Questo studio è stato sostenuto da sovvenzioni a YMS dal National Institutes of Health (CA148828), L&#39;Università del Michigan gastrointestinale Peptide Center e Jeffrey A. Colby Colon Cancer Research e Tom Liu Memorial Fondi della University of Michigan Comprehensive Cancer Center.

1. Tumore del colon Isolamento e dissociazione cellulare <ol><li> Tumori intestinali possono essere isolati da qualsiasi modello di cancro al colon sporadico oppure indotta dal trattamento. I topi devono essere sacrificati con CO 2. I due punti vengono poi raccolti, lavati con freddo PBS (PBS) e aperto longitudinalmente. Identificare le regioni contenenti i tumori utilizzando uno stereomicroscopio, sezionare fuori con un paio di forbici, e lavare con PBS freddo. </li><li> Incubare frammenti intestinali contenenti tumori nel buffer di chelazione EDTA (2 mM EDTA, 5,6 mmol / L di Na 2 HPO 4, 8.0 mmol / L KH 2 PO 4, 96,2 mmol / L di NaCl, 1,6 mmol / L KCl, 43,4 mmol / L di saccarosio, 54.9 mmol / L D-sorbitolo, 0,5 mmol / L di DL-ditiotreitolo in acqua distillata) per 60 min in ghiaccio. </li><li> Dopo chelazione, la maggior parte delle normali cellule epiteliali intestinali sarà rimosso, mentre le cellule tumorali rimane collegata mesenchima. Aspirare il tampone di chelazione contenente cellule epiteliali normalie lavare il tumore residuo frammenta ancora una volta con 5 ml di tampone chelazione freddo. </li><li> Aspirare il tampone di chelazione, lavare frammenti di tumore con 5 ml di freddo 1x PBS. </li><li> Aspirare il PBS 1x off, incubare frammenti di tumore in tampone di digestione (2.5% siero fetale bovino, 1 unità / ml di penicillina, 1 mg / ml di streptomicina, e 2,5 ng / ml di amfotericina B, 200 U / ml di collagenasi di tipo IV, 125 mg / ml di tipo II dispasi in Dulbecco Modified Eagle Medium) per 2 ore a 37 ° C. </li><li> Lasciare che il frammento di tumore a stabilirsi per gravità normale per 1 minuto e raccogliere il surnatante in una provetta Falcon 15. Pellet la singola cellula tumorale sospensione surnatante mediante centrifugazione a 200 xg per 3 min e lavare una volta con 5 ml di PBS mediante centrifugazione a 200 xg per 3 min. </li></ol> 2. Cultura di tumore intestinale <ol><li> Risospendere il pellet di cellule tumorali con 500 microlitri di PBS, contare le cellule tumorali isolate singole utilizzando un emocitometro. </li><li> Pellet cellule tumorali di centorifuging a 200 xg per 3 min, e li risospendere in 5 mg / ml Matrigel su ghiaccio e piastra in piastre da 24 pozzetti a 15.000 cellule per 50 pl di Matrigel per pozzetto. </li><li> Lasciare la Matrigel polimerizzare per 15 min a 37 ° C e aggiungere 500 microlitri / pozzetto basale terreno di coltura (1 unità / ml di penicillina, 1 mg / ml di streptomicina, e 2,5 ng / ml di amfotericina B, 10 mmol / L HEPES , 2mm Glutamax, 1x supplemento N2, 1x