A.S. was partially supported by grants from the National Institutes of Health (NIH) National Institute of Dental and Craniofacial Research (5R03DE021741-02) and the National Cancer Institute (1K08CA154963-01A1).&#160;
The Howard Hughes Medical Institute provided funding for M.L. through the HHMI Medical Research Fellows Program.

דגירה 1. ביצה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להשיג ביצי 8 יום ישנים ספציפיות הפתוגן ללא embryonated. </li><li style=";text-align:right;direction:rtl"> מניחים את הביצים במגש מסתובב ביצה, קצות חותמת יפנו כלפי מעלה, ומניח את המגש מסתובב בתוך חממת ביצה. דגירה ביצים במשך 48 שעות ב 36 מעלות צלזיוס ולחות של 50%. </li></ol> 2. הפלת CAM ופתיחת הביצים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לאסוף ביצים מחממה. ביצי מקום במדף ביצה, חותמת הצד, ולהביא לזרימה למינרית. </li><li style=";text-align:right;direction:rtl"> כבה את האורות בחדר ומכסה מנוע. </li><li style=";text-align:right;direction:rtl"> החזק את הקצה חותם של ביצה קלה לקנדלר הביצה כדי לחשוף את כלי הדם של CAM, כמו גם את שק האוויר. הערה: קנדלר הביצה הוא מקור אור המציג את העובר המתפתח וצק האוויר קשור וכלי הדם. </li><li style=";text-align:right;direction:rtl"> מציב סימן עיפרון בין שני כלי דם גדולים. </li><li style=";text-align:right;direction:rtl"> להדליק את האורות ולהשתמש סיכה לדחוף סטרילי לעשות חור אחד בקצה הביצה מעל (סוף החותמת) שק האוויר וחור אחד בPEסימן ncil. לא לדחוף הסיכה לדחוף לאורך כל הדרך; חור בסביבות 3 מ&quot;מ עמוק בדרך כלל יספיק. </li><li style=";text-align:right;direction:rtl"> לכבות את האורות. לסחוט הנורה בטיחות, לחיצה על הקצה הפתוח בתקיפות נגד החור מעל שק האוויר. החל יניקה לשאוף אוויר אל תוך החור בסימן העיפרון, גורם CAM לרדת מהפגז בסימן העיפרון. </li><li style=";text-align:right;direction:rtl"> בדוק שצק האוויר עבר מהקצה החותמת של הביצה לחור מסומן העיפרון באמצעות קנדלר. אם לא, השתמש בסיכת הדחיפה לעשות שני החורים מעט עמוקים יותר ולאחר מכן להירשם מחדש באמצעות שאיבת הנורה הבטיחות. </li><li style=";text-align:right;direction:rtl"> הדלק את האור. מחזיק את הביצה ביד אחת, להפעיל את הכלי הסיבובי DREMEL עם חתך גלגל 15/16 אינץ &#39;המצורף ולעשות שני חתכים רוחביים רק עמוק מספיק כדי לחתוך דרך הקליפה אבל לא עמוק מספיק כדי לחתוך את כל הדרך עד לדיכאון CAM. להפוך את החתכים סביב 2 סנטימטר באורך 1 סנטימטר ומלבד אחד את השני. הפוך את הקיצוצים רחבים מספיק כך שטבעת סיליקון 1 סנטימטר קוטר יכול לעבור דרךאבל רזה יותר רוחב הסרט דביק לסטנדרטי. </li><li style=";text-align:right;direction:rtl"> בשלב הבא, לעשות חתך אורכי 1 בין ובקצוות של שני חתכים רוחביים בקלילות הנגיעה הגלגל החתוך לקליפה. </li><li style=";text-align:right;direction:rtl"> Slide מלקחיים סטריליות תחת חתיכת קליפה ובמקביל לקליפה. תפוס את חתיכת הקליפה עם המלקחיים ולהסיר את כל החתיכה נקיה. </li><li style=";text-align:right;direction:rtl"> מניחים נייר דבק מעל החלון החדש שנפתח, והקפד לקפל קצה אחד מעל בעצמו על מנת להקל על ההסרה. </li><li style=";text-align:right;direction:rtl"> ביצי מקום בחזרה בחממה במגש הביצה (לא במדפי הביצה מסתובבים) עד הזמן של חיסון. </li></ol> 3. הרכבת חיסון <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> שמור תערובת חלבוני קרום במרתף, כגון Matrigel, על קרח (כדי למנוע פילמור) ומקום במכסת מנוע. </li><li style=";text-align:right;direction:rtl"> השתמש 2 מ&quot;מ EDTA לנתק תאים (4 - 5 מיליליטר לכל בקבוק T75) וצלוחיות מקום בתרבית רקמת חממה במשך 5 דקות. </li><li style=";text-align:right;direction:rtl"> Resuspend התאים המנותק