وقد تم تمويل هذا العمل من قبل معهد أبحاث السرطان أونتاريو (OICR)، ومؤسسة تيري فوكس والرابطة الأمريكية لجراحي الأعصاب.

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الإعلان عن أي تضارب في المصالح.

الخلايا الجذعية السرطانية فرضية 10، على أساس العمل في سرطان الدم 21، 11 و سرطان الثدي سرطان الدماغ 4،5، تشير إلى أن نسبة صغيرة فقط من الخلايا نسبيا في الورم، ووصف الخلايا الجذعية السرطانية، تمتلك القدرة على الانتشار على نطاق واسع والنفس التجديد. معظم ا?...

أورام المخ هي من بين السرطانات الأكثر عدوانية وغير المتجانسة المعروف لدى البشر. على الرغم من أن تم تسهيل الكشف والتشخيص في وقت سابق عن طريق التكنولوجيا الحديثة العصبية التصوير، ونحن لا تزال تفتقر إلى العلاجات العلاجية لعلاج أورام المخ كثيرة، ولا سيما لتلك، ونشر الغازية أو تلك التي تقع في عمق الدماغ. أورام المخ تمثل السبب الرئيسي للوفيات السرطان في الأطفال نظرا لطبيعتها العدوانية للغاية وغير قابل للشفاء كثير من الأحيان. ورم أرومي دبقي (GBM)، والأكثر شيوعا ورم في المخ الأولية في البالغين، هي واحدة من السرطانات الأكثر عدوانية الإنسان، يخشى على التكهن به بشكل موحد قاتلة 1. هذا الورم الخبيث للغاية نجمي (WHO الصف 4) يحدث عادة في البالغين نصفي الكرة المخية، ويمكن أن يحدث أيضا عند الأطفال الصغار والرضع. نموها السريع وغير الارتشاحي، وميزات المرضية التشخيص وتشمل تعدد الأشكال النووية، وانتشار الاوعية الدموية الدقيقة، ونخر 2،3. لأدولTS مع GBM تشخيصها حديثا، ونادرا ما يمتد بقاء متوسط ​​أكثر من 12 شهرا 1، مع استجابات الفقراء عموما لجميع الطرائق العلاجية. لاحظنا أن هناك العديد من أوجه التشابه الوظيفية والوراثية المشتركة من قبل الخلايا الجذعية الجسدية والخلايا السرطانية، والتي غالبا ما dysregulated المسارات الجزيئية التي تنظم نمو الدماغ الطبيعي في السرطان. في تطبيق نماذج بيولوجيا الخلايا الجذعية لدراسة أورام المخ، كنا أول من الباحثين تحديد مستقبلي وتنقية جزء من السكان من الخلايا من GBMS الإنسان التي عرضت خصائص الخلايا الجذعية من، انتشار التجديد الذاتي، والتمايز في المختبر 4 و في فيفو 5. طبقنا شروط الثقافة والمقايسات المستخدمة في الأصل لوصف العادي الخلايا الجذعية العصبية (NSCs) 6،7 في المختبر لعدة أورام الدماغ لدى الأطفال والكبار، وإثراء لهذه الخلايا الجذعية مثل الفرز من قبل الخلية للعلامة السلف سطح الخلية العصبية CD133 8 ،9. وCD133 + ورم في المخ جزء الواردة الخلايا التي كان لها تردد أعلى بكثير من بدء الورم من الكسر-CD133-SCID في NOD الماوس العقول 5،10. هذه المنشأة رسميا أن مجموعة فرعية فقط من الخلايا النادرة ورم في المخ مع خصائص الخلايا الجذعية هي ورم الشروع، مما اكسبها اسم &quot;خلايا ورم في المخ بدء&quot; أو &quot;BTICs&quot;. تحديد رواية BTICs يقدم رؤى جديدة في تكون الأورام الدماغ البشري، وإعطاء دعم قوي للخلية الجذعية السرطانية