Diese Studie wurde unterstützt durch Zuschüsse zu YMS von den National Institutes of Health (CA148828), The University of Michigan Gastrointestinale Peptide Center und Jeffrey A. Colby Colon Cancer Research und der Tom Liu Memorial Funds von der University of Michigan Comprehensive Cancer Center unterstützt.

1. Colon Tumor Isolation und Zelldissoziation <ol><li> Intestinale Tumoren können von jedem sporadische oder Behandlung induzierten Dickdarmkrebs Modell isoliert werden. Die Mäuse sollten mit CO 2 eingeschläfert werden. Doppelpunkte werden dann gesammelt, gespült mit kaltem Phosphat-gepufferte Kochsalzlösung (PBS) und der Länge nach geöffnet. Identifizieren Regionen mit Tumoren mit einem Stereomikroskop, sezieren mit einer Schere, und waschen mit kaltem PBS. </li><li> Inkubieren Darm-Fragmente, die Tumoren in EDTA-Chelat-Puffer (2 mM EDTA, 5,6 mmol / L Na 2 HPO 4, 8,0 mmol / L KH 2 PO 4, 96,2 mmol / L NaCl, 1,6 mmol / L KCl, 43,4 mmol / L Saccharose, 54,9 mmol / l D-Sorbit, 0,5 mmol / l DL-Dithiothreitol in destilliertem Wasser) für 60 min auf Eis. </li><li> Nach Chelat, werden die meisten der normalen intestinalen Epithelzellen abgetrennt werden, während Tumorzellen Mesenchym befestigt bleiben wird. Absaugen die Chelatbildung Puffer mit normalen Epithelzellenund wäscht den Rest Tumorfragmente ein weiteres Mal mit 5 ml kaltem Puffer Chelatbildung. </li><li> Absaugen die Chelatbildung Puffer waschen Tumorfragmente mit 5 ml kaltem 1x PBS. </li><li> Absaugen des 1x PBS, Inkubation Tumorfragmente bei der Verdauung Puffer (2,5% fötalem Rinderserum, 1 Einheit / ml Penicillin, 1 ug / ml Streptomycin und 2,5 ng / ml Amphotericin B, 200 U / ml Kollagenase Typ IV, 125 ug / ml Typ II Dispase in Dulbecco modifiziertem Eagle Medium) für 2 Stunden bei 37 ° C. </li><li> Lassen Sie die Tumorfragment unter normalen Schwerkraft für 1 min absetzen, und den Überstand in ein 15 ml Falcon-Röhrchen. Pellet die einzelne Zelle Tumor Suspension Überstand durch Zentrifugation bei 200 xg für 3 min und waschen einmal mit 5 ml PBS durch Zentrifugation bei 200 xg für 3 min. </li></ol> 2. Kultur Darm Tumor <ol><li> Resuspendieren der Tumorzelle Pellet mit 500 ul PBS, zählen isolierte einzelne Tumorzellen mit einer Zählkammer. </li><li> Pellet Tumorzellen durch centrifuging bei 200 × g für 3 min, und resuspendieren sie in 5 mg / ml Matrigel auf Eis und Platte in Platten mit 24 Vertiefungen mit 15.000 Zellen pro 50 ul pro Vertiefung Matrigel. </li><li> Lassen Sie die Matrigel für 15 min polymerisiert bei 37 ° C, und fügen Sie 500 ul / Vertiefung basalen Kulturmedium (1 Einheit / ml Penicillin, 1 ug / ml Streptomycin und 2,5 ng / ml Amphotericin B, 10 mmol / L HEPES , 2 mM