עבודה זו מומנה על ידי אונטריו המכון לחקר הסרטן (OICR), הטריה פוקס הקרן והאגודה האמריקנית לניתוחים נוירולוגיים.

<ol>
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השערת סרטן תאי גזע 10, המבוססת על עבודה בשנת 21 לוקמיה, סרטן השד והסרטן 11 סרטן המוח 4,5, מצביעה על כך שרק חלק קטן יחסית של תאים בגידול, תאי גזע סרטני המכונים, יש יכולת הנרחבת להתרבות ועצמי חידוש. רוב התאים הסרטניים מאבדים את היכולת להתרבות ולחדש את ע?...

גידולים במוח הם בין סוגי הסרטן האגרסיביים והטרוגנית ביותר הידועים בבני אדם. למרות הגילוי והאבחון המוקדם שלהם כבר בנחייתם של הטכנולוגיה המודרנית נוירו הדמיה, אנחנו עדיין חסרים טיפולים מרפאים עבור גידולים במוח רבים, במיוחד עבור אלה מפוזרים, חודרניים או אלה נמצאים עמוקים במוח. גידולים במוח מייצגים את הגורם המוביל לתמותה מהסרטן בילדים, בשל האופי האגרסיבי מאוד ולעתים בלתי ניתן לריפוי שלהם. גליובלסטומה (GBM), גידול במוח הראשוני הנפוץ ביותר אצל מבוגרים, היא אחד מסוגי הסרטן האגרסיבי ביותר, האדם חרד לסיכויי ההחלמה אחידה הקטלניים 1. גידול זה ממאיר ביותר astrocytic (WHO כיתת 4) מתרחש בדרך כלל באונות המוח של מבוגרים, ויכול להתרחש גם בילדים קטנים ותינוקות. הצמיחה שלו היא מהירה וinfiltrative, ותכונות פתולוגיים כוללות אבחון pleomorphism גרעיני, התפשטות כלי דם, ונימק 2,3. לadults עם GBM שאובחן רק לאחרונה, חציון הישרדות נדירה חורגת 12 חודשים 1, עם תגובות בדרך כלל עניות לכל השיטות הטיפוליות. אנו לציין כי קיימים דמיון רב תפקודי וגנטי משותפים לתאי גזע הסומטיים ותאי סרטן, ושהמסלולים המולקולריים המווסתים את התפתחות מוח נורמלית לעתים קרובות dysregulated בסרטן. ביישום פרדיגמות ביולוגיה של תאי גזע למחקר של גידולים במוח, היינו החוקרים הראשונים ולהבא כדי לזהות ולטהר את תת אוכלוסיות של תאים מGBMs אדם שהציג את המאפיינים של תאי הגזע של התפשטות, התחדשות עצמית, ובידול במבחנה 4 וב vivo 5. אנחנו מוחלים תנאי תרבות ומבחנים ששמשו במקור כדי לאפיין תאי גזע עצביים נורמלים (NSCs) במבחנת 6,7 לגידולי מוח בילדים ומבוגרים רבים, והעשרנו לתאי גזע דמויי תאים אלה על ידי מיון לסמן תא שטח המוליד העצבי CD133 8 ,9. CD133 + שבריר הגידול במוח הכיל תאים שהיו בתדירות גבוהה הרבה יותר מאשר גידול של חניכת CD133-השבריר ב5,10 מוח העכבר NOD-SCID. זה הוקם באופן רשמי כי רק קבוצה נדירה של תאים סרטניים במוח בעלי תכונות של תאי גזע הוא גידול ייזום-, זיכה אותם בשם &quot;גידול במוח ייזום תאים&quot; או &quot;BTICs&quot;. זיהוי הרומן של BTICs מספק תובנה חדשות tumorigenesis המוח האנושי, נותן תמיכה חזקה להשערת סרטן תאי גזע 10-13 כבסיס לגידולים מוצקים רבים, וקובע יעד סלולרי חדשני לטיפול בסרטן יעיל