B27 supplemento, 1 mmol / L N-acetilcisteina in avanzata a Dulbecco Modified Eagle Medium/F12) contenente 50 ng / ml di EGF murino. </li></ol> 3. Manutenzione di organoidi Fondata <ol><li> Cambiare terreno di coltura contenente FEG ogni 2 giorni e passaggio organoidi 01:05 una volta alla settimana. </li><li> Per passaging, sostituire il terreno di coltura con terreno di coltura fresco. Disturbare meccanicamente organoidi e Matrigel utilizzando una pipetta P1000 con punte tagliate e trasferire in una provetta Falcon 15. Un&#39;ulteriore dissociazione meccanica viene ottenuta utilizzando un incendio lucidato Pasteur tubotte. </li><li> Lavare organoidi dissociate con 5 ml di terreno di coltura e centrifugare a 200 xg per 2 min. </li><li> Scartare il surnatante, risospendere il pellet con Matrigel e aggiungere mezzo di coltura come descritto sopra. </li></ol> 4. Conservazione e ripristino di organoidi Fondata <ol><li> Per la conservazione a lungo termine, congelare organoidi a liquido N 2 che sono stabili per almeno 2 anni. Per organoidi congelamento, può interferire con una pipetta P1000 con punte tagliate e trasferire in una provetta Falcon 15. </li><li> Lavare organoidi dissociate con 5 ml di terreno di coltura e centrifugare a 200 xg per 2 min. </li><li> Scartare il surnatante, risospendere il pellet con Dulbecco Modified Eagle Medium (DMEM) contenente 20% di siero fetale bovino (FBS) e 10% di dimetilsolfossido (DMSO). </li><li> Trasferire le cellule in 1,5 ml cryotubes, poi mettere i tubi in un Mr. Frosty contenitore congelamento Nalgene e conservare in un freezer -80 ° C per ottenere una velocità di raffreddamentodi -1 ° C / min. Dopo una notte di incubazione, trasferire i tubi in liquido N 2. </li><li> Per il recupero, i organoidi congelati vengono scongelati rapidamente a 37 ° C a bagnomaria, lavare con terreno di coltura, centrifugare, risospendere in Matrigel e cultura con le condizioni sopra descritte. </li></ol> 5. Estrazione di RNA, estrazione di proteine ​​ed immunoistochimica <ol><li> Estrazione dell&#39;RNA: Le cellule vengono raccolti come descritto sopra per il passaggio. RNA è isolato con PicoPure TM RNA Isolation Kit secondo le istruzioni del produttore. </li><li> Proteine ​​di estrazione: pellet cellulari sono raccolti come descritto sopra e lisate in 100 microlitri tampone radioimmunoprecipitazione (RIPA; 50 mmol / L Tris-HCl pH 7.5, 150 mmol / L di NaCl, 2 mmol / L di EDTA, 1% NP-40, 0,1 % SDS) con 1x inibitore della proteasi. </li><li> Immunoistochimica: Aspirare off mezzo di coltura cellulare, trasferire organoidi tumorali con P1000 pipetta con punte tagliate in Cryo-stampo e congelare immediatamente in tiscitare in giudizio medio congelamento (OCT) con ghiaccio secco. Sezioni congelate sono state tagliate a 7 ​​micron e colorati come descritto in precedenza 26. </li></ol>

