einzurichten, die Zellen verfolgen können<em&gt; In vivo / ex vivo</em&gt; Verwendung von Zeitraffer-Aufnahme an der Ebene einzelner Zellen, um Galvanotaxis / electrotaxis und andere zelluläre Reaktionen untersuchen zu Gleichstrom (DC) elektrische Felder (EF) zu lenken.

Electric Field-gesteuerte gerichtete Wanderung von neuralen Vorläuferzellen in 2D-und 3D-Umgebungen

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 Aufrufe.  Cardiff University. Dieses Protokoll zeigt Methoden verwendet werden, um 2D-und 3D-Umgebungen in speziell angefertigten electrotactic Kammern einzurichten, die Zellen verfolgen können In vivo / ex vivo Verwendung von Zeitraffer-Aufnahme an der Ebene einzelner Zellen, um Galvanotaxis / electrotaxis und andere zelluläre Reaktionen untersuchen zu Gleichstrom (DC) elektrische Felder (EF) zu lenken.

Serie: 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.

Anpassung eines Haptic Robot in einem 3T fMRT

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

Forschung

Biotechnik

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.

Kultivierung humaner neuronaler Vorläuferzellen in einer 3-dimensionalen Self-Montage 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.

Bestellen Einzelzellen und Einzel Embryos in 3D Confinement: Ein neues Gerät für 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 ...

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.

Engineered 3D Silk-Kollagen-basierten Modell von polarisiertem 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 ...

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.

Electro Studium der Lungenkrebszellen unter Verwendung eines Mehrkanal-Zwei elektrischen Feldes Mikrofluidik-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.

Die Verbesserung der 2D- und 3D-Haut<em&gt; In Vitro</em&gt; Modellen mithilfe von 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 ...

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.

2D- und 3D-Matrizen zur Untersuchung der linearen Invadosomenbildung und -aktivität

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.

Zellwanderung in dreidimensionales In Vitro Charakterisierung Wunde Umgebungen

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.

3D Hydrogel Kulturen von primären Gliazellen für In-vitro- Modellierung von Neuroinflammation verbessert

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 von einzelnen neuronalen Stammzellen und Neuronen in Gehirnscheiben mit Robotik-Mikroinjektion

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