Name: establishment of a clinic-based biorepository
Uploaded: 2017-05-29T14:00:00
Description: Watch this Scientific Journal Video about Establishment of a Clinic-based Biorepository at JoVE.com

Diese Arbeit wurde durch Mittel von der Midwestern University College of Health Sciences Research Facilitation Grant, verliehen an EEH und Midwestern University Office of Research und Sponsored Programme Intramural Grant, an KJL vergeben unterstützt. Zusätzliche Unterstützung wurde von angeschlossenen Laboratorien und angeschlossenen Dermatologie zur Verfügung gestellt. Wir danken Sarah Potekhen, Jamie Barto, Stefani Fawks, Cody Jording, Stacie Schimke, Heather Kissel und Ali Zaidi für ihre technische Unterstützung.

Alle Experimente an menschlichen Gewebeproben wurden von Western IRB (WIRB-Protokoll Nr. 20142461) genehmigt. Das einzige Kriterium für die Sammlung von Geweben für diese Verfahren ist eine Biopsie-bewährte Diagnose von SCC. Es wurden keine Ausschlusskriterien im ursprünglichen IRB-Protokoll skizziert, aber Proben von Patienten mit bekannter blutübertragbarer Krankheit wurden nicht verwendet. Informierte Zustimmung wurde vor der Sammlung von Hautgewebe, Blut und Speichel erhalten. Midwestern Institutional Review Bo...

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R., Schwietert, C. W., Jones, A. A., Stern, R. L., Lehman, J., Goldberg, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+methodology+for+sequential+extraction+of+RNA+and+protein+from+small+human+skin+biopsies.">Optimized methodology for sequential extraction of RNA and protein from small human skin biopsies.</a> J Invest Dermatol. 127, 349-353 (2007).</li><li>Schroeder, A., 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=The+RIN:+an+RNA+integrity+number+for+assigning+integrity+values+to+RNA+measurements.">The RIN: an RNA integrity number for assigning integrity values to RNA measurements.</a> BMC Mol Biol. 7, 1-14 (2006).</li><li>Cooper, H. L., 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=Expression+and+glycosylation+of+MUC1+in+epidermolysis+bullosa-associated+and+sporadic+cutaneous+squamous+cell+carcinomas.">Expression and glycosylation of MUC1 in epidermolysis bullosa-associated and sporadic cutaneous squamous cell carcinomas.</a> Br J Dermatol. 151, 540-545 (2004).</li><li>Riihila, P. 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=Complement+factor+H:+a+biomarker+for+progression+of+cutaneous+squamous+cell+carcinoma.">Complement factor H: a biomarker for progression of cutaneous squamous cell carcinoma.</a> J Invest Dermatol. 134, 498-506 (2014).</li><li>Ha Lan, T. 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Nach dem Wissen des Autors ist dieses Protokoll das erste seiner Art, das sich auf die klinische Beschaffung von Hautgewebeproben sowohl in einem kostengünstigen als auch in einem schnellen Ansatz konzentriert. Patienten, die sich einer Mohs-Mikrochirurgie unterziehen, sind typischerweise während bestimmter Zeitblöcke geplant, und die Sammlung ist auf diese Zeiträume beschränkt. Die Probenahme beinhaltet die Anstrengung des medizinischen Assistenten, der an der Patientenversorgung beteiligt ist, der Mohs-Histotechn...

