وقد تم تمويل هذا البحث من قبل دولة بانكهد-كولي الفريق العلمي منحة ولاية فلوريدا (2BT03)، والمعاهد الوطنية لل/ المعهد الوطني للسرطان الصحة (1R21CA164322-01) ومركز موفيت للسرطان علوم فريق غرانت. وقد تم دعم هذا العمل في جزء من مرفق بالحركة البحوث الأساسية في لي موفيت للسرطان ومعهد بحوث H.، وهو مركز السرطان الشامل NCI المعينة (P30-CA076292). تم الحصول على الخلايا الأولية ممكن من خلال بروتوكول رعاية مرضى السرطان إجمالي في مركز موفيت للسرطان.

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P., 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=A+preclinical+assay+for+chemosensitivity+in+multiple+myeloma.">A preclinical assay for chemosensitivity in multiple myeloma.</a> Cancer Res. 74, 56-67 (2014).</li><li>Misund, K., 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=A+method+for+measurement+of+drug+sensitivity+of+myeloma+cells+co-cultured+with+bone+marrow+stromal+cells.">A method for measurement of drug sensitivity of myeloma cells co-cultured with bone marrow stromal cells.</a> J Biomol Screen. 18, 637-646 (2013).</li><li>Ramasamy, K., 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=Fluorescence-based+experimental+model+to+evaluate+the+concomitant+effect+of+drugs+on+the+tumour+microenvironment+and+cancer+cells.">Fluorescence-based experimental model to evaluate the concomitant effect of drugs on the tumour microenvironment and cancer cells.</a> Br J Haematol. 157, 564-579 (2012).</li><li>McMillin, D. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+bone+marrow+plasma+cell+infiltrates+in+multiple+myeloma:+the+added+value+of+CD138+immunohistochemistry.">Assessment of bone marrow plasma cell infiltrates in multiple myeloma: the added value of CD138 immunohistochemistry.</a> Hum Pathol. 38, 1779-1787 (2007).</li><li>Najar, 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=Mesenchymal+stromal+cells+promote+or+suppress+the+proliferation+of+T+lymphocytes+from+cord+blood+and+peripheral+blood:+the+importance+of+low+cell+ratio+and+role+of+interleukin-6.">Mesenchymal stromal cells promote or suppress the proliferation of T lymphocytes from cord blood and peripheral blood: the importance of low cell ratio and role of interleukin-6.</a> Cytotherapy. 11, 570-583 (2009).</li><li>Scott, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+i+trialist.">Phase i trialist.</a> Lancet Oncol. 13, 236 (2012).</li></ol>

The authors have no conflict of interest to disclose.

باختصار، هذا هو وسيلة قوية وعالية الإنتاجية لتحديد chemosensitivity الخلايا MM الابتدائية وخارج الحي الدوائية للأدوية في إعادة بناء نخاع العظم. الخطوات الحاسمة في هذا البروتوكول هي بذر السليم للآبار وتركز كافية (انظر الشكل 2 للنتائج المتوقعة): تأكد من أن الخلاي...