<ol>
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Non abbiamo nulla da rivelare.

Le procedure sperimentali descritte in questo protocollo permetterà di isolamento e la coltura di tumori al colon murini primari. Il protocollo è stato adattato dal lavoro seminale condotto dal Dott. gruppo Clevers 1,24,27. Abbiamo ottimizzato il tempo di digestione e la concentrazione di collagenasi per ottenere una migliore resa di organoidi tumorali. I punti critici sono la digestione delle cellule tumorali in cellule singole, Matrigel risospensione e cultura selettiva. Per la digestione delle cellule tu...

Le cellule epiteliali intestinali proliferano e si girano a una velocità straordinaria, superando tutti gli altri tessuti del corpo dei vertebrati 2,3. Le cellule in divisione, comprese le cellule staminali intestinali (ISC) e cellule di transito-amplificazione si differenziano in due secretoria (calice, Paneth e enteroendocrine) cellule o enterociti 3. L&#39;ISC si trova alla base della cripta. Cellule Paneth spostano verso il fondo delle cripte e sono di lunga durata, mentre altri lignaggi migrano verso l&#39;alto per i villi 3,4. Qui le cellule sono esposte ai contenuti intestinali tra cui microbiota e si staccano dalle punte dei villi, attraverso un meccanismo apoptotico anoikis indotta. Sebbene il colon manca villi e cellule Paneth, il meccanismo per il mantenimento dell&#39;omeostasi è simile 4. La via di segnalazione Wnt è stata implicata nel giocare un ruolo cruciale nella proliferazione intestinale e ISC manutenzione 4. Soppressione del secoloe fattore di trascrizione TCF4, un effettore a valle di segnalazione Wnt, porta alla perdita di cellule staminali intestinali e la successiva ripartizione del tessuto 5. Analogamente, l&#39;espressione transgenica dell&#39;inibitore DKK1 Wnt riduce la proliferazione epiteliale e impoverisce linee cellulari secretorie 6. Viceversa, l&#39;iperespressione di agonista Wnt R-spondina-1 induce potente e rapida proliferazione di cellule intestinali cripta 7. Data l&#39;importanza della segnalazione Wnt per l&#39;omeostasi intestinale, le mutazioni di Wnt sono frequentemente osservate nel cancro del colon 8. Il cancro del colon è la terza causa di morte per cancro negli Stati Uniti 9. Assunzione in eccesso di carne rossa e alcool, ridotta attività fisica, e ha ereditato e mutazioni somatiche sono considerati fattori di rischio di tumore del colon 10,11. La poliposi coli (Apc) gene adenomatosa, un fattore chiave di segnalazione Wnt, è mutato in una maggioranza di papazienti con familiare, sporadica, e il cancro del colon colite associata a 12,13. Le mutazioni di altri fattori coinvolti nella via di segnalazione Wnt compresi Axin2 e β-catenina si osservano anche nel cancro del colon 14,15. Tuttavia, il meccanismo preciso ed efficace terapia per il cancro al colon sono ancora carenti. Per facilitare la ricerca dei meccanismi molecolari per il cancro del colon, le linee cellulari tumorali di colon umano che rappresentano diverse fasi della progressione del cancro da un benigno a un tipo di cellule aggressive sono state stabilite 16-18. Mouse linee cellulari di carcinoma del colon con diverse proprietà metastatiche sono disponibili anche 19,20. Tuttavia, le cellule primarie o culture organoide sono preferite rispetto alle linee cellulari trasformate, perché strettamente imitano lo stato in vivo e generano più fisiologicamente rilevanti di dati 21. Maggior colon-cancro linee cellulari derivate crescere come monostrato fissata sulla piastra o come sospensioni cellulari, Lacking di orientamento apicale-basolaterale e giunzioni strette tra le cellule. Inoltre, le cellule epiteliali intestinali normali e tumorali in vivo sottoposti a una forma spontanea di apoptosi anoikis definito come le cellule differenziate raggiungono le punte dei villi e sono capannone 22. Queste caratteristiche sono difficili da ricapitolare in linee cellulari, ma sono importanti nel processo di sviluppo del cancro al colon 23. Queste caratteristiche vengono mantenute in organoidi primari. Inoltre, le culture organoide tumorali forniscono un sistema efficiente per valutare funzioni autonome di geni tumorali rispetto a studi in vivo. Manipolazione genetica in vivo dell&#39;intestino è un processo che richiede tempo principalmente attraverso la creazione transgenici e / o topi knockout utilizzando driver intestino-specifici. Tuttavia, i organoidi tumorali sono facilmente suscettibili di manipolazioni genetiche virali mediata e quindi un ottimo strumento per valutare i meccanismi molecolari precisi. Primari intestinali tumorali organoide culture have stato dimostrato di essere una tecnica fattibile e potente. Coltura di cellule intestinali primaria può stabilire organoidi intestinali funzionali con struttura cripta-villi in vitro da un singolo adulto LGR5 + cellule staminali 24. Questi organoidi possono essere trapiantati e innestate nel tessuto danneggiato del colon per la rigenerazione 25. Ulteriore adeguamento delle condizioni di coltura aveva fatto organoidi epiteliali simili da colon mouse e piccolo intestino umano e del colon fattibile 1. Per il normale epitelio del colon cultura primaria, terreno di coltura così come fattori di crescita tra cui EGF, Zucca, R-spondina e Wnt3a sono essenziali, mentre è sufficiente per la coltivazione del mouse colon organoidi tumore primario 1 terreno di coltura e di EGF. Qui si descrive un protocollo dettagliato per isolare, la cultura, e generare organoidi del tumore del colon.

