Dieses Projekt wurde von der Deutschen Forschungsgemeinschaft (DFG, HE 4578/3-1) gefördert.

A. Knorpelbiopsie (Operationssälen; Schritte 1-5 in Non-sterile Zubereitung Zimmer) <ol><li> Führen Sie eine letzte Gewichtskontrolle des Kaninchens (New Zealand White Kaninchen, weiblich, 3,5-4,0 kg Körpergewicht, 6 Monate alt), um in der Lage sein Dosis Medikamente richtig und Gewicht im Anschluss an Operation zu überwachen. </li><li> Induce Anästhesie der Kaninchen durch eine intravenöse Injektion von 10 mg / kg Propofol. </li><li> Nach der Intubation Aufrechterhaltung der Narkose mit 1,5 mg / kg / min Propofol und 0,05 mg / kg Fentanyl intravenös. Überwachen Anästhesie mit Kapnographie, Pulsoximetrie und Pulsfrequenz. </li><li> Rasur das Knie auf mit einem elektrischen Haarschneider und Vakuum das Fell betrieben werden. </li><li> Desinfizieren Sie die Knie rasiert gründlich und decken den Rest der Kaninchen mit einem sterilen Verband. </li><li> Ertasten Sie die Patella und führen Sie einen Hautschnitt medial der Patella. </li><li> Öffnen Sie das Kniegelenk durch eine medial parapatellaren Arthrotomie unter sterilen Bedingungen. Versuchen Sie zu vermeiden Schneiden jede kleine suoberflächlichen Blutgefäße. </li><li> Anschließend verdrängt man das Patella seitlich. </li><li> Überprüfen Sie das Kniegelenk für irgendwelche Anomalien und makroskopischen Knorpelschäden. </li><li> Sie vorsichtig kleine Stücke Knorpel (2-3 mm) aus der Trochlea ossis femoris mit einem sterilen Skalpell (Abbildung 1). </li><li> Unmittelbar platzieren diese Knorpel Biopsien in einen 50-ml-Tube mit Vollmedium (DMEM + 10% fötalem Kälberserum (FCS) + 1% Penicillin / Streptomycin (Pen / Strep)) mit der in-vitro-Kultur direkt nach der Operation fortgesetzt. </li><li> Reinigen Sie die Mängel und spülen Sie sie mit steriler Kochsalzlösung. </li><li> Positionieren Sie die Patella in der Trochlea Groove. </li><li> Wundverschluss in Schichten mit einzelnen Knopf Nähte (4-0 Vicryl) und einer fortlaufenden Hautnaht (4-0 Monocryl). Verwenden resorbierbares Nahtmaterial. </li><li> Schließlich versiegeln die Wunde mit einem Spray Dressing wasserdampfdurchlässig. </li></ol> B. In-vitro-Kultur <ol><li> Verarbeiten Sie die Biopsie cartilage Stücke so bald wie möglich nach der Operation in einem sterilen laminaren Strömung Gewebekulturabzug. </li><li> Waschen Sie die sterile geerntet Knorpel 2x in PBS mit anschließender Zentrifugation bei 500 xg für 3 min bei RT. </li><li> Cut Knorpel in Stücke von 1 mm 3, Transfer in ein 50 ml Falcon und verdauen sie auf einem Schüttler für 30 min mit 10 ml Trypsin-EDTA (0,25%) und 10 ml PBS für 30 min </li><li> Nach 30 min stoppen Trypsinierung indem Vollmedium. Dann schütteln die Knorpelstücke für eine andere für 12 Stunden mit Kollagenase A (0,21 U / mg) in serumfreiem Medium (DMEM + 1% Pen / Strep). </li><li> Zentrifugieren der resultierenden Lösung bei 170 × g für 3 min bei RT. </li><li> Resuspendieren Zellpellets in 5 ml Vollmedium. </li><li> Seed die isolierten Chondrozyten auf 25 cm 2 Gewebekulturflaschen in 5 ml Vollmedium und halten in einer befeuchteten Gewebekultur-Inkubator bei 37 ° C, 5% CO 