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

Dette arbeidet ble støttet av midler fra Midwestern University College of Health Sciences Research Facilitation Grant, tildelt EEH, og Midwestern University Office of Research og sponsede programmer Intramural Grant, tildelt KJL. Ytterligere støtte ble gitt av tilknyttede laboratorier og tilknyttet dermatologi. Vi takker Sarah Potekhen, Jamie Barto, Stefani Fawks, Cody Jording, Stacie Schimke, Heather Kissel og Ali Zaidi for teknisk hjelp.

Alle eksperimenter på humane vevsprøver ble godkjent av Western IRB (WIRB Protocol # 20142461). Det enkle kriteriet for oppsamling av vev for disse prosedyrene er en biopsi-bevist diagnose av SCC. Ingen ekskluderingskriterier ble skissert i den opprinnelige IRB-protokollen, men prøver fra pasienter med en kjent blodbåren smittsom sykdom ble ikke brukt. Informert samtykke ble oppnådd før oppsamling av kutant vev, blod og spytt. Midwestern Institutional Review Board godkjente valideringsarbeidet med klinikkbaserte b...

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Forfatterens kunnskap er denne protokollen den første i sitt slag som fokuserer på klinisk anskaffelse av kutane vevsprøver i både en kostnadseffektiv og rask tilnærming. Pasienter som gjennomgår Mohs-mikrokirurgi er typisk planlagt i bestemte tidsblokker, og samlingen er begrenset til disse periodene. Eksempelsamling innebærer innsats fra den medisinske assistenten som er involvert i pasientbehandling, Mohs histoteknologen som behandler svulstprøver, og et utpekt laboratoriemedarbeider som håndterer prøveinng...

