Name: establishment of a clinic-based biorepository
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Detta arbete stöddes av medel från Midwestern University College of Health Sciences Research Facilitation Grant, tilldelat EEH, och Midwestern University Office of Research och sponsrade program Intramural Grant, tilldelat KJL. Ytterligare stöd tillhandahölls av Affiliated Laboratories and Affiliate Dermatology. Vi tackar Sarah Potekhen, Jamie Barto, Stefani Fawks, Cody Jording, Stacie Schimke, Heather Kissel och Ali Zaidi för deras tekniska hjälp.

Allt experiment på humana vävnadsprover godkändes av Western IRB (WIRB Protocol # 20142461). Det enda kriteriet för insamling av vävnader för dessa förfaranden är en biopsi-beprövad diagnos av SCC. Inga uteslutningskriterier beskrivs i det ursprungliga IRB-protokollet, men prover från patienter med en känd överförbar sjukdom i blodet användes inte. Informerat samtycke erhölls före insamling av kutan vävnad, blod och saliv. Midwestern Institutional Review Board godkände valideringsarbetet med klinikbase...

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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+high-risk+squamous+cell+carcinoma+of+the+skin.">Management of high-risk squamous cell carcinoma of the skin.</a> Curr Treat Options Oncol. 17, (2016).</li><li>Rasmussen, C., Thomas-Virnig, C., Allen-Hoffmann, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classical+human+epidermal+keratinocyte+cell+culture.">Classical human epidermal keratinocyte cell culture.</a> Methods Mol Biol. 945, 161-175 (2013).</li><li>Purdie, K. J., Pourreyron, C., South, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+cell+culture:+Methods+and+protocols.">Cancer cell culture: Methods and protocols.</a> , 151-159 (2011).</li><li>Berglund, S. 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. T., 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+of+the+p40+isoform+of+p63+has+high+specificity+for+cutaneous+sarcomatoid+squamous+cell+carcinoma.">Expression of the p40 isoform of p63 has high specificity for cutaneous sarcomatoid squamous cell carcinoma.</a> J Cutan Pathol. 41, 831-838 (2014).</li><li>Alomari, A. K., Glusac, E. J., McNiff, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p40+is+a+more+specific+marker+than+p63+for+cutaneous+poorly+differentiated+squamous+cell+carcinoma.">p40 is a more specific marker than p63 for cutaneous poorly differentiated squamous cell carcinoma.</a> J Cutan Pathol. 41, 839-845 (2014).</li><li>Qiao, B., Johnson, N. W., Gao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-mesenchymal+transition+in+oral+squamous+cell+carcinoma+triggered+by+transforming+growth+factor-beta1+is+Snail+family-dependent+and+correlates+with+matrix+metalloproteinase-2+and+-9+expressions.">Epithelial-mesenchymal transition in oral squamous cell carcinoma triggered by transforming growth factor-beta1 is Snail family-dependent and correlates with matrix metalloproteinase-2 and -9 expressions.</a> Int J Oncol. 37, 663-668 (2010).</li><li>Vaught, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developments+in+biospecimen+research.">Developments in biospecimen research.</a> Br Med Bull. 114, 29-38 (2015).</li></ol>

Författarens vetskap är detta protokoll det första i sitt slag som fokuserar på klinisk upphandling av kutana vävnadsprover både i ett kostnadseffektivt och snabbt angreppssätt. Patienter som genomgår Mohs-mikrokirurgi planeras typiskt under specifika tidsintervaller, och insamlingen är begränsad till dessa perioder. Provuppsamling innebär ansträngning från den medicinska assistenten som är inblandad i patientvård, Mohs-histoteknologen som behandlar tumörproverna och en utsedd laboratoriepersonal som han...