Glutamax, 1x N2 Ergänzung, 1x B27 Ergänzung, 1 mmol / L N-Acetylcystein in Modified Adler Erweiterte Dulbecco Medium/F12) mit 50 ng / ml Maus-EGF. </li></ol> 3. Wartung Gegründet Organoide <ol><li> Ändern basalen Kulturmedium mit EGF alle 2 Tage und Passage Organoiden 01.05 einmal pro Woche. </li><li> Für Passagieren, ersetzen Sie das Kulturmedium durch frisches basalen Kulturmedium. Mechanisch stören Organoiden und Matrigel mit einem P1000 mit Spitzen abgeschnitten und in ein 15 ml Falcon-Röhrchen. Weitere mechanische Dissoziation wird erreicht, indem ein Feuer polierten Pasteur Rohrtte. </li><li> Waschen Sie distanzierte Organoiden mit 5 ml der basalen Kulturmedium und Zentrifuge bei 200 xg für 2 min. </li><li> Überstand verwerfen, das Pellet mit Matrigel und fügen Kulturmedium wie oben beschrieben. </li></ol> 4. Speicher-und Recovery von Gegründet Organoide <ol><li> Für die langfristige Lagerung einfrieren Organoiden in flüssigem N 2, die stabil sind für mindestens 2 Jahre. Zum Einfrieren Organoiden, stören mit einem P1000 mit Spitzen abgeschnitten und in ein 15 ml Falcon-Röhrchen. </li><li> Waschen Sie distanzierte Organoiden mit 5 ml der basalen Kulturmedium und Zentrifuge bei 200 xg für 2 min. </li><li> Der Überstand wird verworfen, das Pellet mit Dulbeccos modifiziertem Eagle Medium (DMEM), enthaltend 20% fötales Rinderserum (FBS) und 10% Dimethylsulfoxid (DMSO). </li><li> Transfer-Zellen in 1,5 ml Kryoröhrchen, dann die Rohre in einer Nalgene Mr. Frosty Einfrieren Behälter und Speicher in einem -80 ° C Gefrierschrank, eine Kühlung zu erzielenvon -1 ° C / min. Nach Inkubation über Nacht übertragen Röhrchen in flüssigem N 2. </li><li> Für die Wiederherstellung werden die gefrorenen Organoiden schnell in einem 37 ° C Wasserbad, waschen mit basalen Kulturmedium, Spin-down, in Matrigel und Kultur mit oben beschriebenen Bedingungen resuspendieren. Aufgetaut </li></ol> 5. RNA-Extraktion, Protein Extraction und Immunhistochemie <ol><li> RNA-Extraktion: Die Zellen werden gesammelt, wie oben beschrieben, für den Durchgang. RNA wird mit PicoPure TM RNA Isolation Kit nach den Anweisungen des Herstellers isoliert. </li><li> Protein Extraction: Zellpellets werden gesammelt wie oben beschrieben und lysiert in 100 ul Assaypuffer Radioimmunfällung (RIPA, 50 mmol / l Tris-HCl pH 7,5, 150 mmol / l NaCl, 2 mmol / l EDTA, 1% NP-40, 0,1 % SDS) mit 1x Proteasehemmer. </li><li> Immunhistochemie: Absaugen Zellkulturmedium, übertragen Tumor Organoiden mit P1000 mit Spitzen aus in Cryo-Form geschnitten und sofort einfrieren in tisverklagen Einfriermedium (OCT) mit Trockeneis. Gefrierschnitte wurden bei 7 um geschnitten und gefärbt, wie zuvor beschrieben 26. </li></ol>

