Wir schätzen Dr. Takeshi Ikeya und technisches Personal in Toyo Gosei, Tokyo, Japan für die Bereitstellung von thermosensitiven mikroKulturPlatten sowie wissenschaftliche Beratung. Wir danken auch Frau Satomi Ogura, Ms. Sae Suzuki, Frau Asuka Miyoshi und Frau Katsue Morii für die technische Unterstützung bei Tierversuchen. Diese Arbeit wurde zum Teil durch die JSPS KAKENHI Grant-in-Aid for Scientific Research unterstützt, das Zentrum der Innovation (COI) Programm und die S- Innovationsprogramm von der Japan Science and Technology Agency (JST) und der JSPS Core zu-Kern-Programm, A. Advanced Research Networks.

Alle tierexperimentellen Studien wurden mit der Zustimmung der Animal Care und Verwenden Committee der University of Tokyo, Tokyo, Japan durchgeführt. 1. Zellpräparation <ol><li> Für primären Hepatozyten, folgen Sie dem Protokoll zur Isolation von Hepatozyten der Ratte durch einen modifizierten zweistufigen Kollagenase Verdauung 13,14. <ol><li> Betäuben Sprague Dawley (SD) Ratten (männlich, 5 Wochen alt) unter Inhalationsnarkose mit Isofluran. Legen Sie eine Ratte in einer Kammer zu einer Anästhesiegerät angeschlossen, um Isofluran für die Kammer bereitzustellen. Nehmen Sie die Ratten nach dem Einschlafen, und legen Sie sie auf dem Operationstisch mit Lüftungs Verwendung einer Maske. Steuern Sie die Isofluran fließen etwa auf 0,4 bis 0,7 l / min, indem Sie die Bedingungen der Ratte. Perfusion der Leber von Sprague Dawley (SD) Ratten (männlich, 5 Wochen alt) von der Pfortader mit einer speziellen Lösung von 8 g / l Natriumchlorid zusammengesetzt ist (NaCl), 400 mg / l Kaliumchlorid (KCl), 78 mg / LNatriumdihydrogenphosphat Dihydrat (NaH 2 PO 4 · 2H 2 O), 151 mg / L Dinatriumhydrogenphosphat Dodecahydrat (Na 2 HPO 4 · 12H 2 O), 2,38 g / L 2- [4- (2-hydroxyethyl) -1 piperazinyl] ethansulfonsäure (HEPES), 190 mg / l Ethylenglykoltetraessigsäure (EGTA), 350 mg / L Natriumhydrogencarbonat (NaHCO 3) und 900 mg / l Glukose. </li><li> Zirkulieren Collagenase-Lösung durch die Leber. HINWEIS: Die Lösung von 500 mg / L Kollagenase, 9,8 g / L Hanks gepufferte Salz, zusammen 2,38 g / ml HEPES, 556 mg / ml Calciumchlorid-Hydrat (CaCl 2 · H 2 O), 350 mg / L NaHCO 3, und 50 mg / l Trypsin-Inhibitor, wobei der pH-Wert auf 7,2 eingestellt. </li><li> Entfernen Sie die Leber sorgfältig durch und Hackfleisch sie sanft auf einem Teller mit einem Skalpell, fügen Dulbecco-modifiziertem Eagle-Medium (DMEM) mit 10% fötalem Rinderserum (FBS) ergänzt, und filtern Sie die Zellsuspension durch ein 100 &amp; mgr; mNylon-Mesh. Um zusätzliche Trümmer zu entfernen, zentrifugieren Sie die Zellsuspension bei 20 × g für 1 min. Bei diesem Schritt werden Hepatocyten im Überstand. </li><li> Wiederholen Sie den Zentrifugationsschritt zweimal (insgesamt drei Zentrifugationen). Schließlich wird abzentrifugiert Hepatozyten bei 50 × g für 3 min, um sie in Form eines Pellets zu gewinnen. </li><li> Re-suspendieren die Hepatocyten in einer Konzentration von 4 × 10 5 Zellen / ml in einem speziellen Nährmedium aus DMEM mit 10% FBS, 1% Pen-Strep-Glut (PSQ), 1% Dimethylsulfoxid (DMSO), 0,1 umol / L Dexamethason, 0,5 ug / ml Insulin, 10 mmol / L Nicotinamid, 0,2 mmol / L phosphorylierten Ascorbat (Asc-2P) und 10 ng / ml humaner epidermaler Wachstumsfaktor (hEGF) 15. Dieses spezielle Medium ist für die Erhaltung der Leberfunktion unter in vitro Bedingungen. </li></ol></li><li> Zum Erhalt Ratte MSCs, einschläfern Sprague Dawley (SD) Ratten (männlich, 5 Wochen alt) durch übermäßige Verabreichung von isoflurane. Resektion der Oberschenkelknochen und Schienbeine, und sammeln Sie die Knochenmark, indem ein 22 G Nadel in den Schaft des Knochens, um es zu spülen, mit 10 