יותר 14-20. טיפולים המתמקדים בלהרוג את חלק הארי של הגידול עלול להחמיץ את החלק היחסי הנדיר כמו גזע, המאפשר גידול להמשיך לגדול. טיפולים המתמקדים בהריגת תאי גזע הסרטן עשוי לספק טיפול ופרוגנוזה טובים יותר עבור חולים עם גידולים במוח. כדי ללמוד אוכלוסיות BTIC, יש לנו מעודן cultuפרוטוקולים מחדש במיוחד כדי לבחור עבור אוכלוסיות תאים בתוך גידולים במוח אדם שיש להם תכונות של תאי גזע. תא סרום חופשי, גזע עצבי (המל&quot;ל) בינונית מאפשר לשמירה על מדינת תאי גזע מובחנת, ותוספת של גורם בסיסי גדילה (bFGF), גורם גדילה באפידרמיס (EGF), וגורם המעכב לוקמיה (LIF) מאפשרים ההפצה, חידוש עצמי רבת ​​עצמה, וtumorspheres אדם להרחבה. כאן, אנו מתארים את השיטות המעורבות בעיבוד של גידולים במוח העיקריים וculturing במדיום המל&quot;ל להעשרה לאוכלוסיות BTIC. אנחנו קראנו מערכת המודל הניסיונית שלנו &quot;מבודדת מטופל BTIC&quot; כדי להדגיש את העובדה שהתאים אלה בתרבית רק מינימאלי תחת גזע תנאי תא כדי לבחור עבור אוכלוסיות תאי גזע. immunolabelling העוקב של אוכלוסיות BTIC לסמני תאי גזע מפתח כגון CD133 וCD15 וניתוח cytometry זרימה הוא גם תארו. אז אנחנו דנים בניתוח הדילול המגביל,אשר מסייע בלימוד פוטנציאל התחדשות העצמית של BTICs. לבסוף, אנו לחקור את ניתוח ביטוי הגנים של תאים הנדירים אלה על ידי מיון תאים בודדים על גבי שקופיות AmpliGrid וביצוע תא בודד RT-PCR. טכניקות אלו חלות גם על גידולי מוח אחרים כגון מדולובלסטומה, ependymoma וגליומות ילדים.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם המגיב </td><td> חברה </td><td> מספר קטלוגים </td></tr><tr><td> 01:01 DMEM: F12 </td><td> Invitrogen </td><td> 11320-082 </td></tr><tr><td> תוסף N2 </td><td> Invitrogen </td><td> 17502-048 </td></tr><tr><td> HEPES 1M </td><td> Wisent </td><td> 330-050-EL </td></tr><tr><td> גלוקוז </td><td> Invitrogen </td><td> 15023-021 </td></tr><tr><td> N-acetylcysteine </td><td> סיגמה אולדריץ </td><td> A9165-25 גרם </td></tr><tr><td> הישרדות עצבית גורם -1 (NSF-1) </td><td> Lonza Clonetics </td><td> CC-4323 </td></tr><tr><td> גורם גדילה באפידרמיס (EGF) </td><td> סיגמה אולדריץ </td><td> E9644 </td></tr><tr><td> גורם גדילה פיברובלסטים בסיסי (bFGF) </td><td> Invitrogen </td><td> PHG0261 </td></tr><tr><td> גורם מעכב לוקמיה (LIF) </td><td> Millipore </td><td> LIF1010 </td></tr><tr><td> אנטיביוטיקה / פטרתי </td><td> Wisent </td><td> 450-115-EL </td></tr><tr><td> Liberase TM </td><td> רוש </td><td> 05 401 119 001 </td></tr><tr><td> פתרון אמוניום כלוריד </td><td> טכנולוגיות תאי גזע </td><td> 07850 </td></tr></tbody></table>