Krebs- und Biomarkerforschung beruht auf einer Versorgung mit hochwertigen menschlichen Gewebeproben, und begrenztes Angebot hat die Forschung behindert 1 , 2 . Viele dermatologische Studien sind durch die unzureichende Versorgung, die variable Qualität und die Kosten im Zusammenhang mit der Verwendung von menschlichem Gewebe begrenzt. Die Kosten für die Gründung einer großen, dedizierten Biobank wurde auf etwa zwei Millionen Dollar 3 geschätzt, und diese Kosten legen die Verwendung von menschlichem Gewebe aus der Reichweite vieler Forscher. Darüber hinaus stellt der Prozess der Erzeugung und Speicherung von Forschungsproben das Risiko dar, klinische Operationen zu beeinträchtigen und die Patientenversorgung zu verzögern, wenn sie nicht sorgfältig durchgeführt wird. Es wurde ein kostengünstiges, klinikbasiertes Biorepository etabliert, das sich auf Hautkrebsproben nach empfohlenen Best Practices und Probenvalidierung 4 , 5 , 6 konzentriert. <pEnglisch: www.tab.fzk.de/en/projekt/zusammenf...ng/ab117.htm Dieses Protokoll wurde in einer Dermatologie-Klinik entwickelt, die ein großes Volumen an Mohs-mikrographischen Operationen durchführt, um Plattenepithelkarzinome (SCC), Basalzellkarzinom (BCC) und Melanom-Hautkrebs zu entfernen. Freiwillige Spender können von dieser Patientenpopulation rekrutiert werden. Es ist wichtig, das Biorepository an der Stelle der Sammlung zu etablieren, um schnell Gewebe und Blut von assoziierten Patienten ohne Verzögerung der Behandlung zu erfassen. Das Sammeln von Proben aus der gleichen Klinik minimiert Variationen in der Sammlungstechniken und minimiert Variationen in der Qualität der Proben, was für die nachgeschalteten Anwendungen problematisch sein kann 7 , 8 . Das Ziel der Mohs mikrographischen Chirurgie Technik ist, um sicherzustellen, dass alle Krebs Gewebe entfernt wird, während die Erhaltung so viel gesundes Gewebe wie möglich. Das Verfahren beinhaltet die fortschreitende Entfernung von dünnen Schichten von Tumorgewebe. Jede aufeinanderfolgende Schicht ist seineTologisch untersucht (nach der Kryosektion des Tumorgewebes und Durchführung von H &amp; E-Färbung) durch einen Dermatologen, um festzustellen, ob alle Krebsgewebe entfernt worden sind. Die Exzision und Untersuchung der nachfolgenden Gewebeschichten erfolgt, während der Patient im Büro bleibt. Diese Technik gilt als die beste Behandlungsoption für SCC 9 . An diesem Punkt wird die Wunde geschlossen und zur Verbesserung der Heilung und des kosmetischen Erscheinungsbildes wird neben normalem Gewebe (ANT) häufig herausgeschnitten. So ist dieses chirurgische Verfahren zur Entfernung eines Tumors ideal geeignet, um histologisch charakterisiertes Gewebe für zukünftige Studien zu sammeln. Das Beschaffungsverfahren zur Gewinnung von Tumorgewebe, angrenzend an normalem Gewebe, Speichel und Blutproben wurde so entworfen, dass es minimale Auswirkungen auf die normalen Personalaufgaben hat (Abbildung 1 ). Medizinische Assistenten führen das Blut zieht bei der Vorbereitung des Patienten für das Verfahren. Nach Beendigung des Mohs-Verfahrens ist das M<html> Ohs Histotechnologe bereitet zusätzliche histologische Folien der Probe vor und überträgt das Gewebe auf das Biorepository. Die Kosten für die Errichtung des Biorepositorys beinhalten den Kauf von Kryokonservierungsgefriergeräten, die Schaffung eines bescheidenen klinischen Laborraums und die Entwicklung eines Inventarverfolgungsprogramms. <img alt="Abbildung 1" src="/files/ftp_upload/55583/55583fig1.jpg" /> Abbildung 1: Reihenfolge der Probenahme und verantwortungsbewusstes Personal Nach dem Check-in des Patienten und dem Erreichen der Zustimmung des Patienten sammelt der medizinische Assistent einen bukkalen Tupfer und führt einen Blutentnahme durch. Der Dermatologe und der medizinische Assistent überholen dann den Tumor und schließen die Wunde, während welcher Zeit SCC- und ANT-Proben gesammelt werden. Ein engagierter Labortechniker verarbeitet das Blut und deckt die SCC- und ANT-Proben für die Gewebekultur, die Konservierung und den Eintritt in das Biorepository ab.Ftp_upload / 55583 / 55583fig1large.jpg &quot;target =&quot; _ blank &quot;&gt; Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen. Die Vielfalt der gesammelten Proben ermöglicht eine Vielzahl von experimentellen Ansätzen (Abbildung 2 ). Proben, die vom Patienten gesammelt wurden, sind bukkale Tupfer (Speichel kann auch gesammelt werden, wenn nötig), Vollblut und ausgeschnittenes Gewebe. Die bukkalen Tupfer und eine Probe aus dem Vollblut werden ohne Verarbeitung für die Genotypisierung und Gewebeanpassung gerettet. Vollblut wird in weiße Blutkörperchen (WBC) und Plasmafraktionen für zukünftige