نود أن نشكر موظفي مرفق الأنسجة المشتريات التابعة لجامعة مينيسوتا للحصول على المساعدة في جمع عينات الأنسجة المريضة. وأيد هذا العمل من قبل برنامج بحوث سرطان المبيض وزارة الدفاع (OCRP) OC093424 لMB، من قبل راندي آلة الحلاقة لأبحاث السرطان وصندوق الجماعة لMB، من خلال مينيسوتا سرطان المبيض التحالف لMB والصندوق أمراض النساء الأورام الإدارات لMB. كان الممولين أي دور في تصميم الدراسة وجمع البيانات وتحليلها، قرار نشر أو إعداد المخطوطة.

بيان الأخلاق وتم الحصول على عينات الصلبة من سرطان المبيض من جامعة مينيسوتا مرفق الأنسجة المشتريات (TPF) عقب اجتماع للجنة مجلس المراجعة المؤسساتي: المجلس موضوع الإنسان (IRB) موافقة. 1. إعداد كاشف <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد كاملة DMEM المتوسطة من خلال استكمال DMEM مع FBS 10٪، و 100 وحدات البنسلين الستربتوميسين. تخزين المتوسطة في 4 درجات مئوية والحارة إلى 37 درجة مئوية قبل استخدامها. </li><li style=";text-align:right;direction:rtl"> قسامة dispase الثاني في مجلدات منفصلة من 5-10 مل لتجنب تجميد متعددة يذوب وتخزينها في -20 درجة مئوية في الفريزر خالية من nonfrost. </li></ol> 2. الأنسجة كوكتيل <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> جمع العينات الصلبة من سرطان المبيض (~ 5 ز في الحجم) في وقت الجراحة من المجالات المحددة ظاهريا كما السرطان من قبل الطبيب الشرعي. يتم إلغاء تحديد العينات السريرية ومسجلة وفقا لبروتوكولات المؤسسية. </li><li style=";text-align:right;direction:rtl"> وضع العينات في العقيمة، والشوريغطاء REW، والبولي بروبيلين حاوية العينات مليئة 30 مل من برنامج تلفزيوني الجليد الباردة. نقل العينات من مختبر علم الأمراض الجراحية إلى مختبر أبحاث للتجهيز على الجليد، وخلال 30 دقيقة من جمع العينات (الشكل 1). </li></ol> 3. معالجة الأنسجة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> العمل تحت ظروف معقمة، ونقل العينات على طبق بيتري (60 ملم × 60 ملم) تحتوي على 10 مل من الطازجة، PBS الجليد الباردة وباستخدام شفرة حلاقة معقمة، وقطع الى مزيد من أصغر قطعة ممكنة (2 ملم أو أقل) ( أرقام 2A و 2 ب). </li><li style=";text-align:right;direction:rtl"> نقل الأنسجة المفروم إلى 15 مل أنبوب مخروطي يحتوي على 10 مل من prewarmed (37 درجة مئوية لمدة 30 دقيقة) dispase الثاني (2.4 U / مل) في DMEM واحتضان عند 5٪ CO 2 و 37 درجة مئوية لمدة 30 دقيقة. لضمان الهضم الأمثل للعينات، تستنهض الهمم يدويا الطين خلية كل 5 دقائق. </li><li style=";text-align:right;direction:rtl"> بعد 30 دقيقة الحضانة، ونقل (باستخدام10 مل ماصة المصلية) الطين خلية على مصفاة الخلية (70 ميكرومتر شبكة) وضعت على رأس أنبوب 50 مل المخروطية وتطبيق ضغط لطيف على شبكة باستخدام المكبس المحاقن. تجاهل أي نوع من الأنسجة undissociated (المتبقية على الجزء العلوي من شبكة) وجمع تعليق خلية تم الحصول عليها في 50 مل العقيمة أنبوب مخروطي. أجهزة الطرد المركزي في 320 x ج لمدة 7 دقائق في 4 درجات مئوية (الشكل 3). </li><li style=";text-align:right;direction:rtl"> تجاهل طاف و resuspend بيليه خلية في 10 مل من DMEM تحتوي على 10٪ FBS. </li><li style=";text-align:right;direction:rtl"> احتضان تعليق خلية في طبق بتري في 5٪ CO 2 و 37 درجة مئوية (الشكل 4). </li><li style=";text-align:right;direction:rtl"> تغيير المتوسطة بعد 24 ساعة من الطلاء الأولي. وهذا يسمح لإزالة الحطام الخلوي والغالبية العظمى من كريات الدم الحمراء موجودة في الثقافة. </li><li style=";text-align:right;direction:rtl"> تغيير المتوسطة كل ثلاثة أيام لمدة أسبوعين التالية، وبعد ذلك ثقافات EOC الأولية مستعدون للتطبيقات المصب. </li></ol>

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والكتاب ليس لديهم ما يكشف.

