نود أن نعترف نادين هين وأرند Heerschap على مساعدتهم القيمة. وأيد هذا العمل من قبل المعهد الهولندي للطب التجديدي (NIRM، FES0908)، وبرنامج الاتحاد الأوروبي FP7 ENCITE (HEALTH-F5-2008-201842) وTargetBraIn (HEALTH-F2-2012-279017)، منحة من مؤسسة فولكس واجن ( I/83 443)، والمنظمة الهولندية للبحوث العلمية (VENI 700.10.409 وفيدي 917.76.363)، ERC (المنحة المتقدم 269019)، ومركز جامعة رادبود نيميجن الطبية (AGIKO 2008-2-4).

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorine+(19F)+MRS+and+MRI+in+biomedicine.">Fluorine (19F) MRS and MRI in biomedicine.</a> NMR Biomed. 24, 114-129 (2011).</li><li>Stoll, G., Basse-Lusebrink, T., Weise, G., Jakob, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+inflammation+using+19F-magnetic+resonance+imaging+and+perfluorocarbons.">Visualization of inflammation using 19F-magnetic resonance imaging and perfluorocarbons.</a> Wires Nanomed. Nanobiol. 4, 438-447 (2012).</li><li>Srinivas, M., Boehm-Sturm, P., Figdor, C. G., de Vries, I. J., Hoehn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labeling+cells+for+in+vivo+tracking+using+(19)F.">Labeling cells for in vivo tracking using (19)F.</a> MRI. Biomaterials. 33, 8830-8840 (2012).</li><li>Ahrens, E. T., Flores, R., Xu, H., Morel, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+platform+for+tracking+immunotherapeutic+cells.">In vivo imaging platform for tracking immunotherapeutic cells.</a> Nat. Biotechnol. 23, 983-987 (2005).</li><li>Srinivas, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Customizable,+multi-functional+fluorocarbon+nanoparticles+for+quantitative+in+vivo+imaging+using+19F+MRI+and+optical+imaging.">Customizable, multi-functional fluorocarbon nanoparticles for quantitative in vivo imaging using 19F MRI and optical imaging.</a> Biomaterials. 31, 7070-7077 (2010).</li><li>Boehm-Sturm, P., Mengler, L., Wecker, S., Hoehn, M., Kallur, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+tracking+of+human+neural+stem+cells+with+19F+magnetic+resonance+imaging.">In vivo tracking of human neural stem cells with 19F magnetic resonance imaging.</a> PLoS One. 6, e29040 (2011).</li><li>Srinivas, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+cytometry+of+antigen-specific+t+cells+using+(19)F.">In vivo cytometry of antigen-specific t cells using (19)F.</a> MRI. Magn. Reson. Med. , (2009).</li><li>Srinivas, M., Morel, P. A., Ernst, L. A., Laidlaw, D. H., Ahrens, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorine-19+MRI+for+visualization+and+quantification+of+cell+migration+in+a+diabetes+model.">Fluorine-19 MRI for visualization and quantification of cell migration in a diabetes model.</a> Magn. Reson. Med. 58, 725-734 (2007).</li><li>Mangala Srinivas, E. T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+labeling+and+quantification+for+nuclear+magnetic+resonance+techniques.">Cellular labeling and quantification for nuclear magnetic resonance techniques.</a> US patent. , (2007).</li><li>Boehm-Sturm, P., Pracht, E. D., Aswendt, M., Henn, N., Hoehn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Proceedings of the International Society for Magnetic Resonance in Medicine. , (2012).</li><li>Insko, E. K., Bolinger, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+the+Radiofrequency+Field.">Mapping of the Radiofrequency Field.</a> J. Magn. Res. A. 103, 82-85 (1993).</li><li>Haacke, E. M., Brown, R. W., Thompson, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Magnetic resonance imaging: physical principles and sequence design. , (1999).</li><li>Scheffler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pictorial+description+of+steady-states+in+rapid+magnetic+resonance+imaging.">A pictorial description of steady-states in rapid magnetic resonance imaging.</a> Concepts Magn. Res. 11, 291-304 (1999).</li><li>Firbank, M. J., Coulthard, A., Harrison, R. M., Williams, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+two+methods+for+measuring+the+signal+to+noise+ratio+on+MR+images.">A comparison of two methods for measuring the signal to noise ratio on MR images.</a> Phys. Med. Biol. 44, 261-264 (1999).</li><li>de Chickera, S. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labelling+dendritic+cells+with+SPIO+has+implications+for+their+subsequent+in+vivo+migration+as+assessed+with+cellular+MRI.">Labelling dendritic cells with SPIO has implications for their subsequent in vivo migration as assessed with cellular MRI.</a> Contrast Media Mol. Imaging. 6, 314-327 (2011).</li></ol>

