תאים משורת תאי כונדרוסרקומה SW1353 נמסרו באדיבותו של פרופ &#39;ד&quot;ר PCW Hogendoorn ופרופ&#39; ד&quot;ר י בובי של אוניברסיטת ליידן, הולנד. אנו מודים לג&#39; וג&#39; Mestach Wagemans לקבלת סיוע טכני מעולה, וג&#39; דה Bruyne לציור המקצועי של הסקירה של הפרוטוקול שלנו.

<ol>
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S., Barretina, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+sarcoma+genomics+and+new+therpeutic+targets.">Advances in sarcoma genomics and new therpeutic targets.</a> Nature Reviews Cancer. 11, 541-557 (2011).</li><li>Skubitz, K. M., D'Adamo, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sarcoma.">Sarcoma.</a> Mayo Clinic Proceedings. 82, 1409-1432 (2007).</li><li>Nielsen, T. O., West, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translating+gene+expression+into+clinical+cara:+sarcomas+as+a+paradigm.">Translating gene expression into clinical cara: sarcomas as a paradigm.</a> Journal of Clinical Oncology. 28, 1796-1805 (2010).</li><li>Vaira, V., Fedele, G., Pyne, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+model+of+organotypic+culture+for+pharmacodynamic+profiling+of+human+tumors.">Preclinical model of organotypic culture for pharmacodynamic profiling of human tumors.</a> Proceedings of the National Academy of Science USA. 107, 8352-8356 (2010).</li><li>DeRose, Y. S., Wang, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+grafts+derived+from+women+with+breast+cancer+authentically+reflect+tumor+pathology,+growth,+metastasis+and+disease+outcomes.">Tumor grafts derived from women with breast cancer authentically reflect tumor pathology, growth, metastasis and disease outcomes.</a> Nature Medicine. 17, 1514-1520 (2011).</li><li>Tentler, J. J., Tan, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+tumour+xenografts+as+models+for+oncology+drug+development.">Patient-derived tumour xenografts as models for oncology drug development.</a> Nature Reviews Clinical Oncology. 9, 338-350 (2012).</li><li>Bertotti, A., Migliardi, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecularly+annotated+platform+of+patient-derived+xenografts+("xenopatients")+identifies+HER2+as+an+effective+therapeutic+target+in+cetuximab-resistant+colorectal+cancer.">A molecularly annotated platform of patient-derived xenografts ("xenopatients") identifies HER2 as an effective therapeutic target in cetuximab-resistant colorectal cancer.</a> Cancer Discovery. 1, 508-523 (2011).</li><li>Decaudin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+human+tumor+xenografted+models+('tumorgrafts')+for+good+management+of+patients+with+cancer.">Primary human tumor xenografted models ('tumorgrafts') for good management of patients with cancer.</a> Anticancer Drugs. 22, 827-841 (2011).</li><li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+cancer:+The+next+generation.">Hallmarks of cancer: The next generation.</a> Cell. 144, 646-674 (2011).</li><li>Sys, G., Van Bockstal, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+grafts+derived+from+sarcoma+patients+tumor+morphology,+viability,+and+invasion+potential+and+indicate+disease+outcomes+in+the+chick+chorioallantoic+membrane+model.">Tumor grafts derived from sarcoma patients tumor morphology, viability, and invasion potential and indicate disease outcomes in the chick chorioallantoic membrane model.</a> Cancer Letters. 326, 69-78 (2012).</li><li>Armstrong, P. B., Quigley, J. P., Sidebottom, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transepithelial+invasion+and+intramesenchymal+infiltration+of+the+chick+embryo+chorioallantois+by+tumor+cell+lines.">Transepithelial invasion and intramesenchymal infiltration of the chick embryo chorioallantois by tumor cell lines.</a> Cancer Research. 42, 1826-1837 (1982).</li><li>Deryugina, E., Quigly, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chick+embryo+chorioallantoic+membrane+model+systems+to+study+and+visualize+human+tumor+cell+metastasis.">Chick embryo chorioallantoic membrane model systems to study and visualize human tumor cell metastasis.</a> Histochemistry and Cell Biology. 130, 1119-1130 (2008).</li><li>Knighton, D., Ausprunk, D., Tapper, D., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avascular+and+vascular+phases+of+tumor+growth+in+the+chick+embryo.">Avascular and vascular phases of tumor growth in the chick embryo.</a> British Journal of Cancer. 35, 347-356 (1977).</li><li>Dohle, D. S., Pasa, S. D., Gustmann, S., Laub, M., Wissler, J. H., Jennissen, H. P., D&#252;nker, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chick+ex+ovo+culture+and+ex+ovo+CAM+assay:+how+it+really+works.">Chick ex ovo culture and ex ovo CAM assay: how it really works.</a> J. Vis. Exp. (33), e1620 (2009).</li><li>Balke, M., Neumann, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphologic+characterization+of+osteosarcoma+growth+on+the+chick+chorioallantoic+membrane.">Morphologic characterization of osteosarcoma growth on the chick chorioallantoic membrane.</a> BMC Research Notes. 3, 58 (2010).</li><li>Hendrix, A., Maynard, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+the+secretory+small+GTPase+Rab27B+on+breast+cancer+growth,+invasion,+and+metastasis.">Effect of the secretory small GTPase Rab27B on breast cancer growth, invasion, and metastasis.</a> Journal of the National Cancer Institute. 102, 866-880 (2010).</li><li>Ausprunk, D., Knighton, D., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularization+of+normal+and+neoplastic+tissues+grafted+to+the+chick+chorioallantois:+role+of+host+and+preexisting+graft+vessels.">Vascularization of normal and neoplastic tissues grafted to the chick chorioallantois: role of host and preexisting graft vessels.</a> American Journal of Pathology. 79, 597-618 (1975).</li><li>Lokman, N. A., Elder, A. S. F., Riciardelli, C., Oehler, M. 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יש מחברים אין לחשוף.