processing of primary brain tumor tissue for stem cell assays and flow sorting

גידולים במוח בדרך כלל מורכבים של תאים מורפולוגית מגוונים שמבטאים מגוון של סמני שושלת עצביות. רק חלק קטן יחסית של תאים בגידול עם מאפיינים של תאי גזע, תאים המכונים מוח סרטני ייזום (BTICs), להחזיק יכולת להבחין לאורך שושלות מרובות, עצמי לחדש, ליזום וגידולים בגוף חי. אנחנו מוחלים תנאי התרבות שמשו במקור לתאים נורמלים עצביים גזע (NSCs) למגוון רחב של גידולים במוח אדם ומצאו ששיטת התרבות זו בוחרת במיוחד עבור אוכלוסיות גזע דמויים. מדיום סרום ללא (המל&quot;ל) מאפשר לתחזוקה של מדינת תאי גזע מובחנת, והתוספת של bFGF וEGF מאפשרת ריבוי, חידוש עצמי רבת ​​עצמה, וtumorspheres להרחבה. כדי להמשיך לאפיין אוכלוסיית BTIC של כל גידול, אנו מעריכים סמנים בתא שטח על ידי cytometry זרימה. אנו עשויים גם למיין אוכלוסיות של עניין לcharacteriz ספציפי יותרation. מבחנים עצמי חידוש מבוצעים על BTICs הבודד ממוינת ל96 גם צלחות; ° C היווצרות tumorspheres לאחר דגירה ב 37 מעידה על קיומו של גזע או תאי אב. מספרים סלולריים מרובים של אוכלוסייה מסוימת גם ניתן למיין בבארות שונות להגבלת ניתוח דילול, כדי לנתח את יכולת התחדשות עצמית. אנחנו יכולים גם ללמוד ביטוי גני ההפרש בתוך אוכלוסיית תאים מסוימות על ידי שימוש בתא בודד RT-PCR. הפרוטוקולים הבאים מתארים את התהליכים שלנו לניתוק וculturing של דגימות אנושיות ראשוניות להעשרה לאוכלוסיות BTIC, כמו גם הניתוק של tumorspheres. כמו כן, נכללו פרוטוקולים להכתמה לניתוח תזרים cytometry או מיון, מבחני התחדשות עצמית, ותא בודד RT-PCR.

Watch this Scientific Journal Video about Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting at JoVE.com

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

זיהוי של תאי מוח סרטני ייזום (BTICs), תאים הנדירים בטווח של תכונות הטרוגניות גידול בעלי תאי גזע, מספק תובנה חדשות על היווצרות גידול במוח האנושית. יש לנו מעודנים תנאי התרבות ספציפיים להעשרה לBTICs, ואנו משתמשים באופן שגרתי cytometry זרימה להעשיר אוכלוסיות אלה עוד יותר. מבחנים עצמי חידוש וניתוח תמליל של התא הבודד RT-PCR לאחר מכן ניתן לבצע בתאים מבודדים.

עיבוד של רקמת גידול במוח העיקרית למבחנים בתאי גזע וזרימת מיון

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

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18.0K Views.  McMaster University. זיהוי של תאי מוח סרטני ייזום (BTICs), תאים הנדירים בטווח של תכונות הטרוגניות גידול בעלי תאי גזע, מספק תובנה חדשות על היווצרות גידול במוח האנושית. יש לנו מעודנים תנאי התרבות ספציפיים להעשרה לBTICs, ואנו משתמשים באופן שגרתי cytometry זרימה להעשיר אוכלוסיות אלה עוד יותר. מבחנ...

Video: Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Preparation of Myeloid Derived Suppressor Cells (MDSC) from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting (AutoMACS)