ml DMEM, ergänzt mit 10% FBS. Sammle die Zellen durch Filtration durch ein 100 um Nylonnetz. </li><li> Saatgut der Zellen auf 10 cm-Kulturschalen mit DMEM, das 10% FBS und 1% Penicillin / Streptomycin. Für die Sphäroid Experimenten verwenden MSCs in 5 Passagen. </li></ol> 2. Herstellung von 3D-Zell Sphäroide <ol><li> Kommerziell zu erhalten mit Mikrokulturplatten, bei denen die Zellklebeflächen regelmäßig bei 100 &amp; mgr; m Durchmesser in einer zweidimensionalen Weise angeordnet sind, durch nicht-klebenden Bereiche durch die PEG-Matrix beschichtet ist, umgeben. HINWEIS: Bei Zelltransplantation notwendig, Zellablösung durch Abkühlen der Platten ermöglichen eine zusätzliche Beschichtung mit dem wärmeempfindlichen Polymer PIPAAm (siehe Schritt 5.1). Temperaturschwankungen können sich Änderungen in der Chemie des Polymers zu induzieren8-10. Bei 37 ° C ist PIPAAm etwas hydrophob, so dass die Zellen, die unter normalen Bedingungen gezüchtet werden. Eine Verminderung der Temperatur unter 32 ° C führt zu einer schnellen Hydratation des Polymers, was zur spontanen Ablösung der Zellen. </li><li> Samen der Hepatozyten oder MSCs auf 12-Well-Mikromuster Platten mit einer Dichte von 4 × 10 5 Zellen / Vertiefung bei 37 ° C andincubate sie in einer feuchten Atmosphäre, die 5% CO 2. Die Zellen werden auf den mikrostrukturierten Haft Bereichen ansammeln und nach und nach in 2 Tagen zu bilden runde Kügelchen. HINWEIS: Für die Herstellung Kontrollzellen in einer Monokultur, benutzen normalen 12-Well-Platten und Samen der Zellen bei einer identischen Dichte, nach einem ähnlichen Verfahren wie oben beschrieben. </li></ol> 3. Herstellung von Polyplex Nanomicelles <ol><li> Synthese eines Blockcopolymers, PEG-PAsp (DET) (Poly [N &#39;- [N - (2-Aminoethyl) -2-aminoethyl] aspartamid]) (PEG Mw = 12,000, Polymerisationsgrad (DP) von PAsp Segment (DET) = 59) und ein Homopolymer, das nur aus dem kationischen segment [PAsp (DET)] (DP = 55) zusammengesetzt ist, nach den Verfahren, die zuvor von den Autoren 11, 16, 17. </li><li> Vorbereitung einer gemischten Lösung des PEG-PAsp (DET) Blockcopolymer und das PAsp (DET) Homopolymer in einem gleichen molaren Verhältnis von restlichen Aminogruppen in 10 mM HEPES-Puffer (pH 7,3) durch Einstellen der Polymer-Konzentration auf 33,3 ug / ml und 19,1 ug / ml. HINWEIS: Die oben beschriebenen Polymerkonzentrationen sind repräsentative Werte für die Vorbereitung nanomicelles mit einer Rest molare Verhältnis der gesamten Aminogruppen in den beiden Polymeren zu den Phosphatgruppen der pDNA (N / P-Verhältnis) von 10 N / P-Verhältnis kann variieren je nach Zelltyp und dem Ziel (für Details siehe Diskussion). Der kombinierte Einsatz der beiden Polymere können sowohl wirksam PEG Abschirmung und Funktionsweise PAsp (DET), um die endosomale verbessern erreichenentkommen (Details siehe Diskussion) 18. </li><li> Bereiten Sie die pDNA kodiert Gaussia-Luciferase, GL4 Luciferase oder Erythropoietin durch Klonierung des Segments Expression der entsprechenden Gene in das pCAG-GS-Plasmid (http://www.cdb.riken.jp/pcs/protocol/vector/map/m36.html ), um die Expression unter der CAG-Promotor / Enhancer mit einem kommerziellen Kit nach Herstellerprotokoll erhalten. Verstärken die pDNA in einem kompetenten Escherichia coli-Stamm und zu reinigen, es mit einem Endotoxin-freiem Plasmid-DNA-Reinigungssystem. Bestimmen die pDNA Konzentration bei einer Absorption von 260 nm, um eine 150 ug / ml-Lösung in 10 mM HEPES-Puffer (pH 7,3) zu erhalten. </li><li> Die polyplex nanomicelles vorzubereiten gründliche Durchmischung der pDNA-Lösung (150 ug / ml in 10 mM HEPES-Puffer) und die vorgemischte Lösung der beiden Polymere in einem Verhältnis