والكتاب ليس لديهم تضارب المصالح في الكشف عنها.

بروتوكول المذكورة هنا يصف الإجراء العام لفي الجسم الحي 19 F التصوير بالرنين المغناطيسي تتبع الخلية. على الرغم من الأسلوب شارك في الحضانة وصفها، هناك عدة بروتوكولات مختلفة لوصفها الخلايا مع 19 وكيل F. ومع ذلك، غالبا ما يتم استخدام المشاركين في الحضانة، ?...

في تتبع الخلايا الحية أمر ضروري لتحسين ورصد العلاجات الخلوية 1. نظرا لطبيعتها موسع، والتصوير يوفر فرصا ممتازة لمراقبة الخلايا في الجسم الحي. التصوير بالرنين المغناطيسي (MRI) هو مستقل من الإشعاعات المؤينة ويسمح القرار التصوير متفوقة والجوهرية النقيض من الأنسجة اللينة. وقد تم بالفعل استخدام تتبع خلية مقرها MRI-سريريا لمتابعة الخلايا الجذعية في مرضى سرطان الجلد 2. ويتم التصوير بالرنين المغناطيسي السريرية التقليدية للخروج على 1 H النواة، موجودة في المياه المتنقلة في الأنسجة. ومن الممكن أيضا لتنفيذ التصوير بالرنين المغناطيسي على نواة نشطة أخرى، مثل C 13 و 19 و 23 F نا. ومع ذلك، فقد تم تطبيقه فقط 19 F التصوير بالرنين المغناطيسي بنجاح في الجسم الحي تتبع الخلية، حيث أنها توفر أعلى حساسية بعد 1 H. غياب التصوير بالرنين المغناطيسي للكشف الذاتية F 19 في الأنسجة يسمح عالية الانتقائية إشارة لالكشف عن عوامل التباين الخارجية 19 F ويسمح الكمي لتركيز الفلور مباشرة من بيانات الصورة. للاطلاع على مناقشة مفصلة عن 19 F التصوير بالرنين المغناطيسي، انظر 3-5. وثمة مسألة رئيسية مع 19 F التصوير بالرنين المغناطيسي هو الحاجة إلى تطوير وتحسين مناسبة تسميات الخلية 19 F، على الرغم من أن وضعت العديد من التسميات، مع وجود اتجاه نحو 6 كلاء المتعدد الوسائط. ويستند البروتوكول وصفنا هنا على الدراسات التي مجموعاتنا 7-9، بما في ذلك أول المقالات التي وصفت في الجسم الحي الكمية 19 خلية مقرها MRI-F تتبع 10،11. وتتلخص الإجراء العام من تتبع الخلية باستخدام التصوير بالرنين المغناطيسي F 19 في الشكل 1. نحن تصف بروتوكول عام لوضع العلامات والتصوير من الخلايا الجذعية (DCS) باستخدام عامل المشبعة النقيض حسب الطلب 8. بروتوكول التصوير تنطبق بصورة عامة على مختلف أنواع الخلايا، والعلامات والنماذج الحيوانية. الوينبغي اتخاذ نوع من الخلايا والحيوانات النموذج الموصوف هنا فقط كمثال، وبالتالي فإننا لا توفر تفاصيل عن العزلة الخلية ووضع العلامات، وإنما تركز على البروتوكول التصوير. وسوف يكون من الضروري إدخال تعديلات على كل تسمية، نوع من الخلايا، نموذج حيواني، والإعداد والتصوير، وهذه يمكن أن تكون موجودة في الأدب أو قد تحتاج إلى أن يكون الأمثل من قبل الباحثين. يتم تضمين بعض التعديلات المشتركة في المناقشة.