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological conditions including parasitic infection, inflammation, traumatic stress, graft-versus-host disease, diabetes and cancer1-7. In mice, MDSC express Mac-1 (CD11b) and Gr-1 (Ly6G and Ly6C) surface antigens7. It is important to note that MDSC are well studied in various tumor-bearing hosts where they are significantly expanded and suppress anti-tumor immune responses compared to na&iuml;ve counterparts7-10. However, depending on the pathological condition, there are different subpopulations of MDSC with distinct mechanisms and targets of suppression11,12. Therefore, effective methods to isolate viable MDSC populations are important in elucidating their different molecular mechanisms of suppression in vitro and in vivo.
Recently, the Ghansah group has reported the expansion of MDSC in a murine pancreatic cancer model. Our tumor-bearing MDSC display a loss of homeostasis and increased suppressive function compared to na&iuml;ve MDSC 13. MDSC percentages are significantly less in lymphoid compartments of na&iuml;ve vs. tumor-bearing mice. This is a major caveat, which often hinders accurate comparative analyses of these MDSC. Therefore, enriching Gr-1+ leukocytes from na&iuml;ve mice prior to Fluorescence Activated Cell Sorting (FACS) enhances purity, viability and significantly reduces sort time. However, enrichment of Gr-1+ leukocytes from tumor-bearing mice is optional as these are in abundance for quick FACS sorting. Therefore, in this protocol, we describe a highly efficient method of immunophenotyping MDSC and enriching Gr-1+ leukocytes from spleens of na&iuml;ve mice for sorting MDSC in a timely manner. Immunocompetent C57BL/6 mice are inoculated with murine Panc02 cells subcutaneously whereas na&iuml;ve mice receive 1XPBS. Approximately 30 days post inoculation; spleens are harvested and processed into single-cell suspensions using a cell dissociation sieve. Splenocytes are then Red Blood Cell (RBC) lysed and an aliquot of these leukocytes are stained using fluorochrome-conjugated antibodies against Mac-1 and Gr-1 to immunophenotype MDSC percentages using Flow Cytometry. In a parallel experiment, whole leukocytes from na&iuml;ve mice are stained with fluorescent-conjugated Gr-1 antibodies, incubated with PE-MicroBeads and positively selected using an automated Magnetic Activated Cell Sorting (autoMACS) Pro Separator. Next, an aliquot of Gr-1+ leukocytes are stained with Mac-1 antibodies to identify the increase in MDSC percentages using Flow Cytometry. Now, these Gr1+ enriched leukocytes are ready for FACS sorting of MDSC to be used in comparative analyses (na&iuml;ve vs. tumor- bearing) in in vivo and in vitro assays.

Preparation of Myeloid Derived Suppressor Cells MDSC from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting AutoMACS

This is a rapid and comprehensive method of immunophenotyping Myeloid Derived Suppressor Cells (MDSC) and enriching Gr-1+ leukocytes from mouse spleens. This method uses flow cytometry and AutoMACS Cell Sorting to enrich for viable Gr-1+ leukocytes prior to FACS sorting of MDSC for use in vivo and in vitro assays.

הכנת מיאלואידית תאים מדכאי נגזרים (MDSC) מן הגידול נושאות עכברים תמימים הלבלב באמצעות cytometry זרימה ומיון אוטומטי מגנטי נייד הופעל (AutoMACS)

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological ...

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events in cardiac myocytes such as proliferation and apoptosis require an accurate identification of cardiac myocyte nuclei to analyze cellular renewal in homeostasis and in pathological conditions2. Here, we provide a method to isolate cardiomyocyte nuclei from post mortem tissue by density sedimentation and immunolabeling with antibodies against pericentriolar material 1 (PCM-1) and subsequent flow cytometry sorting. This strategy allows a high throughput analysis and isolation with the advantage of working equally well on fresh tissue and frozen archival material. This makes it possible to study material already collected in biobanks. This technique is applicable and tested in a wide range of species and suitable for multiple downstream applications such as carbon-14 dating3, cell-cycle analysis4, visualization of thymidine analogues (e.g. BrdU and IdU)4, transcriptome and epigenetic analysis.

Cardiac nuclei are isolated via density sedimentation and immunolabeled with antibodies against pericentriolar material 1 (PCM-1) to identify and sort cardiomyocyte nuclei by flow cytometry.

הבידוד של גרעיני Cardiomyocyte מרקמות שלאחר המוות

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

בידוד, אפיון והשוואתי התמיינות של תאי גזע אנושיים שיניים זולות נגזרה שיניים קבועות על ידי שימוש בשתי שיטות שונות

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

תרבות מטופל נגזר סלולרי ובידוד של CD133<sup&gt; +</sup&gt; תאי גזע סרטני משוערים מלנומה

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

רומן ברזולוציה גבוהה<em&gt; בvivo</em&gt; טכניקת הדמיה כדי לחקור את התגובה דינמית של מבנים תוך גולגולתי לצמיחה גידול וTherapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