von 2: 1 (nach Volumen). </li></ol> 4. Gentransfektion in Spheroids <ol><li> Inkubieren der Zellen (Hepatozytenoder MSCs) 72 Stunden nach dem Aussäen auf die mit Mikroplatten, um die Bildung von reifen Sphäroiden erlauben. Zur Gentransfektion, werden 100 ul des polyplex nanomicelle Lösung (enthaltend 10 ug pDNA) in jede Vertiefung nach dem Ersetzen des Kulturmedium mit 1 ml frischem Medium. Fahren Sie mit der Inkubation mit dem nanomicelle Lösung für 24 Stunden. </li><li> Für eine Steuerung unter Verwendung einer lipidbasierten Transfektionsreagenz mischen pDNA Lösungen mit dem Reagenz in einem Gewichtsverhältnis Reagenz / pDNA der 3. Justieren der endgültigen Dosis der pDNA gleich sowohl für die Lipid basierende Reagenz und nanomicelle Methoden sein. </li></ol> 5. Wiederherstellung und Transplantation von Zell Spheroids <ol><li> Ersetzen Sie das Kulturmedium mit 200 ul PBS gekühlt und legen Sie die Platten auf Eis. HINWEIS: In der Regel können die Sphäroide in etwa 15 min zu lösen, und kann in Form einer Suspension für die Transplantation gewonnen werden. </li><li> Vorsichtig aspirieren der Zellen in 200 &amp; mgr;Suspension mit einer Spritze mit einer 23 G oder 27 G-Nadel für die in vivo-Injektionen. </li></ol> 6. Auswertung der Transgen-Expression <ol><li> Für die in vitro-Bewertung der Expression von Gaussia-Luciferase in das Kulturmedium sezerniert, sammeln 50-100 ul des Mediums genau 24 Stunden nach dem Austausch mit frischem Medium. Schätzung der Luciferase-Expression unter Verwendung eines kommerziellen Renilla-Luciferase-Assay-System und ein Luminometer nach dem Protokoll des Herstellers. HINWEIS: Die Gaussia-Luciferase bleibt in dem Kulturmedium für mehr als eine Woche stabil. So, um die Echtzeit-Wirkungsgrad der Expression des Transgens zu bewerten, das Medium mit frischem man das Sammeln der Probenmedium ersetzen vor. Der Zeitpunkt der Mediumwechsel kann flexibel sein. </li><li> Für die in-vivo-Bewertung der Expression des Transgens in Wirtstieren nach Zelltransplantation, betäuben BALB / c-Nacktmäusen (weiblich, 7 Wochenalt) unter Inhalationsnarkose mit Isofluran. <ol><li> Legen Sie eine Maus in einer Kammer zu einer Anästhesiegerät angeschlossen, um Isofluran für die Kammer bereitzustellen. Nehmen Sie die Maus nach dem Einschlafen, und legen Sie sie auf dem Operationstisch mit Lüftungs Verwendung einer Maske. Steuern Sie die Isofluran fließen etwa auf 0,2 bis 0,5 l / min, indem Sie die Bedingungen der Maus. </li><li> Injizieren 200 ul der Zellsuspension, enthaltend Sphäroiden transfiziert mit den GL4 Luciferase-exprimierenden pDNA (wie in 4.1 beschrieben) in das subkutane Gewebe der Bauchgegend. </li><li> Unmittelbar nach der Injektion von D-Luciferin (150 mg / kg; intravenöse Route) zu messen, die die Luciferase-Expression unter Verwendung IVIS Abbildungssystem nach dem Protokoll des Herstellers. </li></ol></li><li> Zur Bewertung der therapeutischen Wirkungen der Zelltransplantation, injizieren 200 ul der Zellsuspension, enthaltend Sphäroiden mit dem Erythropoietin kodierenden pDNA in die transfiziertesubkutane Gewebe der Bauchregion. <ol><li> Sammeln Blutproben durch Unterkieferspeichel Blutungen auf etwa 200 ul Blut 19 ​​zu erhalten. Messen Sie die Hämoglobin und Hämatokrit mit Hilfe eines Blutprobenanalysator. HINWEIS: Die Zellzahl für die Transplantation ist durch die Anzahl von den Platten ausgesät geregelt. Leider ist es schwierig, die genaue Anzahl von Zellen zu bestimmen, da die Anzahl innerhalb Sphäroide nicht gemessen werden kann. </li></ol></li><li> Nach Experimenten, legen Sie die Mäuse auf einem Heizkissen mit einem Temperaturregler verbunden, bis Erwachen aus der Narkose. </li></ol>