Analysen getrennt. Nach der Mohs-Verarbeitung wird der gefrorene Tumor direkt in flüssigen Stickstoff gegeben und auf einen Gefrierschrank von -80 ° C übertragen. Frisches, lebensfähiges Tumorgewebe und ANT-Proben werden unter Verwendung von Modifikationen der vorherigen Techniken 10 , 11 kultiviert und dann kryokonserviert. Während der Erhebung wird die Anzahl der einzelnen Sample-Typen auf einer Kalkulationstabelle vor dem Eintritt aufgezeichnet<html> Das Inventar-Tracking-Programm zur Erleichterung der genauen Verarbeitung ( Tabelle 1 ). <img alt="Figur 2" src="/files/ftp_upload/55583/55583fig2.jpg" /> Abbildung 2: Überblick über die Klinik-basierte Biorepository Probenahme und -verarbeitung. Ein bukkaler Tupfer und eine Blutprobe werden vom Patienten gesammelt und für die nachgeschaltete Genotypisierung und Gewebeanpassung gespeichert. Vollblut wird für die Isolierung der weißen Blutkörperchen (WBC) und die CTC-Analyse sowie für die Plasmasammlung und die Flüssigbiopsieanalyse weiterverarbeitet. Gewebe, das während des Mohs-Verfahrens ausgeschnitten wird, wird für diagnostische Zwecke histologisch verarbeitet, wonach die histologischen Folien experimentell für weitere immunhistochemische Analysen eingesetzt werden können. Unter der Voraussetzung, dass die exzidierte Gewebeprobe groß genug ist, wird ein Teil des frischen Gewebes entfernt und für die Protein- und RNA-Isolierung geschnitten und für die Herstellung von kultivierten Zelllinien.55583 / 55583fig1large.jpg &quot;target =&quot; _ blank &quot;&gt; Bitte hier klicken um eine größere Version dieser Figur zu sehen. <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Abholtermin: </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td> Patient 1 </td><td> Patient 2 </td><td> Patient 3 </td><td> Patient 4 </td><td> Patient 5 </td></tr><tr><td> Initialen und Geburtsdatum </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Augenfarbe </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Beispielstyp </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Beispiel Ort </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Speichel </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> WLoch Blut </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Plasma </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Tumor lebensfähig </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Gewebe Normal lebensfähig </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Tumor Mohs Flüssiger Stickstoff </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Tissue Normal Liquid Stickstoff </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Rutschen </td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table> Tabelle 1: Checkliste zum Aufnehmen von Sample-Sammlungen. Daten, die mit jeder gesammelten Probe verfolgt und aufgezeichnet wurden, beinhalten Patienteninitialen, Geburtsdatum und Augenfarbe (für die Hauttypisierung) sowie die locatiAuf Probenentfernung Die Anzahl der Speichelproben, die Blutentnahmevolumina und die Anzahl der lebensfähigen und konservierten Gewebeproben, die gesammelt wurden, werden auch als Referenzen für Zuweisungen zu späteren Verwendungen aufgezeichnet. <a href="http://ecsource.jove.com/files/ftp_upload/55583/Table1.xlsx">Bitte klicken Sie hier, um diese Datei herunterzuladen.</a> Um die Probenahmeverfahren zu validieren, wurde jeder Sample-Typ in nachgeschalteten Anwendungen getestet. Unter Verwendung von Modifikationen von früheren Techniken 12 wurden Tumor und ANT erfolgreich in der Protein- und RNA-Isolierung verwendet und können möglicherweise für die DNA-Isolierung verwendet werden. Lebensfähige Explantate aus den Gewebeschnitten wurden durch Mikroskopie ausgewertet, während gespeicherte histologische Objektträger für die Immunhistochemie und Immunfluoreszenz verwendet wurden. Durch das hier beschriebene Protokoll ist es möglich, dieses Modell auf andere Dermatologiekliniken, andere Tumortypen (wie Melanom) zu erweitern,, Und andere chirurgische Spezialitäten und Praktiken, um menschliche Gewebe Proben für vielfältige Forschung in menschlichen Krebs zu liefern. Leichte Modifikationen dieses Protokolls sind wahrscheinlich für andere Praktiken notwendig, aber grundsätzlich ist dieses Protokoll auf jede chirurgische Praxis anwendbar, die routinemäßig Patientenproben, die im Laufe der Patientenbehandlung gesammelt wurden, verwischt.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
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			<td>BD Vacutainer Plastic Blood Collection Tubes with K3EDTA</td>
			<td>ThermoFisher Scientific</td>
			<td>02-685-2B</td>
			<td></td>