זמן של חיסון וקציר תזמון היום של חיסון בוצע באמצעות SAOS2 בECM ג&#39;ל (36 מצלמות) ונע בין יום התפתחות העוברי 5 ו -10. לפני הדגירה יום 9, CAM לא באופן עקבי היה גדול מספיק כדי לתמוך ג&#39;ל ECM אנחנו מוחלים. בקציר, ...

סרקומה היא גידול נדיר של רקמות החיבור עם תמותה גבוהה בשל התנגדות 1,2 הטיפול. התקדמות בהישרדות חולה הקשתה על ידי שכיחותם הנמוכה השנתית, הגיוון הרחב שלהם, ואת העובדה שתאי סרקומה הם דיווחו להיות קשים לתרבות במבחנה 3,4. השימוש בתאים בתרבית להערכת טיפול קליני גילו כי מולקולות חדשות, ככל הנראה פעילה במבחנה לא תמיד משקפות את התוצאות בסביבה הקלינית. יתר על כן, סטיות הגנום מתגלים על ידי מערכי ביטוי גנים הן לא תמיד קשורות למאפייני התנהגות סרטניים בחולה בודדת 5-7. על מנת לנסות ולפתור את הבעיות הללו, רפואה מותאמת אישית צברה בחשיבותו, אשר באה לידי ביטוי בחיפוש המוגבר לדגמי xenograft 8-12. ב assay vivo יש את היתרון של המשקף הגומלין המורכב בין גתאי ancer וסביבת רקמת המארח בגידולים מוצקים, הנדרשים להתפשטות הסרטן והפלישה 13. נכון לעכשיו אנחנו לומדים את השימוש של assay ממברנה Chorio-allantoic (CAM-assay) כמודל לשחזור xenograft סרקומה 14,15. assay זה נעשה שימוש נרחב למחקר של אנגיוגנזה גידול 16,17. בספרות, לעומת זאת, מצאנו פרוטוקולים שונים עבור assay זה, בעוד שמחקרים אחרים שנצפו הבדל משמעותי בצמיחה או אנגיוגנזה על פי פרוטוקולים שונים 18,19. במאמר זה אנו לחקור את ההשפעה של משתנה תנאים של CAM-assay על התנהגות תא סרטניים, באמצעות שתלי השעיות תא בודדות שמקורם בגידול בתרביות תאי סרקומה ומבוססות.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent</td>
			<td>Company</td>
			<td>Catalog Number</td>
		</tr>
		<tr>
			<td>Cell Line Nucleofector Kit V</td>
			<td>Amaxa</td>
			<td>VCA-1003</td>
		</tr>
		<tr>
			<td>collagenase 2 solution (500 U/ml RPMI 1640)</td>
			<td>Sigma Aldrich</td>
			<td>C6885</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>41965-039</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D8418</td>
		</tr>
		<tr>
			<td>Dnase solution</td>
			<td>Sigma Aldrich</td>
			<td>DN25</td>
		</tr>
		<tr>
			<td>G418</td>
			<td>Invitrogen</td>
			<td>11811031</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Sigma-Aldrich</td>
			<td>E1270</td>
		</tr>
		<tr>
			<td>mouse primary monoclonal antibody Ki67</td>
			<td>Dako Denmark</td>
			<td>MIB-1</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Fluka</td>
			<td>D76240</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Invitrogen</td>
			<td>20012019</td>
		</tr>
		<tr>
			<td>PBSD</td>
			<td>Invitrogen</td>
			<td>14040083</td>
		</tr>
		<tr>
			<td>peGFP-C1 vector</td>
			<td>Clontech</td>
			<td>632470</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Invitrogen</td>
			<td>15140163</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Invitrogen</td>