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The authors declare that they have no competing financial interests.

In diesem Protokoll ist es kritisch, um die 3D-Struktur der Sphäroide während der Schritte zur Geneinführung und Sphäroid Erholung aufrechterhalten. Es ist wesentlich, um eine günstige Mikroumgebung aufrechtzuerhalten, dass die Zellen den Zelltod oder den Verlust von Zellaktivität zu vermeiden. Zum Beispiel Albuminsekretion, ein Vertreter angeborene Funktion der Hepatozyten, ist in den Hepatozyten-Sphäroiden erhalten, während die Hepatozyten in der herkömmlichen Monolayer-Kultur schnell ihre sekretorischen Kapa...

Zell-Transplantation Therapie hat große Aufmerksamkeit für die Behandlung von verschiedenen hartnäckigen Krankheiten angezogen. Die Aktivität und die Halbwertszeit des biologisch aktiven Faktoren, die durch die transplantierten Zellen sezerniert werden, sind essentiell für eine verbesserte therapeutische Wirksamkeit eines Zelltransplantationssystem. Genetische Modifikation der Zellen vor der Transplantation ist eine positive Technik zu regulieren und zellulären Funktionen, einschließlich der Sekretion der bioaktiven Faktoren manipulieren. Es ist auch wichtig, um eine günstige Mikroumgebung für die Zellen zur Vermeidung Zelltod oder den Verlust von Zellaktivität aufrechtzuerhalten. Dreidimensionale (3D) Sphäroid Zellkultur, in der Zell-zu-Zell-Wechselwirkungen sind gut erhalten, ist vielversprechend für diesen Zweck, zum Beispiel zur Verbesserung der Albumin-Sekretion aus primären Hepatozyten und Förderung Multilinien-Differenzierung von mesenchymalen Stammzellen (MSCs ) 1-7. In dieser Studie wurde eine neue Kombination System spheroid Kultur und Gentransfektion wird verwendet, um als Plattform für gentechnisch veränderte Zelltransplantation dienen. Für die Erstellung von Sphäroid-Zellen wird ein Sphäroid Kultursystem auf mikrostrukturierten Kulturplatten verwendet. Auf diesen Platten sind die Zelladhäsion Bereiche Durchmesser von 100 um regelmäßig in einer zweidimensionalen Weise angeordnet und werden durch nicht-klebenden Bereiche mit einem PEG-Matrix 3 beschichtet umgeben. Durch Animpfen einer ausreichenden Zahl von Zellen, Anordnungen von 3D Sphäroiden von 100 um im Durchmesser entsprechend dem Mikromuster Kulturbett gebildet. Die Sphäroide werden, ohne ihre 3D-Struktur mit wärmeZellKulturPlatten, die mit einem wärmeempfindlichen Polymer, Poly (iso-propylacrylamid) beschichtet waren (PIPAAm) 8-10 gewonnen. Die mikro Architektur basiert auf den wärmeempfindlichen Platten aufgebaut (custom-built). Einfach durch Absenken der Temperatur der Platten werden die Sphäroide aus dem Kulturbett abgetrennt und zerstreuend in phosphatgepufferter Salzlösung (PBS). Somit kann eine große Anzahl von Sphäroiden mit einer einheitlichen Grße von 100 &amp; mgr; m in der Form einer injizierbaren Suspension, erhalten werden. <img alt="Abbildung 1" p="" src="/files/ftp_upload/52384/52384fig1.jpg" /> Abbildung 1. Schematische Darstellung der Sphäroid-Kultur-System auf einem mikrostrukturierten Platte. Genetische Veränderung wird durch Gentransfektion unter Verwendung der ursprünglichen nicht-viralen Gen-Träger, polyplex nanomicelle erreicht. Es wird Plasmid-DNA (pDNA) und Polyethylenglykol (PEG) -polycation Blockcopolymere 11 zusammengesetzt ist. Diese haben eine charakteristische Kern-Schale-Struktur, die aus einer PEG-Hülle und einem inneren Kern aus kondensierter pDNA, so dass sichere und wirksame Gen Einführung in die Zellen für therapeutische Zwecke 11. <a href="https://www.jove.com/files/ftp_upload/52384/52384fig1large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version zu sehen thi</a>Wert. <img alt="Figur 2" src="/files/ftp_upload/52384/52384fig2.jpg" /> Figur 2. Die Struktur der polyplex nanomicelle durch den Komplex von Nukleinsäuren und PEG-Block-Polykation-Blockcopolymeren gebildet ist. In dieser Studie ist der primäre Vorteil dieses Verfahrens, daß die Sphäroid-Struktur wird während Gentransfektion durch die nanomicelles gestört. Nach nanomicelle vermittelte Transfektionen von primären Ratten Hepatocyten-Spheroiden, verlängert Transgen-Expression von mehr als einem Monat bei kontinuierlicher Albuminsekretion aus der Hepatozyten auf einem Niveau vergleichbar mit der von nicht-transfizierten Sphäroide 12 erhalten. Die Expression des Transgens und Albumin-Sekretion aus den Sphäroiden werden auch nach der Genesung von den wärmeempfindlichen Platten gehalten. Es ist offensichtlich, dass nanomicelles sicher Geneinführung ohne eigene Funktionen des hep beeinträchtigen erleichternatocytes. Somit ist die Kombination von Sphäroid Zellen auf mikrothermosensitiven Platten mit Gen-Einführung mit nanomicelles ist eine vielversprechende Plattform für gentechnisch veränderte Zelltransplantation kultiviert. <a href="https://www.jove.com/files/ftp_upload/52384/52384fig2large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a>

