Este estudo foi financiado por subvenções a YMS do National Institutes of Health (CA148828), The University of Michigan Gastrointestinal Peptide Center, e Jeffrey A. Colby Colon Cancer Research e Tom Liu Memorial fundos do Comprehensive Cancer Center da Universidade de Michigan.

1. Colon isolamento do tumor e dissociação celular <ol><li> Tumores intestinais podem ser isolados a partir de qualquer modelo de cancro do cólon ou esporádica induzida pelo tratamento. Os ratos devem ser sacrificados com CO 2. Dois pontos são então recolhidos, lavados com solução salina fria tamponada com fosfato (PBS) e abertos longitudinalmente. Identificar as regiões que contêm tumores usando um microscópio estereoscópico, dissecar com um par de tesouras, e lave com água fria PBS. </li><li> Incubar fragmentos intestinais tumores em tampão contendo quelante EDTA (EDTA 2 mM, 5,6 mmol / L de Na 2 HPO 4, 8,0 mmol / l de KH 2 PO 4, 96,2 mmol / L de NaCl, 1,6 mmol / L de KCl, 43,4 mmol / L de sacarose, 54,9 mmol / L de D-sorbitol, 0,5 mmol / L de DL-ditiotreitol em água destilada) durante 60 min em gelo. </li><li> Depois de quelação, a maior parte das células epiteliais intestinais normais serão separadas, enquanto que as células tumorais irão permanecer ligada ao mesênquima. Aspirar fora o tampão de quelação contendo células epiteliais normaise lavar o tumor remanescente fragmentos mais uma vez com 5 ml de tampão frio de quelação. </li><li> Aspirar fora do tampão de quelação, lave fragmentos do tumor com 5 ml frio 1x PBS. </li><li> Aspirar fora da 1x PBS, incubar fragmentos tumorais em tampão de digestão (2,5% de soro fetal bovino, 1 unidade / ml de penicilina, 1 ug / ml de estreptomicina, e 2,5 ng / ml de anfotericina B, 200 U / mL de colagenase tipo IV, 125 ug / ml de tipo II dispase em Meio Eagle Modificado por Dulbecco) durante 2 horas a 37 ° C. </li><li> Permitir que o fragmento de tumor que se contentar com a gravidade normal para 1 min, e recolher o sobrenadante em um tubo falcon de 15 ml. Pellet a única célula do tumor suspensão sobrenadante por centrifugação a 200 xg durante 3 minutos e lavar uma vez com 5 ml de PBS por centrifugação a 200 xg durante 3 min. </li></ol> 2. Cultura de tumor intestinal <ol><li> Ressuspender o sedimento de células tumorais com 500 mL PBS, contagem de células tumorais únicas isoladas usando um hemocitômetro. </li><li> Pellet células tumorais por centorifuging a 200 xg durante 3 minutos, e voltar a suspender-los em 5 mg / ml de Matrigel em gelo e a placa em placas de 24 poços a 15.000 células por 50 pi por poço de Matrigel. </li><li> Deixe o Matrigel polimerizar durante 15 minutos a 37 ° C e adicionar 500 uL / ​​poço de meio de cultura basal (1 unidade / ml de penicilina, 1 ug / ml de estreptomicina, e 2,5 ng / ml de anfotericina B, 10 mmol / L de HEPES , Glutamax 2 mM, 1x suplemento N2, 