2. </li><li> Wachsen Chondrozyten bis zu einer Dichte von 80%. </li><li> Lassen Sie die Zellen fROM Kolben durch Waschen in PBS 2x vor der Belichtung bis 0,05% Trypsin-EDTA für 3 min. Sobald Chondrozyten lösen, stoppen Trypsinierung durch Zugabe von 10 ul Vollmedium. </li><li> Zentrifugieren Sie die Suspension bei 300 xg (3 min) und Pellet in Vollmedium. </li><li> Seed Zellen in Gewebekulturflaschen (75 cm 2) und getrennte in einem Verhältnis von 1:3 (~ 5.000 Zellen / cm 2) jeder 5. Tag oder bei 80% Konfluenz. </li><li> Bestimmen Zellzahlen und die Lebensfähigkeit durch Trypanblau-Färbung. </li></ol> C. Zell-Aussaat von der Matrix <ol><li> Vor der Implantation Wasch-und Release-Zellen wie oben beschrieben. Führen Zellzahl und übertragen 5 x 10 4 Zellen in ein 1,5-ml-Röhrchen. Dann bei 500 xg zentrifugiert 5 min bei RT. </li><li> Pellet in 15 ul Komplettmedium für eine vollständige Sättigung der Matrix. </li><li> Schneiden Sie die Doppelschicht Kollagengerüsttechnologie zu den genauen Abmessungen des trochlearen Defekt durch eine sterile BiopsiePunsch. </li><li> Platz Matrix in einem passenden Gewebekulturplatten (z. B. 24-Well-Platten). </li><li> Seed Zellen auf dem porösen, zelladhäsiven Seite der Matrix. Dies hält die Zellwanderung weg von der Matrix. </li><li> Lassen Zelle ausgesät Matrizen ohne weitere Zellkulturmedium in einem Inkubator für 1 Stunde in voller Einhaltung der Chondrozyten zu ermöglichen. </li><li> Dann erfolgt Zell-Matrix-Konstrukte in Kulturbehälter (z. B. 24-Well-Platten) mit frischem vollständigem Medium. Achten Sie darauf, nicht zu waschen alle Zellen aus der Matrix, obwohl starke Haftung wird erwartet. </li><li> Jetzt können Zell-Matrix-Konstrukte der Operation Raum für Re-Implantation entnommen werden. </li></ol> D. Matrix-assistierte autologe Chondrozyten-Transplantation <ol><li> Führen parapatellaren Arthrotomie zur kontralateralen Knie wie oben (A. 1-8) beschrieben. </li><li> Erstellen Sie zwei isolierten Knorpeldefekten im Trochleagrube mit steriler Luft operating Bohrmaschine (3,6 mm Durchmesser). </li><li> Reinigen Sie den Fehler und gründlich mit steriler Kochsalzlösung. </li><li> Es sollte darauf geachtet, nicht die Markhöhle jederzeit öffnen. Falls erforderlich, versiegeln den Boden der Defekte bei elektrischen Brenneisen gegen Blutungen. </li><li> Implantat die Matrizen mit den kultivierten Chondrozyten mit der porösen Seite nach unten (Abbildung 2) ausgesät. Dann in die Bohrlöcher eingepresst und bündig die umgebende Oberfläche. </li><li> Verschließen Sie die implantierten Matrizen mit ein wenig Fibrinkleber (Tissucol Duo S), um eine sichere Befestigung zu gewährleisten. </li><li> Nach Gerinnung, verlagern die Kniescheibe im Trochleagrube und gelten vollen Bewegungsumfang des Kniegelenks ein paar mal. </li><li> Anschließend verdrängt man das Patella seitlich nochmals überprüfen und für jede Veranstaltung oder Zeichen der instabilen Fixierung. Die Matrizen müssen noch an ihrem Platz gehalten werden. </li><li> Ersetzen Sie die Patella wieder und beenden Sie den Vorgang mit Wundverschluss in Schichten und Spray Dressing wie oben beschrieben. </li></ol>