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			<td>Electron Microscopy Sciences Double Edge Blades</td>
			<td>ThermoFisher Scientific</td>
			<td>50-949-411</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Medium Point General Purpose Forceps</td>
			<td>ThermoFisher Scientific</td>
			<td>16-100-110</td>
			<td></td>
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			<td>Premium Microcentrifuge Tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>05-408-138</td>
			<td></td>
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			<td>Lonza Walkersville KGM Keratinocyte Medium</td>
			<td>ThermoFisher Scientific</td>
			<td>NC9791321</td>
			<td></td>
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			<td>Electron Microscopy Sciences Tissue TEK OCT Compound</td>
			<td>ThermoFisher Scientific</td>
			<td>50-363-579</td>
			<td></td>
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			<td>Leica CM1950 Cryostat</td>
			<td>Leica Biosystems</td>
			<td>14047743909</td>
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			<td>Frosted Microscope Slides</td>
			<td>ThermoFisher Scientific</td>
			<td>12-550-343</td>
			<td></td>
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			<td>Shandon Rapid-Chrome H&#38;E Frozen Section Staining Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>99-900-01</td>
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			<td>Nalgene Long-Term Storage Cryogenic Tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>03-337-7X</td>
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			<td>Falcon 15 mL Conical Centrifuge Tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>14-959-53A</td>
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			<td>Boca Scientific BuccalT-Swab&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>NC9679349</td>
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			<td>ThermoFisher Scientific</td>
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			<td>Halt Protease and Phosphatase Inhibitor Cocktail</td>
			<td>ThermoFisher Scientific</td>
			<td>PI78443</td>
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			<td>Eppendorf 5424R Microcentrifuge</td>
			<td>ThermoFisher Scientific</td>
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			<td>ThermoFisher Scientific</td>
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			<td>RNAse Free Pellet Pestle</td>
			<td>ThermoFisher Scientific</td>
			<td>121-141-364</td>
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			<td>Forma SteriCycle CO2 Incubator</td>
			<td>ThermoFisher Scientific</td>
			<td>13-998-089</td>
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			<td>HyClone Fetal Bovine Serum</td>
			<td>ThermoFisher Scientific</td>
			<td>SH30071.02</td>
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		<tr>
			<td>Gibco Advanced DMEM/F-12</td>
			<td>ThermoFisher Scientific</td>
			<td>12-634-028</td>
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			<td>Gibco Penicillin-Streptomycin</td>
			<td>ThermoFisher Scientific</td>
			<td>15140148</td>
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			<td>Gibco 1M Hepes</td>
			<td>ThermoFisher Scientific</td>
			<td>15-630-130</td>
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			<td>Nunc Cell Culture 35 mm with Vent</td>
			<td>ThermoFisher Scientific</td>
			<td>1256591</td>
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			<td>Anti-CFH monoclonal antibody clone OX-24</td>
			<td>Abnova</td>
			<td>MAB12583</td>
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			<td>Anti-p40 (p63 delta) antibody</td>
			<td>Abnova</td>
			<td>ABX-144A</td>
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			<td>Anti MUC-1 antibody</td>
			<td>Santa Cruz Biotech</td>
			<td>sc-7313</td>
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			<td>Anti-Snail + Slug (phospho S246)</td>
			<td>Abcam</td>
			<td>Ab63568</td>
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		<tr>
			<td>Goat anti-rabbit IgG, Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A-11034</td>
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			<td>Alexa Fluor 568 Phallodin</td>
			<td>Molecular Probes&#160;</td>
			<td>A12380</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant with DAPI</td>
			<td>Molecular Probes&#160;</td>
			<td>P36941</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Axio Imager Z1 Microscope with Axiocam camera</td>
			<td>Zeiss</td>
			<td>4300009901</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus IX51 phase contrast with DP72 camera</td>
			<td>Olympus</td>
			<td>IX511F3</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishment of a clinic-based biorepository