בתקשורת ולספור באמצעות hemocytometer וtrypan כחול. השתמש בין 500,000 to 2,000,000 תאים להשתלה על CAM, בהתאם לקו תא. לייעל את המספר הנכון בניסויים ראשוניים. </li><li style=";text-align:right;direction:rtl"> מדד הנדרש למספר תא המתאים וצנטריפוגות ב XG 500 במשך 5 דקות נפח. </li><li style=";text-align:right;direction:rtl"> בטל supernatant ואז resuspend תא גלולה ב 40 μ l של PBS ++ ו- 20 μ l של תערובת קרום במרתף. הערה: בשלב זה, ניתן לטפל בתאים עם סמים או סוכני מבחן אחרים כנדרש לניסויים על ידי resuspending להשעית תערובת קרום תא / מרתף. לחלופין, ניתן pipetted סוכנים על ההשעיה תערובת קרום תא / מרתף לאחר השתלה. </li><li style=";text-align:right;direction:rtl"> אחזר מדף ביצה עם ביצים מן החממה והמקום בזרימה למינרית. </li><li style=";text-align:right;direction:rtl"> קלטת לקלף את החלון, לשלוף טבעת סיליקון 1 מ&quot;מ מהכובע של בקבוקון קריוגני באמצעות מלקחיים מעוקרים, ושחרר את הטבעת דרך החלון. </li><li style=";text-align:right;direction:rtl"> mixtur Pipet פתרון תא l 60 μ של PBS ++ וקרום במרתףדואר ישירות למרכז זירת סיליקון נח על CAM. </li><li style=";text-align:right;direction:rtl"> Re-קלטת החלון ולעבור מדף ביצה חזרה לחממה. בשלב זה, בזהירות בעת הזזת הביצים כטבעת והשעית התא יכול בקלות להיות שנעקר. </li><li style=";text-align:right;direction:rtl"> לאפשר גידולים לגדול במשך 5 עד 7 ימים. </li></ol> 4. גידולי קציר <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מניחים מתחת לכרית במכסת מנוע. הכן 35 x 10 מ&quot;מ צלחות ומכולות paraformaldehyde לפי צורך להיסטולוגיה. שים 1 מיליליטר PBS ++ בכל צלחת 35 מ&quot;מ x 10. </li><li style=";text-align:right;direction:rtl"> מניחים שקית Biohazard במכסת מנוע. קח מספריים לנתיחה סטרילית והכנס את הקצה המחודד לביצה בחור קרוב לסוף החותמת. </li><li style=";text-align:right;direction:rtl"> חותך סביב כל הביצה longitudinally. הערה: CAM והגידול עם הטבעת צריך לדבוק במחצית העליונה של הקליפה עם החלון. </li><li style=";text-align:right;direction:rtl"> בעזרת מספריים לנתיחה ומלקחיים, לנתח את הגידול מCAM ולמקם את הגידול בצלחת 35 מ&quot;מ x 10. למדוד ולשקול גידולים. גידולים קבועים בparaformaldehyדה ולאחר מכן פרפין-להטביע אותם לשימוש בניסויי היסטולוגיה ואימונוהיסטוכימיה. </li></ol> 5. (אופציונאלי): דיסוציאציה תאים סרטניים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> השתמש במספריים לנתיחה כדי לרכך את הגידול בצלחת 35 מ&quot;מ x 10. יוצקים גידול טחון לתוך צינור 15 מיליליטר צנטריפוגות ולהסיר כמה שיותר PBS ++ ככל האפשר באמצעות pipetting. </li><li style=";text-align:right;direction:rtl"> הוסף 2 מיליליטר של collagenase (1 מ&quot;ג / מיליליטר PBS ++), להפוך את הצינור על פני כמה פעמים, ודגירת צינור ב 37 מעלות צלזיוס במשך לפחות 30 דקות (עד כמה שעות). </li><li style=";text-align:right;direction:rtl"> הוסף 5 מיליליטר תקשורת על צינור אחד וresuspend ביסודיות על ידי pipetting למעלה ולמטה 20 - 40X. pipetting היסודי הוא קריטי לתא דיסוציאציה מלאה. </li><li style=";text-align:right;direction:rtl"> לאפשר משקעים להתיישב, ולהעביר את supernatant לצינור טרי. צנטריפוגה XG ב 500 במשך 5 דקות. הסר supernatant, משאיר מאחורי תא גלולה ניתק. הערה: בשלב זה, כדורי תא יכולים לשמש לניסויים שונים (cytometry זרימה, תמוגה לשימוש בסופג מערבי, בידוד RNA, וכו &#39;). ניתן להשיג ספירת תאי גידול כוללת באמצעות hemocytometer וצביעת trypan כחולה. שימו לב שתאים קטנים יותר, סגלגלים הם תאי עוף מCAM ולא צריך להיחשב כתאים סרטניים. לא צריך להיות מספר מינימאלי של זיהום תאים שאינם סרטניים (CAM) אם הגידול הוא גזור מCAM בזהירות. </li></ol>