<ol>
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Orthop. 7, 300-304 (1997).</li><li>Brittberg, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+deep+cartilage+defects+in+the+knee+with+autologous+chondrocyte+transplantation.">Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation.</a> N. Engl. J. Med. 331, 889-895 (1994).</li><li>Peterson, L., Vasiliadis, H. S., Brittberg, M., Lindahl, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+chondrocyte+implantation:+a+long-term+follow-up.">Autologous chondrocyte implantation: a long-term follow-up.</a> Am. J. 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J. Radiol. 57, 24-31 (2006).</li><li>Benya, P. D., Shaffer, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dedifferentiated+chondrocytes+reexpress+the+differentiated+collagen+phenotype+when+cultured+in+agarose+gels.">Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels.</a> Cell. 30, 215-224 (1982).</li><li>Rudert, M., Hirschmann, F., Wirth, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+behavior+of+chondrocytes+on+various+biomaterials.">Growth behavior of chondrocytes on various biomaterials.</a> Orthopade. 28, 68-75 (1999).</li><li>Hsu, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+biodegradable+polyesters+modified+by+type+II+collagen+and+Arg-Gly-Asp+as+Tissue+Engineering+scaffolding+materials+for+cartilage+regeneration.">Evaluation of biodegradable polyesters modified by type II collagen and Arg-Gly-Asp as Tissue Engineering scaffolding materials for cartilage regeneration.</a> Artificial Organs. 30, 42-55 (2006).</li><li>Brun, P., Cortivo, R., Zavan, B., Vecchiato, N., Abatangelo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro++reconstructed+tissues+on+hyaluronan-based+temporary+scaffolding.">In vitro reconstructed tissues on hyaluronan-based temporary scaffolding.</a> J. Mater. Sci. Mater. Med. 10, 683-688 (1999).</li><li>Domm, C., Fay, J., Schunke, M., Kurz, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redifferentiation+of+dedifferentiated+joint+cartilage+cells+in+alginate+culture.+Effect+of+intermittent+hydrostatic+pressure+and+low+oxygen+partial+pressure.">Redifferentiation of dedifferentiated joint cartilage cells in alginate culture. Effect of intermittent hydrostatic pressure and low oxygen partial pressure.</a> Orthopade. 29, 91-99 (2000).</li><li>Kimura, T., Yasui, N., Ohsawa, S., Ono, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondrocytes+embedded+in+collagen+gels+maintain+cartilage+phenotype+during+long-term+cultures.">Chondrocytes embedded in collagen gels maintain cartilage phenotype during long-term cultures.</a> Clin. Orthop. Relat. Res. , 231-239 (1984).</li><li>Kon, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Second-generation+autologous+chondrocyte+implantation:+results+in+patients+older+than+40+years.">Second-generation autologous chondrocyte implantation: results in patients older than 40 years.</a> Am. J. Sports Med. 39, 1668-1675 (2011).</li><li>Gavenis, K., Schmidt-Rohlfing, B., Mueller-Rath, R., Andereya, S., Schneider, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+comparison+of+six+different+matrix+systems+for+the+cultivation+of+human+chondrocytes.">In vitro comparison of six different matrix systems for the cultivation of human chondrocytes.</a> In Vitro Cell Dev. Biol. 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X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+outcomes+of+alginate-embedded+allogenic+mesenchymal+stem+cells+versus+autologous+chondrocytes+for+the+repair+of+focal+articular+cartilage+defects+in+a+rabbit+model.">Treatment outcomes of alginate-embedded allogenic mesenchymal stem cells versus autologous chondrocytes for the repair of focal articular cartilage defects in a rabbit model.</a> The American Journal of Sports Medicine. 40, 83-90 (2012).</li><li>Brittberg, M., Nilsson, A., Lindahl, A., Ohlsson, C., Peterson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rabbit+articular+cartilage+defects+treated+with+autologous+cultured+chondrocytes.">Rabbit articular cartilage defects treated with autologous cultured chondrocytes.</a> Clin. Orthop. Relat. Res. , 270-283 (1996).</li></ol>