Die Inzidenz von Hautkrebs ( z. B. Plattenepithelkarzinom, Basalzellkarzinom und Melanom) hat in den vergangenen Jahren zugenommen. Es wird erwartet, dass es eine parallele Nachfrage nach kutanen Tumorproben für biomedizinische Forschungsstudien gibt. Die Verfügbarkeit der Gewebe ist jedoch aufgrund der Kosten für die Etablierung eines Biorepositorys und des Mangels an Protokollen, die für die Erlangung von klinischen Proben zur Verfügung stehen, die die klinischen Operationen nicht beeinträchtigen, begrenzt. Es wurde ein Protokoll erstellt, um kutane Tumor und assoziierte Blut- und Speichelproben zu sammeln und zu verarbeiten, die minimale Auswirkungen auf routinemäßige klinische Verfahren am Tag einer Mohs-Chirurgie haben. Tumorproben werden gesammelt und verarbeitet von Patienten, die sich ihrer ersten Schicht der Mohs-Chirurgie für biopsie-bewährte Hautmalignitäten durch den Mohs-Histotechnologen unterziehen. Angrenzendes normales Gewebe wird zum Zeitpunkt des chirurgischen Verschlusses gesammelt. Zusätzliche Proben, die gesammelt werden können, sind Vollblut und bukkale Tupfer. Durch die Verwendung von Gewebeproben, die normalerweise verworfen werden, wurde ein Biorepository erzeugt, das mehrere wesentliche Vorteile bietet, indem es in der Klinik gegenüber der Laborumgebung basiert. Dazu gehören eine breite Palette von gesammelten Proben; Zugang zu erkannten Patientenakten, einschließlich Pathologieberichten; Und, für den typischen Spender, Zugang zu zusätzlichen Proben bei Folgebesuchen.