Cancer- och biomarkörsforskning bygger på en leverans av mänskliga vävnadsprover av hög kvalitet, och begränsat utbud har hindrat forskning 1 , 2 . Många dermatologiska studier är begränsade av otillräcklig tillgång, variabel kvalitet och kostnader i samband med användningen av mänsklig vävnad. Kostnaden för att skapa en stor, dedikerad biobank har uppskattats till cirka två miljoner dollar 3 , och dessa kostnader gör användningen av mänsklig vävnad utom räckhåll för många forskare. Vidare utgör processen för att generera och lagra forskningsprover risken för att påverka klinisk verksamhet och fördröja patientvården om den inte genomförs noggrant. En kostnadseffektiv klinikbaserad biorepository har etablerats som fokuserar på hudcancerprover efter rekommenderad bästa praxis och provvalidering 4 , 5 , 6 . <pClass = &quot;jove_content&quot;&gt; Detta protokoll har utvecklats i en dermatologiklinik som utför en stor mängd Mohs mikrographic kirurgi för att avlägsna pladecellscancer (SCC), basalcellkarcinom (BCC) och melanom hudcancer. Frivilliga givare kan rekryteras från denna patientpopulation. Det är viktigt att etablera biorepository på insamlingsstället för att snabbt fånga vävnad och blod från samtyckta patienter utan dröjsmål. Att samla prover från samma klinik minimerar variationer i insamlingstekniker och minimerar variationer i provkvaliteten, vilket kan vara problematiskt för nedströmsapplikationer 7 , 8 . Målet med Mohs mikrografiska kirurgiska tekniken är att säkerställa att all cancervävnad avlägsnas samtidigt som så mycket hälsosam vävnad bevaras som möjligt. Förfarandet innefattar progressiv avlägsnande av tunna skikt av tumörvävnad. Varje på varandra följande skikt är hansTologiskt undersökt (efter kryosektion av tumörvävnaden och utförande av H &amp; E-färgning) av en hudläkare för att bestämma om all cancervävnad har avlägsnats. Undersökningen och undersökningen av efterföljande skikt av vävnader utförs medan patienten förblir på kontoret. Denna teknik anses vara det bästa behandlingsalternativet för SCC 9 . Vid denna tidpunkt är såret stängt och för att förbättra läkning och kosmetisk utseende skärs ofta intilliggande normal vävnad (ANT) ofta ut. Således är detta kirurgiska förfarande för att avlägsna en tumör lämpligt lämpad för att samla histologiskt karakteriserad vävnad för framtida studier. Upphandlingsförfarandet för att erhålla tumörvävnad, intill normal vävnad, saliv och blodprover har utformats för att få minimal inverkan på normala personaluppgifter ( Figur 1 ). Medicinska assistenter utför bloddragningarna medan patienten förbereds för proceduren. Efter avslutad Mohs-procedur, M<html> Ohs histoteknologen förbereder ytterligare histologiska bilder av provet och överför vävnaden till biorepository. Kostnaderna i samband med upprättandet av biorepository inkluderar inköp av kryopreserveringsfrysare, skapandet av blygsamt kliniskt laboratoriumutrymme och utvecklingen av ett inventeringsspårningsprogram. <img alt="Figur 1" src="/files/ftp_upload/55583/55583fig1.jpg" /> Figur 1: Sekvens av provinsamling och ansvarig personal. Vid patientinsamling och uppnående av patientens samtycke samlar den medicinska assistenten en buccal swab och utför en bloddragning. Dermatologen och den medicinska assistenten tar sedan bort tumören och stänger såret, under vilken tid SCC och ANT-prov uppsamlas. En särskild laboratorie tekniker behandlar blodet och delar SCC- och ANT-proverna för vävnadsodling, bevarande och inträde i biorepository.Ftp_upload / 55583 / 55583fig1large.jpg &quot;target =&quot; _ blank &quot;&gt; Vänligen klicka här för att se en större version av denna figur. Mångfalden av samlade prov möjliggör en rad olika experimentella tillvägagångssätt ( Figur 2 ). Prov som samlas in från patienten är buccala swabs (saliv kan också samlas vid behov), helblod och skärad vävnad. Buccal swabs och ett prov från hela blodet sparas, utan behandling, för genotypning och vävnadsmatchning. Hela blodet separeras i vita blodkroppar (WBC) och plasmafraktioner för framtida analyser. Efter Mohs-bearbetning placeras den frusna tumören direkt i flytande kväve och överförs till en -80 ° C frys. Friska, livskraftiga tumörvävnader och ANT-prover odlas med användning av modifikationer av tidigare tekniker 10 , 11 och sedan kryokonserverade. Under samlingen registreras numret på varje provtyp i ett kalkylblad före inmatning i<html> Inventeringsspårningsprogrammet för att underlätta korrekt bearbetning ( tabell 1 ). <img alt="Figur 2" src="/files/ftp_upload/55583/55583fig2.jpg" /> Figur 2: Översikt av klinisk baserad biorepository samling och behandling av prov. En buccal swab och blodprov samlas från patienten och lagras för nedströms genotyping och vävnadsmatchning. Hela blodet bearbetas vidare för isolering av vita blodkroppar (WBC) och CTC, samt för plasmainsamling och flytande biopsianalys. Vävnad som skäras under Mohs-proceduren behandlas histologiskt för diagnostiska ändamål, varefter de histologiska glidbanorna kan användas experimentellt för ytterligare immunohistokemiska analyser. Under förutsättning att det skurna vävnadsprovet är stort nog, avlägsnas en del av färsk vävnad och sektioneras för protein- och RNA-isolering och för etablering av odlade cellinjer.55583 / 55583fig1large.jpg &quot;target =&quot; _ blank &quot;&gt; Vänligen klicka här för att se en större version av denna figur. <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Insamlingsdatum: </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> Initialer och födelsedatum </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Ögonfärg </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Provtyp </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Provplats </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Saliv </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> WHålblod </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> Tumörlivbar </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Vävnad Normal Livskraftig </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Tumor Mohs flytande kväve </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Vävnad Normal flytande kväve </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> slides </td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table> Tabell 1: Checklista för att spela in samplingar. Uppgifter som spåras och spelas in med varje prov som samlats in inkluderar patientinitialer, födelsedatum och ögonfärg (för hudtyp) samt lokaliseringPå avlägsnandet av provet. Antalet salivprover, bloduppsamlingsvolymer och antalet insamlade livsdugliga och konserverade vävnadsprover upptecknas också som referenser för tilldelningar till senare användning. <a href="http://ecsource.jove.com/files/ftp_upload/55583/Table1.xlsx">Vänligen klicka här för att ladda ner den här filen.</a> För att validera provinsamlingsprocedurer har varje provtyp testats i applikationer nedströms. Med användning av modifikationer av tidigare tekniker 12 har tumör och ANT framgångsrikt använts i protein- och RNA-isolering och kan potentiellt användas för DNA-isolering. Livskraftiga explanter som är etablerade från vävnadssektionerna har utvärderats av mikroskopi, medan lagrade histologiska diabilder har använts för immunhistokemi och immunofluorescens. Genom att följa protokollet som beskrivs här är det möjligt att förlänga denna modell till andra dermatologikliniker, andra tumortyper (som melanom), Och andra kirurgiska specialiteter och praxis för att tillhandahålla mänskliga vävnadsprover för mångfacetterad forskning kring humankanker. Små modifikationer av detta protokoll är sannolikt nödvändiga för andra metoder, men i princip är detta protokoll tillämpligt på alla kirurgiska metoder som rutinmässigt kasserar patientprover som samlats under patientbehandling.