فهم أفضل لمسببات سرطان المبيض والتنمية أمر حاسم لتحسين نتائج النساء المتضررات من هذا المرض المدمر. في هذا السياق، واستخدام المنشأة و&quot;المتاحة تجاريا&quot; لقد كان المبيض خط الخلايا السرطانية بلا شك مفيدة بشكل هائل. ومع ذلك، واليوم نحن نعرف أن خطوط الخلايا السرطانية ل...

على الرغم من الإصابة به منخفضة نسبيا، وسرطان المبيض هو الأكثر دموية من الأمراض النسائية والسبب الرئيسي الخامس للوفيات السرطان بين النساء 1،2. ويرجع ذلك أساسا إلى عدم وجود الأدوات والنماذج التي يمكن الاعتماد عليها ألخص بأمانة بدء وتطور المرض 3 هذا. وكان معظم معرفتنا اليوم عن سرطان المبيض ممكن من خلال استخدام الخلايا خلد المبيض الظهاري السطح (IOSEs)، وخطوط الخلايا السرطانية في المبيض وخلايا سرطان المبيض الأساسي تعافى من الاستسقاء السوائل 4-7. للأسف، واستخدامها لديها العديد من القيود بما في ذلك عدد من التغيرات الجينية والمظهرية المرتبطة عملية تخليد أو في الممرات المختبر، وعدم تجانس السكان الناجمة عن إعداد سائل الاستسقاء. وبالتالي، خلايا سرطان المبيض الأولية المستمدة من العينات الصلبة القابلة للتحديد ومحددة من سرطان المبيض تمثل UNIQUأداة ه لدراسة تطور سرطان المبيض. الصعوبات الرئيسية التي تشارك في الحصول على هذه الخلايا الخبيثة هي نتيجة لفرط نمو الخلايا اللحمية أو الخلايا الليفية جنبا إلى جنب مع فقدان الجدوى وعدم وجود سابقة لأوانها القدرة التكاثري في ثقافة هذه الخلايا EOC. توجد حاليا العديد من الطرق لخلق وحيدة الخلية تعليق من الأورام الصلبة، من خلال وسائل ميكانيكية أو تفارق الأنزيمية، ولكن تقنيات معينة تنتج كمية أكبر من نتائج المفضل 8. هنا، وتبين لنا أن الهضم الأنزيمي مع dispase الثاني النتائج في الانتعاش الفعال، والخلايا الليفية EOC خالية قابلة للحياة. ثقافات EOC حتى الحصول هي عرضة للتلاعب الجيني ومفيدة أيضا في اختبارات فحص المخدرات، مشيرا إلى أن هذه الثقافات EOC هي مناسبة للعديد من التطبيقات المصب.