Tumor und ANT wurden erfolgreich in der Proteinisolation eingesetzt und durch Western Blotting getestet. Wie in Abbildung 3 gezeigt , können kutane Plattenepithelkarzinommarker (Muc1 14 , FHL-1 15 und p40 16 , 17 ) bei Patientenproben nachgewiesen und in unserem klinikbasierten Biorepository gespeichert werden. <p class="jove_content" fo:keep-together.within-pa...

Watch this Scientific Journal Video about Establishment of a Clinic-based Biorepository at JoVE.com

Establishment of a Clinic-based Biorepository

Kutane Tumoren werden oft nach Mohs mikrographischen Chirurgie verworfen. Hier wird ein Protokoll beschrieben, das es dem klinischen Hilfspersonal ermöglicht, kutane Tumor- ( zB Plattenepithelkarzinom-, Basalzellkarzinom- und Melanom-) Proben für nachgeschaltete Laboranwendungen effektiv zu verarbeiten und zu speichern, ohne die klinischen Operationen zu beeinträchtigen.

Errichtung eines klinischen Biorepositorys

The incidence of skin cancer (e.g., squamous cell carcinoma, basal cell carcinoma, and melanoma) has been increasing over the past several years. It is ...

The overall goal of this protocol is to enable clinical support staff in a dermatology practice to effectively process and store cutaneous tumor samples for downstream laboratory applications, without interfering with routine clinical operations. This protocol was designed to enable medical assistants and Mohs histotechnicians to collect and store cutaneous tumor samples for future biomedical research projects, as part of their daily clinical operations. The main advantage of this protocol is that excised tissues that are normally discarded and destroyed are instead collected and preserved for research, using procedures, which minimally impact routine clinical operations.The variety of samples collected enables correlations between protein and RNA biomarkers in the tissue and plasma samples with patient outcome data. In principle, this protocol, once slightly modified is applicable to any surgical practice, that routinely discards patient samples, gathered in the course of patient treatment. Demonstrating the procedure will be Stefani Fawks, a Mohs histotechnician, and Dr.Mitchell Manway, a research fellow in our practice.To begin, use the tissue received from the Mohs surgeon and with a scalpel, excise a 10 to 50 milligram sample from the center of the apical side of the tissue. With tweezers, transfer the viable tissue to a labeled 1.5 milliliter tube, prefilled with culture medium. Make sure the tissue is entirely immersed in the medium.Place the sample in a refrigerator and store it at four degrees Celsius, for no longer than 24 hours before further processing. Harvest at least a 10 cubic millimeter tissue fragment from the apical side of the excised tissue, when the tumor sample is large enough, and such removal will not interfere with routine Mohs processing. Next, place the remaining unprocessed tumor sample on a cryostat tissue holder.Embed the tissue in an optimal cutting temperature compound, and freeze it within the holder to prepare histological slides. Using a cryostat, cut the tissue into seven micron thick sections, and layer up to three sections over a slide. After sectioning, fix the tissue by immersing the slides in acetone for one to two seconds.Then, allow the slides to air dry. Keeping the remaining sample in a cryostat chamber, pry the tissue from the cryo embedding medium, and transfer it to a labeled cryogenic tube. Immediately transfer the labeled vial into liquid nitrogen.Transfer of the tissue to a cryo vial after Mohs processing must be performed in the negative 20 degrees Celsius cryostat chamber. If performed properly and the tissue is kept frozen, this step will enable the successful extraction of RNA from post-Mohs tissue samples. Next, obtain adjacent normal tissue from the Mohs surgeon.Excise a sample from the apical side of the adjacent normal tissue. Then, using tweezers, transfer the excised sample to a labeled 1.5 milliliter tube filled with culture medium. Immerse the tissue in the same culture medium.Place it into an appropriate sample bag, and store at four degrees Celsius, until subsequent processing. Collect approximately 10 milliliters of venous blood into EDTA-coated collection tubes. Combine the blood samples in a sterile 15 milliliter centrifuge tube.Then, transfer approximately one milliliter of the blood sample into a 1.5 milliliter cryogenic tube, avoiding bubbles. Store this whole blood sample at minus 80 degrees Celsius, until subsequent genotyping and tissue matching. Next, centrifuge the remaining blood sample at 1000 times G for 30 minutes to obtain blood plasma.After spinning, collect and divide the plasma until one milliliter aliquots in 1.5 milliliter cryogenic tubes. And store the samples at minus 80 degrees Celsius for use during future liquid biopsy analyses. Enter the data from the checklist of patient samples, along with patient information and diagnosis, subtracted from the patient chart to the biorepository database.Finally, record the localization of the de-identified samples in the biorepository database, and move the samples to the research lab. Immediately after, transfer the tissue samples into 1.5 milliliter tubes, prefilled with appropriate extraction solutions, making sure the tissue is completely immersed. Store the samples at minus 80 degrees Celsius until further processing.Presented here are the images of representative Western blots of known squamous cell carcinoma markers, mucin one, FHL-one, and p40, obtained for adjacent normal tissue and a tumor sample. The quality of the RNA isolated from Mohs samples was first assessed using gel electrophoresis, followed by RNA integrity analysis. RNA integrity number values achieved for normal and tumor samples indicate that high quality and intact RNA was obtained.Cytometry profiles of the bands were plotted as fluorescence units versus nucleotide number for representative RNA samples from tumor and normal patient tissue. A squamous cell carcinoma tissue explant was imaged using phase contrast microscopy and shown to be a mixed keratinocyte and fibroblast culture, that might be subcultured or used for future cell line development. Finally, in the squamous cell carcinoma tissue sections, positive staining for the epithelial to mesenchymal transition marker was shown.Once mastered, this protocol will extend the standard work flow of the clinic staff to less than 30 minutes, if it is performed properly. The majority of the steps are performed by the existing clinical staff after patient care is completed. While attempting this protocol, it is important to maintain accurate record keeping, linking the samples with the patient's history and diagnosis.As this information is often needed for downstream applications. This technique will help pave the way for researchers in the field of dermatology for the development of liquid biopsies and biomarkers in squamous cell carcinomas and melanoma. After watching this video, you should have a good understanding of how to implement a clinic-based biorepository for cutaneous cancer samples, generated as part of daily practice.