			<td>22409-015</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Invitrogen</td>
			<td>25300054</td>
		</tr>
		<tr>
			<td>Vybrant cell-labeling DiI</td>
			<td>Lifetechnologies</td>
			<td>22885</td>
		</tr>
		<tr>
			<td>Name of Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
		</tr>
		<tr>
			<td>Countess Automated Cell Counter</td>
			<td>Invitrogen</td>
			<td>C10227</td>
		</tr>
		<tr>
			<td>digital color camera</td>
			<td>Leica</td>
			<td>DFC 340 FX</td>
		</tr>
		<tr>
			<td>Digital Egg Incubator</td>
			<td>Auto Elex Co</td>
			<td>R-COM 50</td>
		</tr>
		<tr>
			<td>FACS</td>
			<td>BD Biosciences</td>
			<td>FACSAriaIII</td>
		</tr>
		<tr>
			<td>Gentlemacs C-Tube</td>
			<td>Miltenyi Biotech</td>
			<td>130-093-237</td>
		</tr>
		<tr>
			<td>Gentlemacs Dissociator</td>
			<td>Miltenyi Biotech</td>
			<td>130-093-235</td>
		</tr>
		<tr>
			<td>Gentlemacs Dissociator User Manual containing h_tumor protocol</td>
			<td>Miltenyi Biotech</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>semipermeable adhesive film (Suprasorb F)</td>
			<td>Lohmann&amp;Rauscher</td>
			<td>20468</td>
		</tr>
		<tr>
			<td>stereo fluorescence microscope</td>
			<td>Leica</td>
			<td>M205 FA</td>
		</tr>
		<tr>
			<td>Tissue-Tek Film automated Coverslipper</td>
			<td>Sakura</td>
			<td>6400</td>
		</tr>
		<tr>
			<td>ultraView Universal DAB Detection Kit</td>
			<td>Ventana Medical Systems Inc</td>
			<td>760-500</td>
		</tr>
		<tr>
			<td>Ventana Automated Slide Stainer</td>
			<td>Ventana Medical Systems</td>
			<td>Benchmark XT</td>
		</tr>
	</tbody>
</table>

the in ovo cam-assay as a xenograft model for sarcoma

סרקומה היא מחלה מאוד נדירה שהיא הטרוגנית בטבע, כל פוגעות פיתוח הטיפולים חדשים. חולי סרקומה הם מועמדים אידיאליים לרפואה אישית לאחר ריבוד, המסבירים את הריבית הנוכחית בפיתוח מודל xenotransplant לשעתק ועלות נמוכה למחלה זו. קרום chorioallantoic החומוס הוא מארח immunodeficient טבעי מסוגלים לקיים רקמות ותאים מורכבים ללא הגבלות מינים ספציפיות. בנוסף, ניתן לגשת בקלות, מניפולציות וצלם באמצעות stereomicroscopy אופטי וקרינה. היסטולוגיה נוספת מאפשרת ניתוח מפורט של אינטראקציות הסלולר heterotypic. פרוטוקול זה מתאר בפירוט באובו השתלה של קרום chorioallantoic עם רקמות טריות סרקומה הנגזרות סרטניים, השעיות התא בודדות שלהם, וקווים קבועים וארעיים שכותרתו fluorescently הוקמו סרקומה סלולרי (Saos-2 וSW1353). עכברוש הישרדות החומוסes הוא עד 75%. המודל משמש ללמוד שתל (כדאיות, Ki67 מדד התפשטות, נמק, חדירה) ומארח (חדירה פיברובלסטים, ingrowth כלי דם) התנהגות. להשתלה מקומית של השעיות תא בודדות, ECM ג&#39;ל מספק יתרונות משמעותיים על פני חומרי בלימה אינרטי. מדד ההתפשטות Ki67 קשור למרחק של התאים מפני שטח של CAM ואת משך הזמן של יישום ברמ&quot;א, שהאחרון קביעת מסגרת זמן לתוספת של מוצרים טיפוליים.