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A.S.&#160;receives&#160;funding from the Advaxis Pharmaceutical company to support an investigator-initiated clinical trial.&#160;

כמה שלבים עיקריים בפרוטוקול זה סביר ביותר להסביר את ההישרדות העוברית המשופרת, כמו גם אמינות מוגברת של צמיחת גידול. הפלת CAM מהפגז על ידי יישום יניקה לצק האוויר היא פחות פולשני יותר משיטות קיימות אחרות (באמצעות מחט כדי להסיר אלבומין מהביצה, לנתיחה בוטה, וכו &#39;). באמצעות ...

קרצינומה hepatocellular (HCC) הוא הגורם המוביל 3 בתמותה מהסרטן בעולם 1. כיום רק 30% מחולי HCC זכאים לטיפולים כירורגיים פוטנציאל מרפא 2, וכימותרפיה מערכתית אינה יעילה 3. לכן, יש צורך דחוף שלא סופקו קליני לטיפולי HCC רומן, והפיתוח של מערכות מודל מתאימות לבדיקות יעילים של סוכנים חדשים. Assay חומוס chorioallantoic קרום (CAM) מספק שיטה לשחזור, חסכונית, מהירה ובינונית תפוקה של בדיקת תרופות אנטי-סרטניים פוטנציאליות in vivo. Assay CAM כבר נעשה שימוש נרחב ללמוד אנגיוגנזה 4. כמו כן פיתח בהצלחה למודל xenograft גידול של סרטן, כולל גליובלסטומה 5, סרטן לבלב 6, מלנומה 7-9, ואוסטאוסרקומה 10-11. שני באובו 12 ולשעבר אובו13 טכניקות כבר נוצלו בספרות, עם פרטים שונים מפרוטוקול לפרוטוקול. אחד אתגרים עיקריים למודל xenograft CAM הוא יחסית השכיחות הגבוהה של מוות עוברי לאחר המניפולציה של הביצה, עם שיעורי תמותה שפורסמו עובר אפרוח החל 25-50 אחוזים 11-14. במאמר זה, אנו מתארים את הפיתוח של מודל באוב xenograft של HCC שמייצר באופן מהימן צמיחה של גידולים תלת-ממדיים, כלי דם שהיסטולוגית דומה HCC מובחן. יש לנו מותאם פרוטוקול שתואר לראשונה על ידי Ossowski et al. 14 והשיגו שיעורי הישרדות חומוס עוברי של עד 93% בקליטתם גידול גבוה מאוד.