1x suplemento de B27, 1 mmol / L de N-acetilcisteína em Avançado do Medium/F12 Eagle modificado por Dulbecco) que continha 50 ng / ml de EGF murino. </li></ol> 3. Manutenção de Organóides Fundada <ol><li> Alterar meio de cultura basal contendo EGF a cada 2 dias e passagem Organóides 01:05 uma vez por semana. </li><li> Para passaging, substituir o meio de cultura com meio de cultura basal fresco. Mecanicamente perturbar Organóides e Matrigel usando uma pipeta P1000 com pontas cortadas e transferir para um tubo falcon de 15 ml. Além disso dissociação mecânica é conseguida usando um tubo de fogo Pasteur polidaTte. </li><li> Lavar Organóides dissociadas com 5 ml de meio de cultura basal e centrifugar a 200 xg durante 2 min. </li><li> Rejeitar o sobrenadante, ressuspender o sedimento com Matrigel e adicionar meio de cultura, tal como descrito acima. </li></ol> 4. Armazenamento e Recuperação de Organóides Fundada <ol><li> Para armazenamento a longo prazo, congelar Organóides em N2 líquido, que são estáveis ​​durante pelo menos 2 anos. Para Organóides congelamento, romper com uma pipeta P1000 com as pontas cortadas e transferir para um tubo falcon de 15 ml. </li><li> Lavar Organóides dissociadas com 5 ml de meio de cultura basal e centrifugar a 200 xg durante 2 min. </li><li> Rejeitar o sobrenadante, ressuspender o sedimento com Meio Eagle Modificado de Dulbecco (DMEM) contendo 20% de soro fetal bovino (FBS) e 10% de dimetilsulfóxido (DMSO). </li><li> Transferir as células em 1,5 ml criotubos, em seguida, colocar os tubos em um Mr. Frosty recipiente de congelamento Nalgene e armazenar em um freezer -80 ° C para atingir a taxa de resfriamentode -1 ° C / min. Após incubação durante a noite, para transferir os tubos N2 líquido. </li><li> Para a recuperação, os Organóides congelados são rapidamente descongeladas em 37 ° C em banho-maria, lave com meio de cultura basal, girar, ressuspender em Matrigel e cultura, com as condições descritas acima. </li></ol> 5. Extração de RNA, extração de proteínas e imuno-histoquímica <ol><li> Extracção de ARN: as células são colhidas como descrito acima para a passagem. O RNA é isolado com o PicoPure RNA Isolation Kit TM de acordo com as instruções do fabricante. </li><li> Extracção de Proteína: As pelotas de células são recolhidas como descrito acima e foram lisadas em 100 ul de tampão de ensaio de radioimunoprecipitação (RIPA, 50 mmol / L Tris-HCl pH 7,5, 150 mmol / l de NaCl, 2 mmol / L de EDTA, 1% de NP-40, 0,1 % SDS), 1x com inibidor da protease. </li><li> Imunohistoquímica: Aspirar off meio de cultura celular, transferir Organóides tumorais com P1000 pipeta com as pontas cortadas em Cryo-mofo e congelar imediatamente em tisprocessar meio de congelamento (OCT) com gelo seco. Secções congeladas foram cortadas em 7 um e coradas como descrito previamente 26. </li></ol>