עיבוד של semiautomated מחזור תא גידול מבחני (CTC) ליישומים קליניים ומחקר הקליני

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem cells&#8221; (CSCs) responsible for tumor initiation, maintenance and relapse following conventional chemotherapies. Identification of ovarian CSCs is therefore an important goal. A commonly used method to assess CSC potential in vitro is the spheres assay in which cells are plated under non-adherent culture conditions in serum-free medium supplemented with growth factors and sphere formation is scored after a few days. Here, we review currently available protocols for human ovarian cancer spheres assays and perform a side-by-side analysis between commonly used multi cell-based assays and a more accurate system based on single cell plating. Our results indicate that both multi cell-based as well as single cell-based spheres assays can be used to investigate sphere formation in vitro. The more laborious and expensive single cell-based assays are more suitable for functional assessment of individual cells and lead to overall more accurate results while multi cell-based assays can be strongly influenced by the density of plated cells and require titration experiments upfront. Methylcellulose supplementation to multi cell-based assays can be effectively used to reduce mechanical artifacts.

In vitro spheres assays are commonly used to identify cancer stem cells. Here we compare single with multi cell-based spheres assays. The more laborious single cell-based assays or methylcellulose supplementation give more accurate results while multi cell-based assays performed in liquid medium can be highly influenced by cell density.

הערכה של מאפייני תא גזע בקרצינומה של תאי השחלות אדם באמצעות Multi ומבחני Spheres מבוסס תא בודדים

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem ...

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.

The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

גידול ראשוני ובידוד תא MEF לחקר ריאות גרורה

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by astrocytes, pericytes and brain endothelial cells (BECs). Here, we detail methods to assess BBB coverage using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. To highlight the applicability of the assays to disease states, we describe the effect of oligomeric amyloid-&#946; (oA&#946;), which is an important contributor to Alzheimer&#39;s disease (AD) progression, on BBB coverage. Further, we utilize the epidermal growth factor (EGF) to illuminate the drug screening potential of the techniques. Our results show that single and triple cultured BECs form meshwork-like structures under basal conditions, and that oA&#946; disrupts this cell meshwork formation and degenerates the preformed mesh structures, but EGF blocks this disruption. Thus, the techniques described are important for dissecting fundamental and disease-relevant processes that modulate BBB coverage.

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Maintaining blood-brain barrier coverage is key for the homeostasis of the central nervous system. This protocol describes in vitro techniques to delineate the fundamental and pathological processes that modulate blood-brain barrier coverage.

ב מבחני מבחנה כדי להעריך דם מוח מחסום כמו רשת כלי שיטפון ושיבוש

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by ...

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of genetically modified pig models, and the often-limited number of available animals of these models, establishment of (biobank) collections of adequately processed tissue samples suited for a broad spectrum of subsequent analyses methods, including analyses not specified at the time point of sampling, represent meaningful approaches to take full advantage of the translational value of the model. With respect to the peculiarities of porcine anatomy, comprehensive guidelines have recently been established for standardized generation of representative, high-quality samples from different porcine organs and tissues. These guidelines are essential prerequisites for the reproducibility of results and their comparability between different studies and investigators. The recording of basic data, such as organ weights and volumes, the determination of the sampling locations and of the numbers of tissue samples to be generated, as well as their orientation, size, processing and trimming directions, are relevant factors determining the generalizability and usability of the specimen for molecular, qualitative, and quantitative morphological analyses. Here, an illustrative, practical, step-by-step demonstration of the most important techniques for generation of representative, multi-purpose biobank specimen from porcine tissues is presented. The methods described here include determination of organ/tissue volumes and densities, the application of a volume-weighted systematic random sampling procedure for parenchymal organs by point-counting, determination of the extent of tissue shrinkage related to histological embedding of samples, and generation of randomly oriented samples for quantitative stereological analyses, such as isotropic uniform random (IUR) sections generated by the &#34;Orientator&#34; and &#34;Isector&#34; methods, and vertical uniform random (VUR) sections.

The practical application and performance of methods for generation of representative tissue samples of porcine animal models for a broad spectrum of downstream analyses in biobank projects are demonstrated, including volumetry, systematic random sampling, and differential processing of tissue samples for qualitative and quantitative morphologic and molecular analyses types.

אסטרטגיות דגימה ועיבוד של דגימות רקמה Biobank של מודלים ביו חזירי

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of ...