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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Premium incubated eggs</td>
			<td style="width:108px;">Charles River</td>
			<td style="width:119px;">N/A</td>
			<td>http://www.criver.com/files/pdfs/avian/av_c_spf_egg_price_list.aspx</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:401px;">Egg incubators</td>
			<td style="width:108px;">GQF</td>
			<td style="width:119px;">Hova-Bator 2362N</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:401px;">Rotating egg trays</td>
			<td style="width:108px;">GQF</td>
			<td style="width:119px;">1611 Automatic Egg Turner</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:401px;">Egg candler</td>
			<td style="width:108px;">Lyon</td>
			<td style="width:119px;">Hi-Power 950-070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Dremel 100 rotary tool with 15/16 cut-off wheel</td>
			<td style="width:108px;">Dremel</td>
			<td style="width:119px;">100-N/7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Sterile forceps, push pin, dissection scissors, Scotch tape</td>
			<td style="width:108px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Matrigel</td>
			<td style="width:108px;">BD Biosciences</td>
			<td style="width:119px;">356234</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Cryogenic vials, external thread with silicone washer</td>
			<td style="width:108px;">Corning</td>
			<td style="width:119px;">430659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Collagenase from Clostridium histolyticum</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:119px;">C9891</td>
			<td></td>
		</tr>
	</tbody>
</table>

the in ovo chick chorioallantoic membrane (cam) assay as an efficient xenograft model of hepatocellular carcinoma

קרום החומוס chorioallantoic (רמ&quot;א) מתחיל לפתח על ידי יום 7 לאחר הפריה ומתבגר ביום 12. CAM הוא באופן טבעי immunodeficient וכלי דם מאוד, מה שהופך את מערכת אידיאלית להשתלת גידול. יתר על כן, CAM מכיל חלבונים תאיים מטריצה ​​כגון פיברונקטין, laminin, קולגן, אלפא integrin (נ) beta3, וMMP-2, מה שהופך את מודל אטרקטיבי ללמוד פלישת גידול וגרור. מדענים רבים ניצלו את הפיסיולוגיה של CAM על ידי שימוש בו כמודל של אנגיוגנזה. לאחרונה, assay CAM שונתה כדי לעבוד כמערכת in vivo xenograft מודל לסוגי סרטן שונים שמגשרת על הפער בין עבודה בסיסית במבחנה ודגמי סרטן בבעלי החיים מורכבים יותר. Assay CAM מאפשר לחקר צמיחת גידול, טיפולים אנטי-סרטניים, ומסלולים מולקולריים פרו-סרטניים במערכת רלוונטית מבחינה ביולוגית שהוא גם עלות וזמן יעיל. כאן, אנו מתארים את ההתפתחות של Xen CAMמודל ograft של קרצינומה hepatocellular (HCC) עם שיעורי הישרדות עובריים של עד 93% וגידול אמין לקחת מוביל לצמיחה של גידולים תלת-ממדיים, כלי דם.

תמונות נציג של שלבים עיקריים בפרוטוקול מוצגות כאן. איור 1 א מדגים את השימוש של קנדלר לדמיין את העובר המתפתח, שק האוויר, וכלי הדם של CAM. איור 1-1C להראות את התהליך של נשירת CAM על ידי ביצוע שני חורים ולאחר מכן הפעלת לחץ שלילי באמצעות הנורה הבטיחות, ו1D א?...