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Os procedimentos experimentais descritos no presente protocolo irá permitir o isolamento e cultura de tumores do cólon de murino primários. O protocolo é uma adaptação do trabalho seminal realizado pelo Dr. grupo Clevers 1,24,27. Nós otimizamos o tempo de digestão e da concentração de colagenase para obter um melhor rendimento de Organóides tumorais. As etapas críticas incluem a digestão tumor de células em células individuais, Matrigel ressuspensão e cultura seletiva. Para a digestão de cél...

As células epiteliais intestinais proliferar e virar a um ritmo extraordinário, superando todos os outros tecidos do corpo dos vertebrados 2,3. As células que se dividem, incluindo as células-tronco intestinais (ISC) e células transit-amplificação diferenciar em qualquer secretora (taça, Paneth e enteroendócrinas) células ou enterócitos 3. A ISC está localizado na base da cripta. Células Paneth mover para a parte inferior das criptas e são de longa duração, ao passo que outras linhagens migrar para cima, para as vilosidades 3,4. Aqui, as células são expostas ao conteúdo, incluindo a microbiota intestinal e são eliminados a partir das pontas das vilosidades, através de um mecanismo de apoptose induzida por anoikis. Embora a falta de vilosidades do cólon e células de Paneth, o mecanismo para manutenção da homeostase é semelhante 4. A via de sinalização Wnt tem sido implicada em desempenhar um papel fundamental na proliferação intestinal e ISC manutenção 4. Supressão do diae factor de transcrição TCF4, um efector a jusante de sinalização de Wnt, leva à perda de células estaminais intestinais e subsequente degradação do tecido 5. Do mesmo modo, a expressão transgénica do DKK1 inibidor Wnt reduz a proliferação epitelial e esgota 6 linhagens de células secretoras. Por outro lado, a superexpressão do agonista R-spondin Wnt-1 induz a proliferação potente e rápida das células cripta intestinais 7. Dada a importância da via de sinalização Wnt para a homeostase intestinal, mutações via WNT são freqüentemente observadas no câncer de cólon 8. O cancro do cólon é a terceira principal causa de morte por câncer nos Estados Unidos 9. Ingestão em excesso de carne vermelha e álcool, atividade física reduzida, e herdou e mutações somáticas são considerados fatores de risco de câncer de cólon 10,11. O gene da polipose adenomatosa coli (APC), um factor de sinalização Wnt-chave, está mutado na maioria dos papacientes com DA familial, esporádico, e colite associada ao câncer de cólon 12,13. Mutações de outros fatores envolvidos na via de sinalização Wnt incluindo Axin2 e β-catenina também são observados no câncer de cólon 14,15. No entanto, o mecanismo preciso e tratamento eficaz para o câncer de cólon ainda estão faltando. Para facilitar a investigação sobre os mecanismos moleculares para o cancro do cólon, as linhas celulares de cancro de cólon humano que representam fases diferentes da progressão de um cancro benigno para um tipo de célula agressivo foram estabelecidas 16-18. Rato linhagens celulares de carcinoma de cólon com diferentes propriedades metastáticas também estão disponíveis 19,20. No entanto, as células primárias ou culturas organóide são preferidos sobre linhas de células transformadas, porque eles imitam de perto o estado em vivo e gerar mais fisiologicamente relevante de dados 21. A maioria das linhas de cancro do cólon, células derivadas crescer como monocamadas ligado à placa ou como suspensões de células, lacking de orientação apical-basolateral e junções apertadas entre as células. Além disso, as células epiteliais intestinais normais e tumorais in vivo, sofrem de forma espontânea de apoptose anoikis denominado como as células diferenciadas atingir as pontas das vilosidades e 22 são eliminados. Estas características são difíceis de recapitular em linhas de células, mas são importantes no processo de desenvolvimento de cancro do cólon 23. Estas características são mantidas em Organóides primários. Além disso, as culturas organóide tumor fornecem um sistema eficiente para avaliar as funções autónomas de genes tumorais em comparação com os estudos in vivo. A manipulação genética in vivo do intestino é um processo demorado, principalmente através da criação de transgênicos e / ou camundongos knockout usando drivers específicos de intestino. No entanto, os Organóides tumorais são facilmente passíveis de manipulações genéticas mediadas virais e, portanto, uma ótima ferramenta para avaliar mecanismos moleculares precisos. Primários intestinais culturas organoide tumorais have foi demonstrado ser uma técnica viável e poderosa. Cultura de células intestinal primária pode estabelecer Organóides intestinais funcionais, com estrutura da cripta-vilosidade in vitro a partir de um único adulto Lgr5 + células-tronco 24. Estes Organóides podem ser transplantadas e enxertados no tecido do cólon danificado para a regeneração 25. Além disso adaptação das condições de cultura fez Organóides epiteliais do cólon semelhantes rato e do intestino delgado e do cólon humano 1 viável. Para a cultura primária de cólon normal, epitélio, meio de cultura basal, assim como fatores de crescimento, incluindo EGF, Noggin, R-spondin e Wnt3a são essenciais, enquanto meio de cultura basal e EGF é suficiente para o cultivo de rato cólon Organóides tumores primários 1. Aqui nós descrevemos um protocolo detalhado para isolar, cultura e gerar Organóides tumor do cólon.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Matrigel Basement Membrane Matrix</td>
			<td>BD Biosciences</td>
			<td>356234</td>
			<td>5 mg/ml</td>
		</tr>
		<tr>
			<td>Collagenase Type IV</td>
			<td>Worthington</td>
			<td>LS004188</td>
			<td>375 U/mg</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Gibco</td>
			<td>17105-041</td>
			<td>1.8 U/mg</td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Invitrogen</td>
			<td>12634010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor (EGF), Murine, Natural</td>
			<td>Invitrogen</td>
			<td>53003-018</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>N2 Supplement</td>
			<td>Invitrogen</td>
			<td>17502-048</td>
			<td>100 x</td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td>50 x</td>
		</tr>
		<tr>
			<td>Glutamax-I</td>
			<td>Gibco</td>
			<td>35050-079</td>
			<td>100x</td>
		</tr>
		<tr>
			<td>N-Acetylcysteine</td>
			<td>Sigma</td>
			<td>A9165-5G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dulbecco's Modified Eagle Medium</td>
			<td>Invitrogen</td>
			<td>11965-092</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PicoPureTM RNA Isolation Kit</td>
			<td>Invitrogen</td>
			<td>KIT0204</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