Die Autoren haben nichts zu offenbaren.

Das vorgestellte Protokoll bietet eine etablierte 9,12,13 und leicht reproduzierbar Technik autologen Chondrozyten für nachfolgende Proliferation und Re-Implantation in künstlich geschaffenen Knorpelschäden in Knie Kaninchen isolieren. Die Verwendung von autologen Chondrozyten für Umbau und Reparatur von Gelenkknorpel Läsionen ist bereits in der klinischen Anwendung bieten befriedigende Langzeitergebnisse 6. Große Probleme wie beispielsweise Periost Hypertrophie u...

<table border="1">
	<tbody>
		<tr>
			<td>Name of reagent/equipment</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Biochrom AG</td>
			<td>F 0415</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase A</td>
			<td>Roche</td>
			<td>10 103 586 001</td>
			<td>0.21 U/mg</td>
		</tr>
		<tr>
			<td>Fetal calf serum (FCS)</td>
			<td>PAN Biotech GmbH</td>
			<td>3702-P103009</td>
			<td></td>
		</tr>
		<tr>
			<td>Propofol</td>
			<td>Fresenius Kabi</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Biochrom AG</td>
			<td>A 2213</td>
			<td>10,000 U/ml/10,000 &mu;g/ml</td>
		</tr>
		<tr>
			<td>PBS Dulbecco (1X)</td>
			<td>Biochrom AG</td>
			<td>L1815</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (70%)</td>
			<td>Merck KgaA</td>
			<td>410230</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.25 %/0.02 %</td>
			<td>Biochrom AG</td>
			<td>L2163</td>
			<td>in PBS w/o Ca2+, Mg2+</td>
		</tr>
		<tr>
			<td>Fentanyl</td>
			<td>Delta Select GmBH</td>
			<td>1819340</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl solution (0.9%)</td>
			<td>Bbraun</td>
			<td>8333A193</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture dishes 100 mm/150 mm</td>
			<td>TPP AG</td>
			<td>93100/93150</td>
			<td>Growth area 60.1 mm2/147.8 mm2</td>
		</tr>
		<tr>
			<td>Tissue culture flasks 25/75 mm2</td>
			<td>TPP AG</td>
			<td>90025/90075</td>
			<td>25 mm2, 75 mm2</td>
		</tr>
		<tr>
			<td>Centrifuge Tubes (50 ml)</td>
			<td>TPP AG</td>
			<td>91050</td>
			<td>Gamma-sterilized</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Brand GmbH+Co KG</td>
			<td>717810</td>
			<td>Neubauer</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution 0.4%</td>
			<td>Sigma-Aldrich</td>
			<td>L8154</td>
			<td></td>
		</tr>
		<tr>
			<td>Spray dressing (OpSite)</td>
			<td>Smith&amp;Nephew</td>
			<td>66004978</td>
			<td>Permeable for water vapor</td>
		</tr>
		<tr>
			<td>Chondro-Gide&Ograve;</td>
			<td>Geistlich Pharma AG</td>
			<td>30915.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy Punch</td>
			<td>pfm medical ag</td>
			<td>48351</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissucol Duo S</td>
			<td>Baxter</td>
			<td>3419627</td>
			<td>0.5 ml</td>
		</tr>
	</tbody>
</table>

matrix-assisted autologous chondrocyte transplantation for remodeling and repair of chondral defects in a rabbit model

Gelenkknorpel Mängel sind ein großes gesundheitliches Problem betrachtet, weil Gelenkknorpel hat eine begrenzte Kapazität für Selbst-Regeneration 1. Unbehandelte Knorpelschäden zu andauernden Schmerz zu führen, was sich negativ auf die Qualität des Lebens und der Prädisposition für Arthrose. In den letzten Jahrzehnten wurden verschiedene chirurgische Techniken entwickelt, um solche Läsionen zu behandeln. Doch bis jetzt war es nicht möglich, eine vollständige Reparatur in Bezug auf die Deckung des Defektes mit hyaline Gelenkknorpel oder eine zufriedenstellende langfristige Erholung 2-4 zu erreichen. Daher bleiben Gelenkknorpel Verletzungen ein primäres Ziel für regenerative Techniken wie Tissue Engineering. Im Gegensatz zu anderen chirurgischen Verfahren, die oft zur Bildung von Faser-oder fibrocartilagineum Gewebe führen soll Tissue Engineering zu vollständigen Wiederherstellung der komplexen Struktur und die Eigenschaften der ursprünglichen Gelenkknorpel unter Verwendung der chondrogenen Potential der transplantierten Zellen. RÜNGSTE Entwicklungen eröffnet vielversprechende Möglichkeiten für regenerative Therapien Knorpel. Die erste Zelle Ansatz für die Behandlung von tiefen osteochondralen Läsionen oder Knorpel wurde 1994 von Lars Peterson und Mats Brittberg die klinische autologe Chondrozyten-Implantation (ACI) 5 Pionierarbeit durchgeführt. Heute ist die Technik in der Klinik zur Behandlung von großen Knorpelschaden Mängel des Knies gut etabliert, das Beibehalten der guten klinischen Ergebnisse auch 10 bis 20 Jahre nach der Implantation 6. In den letzten Jahren erlebte die Implantation von autologen Chondrozyten eine schnelle Progression. Der Einsatz eines künstlichen dreidimensionalen Kollagen-Matrix, auf der Zellen anschließend wieder aufgeforstet werden immer mehr und mehr populär 7-9. MACT besteht aus zwei Operationen: Erstens, um Chondrozyten sammeln muss ein Knorpel-Biopsie von einem nicht belasteten Bereich der Knorpel t durchgeführt werden,er Kniegelenk. Dann werden Chondrozyten, die extrahiert, gereinigt und erweitert werden, um eine ausreichende Anzahl von Zellen in vitro. Chondrozyten werden dann auf eine dreidimensionale Matrix ausgesät und anschließend wieder implantiert werden. Bei der Herstellung einer Tissue-Engineering-Implantat sind Proliferationsrate und Differenzierung Kapazität von entscheidender Bedeutung für eine erfolgreiche Geweberegeneration 10. Verwendung einer dreidimensionalen Matrix als Zellträger wird angenommen, dass diese zellulären Merkmale 11 zu unterstützen. Das folgende Protokoll wird zusammengefasst und zeigen eine Technik zur Isolierung von Chondrozyten aus Knorpel Biopsien, ihre Proliferation in vitro und deren Aussaat auf einer 3D-Matrix (Chondro-Gide, Geistlich Biomaterials, Wollhusen, Schweiz). Schließlich wird die Implantation der Zell-Matrix-Konstrukte in künstlich geschaffenen Knorpeldefekten eines Kaninchens Kniegelenk beschrieben. Diese Technik kann als eine experimentelle Einstellung verwendet werdenfür weitere Experimente von Knorpel zu reparieren.