הערכה של רמ&quot;א שתלי גידול הפכו חסיד לרמ&quot;א (איור 2 א). השעיות תא בודדות מחומר שהמטופל להציג שלט מיובש, מוגבה מעט (איור 2 ד) לעתים קרובות. לאחר הכריתה של רמ&quot;א, סימן קימוט של הקרום מתרחש (איורים 2E ו2F). <p clas...

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The In ovo CAM-assay as a Xenograft Model for Sarcoma

<em&gt; באוב</em&gt; Chorioallantoic קרום (רמ&quot;א) הוא מורכב עם רקמות טריות סרקומה הנגזרות סרטניים, השעיות התא בודדות שלהם, ושורות תאי סרקומה הוקמו כותרתו fluorescently קבועות וארעיות. המודל משמש ללמוד שתל (כדאיות, Ki67 מדד התפשטות, נמק, חדירה) ומארח (חדירה פיברובלסטים, ingrowth כלי דם) התנהגות.

<em&gt; באוב</em&gt; CAM-assay כXenograftmodel לסרקומה

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

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The In ovo CAM-assay as a Xenograft Model for Sarcoma

25.2K Views.  Ghent University Hospital.  באוב Chorioallantoic קרום (רמ"א) הוא מורכב עם רקמות טריות סרקומה הנגזרות סרטניים, השעיות התא בודדות שלהם, ושורות תאי סרקומה הוקמו כותרתו fluorescently קבועות וארעיות. המודל משמש ללמוד שתל (כדאיות, Ki67 מדד התפשטות, נמק, חדירה) ומארח (חדירה פיברובלסטים, ingrowth כלי דם) התנה?...

Video: The In ovo CAM-assay as a Xenograft Model for Sarcoma

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

אסטרטגיה לזהות מוטציות דה נובו בהפרעות נפוצות כגון אוטיזם וסכיזופרניה

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma (glioma) and medulloblastoma. The widely used stereotactic approach only allows for the injection of a single animal at a time, is labor intensive and requires highly specialized equipment. The guide screw method, initially developed by Lal et al.,1 was developed to eliminate cumbersome stereotactic procedures. We now describe a modified guide screw approach that is rapid and exceptionally safe; both of which are critical ethical considerations. Notably, our procedure now incorporates an infusion pump that allows up to 10 animals to be simultaneously injected with tumor cells.
To demonstrate the utility of this procedure, we established human U87MG glioma cells as intracranial xenografts in mice, which were then treated with AMG102; a fully human antibody directed to HGF/scatter factor currently undergoing clinical evaluation2-5. Systemic injection of AMG102 significantly prolonged the survival of all mice with intracranial U87MG xenografts and resulted in a number of complete cures. 
This study demonstrates that the guide screw method is an inexpensive, highly reproducible approach for establishing intracranial xenografts. Furthermore, it provides a relevant physiological model for validating novel therapeutic strategies for the treatment of brain cancers.

In order to evaluate novel therapeutic paradigms for the treatment of glioma, physiological relevant models are essential. We utilize an implantable guide screw procedure for establishment of intracranial xenograft models that is more rapid and safer than stereotactic approaches.

מדריך פשוט בורג שיטה ללימודי xenograft תוך גולגולתי של עכברים

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

האדם תאים סרטניים נוירואנדוקריניים קווים כמו מודל תלת ממדי לחקר הטיפול האנושי גידול נוירואנדוקריניים