in vitro organoid culture of primary mouse colon tumors

Várias linhas celulares de cancro de cólon humano e murino foram estabelecidas, a integridade fisiológica de tumores do cólon, tais como diversas camadas de células, de polaridade apical-basal, a capacidade de diferenciar e anoikis não são mantidos em linhas celulares derivadas do cancro do cólon. O presente estudo demonstra um método para a cultura primária Organóides tumorais de cólon de rato adaptados de Sato T et al. 1, que mantém as características fisiológicas importantes de tumores do cólon. Este método consiste na recolha de tecido de tumor do cólon do rato, do epitélio do cólon normal adjacente dissociação, as células de tumor do cólon de digestão em células individuais, a incorporação de células tumorais de cólon em matrigel e cultura selectivo de acordo com o princípio de que as células tumorais manter o crescimento em condições limitantes de nutrientes em relação ao normal células epiteliais. Os Organóides tumor primário se isolados de camundongos geneticamente modificados fornecer um sistema muito útil para avaliar Esta função autônoma tumorião de genes específicos. Além disso, os Organóides tumorais são passíveis de manipulação genética de vírus meditado entrega gene, portanto vias de sinalização envolvidas na carcinogênese do cólon também pode ser amplamente investigado pela superexpressão ou knockdown. Principal cultura Organóides tumor fornece um meio fisiológicas relevantes e viáveis ​​para estudar os mecanismos e modalidades terapêuticas para tumorigenesis cólon.

O curso de tempo da formação de um tumor do cólon organóide de um período de três meses de idade, Apc min / rato + é mostrada na Figura 1. No dia 0, as células individuais podem ser observadas várias horas após plaqueamento (Figura 1A). No dia 1, sobreviveu cólon células epiteliais tumorais com núcleos refratário pode ser observado. No dia 3, o tamanho das células duplicou. No dia 6, o tamanho da organóide expandida mais do que dez vezes, e...

Watch this Scientific Journal Video about In vitro Organoid Culture of Primary Mouse Colon Tumors at JoVE.com

In vitro Organoid Culture of Primary Mouse Colon Tumors

Um método simples para determinar murino organóide tumor de cólon primário é descrito. Este método utiliza o recurso de que as células tumorais de cólon sobreviver e crescer em Organóides em meios contendo fatores de crescimento limitadas, enquanto cólon normais do epitélio não.

Em organoide Cultura in vitro de primário do mouse tumores do cólon

Several  human and murine colon cancer cell lines have been established, physiologic  integrity of colon tumors such as multiple cell layers, basal-apical ...

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Research

JoVE Journal

Medicine

In vitro Organoid Culture of Primary Mouse Colon Tumors

34.9K visualizações.  University of Michigan. Um método simples para determinar murino organóide tumor de cólon primário é descrito. Este método utiliza o recurso de que as células tumorais de cólon sobreviver e crescer em Organóides em meios contendo fatores de crescimento limitadas, enquanto cólon normais do epitélio não.