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</td>
		 </tr>
		 <tr>
			<td>PBS</td>
			<td>Sigma-Aldrich</td>
			<td>MFCD00131855</td>
			<td></td>
		 </tr>
		 <tr>
			<td>TFA</td>
			<td>Sigma-Aldrich</td>
			<td>76-05-1 </td>
			<td></td>
		 </tr>
		 <tr>
			<td>Ketamine (Ketavet)</td>
			<td>Pfizer</td>
			<td>778-551</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Xylazine (Rompun)</td>
			<td>Bayer</td>
			<td>QN05 cm92</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Ophtosan </td>
			<td>Produlab Pharma</td>
			<td>2702</td>
			<td>eye ointment</td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Material name </td>
		 </tr>
		 <tr>
			<td>MRI scanner</td>
			<td>Bruker Biospec</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>NMR spectrometer</td>
			<td>Bruker Biospec</td>
			<td></td>
			<td></td>
		 </tr>
		</tbody>
 </table>

in vivo 19f mri for cell tracking

في الجسم الحي 19 F التصوير بالرنين المغناطيسي يسمح تتبع الخلية الكمي من دون استخدام الأشعة المؤينة. بل هو أسلوب موسع التي يمكن تطبيقها على البشر. هنا، نحن تصف بروتوكول عام لوضع العلامات الخلية، والتصوير، ومعالجة الصور. تقنية قابلة للتطبيق على مختلف أنواع الخلايا والنماذج الحيوانية، على الرغم من هنا ونحن نركز على نموذج الفأر نموذجية لتتبع الخلايا المناعية الفئران. وصفت أهم القضايا لوضع العلامات الخلية، وهذه هي ذات الصلة لجميع الموديلات. وبالمثل، يتم سرد المعلمات التصوير الأساسية، على الرغم من أن التفاصيل سوف تختلف اعتمادا على نظام التصوير بالرنين المغناطيسي والإعداد الفردية. أخيرا، ونحن تشمل بروتوكول لمعالجة الصور الكمي. الاختلافات لهذا، وأجزاء أخرى من البروتوكول، يتم تقييمها في قسم مناقشة. استنادا إلى إجراءات تفصيلية وصفها هنا، فإن المستخدم بحاجة إلى التكيف مع بروتوكول لكل نوع محدد الخلية، والتسمية الخلية، نموذج حيواني، والإعداد والتصوير. نلاحظ أن عويمكن أيضا أن تتكيف rotocol للاستخدام البشري، طالما تم استيفاء القيود السريرية.

نحن هنا تظهر نتائج نموذجية للبروتوكول التي تنطوي على نقل 19 F الخلايا المسمى صاروخ موجه إلى العقدة الليمفاوية استنزاف. ويبين الشكل 2 طيف 19 F NMR من 10 6 الخلايا المسمى باستخدام مرجع TFA. تم تنفيذ الإعداد التصوير خارج كما هو موضح في البروتوكول (?...

Watch this Scientific Journal Video about In vivo 19F MRI for Cell Tracking at JoVE.com

In vivo 19F MRI for Cell Tracking

نحن تصف بروتوكول العامة لفي الجسم الحي تتبع الخلية باستخدام التصوير بالرنين المغناطيسي في نموذج الفأر مع السابقين الخلايا المسمى الجسم الحي. يتم تضمين بروتوكول نموذجي لوضع العلامات الخلية، وتجهيز الحصول على الصور والكمي.

في الجسم الحي 19 F التصوير بالرنين المغناطيسي لتتبع الخليوي

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Research

JoVE Journal

Medicine

In vivo 19F MRI for Cell Tracking

14.6K Views.  Radboud University Medical Center.  نحن تصف بروتوكول العامة لفي الجسم الحي تتبع الخلية باستخدام التصوير بالرنين المغناطيسي في نموذج الفأر مع السابقين الخلايا المسمى الجسم الحي. يتم تضمين بروتوكول نموذجي لوضع العلامات الخلية، وتجهيز الحصول على الصور والكمي. 