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			<td height="21" style="height:21px;width:275px;">Name of Material/ Equipment</td>
			<td style="width:125px;">Company</td>
			<td style="width:155px;">Catalog Number</td>
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			<td height="21" style="height:21px;">1X PBS, sterile&#160;</td>
			<td style="width:125px;">Invitrogen</td>
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			<td height="40" style="height:40px;">&#160;Screw lid, polypropylene specimen container, sterile (Thermoscientific)</td>
			<td style="width:125px;">Thermoscientific</td>
			<td style="width:155px;">02 1090</td>
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			<td height="21" style="height:21px;">&#160;DMEM medium, sterile&#160;</td>
			<td style="width:125px;">Invitrogen</td>
			<td style="width:155px;">11995-065</td>
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			<td height="42" style="height:42px;">&#160;Fetal bovine serum, sterile</td>
			<td style="width:125px;">Thermo Scientific Hyclone</td>
			<td style="width:155px;">SH30396.03</td>
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			<td height="40" style="height:40px;">&#160;100x penicillin-streptomycin, liquid (Invitrogen)</td>
			<td style="width:125px;">Invitrogen</td>
			<td style="width:155px;">15140-122</td>
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			<td height="21" style="height:21px;"></td>
			<td style="width:125px;"></td>
			<td style="width:155px;"></td>
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			<td height="21" style="height:21px;">&#160;Dispase II, sterile (Roche)</td>
			<td style="width:125px;">Roche</td>
			<td style="width:155px;">04942 078 001</td>
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			<td height="42" style="height:42px;">&#160;Small fine-tip forceps and scalpel blade, sterile</td>
			<td style="width:125px;">Fisher Scientific</td>
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			<td style="width:155px;"></td>
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			<td height="21" style="height:21px;">&#160;Small dissecting scissors, sterile</td>
			<td style="width:125px;">Fisher Scientific</td>
			<td style="width:155px;">08-945</td>
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			<td height="21" style="height:21px;"></td>
			<td style="width:125px;"></td>
			<td style="width:155px;"></td>
			<td></td>
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			<td height="40" style="height:40px;">&#160;15 ml conical capped tubes, sterile (BD Falcon)</td>
			<td style="width:125px;">BD, Falcon</td>
			<td align="right" style="width:155px;">352097</td>
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			<td style="width:125px;"></td>
			<td style="width:155px;"></td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;">50 ml conical capped tubes, sterile (BD Falcon)</td>
			<td style="width:125px;">BD, Falcon</td>
			<td align="right" style="width:155px;">352027</td>
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			<td height="21" style="height:21px;"></td>
			<td style="width:125px;"></td>
			<td style="width:155px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">&#160;10 ml serological pipette, sterile&#160;</td>
			<td style="width:125px;">Corning</td>
			<td style="width:155px;">13-678-11E</td>
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		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:125px;"></td>
			<td style="width:155px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">6 ml syringe plunger, sterile&#160;</td>
			<td style="width:125px;">Tyco Healthcare</td>
			<td align="right" style="width:155px;">8881516911</td>
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			<td height="21" style="height:21px;"></td>
			<td style="width:125px;"></td>
			<td style="width:155px;"></td>
			<td></td>
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			<td height="40" style="height:40px;">Nylon filter (70 &#956;m ) cell strainer, sterile (BD Falcon)</td>
			<td style="width:125px;">BD Falcon</td>
			<td align="right" style="width:155px;">352350</td>
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		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:125px;"></td>
			<td style="width:155px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">5-cm Petri dish, sterile&#160;</td>
			<td style="width:125px;">Corning</td>
			<td align="right" style="width:155px;">430166</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:125px;"></td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10-cm Petri dish, sterile&#160;</td>
			<td style="width:125px;">Corning</td>
			<td align="right" style="width:155px;">430167</td>
			<td></td>
		</tr>
	</tbody>
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method for obtaining primary ovarian cancer cells from solid specimens

هناك حاجة ماسة إلى أدوات موثوقة للتحقيق في المبيض بدء السرطان والتقدم. في حين أن استخدام خطوط الخلايا السرطانية في المبيض لا يزال يشكل أداة قيمة لفهم سرطان المبيض، واستخدامها يملك الكثير من القيود. وتشمل هذه عدم وجود التجانس وعدد كبير من التعديلات الجينية المرتبطة ممتدة الركض في المختبر. نحن هنا وصف الأسلوب الذي يسمح لسرعة إنشاء خلايا سرطان المبيض الأساسي تشكيل العينات السريرية الصلبة التي تم جمعها في وقت الجراحة. يتكون أسلوب إخضاع العينات السريرية للهضم الأنزيمية لمدة 30 دقيقة. ولا يسمح للتعليق خلية معزولة للنمو ويمكن استخدامها لتطبيق المصب بما في ذلك فحص المخدرات. الاستفادة من خطوط الخلايا السرطانية في المبيض الأولية على خطوط الخلايا السرطانية في المبيض أنشأت هي أنها ممثل العينات السريرية محددة الأصلي يتم استخلاصها من ويمكن الحصول عليها من مواقع مختلفة سواء الوجاهةسرطان المبيض راي أو النقيلي.

يتم جمع العينات السريرية جديدة من سرطان المبيض بعد الجراحة (الشكل 1) ومقطعة لأجزاء صغيرة (أرقام 2A و 2B) تشكل الطين الخلية. وهذا يسمح للتعرض الأمثل للعينات لعلاج الأنزيمية. تتعرض الخلية إلى الطين الهضم الأنزيمي والمحتضنة في 37 درجة مئوية لمدة 30...