Vídeo: In vitro Organoid Culture of Primary Mouse Colon Tumors

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

Estabelecimento e propagação de Tumores Humanos em Retinoblastoma Imune camundongos deficientes

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Medicina

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease1. Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms2 and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson's disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.

The fibroblast explant culture protocol from human skin punch biopsies is a technically robust and simple way to derive skin cells within 4-8 weeks for banking of about 15-20 million cells at a low passage number.

Pele da biópsia Explant Cultura para Derivação de primárias fibroblastos humanos

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

Primary Culture of Human Vestibular Schwannomas

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in either sporadic or familial (neurofibromatosis type 2, NF2) forms, both associated with inactivating defects in the NF2 tumor suppressor gene. Treatment for VSs is generally surgical resection or radiosurgery, however the morbidity of such procedures has driven investigations into less invasive treatments. Historically, lack of access to fresh tissue specimens and the fact that schwannoma cells are not immortalized have significantly hampered the use of primary cultures for investigation of schwannoma tumorigenesis. To overcome the limited supply of primary cultures, the immortalized HEI193 VS cell line was generated by transduction with HPV E6 and E7 oncogenes. This oncogenic transduction introduced significant molecular and phenotypic alterations to the cells, which limit their use as a model for human schwannoma tumors. We therefore illustrate a simplified, reproducible protocol for culture of primary human VS cells. This easily mastered technique allows for molecular and cellular investigations that more accurately recapitulate the complexity of VS disease.

Vestibular Schwannomas (VSs) are non-malignant tumors of Schwann cell (SC) origin, associated with mutations in the NF2 tumor suppressor gene. We report a reproducible, efficient protocol for primary human VS cell culture that allows for molecular and cellular experimental manipulation and analysis and recapitulates the heterogeneous nature of human disease.

Cultura primária de Vestibular Humano Schwanomas

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in ...

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e., soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo (fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

Established cancer cell lines and xenografts have been the mainstay of cancer research for the past several decades. However, recent evidence suggests that therapeutic response is greatly influenced by the tumor cell microenvironment. Therefore, we have developed an ex vivo analysis of primary tumor specimens for drug development purposes.

Ex Vivo resposta ao tratamento de tumores primários e / ou Associated Metástases para pré-clínica e clínica de Desenvolvimento Therapeutics

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both ...

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The approach allows unbiased identification and functional characterization of individual colorectal afferents. Extracellular recordings of propagated action potentials (APs) that originate from one or a few afferent (i.e., single-fiber) receptive fields (RFs) in the colorectum are made from teased nerve fiber fascicles. The colorectum is removed with either the pelvic (PN) or lumbar splanchnic (LSN) nerve attached and opened longitudinally. The tissue is placed in a recording chamber, pinned flat and perfused with oxygenated Krebs solution. Focal electrical stimulation is used to locate the colorectal afferent endings, which are further tested by three distinct mechanical stimuli (blunt probing, mucosal stroking and circumferential stretch) to functionally categorize the afferents into five mechanosensitive classes. Endings responding to none of these mechanical stimuli are categorized as mechanically-insensitive afferents (MIAs). Both mechanosensitive and MIAs can be assessed for sensitization (i.e., enhanced response, reduced threshold, and/or acquisition of mechanosensitivity) by localized exposure of RFs to chemicals (e.g., inflammatory soup (IS), capsaicin, adenosine triphosphate (ATP)). We describe the equipment and colorectum&#8211;nerve recording preparation, harvest of colorectum with attached PN or LSN, identification of RFs in the colorectum, single-fiber recording from nerve fascicles, and localized application of chemicals to the RF. In addition, challenges of the preparation and application of standardized mechanical stimulation are also discussed.

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation.

In vitro caracterização funcional de Rato Colorretal Aferente Endings

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The ...