والهدف من هذه الطريقة هو أن تميز حساسية العقاقير من المايلوما المتعددة (MM) الخلايا الأولية إلى لجنة من وكلاء خارج الحي، في أقرب وقت ممكن إلى الظروف الفسيولوجية، وبدقة كافية أن هذه النتائج يمكن استخدامها لتحديد chemoresistant الفرعية السكان داخل عبء الورم و، في نهاية المطاف، إلى بالحدود نماذج حسابية تهدف إلى تقدير استجابة سريرية. النماذج الحسابية هي أدوات قوية لتحليل النظم المعقدة، مثل التفاعلات السرطان في استضافة العلاج. ومع ذلك، ليست سوى نماذج جيدة مثل البيانات المستخدمة لبالحدود لهم. لسوء الحظ، فإن معظم البيانات المتاحة في الأدب لا يمكن استخدامها في شكلها الحالي لبالحدود مثل هذه النماذج، لأنها غالبا ما يتم الحصول عليها في ظروف تجريبية يستبعد بعضها بعضا. وهكذا، غالبا ما يطلب من التجارب الجديدة وذلك بهدف الحصول على مجموعة من المعلمات التجريبية الحاجة. معظم فحوصات جدوى، ومع ذلك، هي مدمرة واللنا تقتصر على عدد قليل من النقاط الزمنية، في كثير من الأحيان واحدة فقط. هذا يعيق كثيرا من استخدام المقايسات chemosensitivity إلى بالحدود النماذج الحسابية، لأن مثل هذه التجارب تفشل في توفير معلومات عن ديناميات الزمنية للنظام. هذا ينطبق بشكل خاص مع الخلايا السرطانية الأولية، والتي غالبا ما تقتصر على بضعة ملايين في العينة، ولها عمر قصير بعد الخزعة، مما يحد من عدد من الظروف التجريبية المتاحة هذا. وبالإضافة إلى ذلك، فإن معظم فحوصات الجدوى تتطلب فصل من السرطان من غير السرطانية (سدى) الخلايا في وقت الكمي، وهو ما يضيف العمل الإضافي للبروتوكول، مما يشوش على الخلايا، ويحد من عدد من التجارب التي يمكن القيام بها . قبل 40 عاما، سمك السلمون والمتعاونين 1 اقترح الشهيرة في المختبر تشكيل مستعمرة الفحص لتقييم chemosensitivity الخلايا السرطانية. للأسف وجدت هذا الاختبار متفاوتة من النجاح نظرا لقلة عدد MYE متعددةLOMA (MM) عينات المريض التي كانت قادرة على تشكيل مستعمرات تحت ظروف التحكم: وتشير التقديرات إلى أنه ليس هناك سوى مجموعة فرعية صغيرة من 0.001٪ إلى 0.1٪ من خلايا MM كانت قادرة على تكرارها، ويجري توصف بأنها خلايا الجذعية المايلوما المتعددة 2. هذه محدودة إلى حد كبير من نسبة نجاح الفحص وعدد من الأدوية التي يمكن اختبارها في وقت واحد، حتى في أكثر النماذج الأخيرة 3. هذه المسألة هي أكثر أهمية بكثير الآن عندما كلاء MM (معيار أو تجريبي) هم أكثر عددا مما كانت عليه قبل أربعة عقود. وجود قيود كبير من هذه المقايسات المبكرة انتاجهم إنقسم: إما المريض &quot;حساسة&quot; أو &quot;مقاومة&quot; لدواء معين. يتم توفير أي معلومات بشأن درجة من الحساسية وعدم تجانس السكان الورم. وبالتالي، سيتم تصنيف المرضى الذين يعانون من جماعات فرعية صغيرة من الخلايا المقاومة سريعة النمو بأنها &quot;حساسة&quot; للعلاج، ولكن الانتكاس قريبا، وبالتالي لا بنefit من العلاج. ونظرا لأهمية مدة الاستجابة في البقاء على قيد الحياة (OS) من مرضى السرطان 4،5، فمن الواضح كيف كانت هذه المقايسات غير قادرة على تقدير OS صحيح على الدوام. من أجل الالتفاف على هذه القيود، وتكون قادرة على توليد نماذج حسابية المريض محددة من شأنها أن تقدير استجابة سريرية شخصية إلى لجنة المخدرات، قمنا بتطوير طريقة لحساسية غير المدمر اختبار المخدرات من المايلوما المتعددة (MM) خطوط الخلايا