فرضية 10-13 كأساس لكثير من الأورام الصلبة، ويحدد هدفا لمزيد من رواية الخلوية علاجات السرطان فعالة 14-20. العلاجات التي تركز على الجزء الأكبر من قتل الورم قد يغيب عن الجذعية نادرة مثل الكسر، والسماح للورم أن تستمر في النمو. العلاجات التي تركز على قتل الخلايا الجذعية السرطانية قد توفر أفضل العلاج والتشخيص للمرضى المصابين بأورام الدماغ. من أجل دراسة السكان BTIC، قد حسنت نحن لدينا cultuإعادة البروتوكولات لتحديد على وجه التحديد بالنسبة للسكان داخل الخلية البشرية أورام المخ التي تمتلك خصائص الخلايا الجذعية. مصل خالية، الخلايا الجذعية العصبية (NSC) متوسطة يسمح للحفاظ على حالة الخلايا الجذعية غير المتمايزة، وإضافة عامل نمو الخلايا الليفية الأساسية (bFGF)، عامل نمو البشرة (EGF)، وعامل مثبط اللوكيميا (LIF) يسمح لل انتشار قوة متعددة، وتجديد الذات، وقابلة للتوسيع tumorspheres الإنسان. هنا، نحن تصف أساليب المشاركة في معالجة أورام الدماغ الأولية والمتوسطة في زراعة لهم NSC لإثراء للسكان BTIC. لقد دعا نظامنا نموذج تجريبي &quot;عزلة المريض BTIC&quot; للتأكيد على حقيقة أن الحد الأدنى فقط مثقف هذه الخلايا الجذعية تحت خلية لتحديد الظروف للسكان الخلايا الجذعية. يوصف أيضا immunolabelling اللاحقة للسكان BTIC لمفتاح علامات الخلايا الجذعية مثل CD133 CD15 وتحليل وتدفق الخلوي. ثم نناقش تحليل تخفيف الحد،الذي يساعد في دراسة إمكانية تجديد الذاتي BTICs. وأخيرا، فإننا استكشاف تحليل التعبير الجيني من هذه الخلايا النادرة عن طريق الفرز الخلايا واحدة على الشرائح AmpliGrid وأداء خلية واحدة RT-PCR. هذه التقنيات تنطبق أيضا على أورام المخ الأخرى مثل بطاني عصبي، والاورام الدبقية نخاعي للأطفال.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> كتالوج رقم </td></tr><tr><td> 01:01 DMEM: F12 </td><td> إينفيتروجن </td><td> 11320-082 </td></tr><tr><td> N2 الملحق </td><td> إينفيتروجن </td><td> 17502-048 </td></tr><tr><td> 1M HEPES </td><td> Wisent </td><td> 330-050-EL </td></tr><tr><td> جلوكوز </td><td> إينفيتروجن </td><td> 15023-021 </td></tr><tr><td> N-أسيتيل </td><td> سيغما الدريتش </td><td> A9165-25G </td></tr><tr><td> -1 العصبية بقاء عامل (NSF-1) </td><td> Lonza Clonetics </td><td> CC-4323 </td></tr><tr><td> عامل نمو البشرة (EGF) </td><td> سيغما الدريتش </td><td> E9644 </td></tr><tr><td> عامل نمو الخلايا الليفية الأساسية (bFGF) </td><td> إينفيتروجن </td><td> PHG0261 </td></tr><tr><td> عامل مثبط اللوكيميا (LIF) </td><td> ميليبور </td><td> LIF1010 </td></tr><tr><td> المضادات الحيوية / الفطرية </td><td> Wisent </td><td> 450-115-EL </td></tr><tr><td> Liberase TM </td><td> روش </td><td> 05 401 119 001 </td></tr><tr><td> كلوريد الأمونيوم الحل </td><td> وقف تقنيات الخليوي </td><td> 07850 </td></tr></tbody></table>