Die beschriebene Operationstechnik ermöglicht eine erfolgreiche Isolierung und die Implantation von autologen Chondrozyten in ein künstliches Knorpeldefekt. Der experimentelle Aufbau ergab eine erfolgreiche Integration des Implantates in den umgebenden Knorpel. Nach 12 Wochen in vivo wurde der Knorpeldefekt durch Reparatur Gewebe mit einer homogenen und intakte Oberfläche, die Schubspannung und Schäden an dem Implantat (4) reduziert gefüllt. Außerdem wurden ke...

Watch this Scientific Journal Video about Matrix-assisted Autologous Chondrocyte Transplantation for Remodeling and Repair of Chondral Defects in a Rabbit Model at JoVE.com

Matrix-assisted Autologous Chondrocyte Transplantation for Remodeling and Repair of Chondral Defects in a Rabbit Model

Ein experimentelles Verfahren für die Behandlung von Knorpeldefekten in das Kaninchen Kniegelenk beschrieben. Die Implantation von autologen Chondrozyten auf einer Matrix ausgesät ist eine gut akzeptierte Methode für den Umbau und die Reparatur von Gelenkknorpelläsionen Bereitstellung befriedigend langfristige Ergebnisse. Matrix-assistierte autologe Chondrozyten-Transplantation (MACT) bietet eine standardisierte und klinisch etablierte Methode der Implantation.

Matrix-assistierte autologe Chondrozyten-Transplantation für Umbau und Reparatur von Knorpeldefekten in einem Kaninchen-Modell

Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration 1. Untreated ...

matrix-assisted-autologous-chondrocyte-transplantation-for-remodeling

Research

JoVE Journal

Medicine

14.3K Aufrufe.  Klinikum rechts der Isar der Technischen Universität München. Ein experimentelles Verfahren für die Behandlung von Knorpeldefekten in das Kaninchen Kniegelenk beschrieben. Die Implantation von autologen Chondrozyten auf einer Matrix ausgesät ist eine gut akzeptierte Methode für den Umbau und die Reparatur von Gelenkknorpelläsionen Bereitstellung befriedigend langfristige Ergebnisse. Matrix-assistierte autologe Chondrozyten-Transplantation (MACT) bietet eine standardisierte und klinisch etablierte Methode der Implantation.