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Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

توضح هذه الدراسة طريقة مفصلة لعزل وتوصيف خلايا سرطان المبيض الأولي من العينات السريرية الصلبة. تخضع سرطان المبيض العينات السريرية للهضم الأنزيمية للحصول قابلة للحياة، وخالية من الخلايا الليفية سرطان المبيض الظهاري (EOC) الخلايا مناسبة تماما للتطبيقات المصب.

طريقة الحصول على خلايا سرطان المبيض الابتدائية من العينات الصلبة

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

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13.4K Views.  University of Minnesota.  توضح هذه الدراسة طريقة مفصلة لعزل وتوصيف خلايا سرطان المبيض الأولي من العينات السريرية الصلبة. تخضع سرطان المبيض العينات السريرية للهضم الأنزيمية للحصول قابلة للحياة، وخالية من الخلايا الليفية سرطان المبيض الظهاري (EOC) الخلايا مناسبة تما...

Video: Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients. Thus, novel and reliable methods for evaluating drug efficacy are warranted in pre-clinical phase. In vitro culture of tumor tissues, including cell lines, has substantial phenotypic, genetic, and epigenetic alterations of cancer cells caused by artificial environment of cell culture, which may not reflect the biology of original tumors in situ. Xenograft models with the immunodeficient mice also have limitations, i.e., the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment. Here, we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects. To validate this method, data with the current first-line chemotherapeutic agent, temozolomide (TMZ), are described.


We used the freshly-removed surgical specimen of malignant glioma for our experiments. We performed intratumoral injection of TMZ or other drug candidates, followed by incubation and analysis on surgical specimens. Here, we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti-cancer therapies so that we may be able to overcome, at least, some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient's tumor. This method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.

Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed &#x201C;tumor explant method&#x201D;. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide.

أسلوب الرواية تطوير أدوية لمكافحة السرطان باستخدام Explants ورم العينات الجراحية


The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined ...

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome compared to matched normal tissue. Based on these analyses it is evident that there are many undiscovered genetic drivers of cancer1. Unfortunately these drivers are hidden within a much larger number of passenger anomalies in the genome that do not directly contribute to tumor formation. Another aspect of the cancer genome is that there is considerable genetic heterogeneity within similar tumor types. Each tumor can harbor different mutations that provide a selective advantage for tumor formation2. Performing an unbiased forward genetic screen in mice provides the tools to generate tumors and analyze their genetic composition, while reducing the background of passenger mutations. The Sleeping Beauty (SB) transposon system is one such method3. The SB system utilizes mobile vectors (transposons) that can be inserted throughout the genome by the transposase enzyme. Mutations are limited to a specific cell type through the use of a conditional transposase allele that is activated by Cre Recombinase. Many mouse lines exist that express Cre Recombinase in specific tissues. By crossing one of these lines to the conditional transposase allele (e.g. Lox-stop-Lox-SB11), the SB system is activated only in cells that express Cre Recombinase. The Cre Recombinase will excise a stop cassette that blocks expression of the transposase allele, thereby activating transposon mutagenesis within the designated cell type. An SB screen is initiated by breeding three strains of transgenic mice so that the experimental mice carry a conditional transposase allele, a concatamer of transposons, and a tissue-specific Cre Recombinase allele. These mice are allowed to age until tumors form and they become moribund. The mice are then necropsied and genomic DNA is isolated from the tumors. Next, the genomic DNA is subjected to linker-mediated-PCR (LM-PCR) that results in amplification of genomic loci containing an SB transposon. LM-PCR performed on a single tumor will result in hundreds of distinct amplicons representing the hundreds of genomic loci containing transposon insertions in a single tumor4. The transposon insertions in all tumors are analyzed and common insertion sites (CISs) are identified using an appropriate statistical method5. Genes within the CIS are highly likely to be oncogenes or tumor suppressor genes, and are considered candidate cancer genes. The advantages of using the SB system to identify candidate cancer genes are: 1) the transposon can easily be located in the genome because its sequence is known, 2) transposition can be directed to almost any cell type and 3) the transposon is capable of introducing both gain- and loss-of-function mutations6. The following protocol describes how to devise and execute a forward genetic screen using the SB transposon system to identify candidate cancer genes (Figure 1).