<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

Sono state create diverse linee di cellule tumorali di colon umano e murino, l&#39;integrità fisiologica dei tumori del colon, come gli strati multipli delle cellule basali, polarità-apicale, la capacità di differenziare e anoikis non vengono mantenute nel cancro linee cellulari derivate colon. Il presente studio dimostra un metodo per la coltura principale del mouse colon organoidi tumorali adattato da Sato T et al. 1, che conserva importanti funzioni fisiologiche di tumori del colon. Questo metodo consiste colon raccolta topo tessuto tumorale, adiacente normale epitelio dissociazione colon, colon cellule tumorali digestione in singole cellule, incorporando le cellule tumorali del colon in matrigel, e la coltura selettivo basato sul principio che le cellule tumorali mantenere la crescita sul limitare condizioni di nutrienti rispetto al normale cellule epiteliali. Le organoidi tumore primario, se isolate da topi geneticamente modificati, forniscono un sistema molto utile per valutare Funz autonomo tumoreione di geni specifici. Inoltre, i organoidi tumorali sono suscettibili di manipolazione genetica da virus meditato la consegna del gene; percorsi quindi di segnalazione coinvolte nella tumorigenesi del colon potrebbero essere ampiamente studiati da sovraespressione o atterramento. Primaria cultura organoidi tumorale fornisce un fisiologici relativi mezzi e fattibile per studiare i meccanismi e le modalità terapeutiche per la tumorigenesi del colon.

La durata di un tumore del colon organoide da una formazione di tre mesi Apc min / + topo è mostrato in Figura 1. Al giorno 0, singole cellule potrebbero essere osservati diverse ore dopo la placcatura (Figura 1A). Al giorno 1, sopravvissero colon cellule epiteliali tumorali con nuclei refrattaria poteva essere rispettato. Al giorno 3, la dimensione delle cellule raddoppiato. Al giorno 6, il formato di organoide espansa più di dieci volte e ha mostrato segni di apo...

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

Un metodo semplice per determinare primario murino organoide tumore colon è descritto. Questo metodo utilizza la funzione che le cellule tumorali del colon sopravvivere e crescere in organoidi in terreni contenenti fattori di crescita limitate, mentre colon normale epiteliale non lo fanno.

In vitro organoide Cultura di primari tumori del colon mouse

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 Views.  University of Michigan. Un metodo semplice per determinare primario murino organoide tumore colon è descritto. Questo metodo utilizza la funzione che le cellule tumorali del colon sopravvivere e crescere in organoidi in terreni contenenti fattori di crescita limitate, mentre colon normale epiteliale non lo fanno.

Video: 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.

Istituzione e Propagazione dei Tumori retinoblastoma umani in topi privi di sistema immunitario

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

Ricerca

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.

Pelle Punch Biopsia Espianto Cultura per la derivazione di fibroblasti umani primari

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 primario di Human vestibolare Schwannomi

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 risposta al trattamento di tumori primari e / o associati per metastasi preclinici e sviluppo clinico di 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 Caratterizzazione funzionale del topo colorettale Afferent 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.

Analisi clinicopatologica di miRNA espressione in Breast Cancer tessuti mediante l&#39;utilizzo di miRNA<em&gt; In Situ</em&gt; ibridazione

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 coltura di cellule endoteliali primarie dalle arterie e vene Canine

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.

Forskolin indotta Gonfiore intestinale organoidi: An<em&gt; In Vitro</em&gt; Test per la valutazione della risposta farmacologica nella fibrosi cistica pazienti

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.

Squilibri muscolari: Test e formazione forza eccentrica funzionale del tendine del ginocchio nelle popolazioni di atletiche

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.

Campionamento ed estrazione in loco di tumori cerebrali per l'analisi metabolomica e lipidomica

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