establishment-of-a-clinic-based-biorepository

Research

JoVE Journal

Medicine

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

Minimal Invasive Gründung von murinen orthotopen Blasen Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

Forschung

Medizin

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

Gründung des Menschen Epithelial Enteroids und Colonoids von Whole Gewebe und Biopsie

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

Aufbau und Charakterisierung von UTI und CAUTI in einem Mausmodell

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a model organism, the mouse has already been widely used in bone-related research. Large diaphyseal bone defect models in mice, however, are sparse and often use bone fixation which fills the bone marrow cavity and does not provide optimal mechanical stability. The objectives of the current study were to develop a critical-size, segmental, femoral defect in nude mice. A 3.5-mm mid-diaphyseal femoral ostectomy (approximately 25% of the femur length) was performed using a dedicated jig, and was stabilized with an anterior located locking plate and 4 locking screws. The bone defect was subsequently either left empty or filled with a bone substitute (syngenic bone graft or coralline scaffold). Bone healing was monitored noninvasively using radiography and in vivo micro-computed-tomography and was subsequently assessed by ex vivo micro-computed-tomography and undecalcified histology after animal sacrifice, 10 weeks postoperatively. The recovery of all mice was excellent, a full-weight-bearing was observed within one day following the surgical procedure. Furthermore, stable bone fixation and consistent fixation of the implanted materials were achieved in all animals tested throughout the study. When the bone defects were left empty, non-union was consistently obtained. In contrast, when the bone defects were filled with syngenic bone grafts, bone union was always observed. When the bone defects were filled with coralline scaffolds, newly-formed bone was observed in the interface between bone resection edges and the scaffold, as well as within a short distance within the scaffold. 
The present model describes a reproducible critical-size femoral defect stabilized by plate osteosynthesis with low morbidity in mice. The new load-bearing segmental bone defect model could be useful for studying the underlying mechanisms in bone regeneration pertinent to orthopaedic applications.

Although mouse models are invaluable tools for bone tissue engineering, models of long bone defects are sparse. This need motivated development of the present protocol which uses a locking plate with four screws and a dedicated jig to perform and stabilize a reproducible, femoral, critical-size defect with low morbidity.

Einrichtung eines segmentalen Femoral Critical-size Defektmodell in Mäusen durch Plattenosteosynthese Stabilisierte

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a ...

Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic step. Despite advances in our understanding of DED, significant knowledge gaps remain. Advances are limited in part due to the lack of informative animal models. The authors recently reported on a method of DED induced by injecting all orbital lacrimal gland (LG) tissues with the lectin concanavalin A. Here, we report a novel model of aqueous-deficient DED based on the surgical resection of all orbital LG (dacryoadenectomy) tissues. Both methods use rabbits because of their similarity to human eyes in terms of the size and structure of the ocular surface. One week after removal of the nictitating membrane, the orbital superior LG was surgically removed under anesthesia, followed by removal of the palpebral superior LG, and finally removal of the inferior LG. Dacryoadenectomy induced severe DED, evidenced by a marked reduction in the tear break up time test and the Schirmer&#39;s tear test, and significantly increased tear osmolarity and rose bengal staining. Dacryoadenectomy-induced DED lasted at least eight weeks. There were no complications and animals tolerated the procedure well. The technique can be mastered relatively easily by those with adequate surgical experience and appreciation of the relevant rabbit anatomy. Since this model recapitulates the features of human aqueous-deficient DED, it is suitable for studies of ocular surface homeostasis, DED, and candidate therapeutics.

A novel approach is presented to induce chronic dry eye disease in rabbits by surgically removing all orbital lacrimal glands. This method, distinct from those previously reported, produces a stable, reproducible model of aqueous deficient dry eye well suited to study tear physiology and pathophysiology and ocular therapeutics.

Etablierung eines schweren Trockenauges modell mit vollständiger Dakryoadenektomie bei Kaninchen

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic ...