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

A method of identifying unknown drivers of carcinogenesis using an unbiased approach is described. The method uses the Sleeping Beauty transposon as a random mutagen directed to specific tissues. Genomic mapping of transposon insertions that drive tumor formation identifies novel oncogenes and tumor suppressor genes

تحديد<em&gt; الجمال النائم</em&gt; الإدراج ينقول في الأورام الصلبة باستخدام رابط بوساطة PCR

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

الوقت الفاصل بين التصوير من الخلايا الثديية سابق للورم ظهاري الأولية المستمدة من نماذج الماوس المعدلة وراثيا لسرطان الثدي

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

A Sensitive Method to Quantify Senescent Cancer Cells

Human cells do not indefinitely proliferate. Upon external and/or intrinsic cues, cells might die or enter a stable cell cycle arrest called senescence. Several cellular mechanisms, such as telomere shortening and abnormal expression of mitogenic oncogenes, have been shown to cause senescence. Senescence is not restricted to normal cells; cancer cells have also been reported to senesce.
	Chemotherapeutical drugs have been shown to induce senescence in cancer cells. However, it remains controversial whether senescence prevents or promotes tumorigenesis. As it might eventually be patient-specific, a rapid and sensitive method to assess senescence in cancer cell will soon be required.
	To this end, the standard &beta;-galactosidase assay, the currently used method, presents major drawbacks: it is time consuming and not sensitive. We propose here a flow cytometry-based assay to study senescence on live cells. This assay offers the advantage of being rapid, sensitive, and can be coupled to the immunolabeling of various cellular markers.

Whether senescence prevents or promotes tumorigenesis remains controversial. Since chemotherapeutical drugs can induce cancer cells to senesce, studying senescence is essential for proposing new therapies. However, the standard and broadly used &beta;-galactosidase assay presents major drawbacks. We propose here a rapid and sensitive flow cytometry-based assay to quantify senescence.

طريقة حساسة لقياس خلايا السرطان هرمة

Human cells do not indefinitely proliferate. Upon external and/or intrinsic  cues, cells might die or enter a stable cell cycle arrest called senescence. ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

عزل الخلايا الجذعية للسرطان من العينات البشرية سرطان البروستاتا

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell culture systems arising from human endometrial samples are no exception. Applications range from normal cyclic physiological processes to endometrial pathologies such as gynecological cancers, infectious diseases, and reproductive deficiencies. Here, we provide two methods for establishing primary endometrial stromal cells from surgically resected endometrial hysterectomy specimens. The first method is referred to as &#8220;the scraping method&#8221; and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed &#8220;the trypsin method.&#8221; This latter method uses the enzymatic activity of trypsin to promote the separation of cells and primary cell outgrowth. We illustrate step-by-step methodology through digital images and microscopy. We also provide examples for validating endometrial stromal cell lines via quantitative real time polymerase chain reactions (qPCR) and immunofluorescence (IF).

Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human patients.&#160;

طريقتين لإنشاء أولي خلايا وانسجة بطانة الرحم الإنسان من استئصال الرحم العينات

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

تقييم سرطان المبيض كروي مرفق وغزو الخلايا الظهارية في الوقت الحقيقي

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of care consisting of surgical debulking and combination chemotherapy consisting of platinum and taxane compounds, almost 90% of patients recur within a few years. In these patients the development of chemoresistant disease limits the efficacy of currently available chemotherapy agents and therefore contributes to the high mortality. To discover novel therapy options that can target recurrent disease, appropriate animal models that closely mimic the clinical profile of patients with recurrent ovarian cancer are required. The challenge in monitoring intra-peritoneal (i.p.) disease limits the use of i.p. models and thus most xenografts are established subcutaneously. We have developed a sensitive optical imaging platform that allows the detection and anatomical location of i.p. tumor mass. The platform includes the use of optical reporters that extend from the visible light range to near infrared, which in combination with 2-dimensional X-ray co-registration can provide anatomical location of molecular signals. Detection is significantly improved by the use of a rotation system that drives the animal to multiple angular positions for 360 degree imaging, allowing the identification of tumors that are not visible in single orientation. This platform provides a unique model to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention or treatment of recurrent ovarian cancer.

We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer.

نموذج الفئران التصوير غير الغازية لكشف ورصد سرطان المبيض تكرار

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

لتخصيب Chemoresistant سرطان المبيض الخلايا الجذعية من خطوط الخلايا البشرية

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.

In-depth analyses of cancer cell proteomes facilitate identification of novel drug targets and diagnostic biomarkers. We describe an experimental workflow for quantitative analysis of (phospho-)proteomes in cancer cell subpopulations derived from liquid and solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry.

الكمي قداس الطيفية من التنميط Proteomes خلايا السرطان المستمدة من السائل والأورام الصلبة

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic ...