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

Este trabalho foi apoiado por fundos da Midwestern University College de Ciências da Saúde Research Facilitation Grant, concedido à EEH, e Midwestern University Office of Research and Sponsored Programs Intramural Grant, atribuído à KJL. O suporte adicional foi fornecido por Affiliated Laboratories e Affiliated Dermatology. Agradecemos a Sarah Potekhen, Jamie Barto, Stefani Fawks, Cody Jording, Stacie Schimke, Heather Kissel e Ali Zaidi por sua assistência técnica.

Todas as experiências em amostras de tecido humano foram aprovadas pelo Western IRB (Protocolo WIRB # 20142461). O único critério para a coleta de tecidos para esses procedimentos é um diagnóstico de SCC comprovado por biópsia. Não foram descritos critérios de exclusão no protocolo IRB original, mas não foram utilizadas amostras de doentes com uma doença transmissível transmitida pelo sangue conhecida. O consentimento informado foi obtido antes da coleta de tecido cutâneo, sangue e saliva. O Midwestern Inst...

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Para o conhecimento do autor, este protocolo é o primeiro de seu tipo que se concentra na aquisição clínica de amostras de tecido cutâneo, tanto em uma abordagem de custo eficaz e rápido. Pacientes submetidos a microcirurgia de Mohs são tipicamente agendados durante blocos específicos de tempo, ea coleta é limitada a esses períodos. A colheita de amostras envolve esforços do assistente médico envolvido na assistência ao paciente, o histotechnologist de Mohs que processa as amostras de tumor, e um membro do ...