<ol>
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Wir haben nichts zu offenbaren.

Die experimentellen Verfahren in diesem Protokoll beschrieben wird zur Isolierung und Kultivierung von primären murinen Kolontumoren ermöglichen. Das Protokoll wird von bahnbrechenden Arbeit von Dr. Clevers Gruppe 1,24,27 getan angepasst. Wir optimierten die Verdauung Zeit und Kollagenasekonzentration um eine bessere Ausbeute des Tumors Organoiden bekommen. Die kritischen Schritte umfassen Tumorzelle Verdauung in einzelne Zellen, Matrigel Resuspension und selektive Kultur. Für Tumorzellen Verdauung, um ein...

Die intestinalen Epithelzellen vermehren und drehen auf einer außerordentlichen Geschwindigkeit und übertraf damit alle anderen Geweben im Körper Wirbeltier 2,3. Die teilenden Zellen, einschließlich Darm-Stammzellen (ISC) und Transit-Verstärkung Zellen differenzieren entweder in sekretorischen (Kelch, Paneth und enteroendokrinen) Zellen oder Enterozyten 3. Das ISC ist an der Basis der Krypta. Panethzellen nach unten auf den Boden des Krypten und sind langlebig, während andere Linien nach oben wandern, um den Darmzotten 3,4. Hier werden die Zellen in den Darm Inhalte einschließlich Mikroflora ausgesetzt und werden von den villus Tipps durch eine anoikis-induzierte Apoptose-Mechanismus zu vergießen. Obwohl die Doppelpunkt fehlt Zotten und Panethzellen, ist der Mechanismus für die Aufrechterhaltung der Homöostase ähnlich 4. Der Wnt-Signalweg ist in eine entscheidende Rolle in der intestinalen Proliferation und ISC Wartung 4 gebracht worden. Löschung von the Transkriptionsfaktor TCF4, ein nachgeschalteter Effektor der Wnt-Signalweg, führt zum Verlust der intestinalen Stammzellen und die anschließende Aufteilung des Gewebes 5. Ebenso reduziert transgene Expression der Wnt-Inhibitor DKK1 die epithelialen Proliferation und erschöpft sekretorischen Zelllinien 6. Umgekehrt induziert die Überexpression des Wnt-Agonisten R-spondin-1 potente und rasche Verbreitung der intestinalen Kryptenzellen 7. Angesichts der Bedeutung der Wnt-Signalweg für intestinale Homöostase, sind Wnt-Signalweg Mutationen häufig in Darmkrebs 8 beobachtet. Darmkrebs ist die dritthäufigste Todesursache durch Krebs in den Vereinigten Staaten 9. Excess Nahrungsaufnahme mit rotem Fleisch und Alkohol, Bewegungsmangel und geerbt und somatische Mutationen gelten als Risikofaktoren für Darmkrebs 10,11 sein. Die adenomatöse Polyposis coli (APC)-Gen, ein Schlüssel Wnt-Faktor ist in der Mehrzahl der pa mutiertenPatienten mit familiärer, sporadisch, und Colitis-assoziierten Darmkrebs 12,13. Mutationen von anderen Faktoren in Wnt-Signalweg einschließlich Axin2 und β-Catenin beteiligt sind auch bei Darmkrebs 14,15 beobachtet. Jedoch ist die genaue Mechanismus und wirksame Therapie für Darmkrebs noch aus. Um die Untersuchung der molekularen Mechanismen für Darmkrebs zu erleichtern, haben menschliche Darmkrebs-Zelllinien, die verschiedene Stufen der Tumorprogression von einer gutartigen zu einer aggressiven Zelltyp wurden 16-18 etabliert. Maus Kolonkarzinomzelllinien mit verschiedenen metastasierenden Eigenschaften sind auch 19,20. Dennoch sind Primärzellen oder organoide Kulturen über transformierten Zelllinien bevorzugt, weil sie genau imitieren die in vivo-Zustand und erzeugen mehr physiologisch relevanten Daten 21. Die meisten Dickdarm-Krebs-Zelllinien wachsen als Monolayer an der Platte befestigt oder als Zellsuspensionen lacking apikalen basolateralen Orientierung und Tight Junctions zwischen den Zellen. Außerdem unterziehen normalen und Tumor-intestinalen Epithelzellen in vivo eine spontane Form der Apoptose bezeichnet anoikis wie die differenzierten Zellen die villus Tipps erreichen und Schuppen 22. Diese Funktionen sind nur schwer in Zelllinien rekapitulieren, sind aber in den Entwicklungsprozess von Darmkrebs 23 wichtig. Diese Funktionen werden in primäre Organoiden gepflegt. Darüber hinaus bieten die Tumorzellen organoiden Kulturen ein wirksames System zur Tumor autonomen Funktion der Gene im Vergleich zu in-vivo-Studien zu beurteilen. Genetische Manipulation in vivo des Darms ist ein zeitaufwendiger Prozess vor allem durch die Schaffung von transgenen und / oder Knockout-Mäusen mit Darm-spezifischen Treibern. Allerdings sind die Tumor Organoiden leicht zugänglich, um virale vermittelte genetische Manipulationen und damit ein großes Werkzeug für die Beurteilung der genauen molekularen Mechanismen. Primäre Darmtumor organoide Kulturen have demonstriert worden, um eine praktikable und leistungsfähige Technik sein. Primäre Darm Zellkultur funktionelle Darm Organoiden mit Krypta-Darmzotten Struktur in vitro herzustellen aus einer einzigen Erwachsenen Lgr5 + Stammzellen 24. Diese Organoiden können transplantiert werden und eingepflanzt in ein beschädigtes Gewebe zur Regeneration Doppelpunkt 25. Weitere Anpassung der Kulturbedingungen hatte ähnliche epithelialen Organoiden von Maus Doppelpunkt und menschlichen Dünndarm und Dickdarm möglich 1 gemacht. Für primären normalen Kolonepithel Kultur, basale Kulturmedium sowie Wachstumsfaktoren, einschließlich EGF, Noggin, R-und spondin Wnt3a sind unerlässlich, während basale Kulturmedium und EGF ist ausreichend für wachsende primäre Maustaste Kolontumor Organoiden 1. Hier beschreiben wir ein ausführliches Protokoll zu isolieren, Kultur und erzeugen Kolontumor Organoiden.