Watch this Scientific Journal Video about The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma at JoVE.com

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 ...

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Research

JoVE Journal

Medicine

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

34.9K Views.  Icahn School of Medicine at Mount Sinai. 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.

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

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Multi-פוטון הדמיה של הפלישה תאים סרטניים במודל עכבר orthotopic של קרצינומה של תאים קשקשיים אוראלי

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer cells into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening and evaluation of new therapeutic treatments. There are a number of advantages to using a subcutaneous model; 1) rapid, 2) reproducible, and 3) tumor establishment, growth, and response to therapeutic agents may be monitored by visual inspection. However, substantial evidence has shed light on the short-comings of subcutaneous xenograft models1-3. For instance, medicinal treatments demonstrating curative properties in subcutaneous xenograft models often have no notable impact on the human disease. The microenvironment of the site of xenographic transplantation or injection lies at the heart of this dissimilarity.
Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease. The advantages of implanting diseased cells or tissue into their anatomical origin equivalent within a host animal includes a suitable site for tumor-host interactions, development of disease-related metastases and the ability to examine site-specific influence on investigational therapeutic remedies. Therefore, orthotopic xenograft models harbor far more clinical value because they closely reproduce human disease. For these reasons, a number of groups have taken advantage of an orthotopic thyroid cancer model as a research tool4-7.
Here, we describe an approach that establishes an orthotopic model for the study of anaplastic thyroid carcinoma (ATC), which is highly invasive, resists treatment, and is virtually fatal in all diagnosed patients. Cultured ATC cells are prepared as a dissociated cellular suspension in a solution containing a basement membrane matrix. A small volume is slowly injected into the right thyroid gland. Overall appearance and health of the mice are monitored to ensure minimal post-operative complications and to gauge pathological penetrance of the cancer. Mice are sacrificed at 4 weeks, and tissue is collected for histological analysis. Animals may be taken at later time-points to examine more advance progression of the disease. Production of this orthotopic mouse model establishes a platform that accomplishes two objectives: 1) further our understanding of ATC pathology, and 2) screen current and future therapeutic agents for efficacy in combating ATC.

Generation of an orthotopic mouse model of anaplastic thyroid carcinoma is described here. This technique employs surgical placement of human anaplastic thyroid cancer cells into the thyroid of immunodeficient mice, thus creating a more clinically relevant setting to study disease progression as well as screen innovative therapeutic interventions.

מודל עכבר orthotopic של קרצינומה של בלוטת התריס אנפלסטית

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer ...

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

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

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

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

<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 ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

דור של תת עורי וintrahepatic אדם xenografts קרצינומה hepatocellular בעכברים עם מערכת חיסון חלשים

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

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 ...

Confocal Time Lapse Imaging as an Efficient Method for the Cytocompatibility Evaluation of Dental Composites

It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The objective of this study was to use 3D CLSM time lapse confocal imaging to assess the in vitro biocompatibility of dental composites. This method provides an accurate and sensitive indication of viable cell rate in contact with dental composite extracts. The ELS extra low shrinkage, a dental composite used for direct restoration, has been taken as example. In vitro assessment was performed on cultured primary human gingival fibroblast cells using Live/Dead staining. Images were obtained with the FV10i confocal biological inverted system and analyzed with the FV10-ASW 3.1 Software. Image analysis showed a very slight cytotoxicity in the presence of the tested composite after 5 hours of time lapse. A slight decrease of cell viability was shown in contact with the tested composite extracts compared to control cells. The findings highlighted the use of 3D CLSM time lapse imaging as a sensitive method to qualitatively and quantitatively evaluate the biocompatibility behavior of dental composites.