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

وتتألف عادة من خلايا أورام المخ المختلفة التي تعبر عن شكليا مجموعة متنوعة من علامات النسب العصبية. سوى جزء صغير نسبيا من الخلايا في الورم مع خصائص الخلايا الجذعية، ووصف الخلايا السرطانية الدماغ بدء (BTICs)، تمتلك القدرة على التفريق على طول الأنساب متعددة، صاحبة تجديد، والشروع في الأورام في الجسم الحي. طبقنا شروط الثقافة في الأصل تستخدم لطبيعية الخلايا الجذعية العصبية (NSCs) لمجموعة متنوعة من الأورام الدماغ البشري، ووجدت أن هذه الطريقة الثقافة يختار خصيصا لالجذعية مثل السكان. مصل خالية المتوسطة (NSC) يسمح للحفاظ على حالة الخلايا الجذعية غير المتمايزة، وإضافة bFGF وEGF يسمح لانتشار قوة متعددة، وتجديد الذات، وقابلة للتوسيع tumorspheres. لمزيد من تميز كل السكان الورم BTIC، نقيم علامات سطح الخلية بواسطة التدفق الخلوي. ونحن قد ترتب أيضا السكان من الاهتمام لcharacteriz أكثر تحديداأوجه. يتم تنفيذ الذاتي تجديد المقايسات على BTICs واحد مصنفة في 96 لوحات جيدة، وتشكيل tumorspheres بعد الحضانة عند 37 درجة مئوية يدل على وجود ساق أو خلية السلف. ويمكن أيضا أعداد الخلايا متعددة من السكان خاصة في الآبار يمكن فرز مختلفة للحد من تحليل التخفيف، لتحليل الذاتي تجديد القدرات. يمكننا أيضا دراسة التعبير الجيني الفرق بين السكان خلية معينة باستخدام خلية واحدة RT-PCR. البروتوكولات التالية تصف إجراءاتنا لتفارق وزراعة العينات الأولية لتخصيب الإنسان للسكان BTIC، فضلا عن تفكك tumorspheres. كما تشمل بروتوكولات لتلطيخ للتحليل التدفق الخلوي أو الفرز، المقايسات الذاتي تجديد، وخلية واحدة 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

تحديد خلايا ورم في المخ بدء (BTICs)، وخلايا نادرة داخل الورم غير متجانسة تمتلك خصائص الخلايا الجذعية، ويقدم رؤى جديدة في الدماغ البشري المرضية الورم. صقلنا ظروف محددة لإثراء الثقافة لBTICs، والتي نستخدمها بشكل روتيني التدفق الخلوي لإثراء هؤلاء السكان. يمكن في وقت لاحق الذاتي تجديد المقايسات وتحليل نص واحد بواسطة PCR-RT الخلية سيتم تنفيذها على هذه الخلايا المعزولة.

تجهيز الدماغ الأولية للفحوصات الأنسجة السرطانية والخلايا الجذعية تدفق الفرز

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. تحديد خلايا ورم في المخ بدء (BTICs)، وخلايا نادرة داخل الورم غير متجانسة تمتلك خصائص الخلايا الجذعية، ويقدم رؤى جديدة في الدماغ البشري المرضية الورم. صقلنا ظروف محددة لإثراء الثقافة لBTICs، والتي نستخدمها بشكل روتيني التدفق الخلوي لإثراء هؤلاء السكان. يمكن في وقت لا?...

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.

إعداد الخلايا متعلق بالنخاع الشوكي المكثف المشتقة (MDSC) من الفئران الحاملة للورم ساذجة والبنكرياس باستخدام التدفق الخلوي والفرز الآلي المغناطيسي خلية المنشط (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.

العزلة نوى Cardiomyocyte من الأنسجة بعد الوفاة

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.

العزلة، وتوصيف المقارن التمييز بين الخلايا الجذعية البشرية الأسنان اللب المستمدة من الأسنان الدائمة باستخدام طريقتين مختلفتين

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).

زراعة الخلايا المشتقة المريض وعزل CD133<sup&gt; +</sup&gt; خلايا الجذعية السرطانية المزعومين من الميلانوما

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.

رواية عالية الدقة<em&gt; في الجسم الحي</em&gt; تقنية التصوير لدراسة الاستجابة الديناميكية للمنشآت داخل القحف لنمو الورم والتداوي

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.

التكيف من Semiautomated اعارة ورم الخلية (CTC) فحوصات للتطبيقات السريرية وقبل السريرية البحوث

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.

تقييم خصائص الخلايا الجذعية في الإنسان خلايا سرطان المبيض عن طريق متعدد واحدة على أساس خلية المجالات فحوصات

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.

ورم الابتدائي وMEF عزل الخلايا لدراسة الرئة الانبثاث

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.

في مقايسات المختبر لتقييم الحاجز الدموي الدماغي تشبه تشكيل سفينة وتشوه

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.

استراتيجيات أخذ العينات وتجهيزها لعينات الأنسجة المصرف البيولوجي من النماذج الطبية الخنزير

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