Video: In vivo 19F MRI for Cell Tracking

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

تسلسلي<em&gt; في الجسم الحي</em&gt; التصوير من الخلايا الجذعية المكونة للعظم / السلف خلال اصلاح الكسر

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans biofilms have been studied predominantly in vitro but there is a crucial need for better understanding of this dynamic process under in vivo conditions. We developed an in vivo subcutaneous rat model to study C. albicans biofilm formation. In our model, multiple (up to 9) Candida-infected devices are implanted to the back part of the animal. This gives us a major advantage over the central venous catheter model system as it allows us to study several independent biofilms in one animal. Recently, we adapted this model to study C. albicans biofilm development in BALB/c mice. In this model, mature C. albicans biofilms develop within 48 hr and demonstrate the typical three-dimensional biofilm architecture. The quantification of fungal biofilm is traditionally analyzed post mortem and requires host sacrifice. Because this requires the use of many animals to perform kinetic studies, we applied non-invasive bioluminescence imaging (BLI) to longitudinally follow up in vivo mature C. albicans biofilms developing in our subcutaneous model. C. albicans cells were engineered to express the Gaussia princeps luciferase gene (gLuc) attached to the cell wall. The bioluminescence signal is produced by the luciferase that converts the added substrate coelenterazine into light that can be measured. The BLI signal resembled cell counts obtained from explanted catheters. Non-invasive imaging for quantifying in vivo biofilm formation provides immediate applications for the screening and validation of antifungal drugs under in vivo conditions, as well as for studies based on host-pathogen interactions, hereby contributing to a better understanding of the pathogenesis of catheter-associated infections.

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.

المبيضات البيض بيوفيلم التنمية على الهيئات الأجنبية طبيا ذات الصلة في الماوس تحت الجلد نموذج تلاه ضوء بارد التصوير

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans ...

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (&#60;10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.
This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

في فيفو التصوير وتتبع التكنيتيوم-99m إعتبر نخاع العظم الوسيطة الخلايا الجذعية في فرسي Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

استخدام فائقة MRI الميداني في القوارض الصغيرة نماذج من مرض الكلى المتعدد الكيسات ل<em&gt; في فيفو</em&gt; Phenotyping والدواء المراقبة

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

دراسة سرطان البنكرياس الجذعية خصائص الخلايا لتطوير استراتيجيات علاجية جديدة

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

نضوب خلايا الفأر من Xenografts ورم الإنسان يحسن بشكل ملحوظ تحليل المصب من الخلايا المستهدفة

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of cerebral malaria can be difficult. We present an experimental mouse model of cerebral malaria that shares multiple features of the human disease, including edema and microvascular pathology. Using magnetic resonance imaging (MRI), we can detect and track the blood-brain barrier disruption, edema development, and subsequent brain swelling. We describe multiple MRI techniques that can visualize these pertinent pathological changes. Thus, we show that MRI represents a valuable tool to visualize and track pathological changes, such as edema, brain swelling, and microvascular pathology, in vivo.

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging.

في فيفو تتبع من تطوير وذمة والأمراض الوعائية الوعائية في نموذج من الملاريا الدماغية التجريبية باستخدام التصوير بالرنين المغناطيسي

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The amount of the different tissues in the callus provides important information on the fracture healing progress. Available in vivo techniques to longitudinally monitor the callus tissue development in preclinical fracture-healing studies using small animals include digital radiography and &#181;CT imaging. However, both techniques are only able to distinguish between mineralized and non-mineralized tissue. Consequently, it is impossible to discriminate cartilage from fibrous tissue. In contrast, magnetic resonance imaging (MRI) visualizes anatomical structures based on their water content and might therefore be able to noninvasively identify soft tissue and cartilage in the fracture callus. Here, we report the use of an MRI-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice. The experiments demonstrated that the fixator and a custom-made mounting device allow repetitive MRI scans, thus enabling longitudinal analysis of fracture-callus tissue development.

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

The evaluation of tissue development in the fracture callus during endochondral bone healing is essential to monitor the healing process. Here, we report the use of a magnetic resonance imaging (MRI)-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice.