Serie: Matrix-assisted Autologous Chondrocyte Transplantation for Remodeling and Repair of Chondral Defects in a Rabbit Model

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

Einem Mausmodell der In Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

Forschung

Medizin

Development of Obliterative Bronchiolitis in a Murine Model of Orthotopic Lung Transplantation

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and standardized not only for the study of allo-rejection but also between syngeneic strains for examining mechanisms of ischemia-reperfusion injury after lung transplantation. Although the application of the rat and other large animal model 2 contributed significantly to the elucidation of these studies, the scope of those investigations is limited by the scarcity of knockout and transgenic rats. Due to no effective therapies for obliterative bronchiolitis, the leading cause of death in lung transplant patients, there has been an intensive search for pre-clinical models that replicate obliterative bronchiolitis. The tracheal allograft model is the most widely used and may reproduce some of the histopathologic features of obliterative bronchiolitis 3. However, the lack of an intact vasculature with no connection to the recipient's conducting airways, and incomplete pathologic features of obliterative bronchiolitis limit the utility of this model 4. Unlike transplantation of other solid organs, vascularized mouse lung transplants have only recently been reported by Okazaki and colleagues for the first time in 2007 5. Applying the basic principles of the rat lung transplant, our lab initiated the obliterative bronchiolitis model using minor histoincompatible antigen murine orthotopic single-left lung transplants which allows the further study of obliterative bronchiolitis immunopathogenesis6.

Obliterative bronchiolitis is the key impediment to the long-term survival of lung transplant recipients and the lack of a robust preclinical model precludes examining obliterative bronchiolitis immunopathogenesis. Unlike other solid organ transplants, vascularized mouse lung transplantation has only recently been developed. Here we show our independently developed obliterative bronchiolitis model after murine orthotopic single-lung transplantation.

Entwicklung einer Bronchiolitis obliterans in einem Mausmodell der orthotopen Lungentransplantation

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and ...

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

Eine Maus fetalen Hautmodell von Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

A Rat Carotid Balloon Injury Model to Test Anti-vascular Remodeling Therapeutics

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is also a valuable model system to test, and to evaluate therapeutics and drugs that negate maladaptive remodeling in the vessel. The injury, or barotrauma, in the vessel lumen caused by an inflated balloon via an inserted catheter induces subsequent neointimal growth, often leading to hyperplasia or thickening of the vessel wall that narrows, or obstructs the lumen. The method described here is sufficiently sensitive, and the results can be obtained in relatively short time (2 weeks after the surgery). The efficacy of the drug or therapeutic against the induced-remodeling can be evaluated either by the post-mortem pathological and histomorphological analysis, or by ultrasound sonography in live animals. In addition, this model system has also been used to determine the therapeutic window or the time course of the administered drug. These studies can leadto the development of a better administrative strategy and a better therapeutic outcome. The procedure described here provides a tool for translational studies that bring drug and therapeutic candidates from bench research to clinical applications.

The rat carotid balloon injury model described below allows researchers to evaluate drugs or therapeutics that negate injury-induced arterial hyperplasia. Detailed pre-surgical preparation, surgical procedure, and post-surgical cares of the animal are described.

Eine Ratte Karotis-Ballon-Verletzung Modell Anti-vaskuläre Remodeling Therapeutics zu Test

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is ...

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the treatment of many hematopoietic diseases in humans, as their use offers stable transgene expression due to the vector&#39;s ability to integrate into the host genome. This method demonstrates the application of ex vivo gene therapy of hepatocytes to a large animal model of hereditary tyrosinemia type I. This process consists of 1) isolation of primary hepatocytes from the autologous donor/recipient animal, 2) ex vivo gene delivery via hepatocyte transduction with a lentiviral vector, and 3) autologous transplant of corrected hepatocytes via portal vein injection. Success of the method generally relies upon efficient and sterile removal of the liver resection, careful handling of the excised specimen for isolation of viable hepatocytes sufficient for re-engrafting, high-percentage transduction of the isolated cells, and aseptic surgical procedures throughout to prevent infection. Technical failure at any of these steps will result in low yield of viable transduced hepatocytes for autologous transplant or infection of the donor/recipient animal. The pig model of human type 1 hereditary tyrosinemia (HT-1) chosen for this approach is uniquely amenable to such a method, as even a small percentage of engraftment of corrected cells will lead to repopulation of the liver with healthy cells based on a powerful selective advantage over native-diseased hepatocytes. Although this growth selection will not be true for all indications, this approach is a foundation for expansion into other indications and allows for manipulation of this environment to address additional diseases, both within the liver and beyond, while controlling for exposure to viral vector and opportunity for off-target toxicity and tumorigenicity.

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

This protocol is intended to describe porcine hepatocyte isolation and ex vivo gene delivery to cure models of metabolic diseases via autologous cell transplantation. Although this particular model enjoys unique advantages that favor successful therapy, the application is a relevant foundation to address additional diseases and indications.