A pesquisa com câncer e biomarcadores depende de uma oferta de amostras de tecido humano de qualidade, e a oferta limitada tem dificultado a pesquisa 1 , 2 . Muitos estudos dermatológicos são limitados pela oferta inadequada, qualidade variável e custos associados ao uso de tecido humano. O custo de estabelecer um grande biobank dedicado foi estimado em aproximadamente dois milhões de dólares 3 , e esses custos colocam o uso de tecido humano fora do alcance de muitos pesquisadores. Além disso, o processo de geração e armazenamento de amostras de pesquisa apresenta o risco de afetar as operações clínicas e retardar o atendimento ao paciente se não for executado cuidadosamente. Um biorepository custo-eficaz, clínico-baseado foi estabelecido que se centre em amostras do cancro da pele que seguem as mais melhores práticas recomendadas e a validação da amostra 4 , 5 , 6 . <pEsse protocolo foi desenvolvido em uma clínica de dermatologia que realiza um grande volume de cirurgias micrográficas de Mohs para remover carcinoma de células escamosas (SCC), carcinoma basocelular (CBC) e câncer de pele de melanoma. Os doadores voluntários podem ser recrutados a partir desta população de doentes. É importante estabelecer o biorrepositório no local de coleta para capturar rapidamente tecido e sangue de pacientes autorizados sem atrasar o tratamento. A colheita de amostras da mesma clínica minimiza as variações nas técnicas de coleta e minimiza as variações na qualidade das amostras, o que pode ser problemático para aplicações a jusante 7 , 8 . O objetivo da técnica de cirurgia micrográfica Mohs é garantir que todo o tecido do câncer é removido, preservando o máximo de tecido saudável possível. O procedimento envolve a remoção progressiva de camadas finas de tecido tumoral. Cada camada sucessiva é suaExaminado toxicologicamente (após criosecção do tecido tumoral e realização de coloração de H &amp; E) por um dermatologista para determinar se todo o tecido de câncer foi removido. A excisão e exame de camadas subsequentes de tecidos é executado enquanto o paciente permanece no consultório. Esta técnica é considerada a melhor opção de tratamento para SCC 9 . Neste ponto, a ferida é fechada e, para melhorar a cicatrização e aparência cosmética, o tecido normal adjacente (ANT) é frequentemente excisado. Deste modo, este procedimento cirúrgico para remover um tumor é idealmente adequado para a recolha de tecido histologicamente caracterizado para futuros estudos. O procedimento de aquisição para obtenção de tecido tumoral, tecidos normais adjacentes, saliva e amostras de sangue foi concebido para ter um impacto mínimo sobre as tarefas normais do pessoal ( Figura 1 ). Assistentes médicos realizar o sangue desenha durante a preparação do paciente para o procedimento. Após a conclusão do procedimento de Mohs, o M<html> Ohs histotechnologist prepara slides histológicos adicionais do espécime e transfere o tecido para o biorepository. Os custos associados ao estabelecimento do biorrepositório incluem a compra de congeladores de criopreservação, a criação de modestos laboratórios clínicos e o desenvolvimento de um programa de rastreamento de inventário. <img alt="figura 1" src="/files/ftp_upload/55583/55583fig1.jpg" /> Figura 1: Seqüência de coleta de amostras e pessoal responsável. Após o check-in do paciente e a obtenção do consentimento do paciente, o assistente médico coleta um cotonete bucal e realiza uma extração de sangue. O dermatologista e assistente médico, em seguida, excise o tumor e fechar a ferida, durante o qual tempo SCC e ANT espécimes são coletados, respectivamente. Um técnico de laboratório dedicado processa o sangue e separa os espécimes de SCC e ANT para cultura de tecidos, preservação e entrada no biorepositório.Ftp_upload / 55583 / 55583fig1large.jpg &quot;target =&quot; _ blank &quot;&gt; Clique aqui para ver uma versão ampliada desta figura. A diversidade de amostras coletadas possibilita uma variedade de abordagens experimentais ( Figura 2 ). As amostras coletadas do paciente são cotonetes bucais (saliva também pode ser coletada se necessário), sangue total e tecido excisado. Os swabs bucais e uma amostra do sangue total são salvos, sem processamento, para genotipagem e correspondência de tecido. O sangue completo é separado em glóbulos brancos (WBC) e frações de plasma para futuras análises. Após o processamento de Mohs, o tumor congelado é colocado directamente em azoto líquido e transferido para um congelador a -80 ° C. As amostras de tecido de tumor fresco e viável e ANT são cultivadas utilizando modificações das técnicas anteriores 10 , 11 e depois criopreservadas. Durante a coleta, o número de cada tipo de amostra é registrado em uma planilha antes da<html> O programa de rastreamento de inventário para facilitar o processamento preciso ( Tabela 1 ). <img alt="Figura 2" src="/files/ftp_upload/55583/55583fig2.jpg" /> Figura 2: Esboço de coleta e processamento de amostras de biorrepositores baseados em clínicas. Um cotonete bucal e uma amostra de sangue são recolhidos do doente e armazenados para genotipagem a jusante e correspondência de tecido. O sangue total é processado para o isolamento de glóbulos brancos (WBC) e análise de CTC, bem como para a coleta de plasma e análise de biópsia líquida. O tecido excisado durante o procedimento de Mohs é processado histologicamente para fins de diagnóstico, após o que as lâminas histológicas podem ser usadas experimentalmente para análises imunoistoquímicas adicionais. Desde que a amostra de tecido excisada seja suficientemente grande, uma porção de tecido fresco é removida e seccionada para isolamento de proteína e ARN e para o estabelecimento de linhas de células cultivadas.55583 / 55583fig1large.jpg &quot;target =&quot; _ blank &quot;&gt; Clique aqui para ver uma versão ampliada desta figura. <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Data da coleta: </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td> Paciente 1 </td><td> Paciente 2 </td><td> Paciente 3 </td><td> Paciente 4 </td><td> Paciente 5 </td></tr><tr><td> Iniciais e data de nascimento </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Cor dos olhos </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Tipo de amostra </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Localização da amostra </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Saliva </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> WSangue do buraco </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 viável </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Tecido Normal Viable </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Tumor Mohs Nitrogênio Líquido </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Tecido Nitrogênio Líquido Normal </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> Tabela 1: Lista de verificação para registro de coletas de amostras. Os dados registados e registados com cada amostra recolhida incluem iniciais do doente, data de nascimento e cor dos olhos (para a tipificação da pele), bem como a localizaçãoNa remoção da amostra. O número de amostras de saliva, volumes de coleta de sangue e o número de espécimes de tecido viáveis ​​e conservados coletados também são registrados como referências para alocações para usos posteriores. <a href="http://ecsource.jove.com/files/ftp_upload/55583/Table1.xlsx">Clique aqui para baixar este arquivo.</a> Para validar procedimentos de recolha de amostras, cada tipo de amostra foi testado em aplicações a jusante. Utilizando modificações das técnicas anteriores 12 , o tumor e a ANT foram utilizados com sucesso no isolamento de proteínas e ARN e podem potencialmente ser utilizados para o isolamento do ADN. Os explantes viáveis ​​estabelecidos a partir das secções de tecido foram avaliados por microscopia, enquanto as lâminas histológicas armazenadas foram utilizadas para imuno-histoquímica e imunofluorescência. Seguindo o protocolo aqui descrito, é possível estender este modelo para outras clínicas de dermatologia, outros tipos de tumores (tais como melanoma), E outras especialidades cirúrgicas e práticas para fornecer amostras de tecido humano para a investigação multifacetada em cancros humanos. Podem ser necessárias ligeiras modificações deste protocolo para outras práticas, mas, em princípio, este protocolo é aplicável a qualquer prática cirúrgica que descarte rotineiramente amostras de pacientes recolhidas no decurso do tratamento do doente.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>BD Vacutainer Plastic Blood Collection Tubes with K3EDTA</td>
			<td>ThermoFisher Scientific</td>
			<td>02-685-2B</td>
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			<td>Electron Microscopy Sciences Double Edge Blades</td>
			<td>ThermoFisher Scientific</td>
			<td>50-949-411</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>
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			<td>Electron Microscopy Sciences Tissue TEK OCT Compound</td>
			<td>ThermoFisher Scientific</td>
			<td>50-363-579</td>
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			<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>
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			<td>ThermoFisher Scientific</td>
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			<td>ThermoFisher Scientific</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>Cell Signaling Technology 10x RIPA Buffer</td>
			<td>ThermoFisher Scientific</td>
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			<td>ThermoFisher Scientific</td>
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			<td>Eppendorf 5424R Microcentrifuge</td>
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			<td>Pellet Morter Cordless Homogenizer</td>
			<td>ThermoFisher Scientific</td>
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			<td>RNAse Free Pellet Pestle</td>
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			<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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		<tr>
			<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>
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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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		<tr>
			<td>Zeiss Axio Imager Z1 Microscope with Axiocam camera</td>
			<td>Zeiss</td>
			<td>4300009901</td>
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		<tr>
			<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