Kreft- og biomarkørforskning er avhengig av forsyning av humane vevsprøver av høy kvalitet, og begrenset tilgang har hindret forskningen 1 , 2 . Mange dermatologiske studier er begrenset av utilstrekkelig tilførsel, variabel kvalitet og kostnader forbundet med bruk av humant vev. Kostnaden ved å etablere en stor, dedikert biobank har blitt anslått til om lag to millioner dollar 3 , og disse kostnadene legger bruken av menneskelig vev utenfor rekkevidde av mange forskere. Videre utgjør prosessen med å generere og lagre forskningsprøver risikoen for å påvirke klinisk operasjon og forsinke pasientomsorgen dersom den ikke utføres forsiktig. En kostnadseffektiv klinisk-basert biorepository er etablert som fokuserer på hudkreftprøver etter anbefalte beste praksis og prøvevalidering 4 , 5 , 6 . <pClass = &quot;jove_content&quot;&gt; Denne protokollen er utviklet i en dermatologi klinikk som utfører et stort antall Mohs mikrographic operasjoner for å fjerne squamous cell carcinoma (SCC), basal celle karsinom (BCC) og melanom hudkreft. Frivillige givere kan rekrutteres fra denne pasientpopulasjonen. Det er viktig å etablere biorepository på innsamlingsstedet for raskt å fange vev og blod fra samtykkede pasienter uten forsinkelse av behandlingen. Å samle prøver fra samme klinikk minimerer variasjoner i innsamlingsteknikker og minimerer variasjoner i kvaliteten på prøvene, noe som kan være problematisk for nedstrømsapplikasjoner 7 , 8 . Målet med Mohs mikrografisk kirurgi teknikk er å sikre at alt kreftvev blir fjernet, samtidig som det opprettholder så mye sunt vev som mulig. Prosedyren innebærer progressiv fjerning av tynne lag av tumorvev. Hvert påfølgende lag er hansTologisk undersøkt (etter kryoseksjonering av tumorvevet og utførelse av H &amp; E-farging) av en hudlege for å avgjøre om alt kreftvev har blitt fjernet. Excisionen og undersøkelsen av påfølgende lag av vev utføres mens pasienten forblir på kontoret. Denne teknikken anses som det beste behandlingsalternativet for SCC 9 . På dette tidspunktet er såret lukket, og for å forbedre helbredelse og kosmetisk utseende, blir nærliggende normalt vev (ANT) ofte skåret ut. Dermed er denne kirurgiske prosedyren for å fjerne en svulst ideell til å samle histologisk karakterisert vev for fremtidige studier. Anskaffelsesprosedyren for å skaffe svulstvev, tilstøtende normalt vev, spytt og blodprøver, er utformet for å få minimal innvirkning på vanlige ansatteoppgaver ( figur 1 ). Medisinske assistenter utfører bloddragene mens du forbereder pasienten for prosedyren. Etter fullføring av Mohs-prosedyren, M<html> Åh histoteknologen forbereder ytterligere histologiske lysbilder av prøven og overfører vevet til biorepositoryet. Kostnader knyttet til etablering av biorepository inkluderer kjøp av kryopreserveringsfrysere, opprettelse av beskjedent klinisk laboratorierom og utvikling av et oppfølgingssporingsprogram. <img alt="Figur 1" src="/files/ftp_upload/55583/55583fig1.jpg" /> Figur 1: Sekvens av prøveinnsamling og ansvarlig personale. Ved pasientens innsjekking og oppnåelse av pasientens samtykke samler den medisinske assistenten en bukkalpinne og utfører en blodtrekk. Dermatologen og den medisinske assistenten beskytter deretter svulsten og lukker såret, i løpet av hvilken tid SCC og ANT prøver samles, henholdsvis. En dedikert laboratorie tekniker behandler blodet og deler SCC og ANT prøver for vevskultur, bevaring og inngang i biorepository.Ftp_upload / 55583 / 55583fig1large.jpg &quot;target =&quot; _ blank &quot;&gt; Vennligst klikk her for å se en større versjon av denne figuren. Mangfoldet av samlede prøver muliggjør en rekke eksperimentelle tilnærminger ( figur 2 ). Prøver oppsamlet fra pasienten er buccal swabs (spytt kan også samles om nødvendig), helblod og skåret vev. Buccal swabs og en prøve fra hele blodet blir lagret, uten behandling, for genotyping og vevsmatching. Hele blodet separeres i hvite blodlegemer (WBC) og plasmafraksjoner for fremtidige analyser. Etter Mohs-prosessering, plasseres den frosne svulsten direkte i flytende nitrogen og overføres til en -80 ° C fryser. Friskt, levedyktig tumorvev og ANT-prøver dyrkes ved å bruke modifikasjoner av tidligere teknikker 10 , 11 og deretter kryokonservert. Under samlingen registreres nummeret på hver prøvetype på et regneark før inngangen til<html> Inventar sporing programmet for å lette nøyaktig bearbeiding ( tabell 1 ). <img alt="Figur 2" src="/files/ftp_upload/55583/55583fig2.jpg" /> Figur 2: Oversikt over klinikkbasert biorepositorprøveinnsamling og -behandling. En bukkalpinne og blodprøve oppsamles fra pasienten og lagres for nedstrøms genotyping og vevsmatching. Hele blodet blir videre behandlet for isolering av hvite blodlegemer og CTC-analyser, samt for plasmainnsamling og væskebiopsianalyse. Vev utskåret under Mohs prosedyren er histologisk behandlet for diagnostiske formål, hvoretter de histologiske lysbildene kan brukes eksperimentelt for ytterligere immunhistokemiske analyser. Forutsatt at den utskårne vevsprøven er stor nok, fjernes en del av ferskt vev og deles for protein- og RNA-isolasjon og for etablering av dyrkede cellelinjer.55583 / 55583fig1large.jpg &quot;target =&quot; _ blank &quot;&gt; Vennligst klikk her for å se en større versjon av denne figuren. <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Samlingsdato: </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td> Pasient 1 </td><td> Pasient 2 </td><td> Pasient 3 </td><td> Pasient 4 </td><td> Pasient 5 </td></tr><tr><td> Initialer og fødselsdato </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Øyenfarge </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Eksempel Type </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Eksempelplassering </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Spytt </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> WHull blod </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 levedyktig </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Vev Normal Levende </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Tumor Mohs Flytende nitrogen </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Vev Normal Flytende Kväve </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> lysbilder </td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table> Tabell 1: Sjekkliste for å registrere samlinger. Data spores og registreres med hver samplet samling inkluderer pasientens initialer, fødselsdato og øyenfarge (for hudtyping), så vel som lokaliseringenPå prøvefjerning. Antall spyttprøver, blodoppsamlingsvolumer og antall levedyktige og konserverte vevsprøver samlet inn registreres også som referanser for tildelinger til senere bruk. <a href="http://ecsource.jove.com/files/ftp_upload/55583/Table1.xlsx">Vennligst klikk her for å laste ned denne filen.</a> For å validere prøveinnsamlingsprosedyrer, har hver prøvetype blitt testet i nedstrømsapplikasjoner. Ved bruk av modifikasjoner av tidligere teknikker 12 har tumor og ANT blitt vellykket brukt i protein- og RNA-isolasjon og kan potensielt brukes til DNA-isolasjon. Levende eksplanter som er etablert fra vevseksjonene, er blitt evaluert ved mikroskopi, mens lagrede histologiske lysbilder har blitt brukt til immunhistokjemi og immunfluorescens. Ved å følge protokollen beskrevet her, er det mulig å utvide denne modellen til andre dermatologiklinikker, andre svulstetyper (som melanom), Og andre kirurgiske spesialiteter og praksiser for å gi humane vevsprøver for mangfoldig forskning i humane kreftformer. Små modifikasjoner av denne protokollen vil trolig være nødvendig for annen praksis, men i prinsippet er denne protokollen gjeldende for enhver kirurgisk praksis som rutinemessig sletter pasientprøver samlet i løpet av pasientbehandling.