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

הפעלת יתר רעיל גירוי של המוח כMicroslices<em&gt; במבחנה</em&gt; דגם של שבץ

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina

Neuroprotection has been a strong field of investigation in ophthalmological research in the past decades and affects diseases such as glaucoma, retinal vascular occlusion, retinal detachment, and diabetic retinopathy. It was the object of this study to introduce a standardized stress model for future preclinical therapeutic testing.
Bovine retinas were prepared and perfused with an oxygen saturated standard solution, and the ERG was recorded. After recording stable b-waves, hypoxia (pure N2) or glutamate stress (250 &#181;m glutamate) was exerted for 45 min. To investigate the effects on photoreceptor function alone, 1 mM aspartate was added to obtain a-waves. ERG-recovery was monitored for 75 min.
For hypoxia, a decrease in a-wave amplitude of 87.0% was noted (p &#60;0.01) after an exposition time of 45 min (decrease of 36.5% after the end of the washout p = 0.03). Additionally, an initial decrease in b-wave amplitudes of 87.23% was recorded, that reached statistical significance (p &#60;0.01, decrease of 25.5% at the end of the washout, p = 0.03).
For 250 &#181;m glutamate, an initial 7.8% reduction of a-wave amplitudes (p &#62;0.05) followed by a reduction of 1.9% (p &#62;0.05). A reduction of 83.7% of b-wave amplitudes (p &#60;0.01) was noted; after a washout of 75 min the reduction was 2.3% (p = 0.62). In this study, a standardized stress model is presented that may be useful to identify possible neuroprotective effects in the future.

With this study, we introduce a standardized stress model for the isolated superfused bovine retina for future preclinical therapeutic testing. The effect of either hypoxia (pure N2) or glutamate stress (250 &#181;M glutamate) on retinal function represented by a- and b-wave amplitudes was evaluated.

גלוטמט והיפוקסיה כמודל מתח לperfused חוליות הרשתית מבודדת

Neuroprotection has been a strong field of investigation in ophthalmological research in the past decades and affects diseases such as glaucoma, retinal ...

The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and highly vascularized, making it an ideal system for tumor implantation. Furthermore, the CAM contains extracellular matrix proteins such as fibronectin, laminin, collagen, integrin alpha(v)beta3, and MMP-2, making it an attractive model to study tumor invasion and metastasis. Scientists have long taken advantage of the physiology of the CAM by using it as a model of angiogenesis. More recently, the CAM assay has been modified to work as an in vivo xenograft model system for various cancers that bridges the gap between basic in vitro work and more complex animal cancer models. The CAM assay allows for the study of tumor growth, anti-tumor therapies, and pro-tumor molecular pathways in a biologically relevant system that is both cost- and time-effective. Here, we describe the development of CAM xenograft model of hepatocellular carcinoma (HCC) with embryonic survival rates of up to 93% and reliable tumor take leading to growth of three-dimensional, vascularized tumors.

The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay.

<em&gt; באוב</emAssay&gt; אפרוח chorioallantoic ממברנה (CAM) כמודל xenograft יעיל של Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

הקמת מודל xenograft מיאלומה נפוצה אדם בעוף לחקר צמיחת גידול, פלישה ואנגיוגנזה

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

Oleic Acid-Injection in Pigs As a Model for Acute Respiratory Distress Syndrome

The acute respiratory distress syndrome is a relevant intensive care disease with an incidence ranging between 2.2% and 19% of intensive care unit patients. Despite treatment advances over the last decades, ARDS patients still suffer mortality rates between 35 and 40%. There is still a need for further research to improve the outcome of patients suffering from ARDS. One problem is that no single animal model can mimic the complex pathomechanism of the acute respiratory distress syndrome, but several models exist to study different parts of it. Oleic acid injection (OAI)-induced lung injury is a well-established model for studying ventilation strategies, lung mechanics and ventilation/perfusion distribution in animals. OAI leads to severely impaired gas exchange, deterioration of lung mechanics and disruption of the alveolo-capillary barrier. The disadvantage of this model is the controversial mechanistic relevance of this model and the necessity for central venous access, which is challenging especially in smaller animal models. In summary, OAI-induced lung injury leads to reproducible results in small and large animals and hence represents a well-suited model for studying ARDS. Nevertheless, further research is necessary to find a model that mimics all parts of ARDS and lacks the problems associated with the different models existing today.

In this article, we present a protocol to induce acute lung injury in pigs by central-venous injection of oleic acid. This is an established animal model for studying the acute respiratory distress syndrome (ARDS).

הזרקת חומצה אולאית חזירים כמודל עבור תסמונת מצוקה נשימתית חריפה

The acute respiratory distress syndrome is a relevant intensive care disease with an incidence ranging between 2.2% and 19% of intensive care unit ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Contractility איזומטרי מדידה של עורק מצע המעי העליון העכבר באמצעות חוט Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

אפיתל שחיקה הקרנית עם עינית Burr כמודל עבור קרנית ריפוי פצעים

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...