Establishment and Evaluation of a Porcine Vein Graft Disease Model

Venous graft disease (VGD) is the leading cause of coronary artery bypass graft (CABG) failure. Large animal models of CABG-VGD are needed for the investigation of disease mechanisms and the development of therapeutic strategies. 
To perform the surgery, we enter the cardiac chamber through the third intercostal space and carefully dissect the internal mammary vein and immerse it in normal saline. The right main coronary artery is then treated for ischemia. The target vessel is incised, a shunt plug is placed, and the distal end of the graft vein is anastomosed. The ascending aorta is partially blocked, and the proximal end of the graft vein is anastomosed after perforation. The graft vein is checked for patency, and the proximal right coronary artery is ligated. 
CABG surgery is performed in minipigs to harvest the left internal mammary vein for its use as a vascular graft. Serum biochemical tests are used to evaluate the physiological status of the animals after surgery. Ultrasound examination shows that the proximal, middle, and distal end of the graft vessel are unobstructed. In the surgical model, turbulent blood flow in the graft is observed upon histological examination after the CABG surgery, and venous graft stenosis associated with intimal hyperplasia is observed in the graft. The study here provides detailed surgical procedures for the establishment of a repeatable CABG-induced VGD model.

In this protocol, novel pig vein bypass grafting was performed through a small incision in the left chest wall without cardiopulmonary bypass. A postoperative pathology study was done, which showed intimal thickening.

Etablierung und Evaluierung eines Porcine Vent Graft Disease Modells

Venous graft disease (VGD) is the leading cause of coronary artery bypass graft (CABG) failure. Large animal models of CABG-VGD are needed for the ...

Establishment of a Simple and Effective Rat Model for Intraoperative Parathyroid Gland Imaging

Parathyroid gland (PG) identification is a critical unmet need in thyroidectomy. The identification of the PG is challenging in thyroid surgery as it is similar in color to the thyroid gland. The lack of effective animal models in preclinical research is a severe limitation for the development of PG identification techniques. This protocol allows for the establishment of a simple and effective rat model for PG identification. In this model, black iron oxide nanoparticles (IONPs) are injected locally in the thyroid gland and rapidly diffuse within the thyroid gland but not the PG. A negatively stained PG and a positively stained thyroid gland can be easily identified by the naked eye without requiring external microscopes. The position of the PG can be identified by increasing the color contrast between the thyroid gland and the PG, based on the color of the black IONPs. This rat model is low-cost and convenient for PG identification, and the IONPs are a novel PG contrast agent.

To date, the development of parathyroid gland (PG) identification methods is limited by the lack of animal models in preclinical research. Here, we establish a simple and effective rat model for intraoperative PG imaging and evaluate its effectiveness by using iron oxide nanoparticles as a novel PG contrast agent.

Etablierung eines einfachen und effektiven Rattenmodells für die intraoperative Bildgebung der Nebenschilddrüse

Parathyroid gland (PG) identification is a critical unmet need in thyroidectomy. The identification of the PG is challenging in thyroid surgery as it is ...

Modifizierte arteriovenöse Fistelchirurgie zur Etablierung des RV-Volumenüberlastungs-Mausmodells

Establishment and Confirmation of a Postnatal Right Ventricular Volume Overload Mouse Model

Right ventricular (RV) volume overload (VO) is common in children with congenital heart disease. In view of distinct developmental stages,the RV myocardium may respond differently to VO in children compared to adults. The present study aims to establish a postnatal RV VO model in mice using a modified abdominal arteriovenous fistula. To confirm the creation of VO and the following morphological and hemodynamic changes of the RV, abdominal ultrasound, echocardiography, and histochemical staining were performed for 3 months. As a result, the procedure in postnatal mice showed an acceptable survival and fistula success rate. In VO mice, the RV cavity was enlarged with a thickened free wall, and the stroke volume was increased by about 30%-40% within 2 months after surgery. Thereafter, the RV systolic pressure increased, corresponding pulmonary valve regurgitation was observed, and small pulmonary artery remodeling appeared. In conclusion, modified arteriovenous fistula (AVF) surgery is feasible to establish the RV VO model in postnatal mice. Considering the probability of fistula closure and elevated pulmonary artery resistance, abdominal ultrasound and echocardiography must be performed to confirm the model status before application.

Autor im Rampenlicht: Etablierung und Bestätigung eines postnatalen Mausmodells für rechtsventrikuläre Volumenüberlastung  

This protocol presents the establishment and confirmation of a postnatal right ventricular volume overload (VO) model in mice with abdominal arteriovenous fistula (AVF), which can be applied to investigate how VO contributes to postnatal heart development.