<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>
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			<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>
			<td></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>
			<td></td>
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			<td>Nalgene Long-Term Storage Cryogenic Tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>03-337-7X</td>
			<td></td>
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			<td>Falcon 15 mL Conical Centrifuge Tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>14-959-53A</td>
			<td></td>
		</tr>
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			<td>Boca Scientific BuccalT-Swab&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>NC9679349</td>
			<td></td>
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			<td>Cell Signaling Technology 10x RIPA Buffer</td>
			<td>ThermoFisher Scientific</td>
			<td>50-195-822</td>
			<td></td>
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			<td>Halt Protease and Phosphatase Inhibitor Cocktail</td>
			<td>ThermoFisher Scientific</td>
			<td>PI78443</td>
			<td></td>
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			<td>Eppendorf 5424R Microcentrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>05-401-203</td>
			<td></td>
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			<td>Pellet Morter Cordless Homogenizer</td>
			<td>ThermoFisher Scientific</td>
			<td>12-141-3</td>
			<td></td>
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			<td>RNAse Free Pellet Pestle</td>
			<td>ThermoFisher Scientific</td>
			<td>121-141-364</td>
			<td></td>
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			<td>Forma SteriCycle CO2 Incubator</td>
			<td>ThermoFisher Scientific</td>
			<td>13-998-089</td>
			<td></td>
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			<td>HyClone Fetal Bovine Serum</td>
			<td>ThermoFisher Scientific</td>
			<td>SH30071.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Advanced DMEM/F-12</td>
			<td>ThermoFisher Scientific</td>
			<td>12-634-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Penicillin-Streptomycin</td>
			<td>ThermoFisher Scientific</td>
			<td>15140148</td>
			<td></td>
		</tr>
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			<td>Gibco 1M Hepes</td>
			<td>ThermoFisher Scientific</td>
			<td>15-630-130</td>
			<td></td>
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			<td>Nunc Cell Culture 35 mm with Vent</td>
			<td>ThermoFisher Scientific</td>
			<td>1256591</td>
			<td></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>
			<td></td>
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			<td>Anti MUC-1 antibody</td>
			<td>Santa Cruz Biotech</td>
			<td>sc-7313</td>
			<td></td>
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			<td>Anti-Snail + Slug (phospho S246)</td>
			<td>Abcam</td>
			<td>Ab63568</td>
			<td></td>
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			<td>Goat anti-rabbit IgG, Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A-11034</td>
			<td></td>
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			<td>Alexa Fluor 568 Phallodin</td>
			<td>Molecular Probes&#160;</td>
			<td>A12380</td>
			<td></td>
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			<td>ProLong Gold Antifade Mountant with DAPI</td>
			<td>Molecular Probes&#160;</td>
			<td>P36941</td>
			<td></td>
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			<td>Zeiss Axio Imager Z1 Microscope with Axiocam camera</td>
			<td>Zeiss</td>
			<td>4300009901</td>
			<td></td>
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			<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