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Pen-Strep-Glut</td>
			<td>GIBCO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Wako Pure Chemical Industries</td>
			<td>041-18861</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Wako Pure Chemical Industries</td>
			<td>141-01202</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;s buffered salt and L-ascorbic acid 2-phosphate (Asc-2P)</td>
			<td>Sigma-Aldrich</td>
			<td>A8960</td>
			<td></td>
		</tr>
		<tr>
			<td>Human epidermal growth factor (hEGF)</td>
			<td>Toyobo</td>
			<td>PT10015</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell-able multi-well plates</td>
			<td>Toyo Gosei</td>
			<td>PP-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermosensitive cell culture plates (Upcell)</td>
			<td>CellSeed Inc</td>
			<td></td>
			<td>The micropatterned architecture is constructed on the thermosensitive plates (custom-built by Toyo Gosei)</td>
		</tr>
		<tr>
			<td>Lipid-based transfection reagent (FuGENE HD)</td>
			<td>Promega</td>
			<td>E2311</td>
			<td></td>
		</tr>
		<tr>
			<td>Renilla Luciferase Assay System</td>
			<td>Promega</td>
			<td>E2810</td>
			<td></td>
		</tr>
		<tr>
			<td>pGL4 Luciferase Reporter Vector</td>
			<td>Promega</td>
			<td>E6651</td>
			<td></td>
		</tr>
		<tr>
			<td>pDNA expressing Gaussia luciferase</td>
			<td>New England BioLabs</td>
			<td>N8082S</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse erhthropoietin-expressing vector</td>
			<td>Origene</td>
			<td>MC208445</td>
			<td></td>
		</tr>
		<tr>
			<td>pCAG-GS</td>
			<td></td>
			<td></td>
			<td>Kindly provided by Laboratory for Pluripotent Cell Studies, Center for Developmental Biology, RIKEN</td>
		</tr>
		<tr>
			<td>Escherichia coli DH5&#945; competent cells</td>
			<td>Takara</td>
			<td>9057</td>
			<td></td>
		</tr>
		<tr>
			<td>Endotoxin-free plasmid DNA purification system</td>
			<td>Nippon Genetics</td>
			<td>NucleoBond Xtra EF</td>
			<td></td>
		</tr>
		<tr>
			<td>collagenase</td>
			<td>Wako Pure Chemical Industries</td>
			<td>639-00951</td>
			<td></td>
		</tr>
		<tr>
			<td>trypsin inhibitor</td>
			<td>GIBCO</td>
			<td>R-007-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminometer</td>
			<td>Promega</td>
			<td>GloMax&#8482; 96 Microplate Luminometer</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Imaging System</td>
			<td>Xenogen Corp.</td>
			<td>Xenogen IVIS Spectrum in vivo imaging system</td>
			<td></td>
		</tr>
		<tr>
			<td>blood sample analyzer</td>
			<td>Sysmex</td>
			<td>pocH-100i Automated Hematology Analyzer</td>
			<td></td>
		</tr>
	</tbody>
</table>