The most studied aspect of the biocompatibility of dental composites is their cytotoxicity 3D CLSM time lapse imaging combined with fluorescent signal quantification allows sensitive evaluation of the cell fate over time. Utilizing this method could be an efficient tool to assess the cytocompatibility of dental composites.

ההדמיה confocal זמן הפקיעה הוא אמצעי יעיל להערכת Cytocompatibility מרוכבי שיניים

It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

A &quot;Patient-Like&quot; Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

&quot;מטופל כמו&quot; Orthotopic syngeneic עכבר הדגם של Hepatocellular Carcinoma גרורה

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

Designing CAD/CAM Surgical Guides for Maxillary Reconstruction Using an In-house Approach

Computer-aided design/computer-assisted manufacturing (CAD/CAM) is now being evaluated as a preparative technique for maxillofacial surgery. Because this technique is expensive and available in only limited areas of the world, we developed a novel CAD/CAM surgical guide using an in-house approach. By using the CAD software, the maxillary resection area and cutting planes and the fibular cutting planes and angles are determined. Once the resection area is decided, the necessary faces are extracted using a Boolean modifier. These superficial faces are united to fit the surface of the bones and thickened to stabilize the solids. Not only the cutting guides for the fibula and maxilla but also the location arrangement of the transferred bone segments is defined by thickening the superficial faces. The CAD design is recorded as .stl files and three-dimensionally (3-D) printed as actual surgical guides. To check the accuracy of the guides, model surgery using 3-D-printed facial and fibular models is performed. These methods may be used to assist surgeons where commercial guides are not available.

Methods for designing a computer-aided design/computer-aided manufacturing (CAD/CAM) surgical guide are shown. Cutting planes are separated, united, and thickened to easily visualize the necessary bone transfer. These designs can be three-dimensional printed and checked for accuracy.

תכנון CAD/CAM מדריכים כירורגית בלסת העליונה שחזור באמצעות גישה ללא צורך במיקור חוץ

Computer-aided design/computer-assisted manufacturing (CAD/CAM) is now being evaluated as a preparative technique for maxillofacial surgery. Because this ...

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 ...

Predicting Treatment Response to Image-Guided Therapies Using Machine Learning: An Example for Trans-Arterial Treatment of Hepatocellular Carcinoma

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. The objective of this study was to develop a method to predict response to intra-arterial treatment prior to intervention.
The method provides a general framework for predicting outcomes prior to intra-arterial therapy. It involves pooling clinical, demographic and imaging data across a cohort of patients and using these data to train a machine learning model. The trained model is applied to new patients in order to predict their likelihood of response to intra-arterial therapy.
The method entails the acquisition and parsing of clinical, demographic and imaging data from N patients who have already undergone trans-arterial therapies. These data are parsed into discrete features (age, sex, cirrhosis, degree of tumor enhancement, etc.) and binarized into true/false values (e.g., age over 60, male gender, tumor enhancement beyond a set threshold, etc.). Low-variance features and features with low univariate associations with the outcome are removed. Each treated patient is labeled according to whether they responded or did not respond to treatment. Each training patient is thus represented by a set of binary features and an outcome label. Machine learning models are trained using N - 1 patients with testing on the left-out patient. This process is repeated for each of the N patients. The N models are averaged to arrive at a final model.
The technique is extensible and enables inclusion of additional features in the future. It is also a generalizable process that may be applied to clinical research questions outside of interventional radiology. The main limitation is the need to derive features manually from each patient. A popular modern form of machine learning called deep learning does not suffer from this limitation, but requires larger datasets.

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. A method for predicting response to these therapies is proposed. The technique uses pre-procedural clinical, demographic, and imaging information to train machine learning models capable of predicting response prior to treatment.

חיזוי טיפול בתגובה תמונה מונחה טיפולים באמצעות למידה חישובית: דוגמא לטיפול קרצינומה Hepatocellular הטרנס-עורקים

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. The objective of this ...