Lentivirale Vektor-vermittelte Gentherapie von Hepatozyten Ex Vivo für autologe Transplantation bei Schweinen

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the ...

Transthoracic Echocardiographic Examination in the Rabbit Model

Large animal models such as the rabbit are valuable for translational preclinical research. Rabbits have a similar cardiac electrophysiology compared to that of humans and that of other large animal models such as dogs and pigs. However, the rabbit model has the additional advantage of lower maintenance costs compared to other large animal models. The longitudinal evaluation of cardiac function using echocardiography, when appropriately implemented, is a useful methodology for preclinical assessment of novel therapies for heart failure with reduced ejection fraction (e.g. cardiac regeneration). The correct use of this non-invasive tool requires the implementation of a standardized examination protocol following international guidelines. Here we describe, step by step, a detailed protocol supervised by veterinary cardiologists for performing echocardiography in the rabbit model, and demonstrate how to correctly obtain the different echocardiographic views and imaging planes, as well as the different imaging modes available in a clinical echocardiography system routinely used in human and veterinary patients.

Here we describe, step by step, a detailed protocol for performing echocardiography in the rabbit model. We show how to correctly obtain the different echocardiographic views and imaging planes, as well as the different imaging modes available in a clinical echocardiography system routinely used in human and veterinary patients.

Transthorakale Echokardiographische Untersuchung im Kaninchenmodell

Large animal models such as the rabbit are valuable for translational preclinical research. Rabbits have a similar cardiac electrophysiology compared to ...

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These conditions include retinitis pigmentosa (RP) and advanced forms of age-related macular degeneration (AMD), such as geographic atrophy (GA). Together, these disorders represent a significant proportion of currently untreatable blindness globally. These unmet medical needs have generated heightened academic interest in developing methods of RPE replacement. Among the animal models commonly utilized for preclinical testing of therapeutics, the non-human primate (NHP) is the only animal model that has a macula. As it shares this anatomical similarity with the human eye, the NHP eye is an important and appropriate preclinical animal model for the development of advanced therapy medicinal products (ATMPs) such as RPE cell therapy.
This manuscript describes a method for the submacular transplantation of an RPE monolayer, cultured on a polyethylene terephthalate (PET) cell carrier, underneath the macula onto a surgically created RPE wound in immunosuppressed NHPs. The fovea-the central avascular portion of the macula-is the site of the greatest mechanical weakness during the transplantation. Foveal trauma will occur if the initial subretinal fluid injection generates an excessive force on the retina. Hence, slow injection under perfluorocarbon liquid (PFCL) vitreous tamponade is recommended with a dual-bore subretinal injection cannula at low intraocular pressure (IOP) settings to create a retinal bleb. 
Pretreatment with an intravitreal plasminogen injection to release parafoveal RPE-photoreceptor adhesions is also advised. These combined strategies can reduce the likelihood of foveal tears when compared to conventional techniques. The NHP is a key animal model in the preclinical phase of RPE cell therapy development. This protocol addresses the technical challenges associated with the delivery of RPE cellular therapy in the NHP eye.

The non-human primate (NHP) is an ideal model for studying human retinal cellular therapeutics due to the anatomical and genetic similarities. This manuscript describes a method for submacular transplantation of retinal pigment epithelial cells in the NHP eye and strategies to prevent intraoperative complications associated with macular manipulation.

Retinale Pigmentepitheltransplantation in einem nicht-menschlichen Primatenmodell für degenerative Netzhauterkrankungen

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These ...

A Simplified Model for Heterotopic Heart Valve Transplantation in Rodents

There is an urgent clinical need for heart valve replacements that can grow in children. Heart valve transplantation is proposed as a new type of transplant with the potential to deliver durable heart valves capable of somatic growth with no requirement for anticoagulation. However, the immunobiology of heart valve transplants remains unexplored, highlighting the need for animal models to study this new type of transplant. Previous rat models for heterotopic aortic valve transplantation into the abdominal aorta have been described, though they are technically challenging and costly. For addressing this challenge, a renal subcapsular transplant model was developed in rodents as a practical and more straightforward method for studying heart valve transplant immunobiology. In this model, a single aortic valve leaflet is harvested and inserted into the renal subcapsular space. The kidney is easily accessible, and the transplanted tissue is securely contained in a subcapsular space that is well vascularized and can accommodate a variety of tissue sizes. Furthermore, because a single rat can provide three donor aortic leaflets and a single kidney can provide multiple sites for transplanted tissue, fewer rats are required for a given study. Here, the transplantation technique is described, providing a significant step forward in studying the transplant immunology of heart valve transplantation.