في فيفو تقييم التنمية دشبذ الكسر أثناء التئام العظام في الفئران باستخدام جهاز الصدمة المتوافقة مع التصوير بالرنين المغناطيسي لعظم الفخذ الماوس

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...

Whole-brain Segmentation and Change-point Analysis of Anatomical Brain MRI&#8212;Application in Premanifest Huntington's Disease

Recent advances in MRI offer a variety of useful markers to identify neurodegenerative diseases. In Huntington&#39;s disease (HD), regional brain atrophy begins many years prior to the motor onset (during the &#34;premanifest&#34; period), but the spatiotemporal pattern of regional atrophy across the brain has not been fully characterized. Here we demonstrate an online cloud-computing platform, &#34;MRICloud&#34;, which provides atlas-based whole-brain segmentation of T1-weighted images at multiple granularity levels, and thereby, enables us to access the regional features of brain anatomy. We then describe a regression model that detects statistically significant inflection points, at which regional brain atrophy starts to be noticeable, i.e. the &#34;change-point&#34;, with respect to a disease progression index. We used the CAG-age product (CAP) score to index the disease progression in HD patients. Change-point analysis of the volumetric measurements from the segmentation pipeline, therefore, provides important information of the order and pattern of structural atrophy across the brain. The paper illustrates the use of these techniques on T1-weighted MRI data of premanifest HD subjects from a large multicenter PREDICT-HD study. This design potentially has wide applications in a range of neurodegenerative diseases to investigate the dynamic changes of brain anatomy.

Whole-brain Segmentation and Change-point Analysis of Anatomical Brain MRI&amp;#8212;Application in Premanifest Huntington&#039;s Disease

This paper describes a statistical model for volumetric MRI data analysis, which identifies the &#34;change-point&#34; when brain atrophy begins in premanifest Huntington&#39;s disease. Whole-brain mapping of the change-points is achieved based on brain volumes obtained using an atlas-based segmentation pipeline of T1-weighted images.

الدماغ كل تجزئة وتحليل التغيير-نقطة من الدماغ التشريحية التصوير بالرنين المغناطيسي – التطبيق في مرض هنتنغتون بريمانيفيست

Recent advances in MRI offer a variety of useful markers to identify neurodegenerative diseases. In Huntington&#39;s disease (HD), regional brain atrophy ...

An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No current animal models of diabetes and oxygen-induced retinopathy develop the full-range progressive changes manifested in human proliferative diabetic retinopathy (PDR). Therefore, understanding of the disease pathogenesis and pathophysiology has relied largely on the use of histological sections and vitreous samples in approaches that only provide steady-state information on the involved pathogenic factors. Increasing evidence indicates that dynamic cell-cell and cell-extracellular matrix (ECM) interactions in the context of three-dimensional (3D) microenvironments are essential for the mechanistic and functional studies towards the development of new treatment strategies. Therefore, we hypothesized that the pathological fibrovascular tissue surgically excised from eyes with PDR could be utilized to reliably unravel the cellular and molecular mechanisms of this devastating disease and to test the potential for novel clinical interventions. Towards this end, we developed a novel method for 3D ex vivo culture of surgically-excised patient-derived fibrovascular tissue (FT), which will serve as a relevant model of human PDR pathophysiology. The FTs are dissected into explants and embedded in fibrin matrix for ex vivo culture and 3D characterization. Whole-mount immunofluorescence of the native FTs and end-point cultures allows thorough investigation of tissue composition and multicellular processes, highlighting the importance of 3D tissue-level characterization for uncovering relevant features of PDR pathophysiology. This model will allow the simultaneous assessment of molecular mechanisms, cellular/tissue processes and treatment responses in the complex context of dynamic biochemical and physical interactions within the PDR tissue architecture and microenvironment. Since this model recapitulates PDR pathophysiology, it will also be amenable for testing or developing new treatments.

Here, we present a protocol to study the pathophysiology of proliferative diabetic retinopathy by using patient-derived, surgically-excised, fibrovascular tissues for three-dimensional native tissue characterization and ex vivo culture. This ex vivo culture model is also amenable for testing or developing new treatments.

نموذج زراعة الأنسجة فيفو السابقين لمضاعفات فيبروفاسكولار في اعتلال الشبكية السكري التكاثري

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No ...