gene transfection toward spheroid cells on micropatterned culture plates for genetically-modified cell transplantation

Zur Verbesserung der therapeutischen Wirksamkeit der Zelltransplantation wurde ein Transplantationssystem von gentechnisch veränderten, injizierbare Sphäroiden entwickelt. Die Zellkügelchen werden in einem Kultursystem über mikroPlatten mit einer wärmeempfindlichen Polymer überzogen hergestellt. Eine Anzahl von Sphäroiden werden auf den Platten gebildet sind, entsprechend den Zelladhäsion Bereiche von 100 um Durchmesser, die regelmäßig in einer zweidimensionalen Weise angeordnet sind, die von nicht-klebenden Bereiche umgeben, die von einem Polyethylenglykol (PEG) Matrix beschichtet sind. Die Sphäroide können leicht als eine flüssige Suspension durch Absenken der Temperatur der Platten wiederhergestellt werden, und ihre Struktur ist gut, indem sie durch Injektionsnadeln mit einer ausreichend großen Kalibers (über 27 G) gehalten. Genetische Modifizierung durch Gentransfektion unter Verwendung des ursprünglichen nicht-virale Gen-Träger polyplex nanomicelle, die geeignet ist zur Einführung von Genen in Zellen ohne Störung des Sphäroids Struktur erreicht wird. Für primary Hepatozyten-Sphäroiden mit einem Luciferase-exprimierenden Gen transfiziert wird die Luciferase nachhaltig in transplantierten Tieren erhalten, zusammen mit erhaltenen Hepatozyten-Funktion, wie Albumin Ausdruck angegeben. Dieses System kann auf einer Vielzahl von Zelltypen, einschließlich von mesenchymalen Stammzellen angewendet werden.

Gentransfektion der Gaussia-Luciferase-exprimierenden pDNA in den durch die Hepatozyten oder MSCs mit polyplex nanomicelles oder die Kontrolle auf Lipidbasis Transfektionsreagenz 12 gebildeten Sphäroiden geführt. Die nanomicelles induzierte fast keine Veränderung des Sphäroids Struktur im Vergleich mit nicht-transfizierten Sphäroiden auf die mikroPlatten, wohingegen das Kontrollreagens täglich erheblich gestört, die die Struktur nach der Transfektion (Abbildung 3). Nach der Transfektio...

Watch this Scientific Journal Video about Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation at JoVE.com

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

Gentransfektion in Richtung Spheroid Zellen auf mikrostrukturierten Kulturplatten für genetisch veränderte Zelltransplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

gene-transfection-toward-spheroid-cells-on-micropatterned-culture

Research

JoVE Journal

Bioengineering

8.7K Aufrufe.  The University of Tokyo. This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

Serie: Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Mikrostrukturierten Oberflächen zum Studium Hyaluronsäure Wechselwirkungen mit Krebszellen

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

Forschung

Biotechnik

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

Verbesserte Visualisierung und quantitative Analyse von Arzneimittelwirkungen mit mikrostrukturierten Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

Herstellung von mikrostrukturierten Hydrogele für Neural Kultur-Systeme mit Dynamic Mask Projektion Photolithographie

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

Rotierende Cell Culture Systems für Human Cell Culture: Human Trophoblastzellen als Modell

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

Messe Cytometry: Protokoll für die tägliche Tuning und Ausführen von Zellproben auf einem CyTOF Messe Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Transplantation von induzierten pluripotenten Stammzellen-abgeleiteten mesoangioblast-ähnlichen myogenen Vorläufern in Mausmodellen der Muskelregeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Stretching Micropatterned Cells on a PDMS Membrane