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			<td>Molecular Probes&#160;</td>
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			<td>Molecular Probes&#160;</td>
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			<td>Zeiss</td>
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			<td>Olympus IX51 phase contrast with DP72 camera</td>
			<td>Olympus</td>
			<td>IX511F3</td>
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establishment of a clinic-based biorepository

Förekomsten av hudcancer ( t.ex. plavocellkarcinom, basalcellkarcinom och melanom) har ökat under de senaste åren. Det förväntas att det kommer att finnas en parallell efterfrågan på kutana tumörprover för biomedicinska forskningsstudier. Tissue tillgänglighet är dock begränsad på grund av kostnaden för att etablera ett biorepository och avsaknaden av protokoll som är tillgängliga för att erhålla kliniska prover som inte stör klinisk verksamhet. Ett protokoll upprättades för att samla och bearbeta kutan tumör och associerade blod- och salivprover som har minimal inverkan på rutinmässiga kliniska förfaranden vid datumet för en Mohs-operation. Tumörprover samlas in och behandlas från patienter som genomgår Mohs-histoteknologins första lager av Mohs-kirurgi för biopsi-beprövade kutana maligniteter. Intilliggande normal vävnad uppsamlas vid kirurgisk tillslutning. Ytterligare prov som kan samlas är helblod och buccal swabs. Genom att använda vävnadsprover som normalt kasseras, genererades en biorepository som erbjuder flera viktiga fördelar genom att vara baserad i kliniken jämfört med laboratorieinställningen. Dessa inkluderar ett brett spektrum av samlade prover; Tillgång till identifierade patientjournaler, inklusive patologiska rapporter Och för den typiska givaren tillgång till ytterligare prover under uppföljningsbesök.

Tumör och ANT har använts framgångsrikt i proteinisolering och testats med Western blotting. Såsom visas i figur 3 kan kutana skivformiga cellkarcinommarkörer (Mucl 14 , FHL-1 15 och p40 16 , 17 ) detekteras i patientprover uppsamlade och lagrade i vår klinikbaserade biorepository. <img alt="Figur 3"...

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

Establishment of a Clinic-based Biorepository

Kutana tumörer kasseras ofta efter Mohs mikrografisk kirurgi. Ett protokoll beskrivs här som gör det möjligt för klinisk supportpersonal att effektivt bearbeta och lagra kutan tumör ( t.ex. pladecells carcinom, basalcellkarcinom och melanom) prover för nedströms laboratorieapplikationer utan att störa kliniska operationer.

Inrättande av ett klinikbaserat 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.

Minimalinvasiva Etablering av murina Orthotopic Bladder implanterad

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 Human Epithelial Enteroids och Colonoids från Whole Tissue och 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.

Upprättande och karakterisering av UTI och CAUTI i en musmodell

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 segment Femoral kritisk storlek Defekt modell hos möss som stabiliseras av Plate Osteosyntes

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.

Fastställande av en svår torr ögon modell med fullständig Dacryoadenektomi hos 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.

Upprättande och utvärdering av en modell för svinvenstransplantatsjukdom

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 och effektiv råttmodell för intraoperativ bisköldkörtelavbildning

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

Modifierad arteriovenös fistelkirurgi för att fastställa RV Volume Overload Mouse-modellen

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.

Författare i fokus: Etablering och bekräftelse av en postnatal musmodell för volymöverbelastning av höger kammare  

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.