Forekomsten av hudkreft ( f. Eks. Squamisk cellekarcinom, basalcellekarcinom og melanom) har økt de siste årene. Det forventes at det vil være en parallell etterspørsel etter kutane tumorprøver for biomedisinsk forskning. Vevtilgjengelighet er imidlertid begrenset på grunn av kostnaden ved å etablere en biorepository og mangelen på protokoller tilgjengelig for å oppnå kliniske prøver som ikke forstyrrer klinisk operasjon. En protokoll ble opprettet for å samle og behandle kutan tumor og tilhørende blod- og spyttprøver som har minimal innvirkning på rutinemessige kliniske prosedyrer på datoen for en Mohs-kirurgi. Tumorprøver oppsamles og behandles fra pasienter som gjennomgår Mohs-histoteknologens første lag med Mohs-kirurgi for biopsi-beprøvde kutane maligniteter. Tilstøtende normalt vev oppsamles ved kirurgisk lukning. Ytterligere prøver som kan samles er helblod og buccal swabs. Ved å bruke vevsprøver som normalt kasseres, ble det utviklet en biorepository som tilbyr flere viktige fordeler ved å være basert i klinikken versus laboratorieinnstillingen. Disse inkluderer et bredt spekter av innsamlede prøver; Tilgang til deidentifiserte pasientopptegnelser, inkludert patologiske rapporter; Og, for den typiske giveren, tilgang til ytterligere prøver under oppfølgingsbesøk.

Tumor og ANT har blitt brukt med suksess i proteinisolasjon og testet ved Western blotting. Som vist i figur 3 kan kutane skivepitelcellekarcinommarkører (Muc1 14 , FHL-1 15 og p40 16 , 17 ) detekteres i pasientprøver oppsamlet og lagret i vår klinikkbaserte biorepository. <img alt="Figur 3" src="/files...

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

Establishment of a Clinic-based Biorepository

Kutane svulster blir ofte kastet etter Mohs mikrografisk kirurgi. En protokoll er beskrevet her som gjør det mulig for klinisk hjelpepersonell å effektivt behandle og lagre kutan tumor ( f.eks. Squamous cell carcinoma, basal cell carcinoma og melanoma) prøver for nedstrøms laboratorieapplikasjoner uten å forstyrre klinisk operasjon.

Etablering av et klinisk-basert biorepository

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 Etablering av Murint orthotopic Blære xenografts

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

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.

Etablering av Menneskelig Epithelial Enteroids og Colonoids fra Whole Tissue og Biopsi

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.

Etablering og karakterisering av UTI og CAUTI i en musemodell

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.

Etablering av en Segmental Femoral kritisk størrelse Defect Modell i Mus stabilisert av Plate osteosintese

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.

Etablering av en alvorlig tørr øye modell bruke komplett Dacryoadenectomy i kaniner

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.

Etablering og evaluering av en modell for transplantatsykdom hos svin

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.

Etablering av en enkel og effektiv rottemodell for intraoperativ avbildning av biskjoldbruskkjertelen

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

Modifisert arteriovenøs fistelkirurgi for å etablere RV-volumoverbelastningsmusemodellen

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.

Forfatter i søkelyset: Etablering og bekreftelse av en postnatal musemodell for overbelastning av høyre ventrikkelvolum  

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.