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA (Locked Nucleic Acid) probe. The probe was recognized by anti-DIG alkaline phosphatase antibodies and later developed using alkaline phosphatase substrate producing fluorescence signals. Here we utilized miRNA in situ hybridization (MISH) technique to analyze expression of miR-489 in tissues from breast cancer patients. This technique can detect the localization of miRNA of interest in individual tissue samples. This technique can be used to compare the expression of desired miRNA in tumor tissue with that in adjacent normal tissue and to identify the specific structures responsible for expressing this miRNA. This technique can be very useful in answering certain clinical questions, such as role of specific miRNA in the development of cancer. Our results indicate that mammary epithelial cells express significantly higher levels of miR-489 than adjacent tumor cells.

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

Here we present a protocol to detect miRNA expression in breast cancer patient samples using miRNA in situ hybridization.

Análise clínico-patológico de miRNA Expressão em cancro da mama tecidos usando miRNA<em&gt; In Situ</em&gt; Hibridização

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

Isolamento e cultura de células endoteliais primárias de Artérias e Veias canina

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

Inchaço na Organóides intestinal induzida por forscolina: An<em&gt; In Vitro</em&gt; Ensaio para a Avaliação de Medicamentos Response na Fibrose Cística Os pacientes

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Muscle Imbalances: Testing and Training Functional Eccentric Hamstring Strength in Athletic Populations

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite of these eccentric hamstring actions are concentric quadriceps actions, where the larger and likely stronger quadriceps straighten the knee. Therefore, to stabilize the lower limbs during movement, the hamstrings must eccentrically combat against the strong knee-straightening torque of the quadriceps. As such, eccentric hamstring strength expressed relative to concentric quadricep strength is commonly referred to as the &#34;functional ratio&#34; as most movements in sports require simultaneous concentric knee extension and eccentric knee flexion. To increase the strength, resiliency, and functional performance of the hamstrings, it is necessary to test and train the hamstrings at different eccentric speeds. The main purpose of this work is to provide instructions for measuring and interpreting eccentric hamstring strength. Techniques for measuring the functional ratio using isokinetic dynamometry are provided and sample data will be compared. Additionally, we briefly describe how to address hamstring strength deficiencies or unilateral strength differences using exercises that specifically focus on increasing eccentric hamstring strength.

The hamstrings are a group of muscles that are sometimes problematic for athletes, resulting in soft tissue injury in the lower limbs. To prevent such injuries, functional training of the hamstrings requires intensive eccentric contractions. Additionally, hamstring function should be tested in relation to quadricep function at different contraction speeds.

Desequilíbrios musculares: Teste e treinamento de força funcional da limitação excêntrico em populações atléticas

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite ...

On-Site Sampling and Extraction of Brain Tumors for Metabolomics and Lipidomics Analysis

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still in need. In recent years, mass spectrometry has become a method of choice for untargeted profiling of discriminatory compound as potential biomarkers of a disease. Biofluids are generally considered as preferable matrices given their accessibility and easier sample processing while direct tissue profiling provides more selective information about a given cancer. Preparation of tissues for the analysis via traditional methods is much more complex and time-consuming, and, therefore, not suitable for fast on-site analysis. The current work presents a protocol combining sample preparation and extraction of small molecules on-site, immediately after tumor resection. The sampling device, which is of the size of an acupuncture needle, can be inserted directly into the tissue and then transported to the nearby laboratory for instrumental analysis. The results of metabolomics and lipidomics analyses demonstrate the capability of the approach for the establishment of phenotypes of tumors related to the histological origin of the tumor, malignancy, and genetic mutations, as well as for the selection of discriminating compounds or potential biomarkers. The non-destructive nature of the technique permits subsequent performance of routinely used tests e.g., histological tests, on the same samples used for SPME analysis, thus enabling attainment of more comprehensive information to support personalized diagnostics.

The manuscript presents on-site sampling of human brain tumors with solid phase microextraction followed by their biochemical profiling towards the discovery of biomarkers.

Amostragem e Extração de Tumores Cerebrais no local para análise metabolômica e lipidômica

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still ...