Mechanical forces exerted on cells and/or tissues play a major role in numerous processes. We have developed a device to stretch cells plated on a PolyDiMethylSiloxane (PDMS) membrane, compatible with imaging. This technique is reproducible and versatile. The PDMS membrane can be micropatterned in order to confine cells or tissues to a specific geometry. The first step is to print micropatterns onto the PDMS membrane with a deep UV technique. The PDMS membrane is then mounted on a mechanical stretcher. A chamber is bound on top of the membrane with biocompatible grease to allow gliding during the stretch. The cells are seeded and allowed to spread for several hours on the micropatterns. The sample can be stretched and unstretched multiple times with the use of a micrometric screw. It takes less than a minute to apply the stretch to its full extent (around 30%). The technique presented here does not include a motorized device, which is necessary for applying repeated stretch cycles quickly and/or computer controlled stretching, but this can be implemented. Stretching of cells or tissue can be of interest for questions related to cell forces, cell response to mechanical stress or tissue morphogenesis. This video presentation will show how to avoid typical problems that might arise when doing this type of seemingly simple experiment.

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching.

Stretching-Cells auf einer mikrostrukturierten PDMS-Membran

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular behaviors on disease-relevant time scales. Previously, microcontact printing has been used to fabricate two-dimensional functional arterial mimics through patterning of extracellular matrix protein as guidance cues for tissue organization. Vascular muscular thin films utilized these mimics to assess functional contractility. However, the microcontact printing fabrication technique used typically incorporates hydrophobic PDMS substrates. As the tissue turns over the underlying extracellular matrix, new proteins must undergo a conformational change or denaturing in order to expose hydrophobic amino acid residues to the hydrophobic PDMS surfaces for attachment, resulting in altered matrix protein bioactivity, delamination, and death of the tissues.
Here, we present a microfluidic deposition technique for patterning of the crosslinker compound genipin. Genipin serves as an intermediary between patterned tissues and PDMS substrates, allowing cells to deposit newly-synthesized extracellular matrix protein onto a more hydrophilic surface and remain attached to the PDMS substrates. We also show that extracellular matrix proteins can be patterned directly onto deposited genipin, allowing dictation of engineered tissue structure. Tissues fabricated with this technique show high fidelity in both structural alignment and contractile function of vascular smooth muscle tissue in a vascular muscular thin film model. This technique can be extended using other cell types and provides the framework for future study of chronic tissue- and organ-level functionality.

We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses.

Mikrofluidischen Genipin Abscheidetechnik für Extended Kultur von mikrostrukturierten Vascular Muscular Thin Films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular ...

Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and multidirectional shear stresses have all been postulated to stimulate a pro-atherosclerotic phenotype in endothelial cells, whereas high magnitude and unidirectional or uniaxial shear are thought to promote endothelial homeostasis. These hypotheses require further investigation, but traditional in vitro techniques have limitations, and are particularly poor at imposing multidirectional shear stresses on cells. 
One method that is gaining increasing use is to culture endothelial cells in standard multi-well plates on the platform of an orbital shaker; in this simple, low-cost, high-throughput and chronic method, the swirling medium produces different patterns and magnitudes of shear, including multidirectional shear, in different parts of the well. However, it has a significant limitation: cells in one region, exposed to one type of flow, may release mediators into the medium that affect cells in other parts of the well, exposed to different flows, hence distorting the apparent relation between flow and phenotype. 
Here we present an easy and affordable modification of the method that allows cells to be exposed only to specific shear stress characteristics. Cell seeding is restricted to a defined region of the well by coating the region of interest with fibronectin, followed by passivation using passivating solution. Subsequently, the plates can be swirled on the shaker, resulting in exposure of cells to well-defined shear profiles such as low magnitude multidirectional shear or high magnitude uniaxial shear, depending on their location. As before, the use of standard cell-culture plasticware allows straightforward further analysis of the cells. The modification has already allowed the demonstration of soluble mediators, released from endothelium under defined shear stress characteristics, that affect cells located elsewhere in the well.

This protocol describes a coating method to restrict endothelial cell growth to a specific region of a 6-well plate for shear stress application using the orbital shaker model.

Segmentierung des Wachstums von Endothelzellen in 6-Well-Platten auf einem Orbitalschüttler für mechanobiologische Studien

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

Eine Kulturmethode zur Erhaltung von quiescent Human Hematopoietic Stammzellen

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...