A incidência de cancro da pele ( por exemplo, carcinoma epidermóide, carcinoma basocelular e melanoma) tem vindo a aumentar nos últimos anos. Espera-se que haja uma demanda paralela de amostras tumorais cutâneas para estudos de pesquisa biomédica. A disponibilidade de tecidos, no entanto, é limitada devido ao custo de estabelecimento de um biorrepositório e à falta de protocolos disponíveis para a obtenção de amostras clínicas que não interfiram com as operações clínicas. Um protocolo foi estabelecido para coletar e processar tumores cutâneos e sangue associado e amostras de saliva que tem impacto mínimo em procedimentos clínicos de rotina na data de uma cirurgia de Mohs. As amostras de tumor são recolhidas e processadas a partir de pacientes submetidos à sua primeira camada de cirurgia de Mohs para neoplasias cutâneas comprovadas por biopsia pelo histotecnista de Mohs. O tecido normal adjacente é recolhido no momento do fecho cirúrgico. As amostras adicionais que podem ser coletadas são swabs de sangue total e bucal. Utilizando amostras de tecido que são normalmente descartadas, um biorrepositório foi gerado que oferece várias vantagens fundamentais por estar baseado na clínica versus o laboratório. Estes incluem uma vasta gama de amostras recolhidas; Acesso a registros de pacientes não identificados, incluindo relatórios de patologia; E, para o doador típico, o acesso a amostras adicionais durante as visitas de acompanhamento.

O tumor e a ANT foram utilizados com sucesso na isolação de proteínas e testados por Western blotting. Como mostrado na Figura 3 , os marcadores cutâneos de carcinoma de células escamosas (Muc1 14 , FHL- 15 e p40 16 , 17 ) podem ser detectados em amostras de pacientes coletadas e armazenadas em nosso biorrepositório clínico. <p class="jove_content" fo:kee...

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

Establishment of a Clinic-based Biorepository

Os tumores cutâneos são frequentemente descartados após a cirurgia micrográfica de Mohs. Descreve-se aqui um protocolo que permite que a equipe de suporte clínico processe e armazene eficazmente amostras de tumores cutâneos ( por exemplo, carcinoma de células escamosas, carcinoma basocelular e melanoma) para aplicações laboratoriais a jusante sem interferir com as operações clínicas.

Estabelecimento de um Biorepository baseado em Clínica

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.

Minimamente Invasiva Estabelecimento de murino ortotópico bexiga Xenoenxertos

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

Medicina

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.

Estabelecimento de epiteliais humanas Enteroids e Colonoids de tecido inteiro e biópsia

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.

Criação e Caracterização da UTI e CAUTI em um modelo de rato

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.

Estabelecimento de um Defeito Modelo segmentar Femoral Critical-size em Ratos estabilizada por osteossíntese Placa

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.

Estabelecimento de um modelo severo do olho seco usando dacryoadenectomy completo nos coelhos

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.

Estabelecimento e Avaliação de um Modelo de Enxerto de Veia Suína

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.

Estabelecimento de um Modelo de Rato Simples e Eficaz para Imagem Intraoperatória da Glândula Paratireoide

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

Cirurgia de fístula arteriovenosa modificada para estabelecer o modelo de camundongo de sobrecarga de volume do VD

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 em destaque: Estabelecimento e confirmação de um modelo de camundongo com sobrecarga de volume do ventrículo direito pós-natal  

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.