<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

Mehrere Menschen und der Maus Darmkrebs-Zelllinien etabliert haben, sind physiologische Integrität Kolontumoren wie mehrere Zellschichten, Basal-apikale Polarität, die Fähigkeit, zu differenzieren und nicht in anoikis Darmkrebs abgeleiteten Zelllinien erhalten. Die vorliegende Studie zeigt, ein Verfahren zur Kultivierung von primären Maus Kolontumors organoids von Sato T et al. 1, die wichtige physiologische Funktionen Kolontumoren behält angepasst. Dieses Verfahren besteht aus Maus Colontumorgewebe Sammlung, neben normalen Kolonepithel Dissoziation Kolontumorzellen Verdauung in einzelne Zellen, die Einbettung Kolontumorzellen in Matrigel und selektive Kultur beruht auf dem Grundsatz, dass Tumorzellen das Wachstum auf limitierende Nährstoff Bedingungen gewährleisten im Vergleich zu normalen Epithelzellen. Der Primärtumor Organoiden wenn aus genetisch veränderten Mäusen isoliert bieten einen sehr nützlichen System Tumor autonomen Funkt beurteilenIonen bestimmter Gene. Darüber hinaus sind die Tumor organoids zugänglich genetische Manipulation von Virus nachgedacht Gentransfer; daher Signalwege im Dickdarm Tumorentstehung beteiligt könnte auch weitgehend durch Überexpression oder Knockdown untersucht werden. Primärtumor Organoiden Kultur bietet eine physiologische relevant und machbar Mittel, um die Mechanismen und therapeutischen Modalitäten für Doppelpunkt Tumorentstehung zu untersuchen.

Der zeitliche Verlauf eines Kolontumor organoide Bildung von einem drei Monate alten ApcMin / + Maus ist in Abbildung 1 dargestellt. Am Tag 0, könnten einzelne Zellen mehrere Stunden nach der Beschichtung (Abbildung 1A) beobachtet werden. Am Tag 1, überlebten Kolontumors Epithelzellen mit feuerfesten Kerne beobachtet werden. Am Tag 3, verdoppelte sich die Größe der Zellen. Am Tag 6, erweitert die Größe der organoide mehr als das Zehnfache und zeigte Anzeichen v...

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

Eine einfache Methode zur primären murinen Kolontumor organoide etablieren beschrieben. Dieses Verfahren nutzt die Eigenschaft, dass Kolontumorzellen überleben und wachsen in Organoiden in Medien, die begrenzte Wachstumsfaktoren, während normale Kolonepithelzellen nicht tun.

In-vitro-Kultur von Organoide Primäre Maus Kolontumoren

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

in-vitro-organoid-culture-of-primary-mouse-colon-tumors

Research

JoVE Journal

Medicine

In vitro Organoid Culture of Primary Mouse Colon Tumors

34.9K Aufrufe.  University of Michigan. Eine einfache Methode zur primären murinen Kolontumor organoide etablieren beschrieben. Dieses Verfahren nutzt die Eigenschaft, dass Kolontumorzellen überleben und wachsen in Organoiden in Medien, die begrenzte Wachstumsfaktoren, während normale Kolonepithelzellen nicht tun.

Serie: 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.

Aufbau und Verbreitung der Menschenrechte Retinoblastoma Tumore in immundefizienten Mäuse

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

Forschung

Medizin

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.

Haut Stanzbiopsie Explantatkultur für die Ableitung des primären humanen Fibroblasten

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.

Primäre Kultur der Menschen vestibuläre Schwannome

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-Ansprechen auf die Behandlung von Primärtumoren und / oder assoziierten Metastasen für die präklinische und klinische Entwicklung von Therapeutika

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-Funktionelle Charakterisierung von Maus Colorectal Afferente 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.

Clinicopathological Analyse von miRNA Expression in Mammakarzinomen von miRNA Verwendung<em&gt; In Situ</em&gt; Die Hybridisierung

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.

Isolierung und Kultur von primären Endothelzellen von Canine Arterien und der

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-induzierte Quellung in Intestinale Organoide: Ein<em&gt; In Vitro</em&gt; Assay für Fibrosis Patienten in Cystic Drug Response-Beurteilung

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.

Muskel-Ungleichgewichte: Testen und trainieren funktionale exzentrische Muskelfaserriss Stärke in Athletic Populationen

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.

Vor-Ort-Probenahme und Extraktion von Hirntumoren für die Metabolomik- und Lipidomik-Analyse

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