This protocol describes a simple and efficient method for the transplantation of aortic valve leaflets under the renal capsule to allow for the study of alloreactivity of heart valves.

Ein vereinfachtes Modell für die heterotope Herzklappentransplantation bei Nagetieren

There is an urgent clinical need for heart valve replacements that can grow in children. Heart valve transplantation is proposed as a new type of ...

Left Lung Orthotopic Transplantation in a Juvenile Porcine Model for ESLP

Lung transplantation is the gold-standard treatment for end-stage lung disease, with over 4,600 lung transplantations performed worldwide annually. However, lung transplantation is limited by a shortage of available donor organs. As such, there is high waitlist mortality. Ex situ lung perfusion (ESLP) has increased donor lung utilization rates in some centers by 15%-20%. ESLP has been applied as a method to assess and recondition marginal donor lungs and has demonstrated acceptable short- and long-term outcomes following transplantation of extended criteria donor (ECD) lungs. Large animal (in vivo) transplantation models are required to validate ongoing in vitro research findings. Anatomic and physiologic differences between humans and pigs pose significant technical and anesthetic challenges. An easily reproducible transplant model would permit the&#160;in vivo&#160;validation of current ESLP strategies and the preclinical evaluation of various interventions designed to improve donor lung function. This protocol describes a porcine model of orthotopic left lung allotransplantation. This includes anesthetic and surgical techniques, a customized surgical checklist, troubleshooting, modifications, and the benefits and limitations of the approach.

This protocol describes a juvenile porcine model of orthotopic left lung allotransplantation designed for use with ESLP research. Focus is made on anesthetic and surgical techniques, as well as critical steps and troubleshooting.

Orthotope Transplantation der linken Lunge in einem juvenilen Schweinemodell für ESLP

Lung transplantation is the gold-standard treatment for end-stage lung disease, with over 4,600 lung transplantations performed worldwide annually. ...

Transcatheter Pulmonary Valve Replacement from Autologous Pericardium with a Self-Expandable Nitinol Stent in an Adult Sheep Model

Transcatheter pulmonary valve replacement has been established as a viable alternative approach for patients suffering from right ventricular outflow tract or bioprosthetic valve dysfunction, with excellent early and late clinical outcomes. However, clinical challenges such as stented heart valve deterioration, coronary occlusion, endocarditis, and other complications must be addressed for lifetime application, particularly in pediatric patients. To facilitate the development of a lifelong solution for patients, transcatheter autologous pulmonary valve replacement was performed in an adult sheep model. The autologous pericardium was harvested from the sheep via left anterolateral minithoracotomy under general anesthesia with ventilation. The pericardium was placed on a 3D shaping heart valve model for non-toxic cross-linking for 2 days and 21 h. Intracardiac echocardiography (ICE) and angiography were performed to assess the position, morphology, function, and dimensions of the native pulmonary valve (NPV). After trimming, the crosslinked pericardium was sewn onto a self-expandable Nitinol stent and crimped into a self-designed delivery system. The autologous pulmonary valve (APV) was implanted at the NPV position via left jugular vein catheterization. ICE and angiography were repeated to evaluate the position, morphology, function, and dimensions of the APV. An APV was successfully implanted in&#160;sheep J. In this paper, sheep J was selected to obtain representative results. A 30 mm APV with a Nitinol stent was accurately implanted at the NPV position without any significant hemodynamic change. There was no paravalvular leak, no new pulmonary valve insufficiency, or stented pulmonary valve migration. This study demonstrated the feasibility and safety, in a long-time follow-up, of developing an APV for implantation at the NPV position with a self-expandable Nitinol stent via jugular vein catheterization in an adult sheep model.

This study demonstrates the feasibility and safety of developing an autologous pulmonary valve for implantation at the native pulmonary valve position by using a self-expandable Nitinol stent in an adult sheep model. This is a step toward developing transcatheter pulmonary valve replacement for patients with right ventricular outflow tract dysfunction.

Transkatheter-Pulmonalklappenersatz aus autologem Perikard mit einem selbstexpandierenden Nitinol-Stent in einem erwachsenen Schafmodell

Transcatheter pulmonary valve replacement has been established as a viable alternative approach for patients suffering from right ventricular outflow ...