والخلايا MM الأولية في إعادة الإعمار خارج الحي من نخاع العظام المكروية، بما في ذلك المصفوفة خارج الخلية وسدى 6. هذا الاختبار، ومع ذلك، كان الحد من الاعتماد على شريحة معينة ميكروفلويديك التجارية، أبعادها وتكلفة تقييد عدد من التجارب أو الأدوية التي يمكن أن يؤديها في وقت واحد في قطعة معينة من المعدات. النظام هنا وصف يمتد هذا الاختبار الأصلي إلى أعلى-throughput عضوي النمط منصة الاستجابة للجرعة، لفي المختبر فحص المخدرات، استنادا إلى خوارزمية تحليل الصور الرقمية لغير المدمر تحديد بقاء الخلية. كل بئر في 384 أو 1536-جيدا لوحات هي إعادة الإعمار 3D من نخاع العظام المكروية، بما في ذلك الخلايا الأولية MM، المصفوفة خارج الخلية، وسدى ونمو العوامل المستمدة من المريض. يتم استخدام المجهر الحية وتحليل الصور الرقمية للكشف عن أحداث موت الخلايا في تركيزات المخدرات المختلفة، والتي تستخدم لتوليد السطوح الاستجابة للجرعة. من البيانات في المختبر، ويحدد نموذج رياضي حجم وchemosensitivity السكان فرعية ضمن عبء ورم المريض، ويمكن استخدامها لمحاكاة كيفية الورم سوف يستجيب للدواء (ق) في الظروف الفسيولوجية في نظام السريري 6 ( الشكل 1). الابتكارات الرئيسية من هذه المنصة هي: (أ) عدد قليل من الخلايا السرطانية المطلوبة (1،000-10،000 في تناسق المخدراتالتموينية)؛ (ب) تقييم نجاعة الأدوية في الظروف الفسيولوجية (المصفوفة خارج الخلية، سدى، وعوامل النمو المشتق المريض)؛ يستخدم (ج) لا سمية من علامات الجدوى 7 منذ التصوير حقل مشرق فقط، وبالتالي لا حاجة ل transfect الخلايا مع مضان 8 أو 9 تلألؤ بيولوجي. (د) على التصوير المستمر تأثير المخدرات بوصفها وظيفة من التركيز ومدة التعرض (الدوائية)؛ و (ه) التكامل بين في المختبر ونماذج تطورية الحسابية، لتقدير نتائج سريرية 6 (سيلفا وآخرون، قيد الإعداد). العامل الرئيسي الذي يسمح للخوارزمية تحليل الصور الرقمية لتبين السرطان من الخلايا اللحمية هو تقارب مختلف بهم إلى قاع البئر: خلايا MM لا تلتزم، ويبقى معلق في المصفوفة، في حين أن الخلايا اللحمية تلتزم الجزء السفلي من جيدا ثم تمتد. يحدث هذا الفصل على الرغم من MM وسدى لإعادة المصنف في نفس الوقت، ويحدث أثناء O / N عملية الحضانة. يسرع البذر الغزل عليها في أجهزة الطرد المركزي التالي من لوحة مزيد من هذه العملية. ونتيجة لذلك، تظهر خلايا MM في الصورة كأقراص مشرق مستديرة تحيط بها حلقة مظلمة، في حين يحتفظ سدى عن الانظار وأغمق (الشكل 2). وهكذا، فإن فحص في شكله الحالي هو الانسب لخلايا السرطان غير ملتصقة، على الرغم من التعديلات في الخوارزمية يمكن القيام به لسرطان منفصل وسدى على أساس الخصائص الشكلية الأخرى، مثل الحجم والشكل. في هذا البروتوكول وصفنا نوعين من المصفوفة خارج الخلية (الكولاجين والطابق السفلي مصفوفة الغشاء)، فضلا عن نوعين من المشترك الثقافات، مع (نخاع المستمدة الخلايا الجذعية الوسيطة العظام) BMSC وHUVECS، التي تمثل منافذ فراغي وحول الأوعية من نخاع العظام ، على التوالي. باستخدام لوحة 384 جيدا، 5 تركيزات في المخدرات واثنين من مكررات، كنا قادرين على قسم التدريب والامتحاناتتي تصل إلى 31 الأدوية المختلفة مع عينة من مريض واحد. في لوحات 1536 جيدا هذا الرقم 127. الشكل 3 يصور تخطيط المستخدمة حاليا اليدوي للزرع الخلايا، في حين يمثل التكميلي الشكل 1 التوزيع الأمثل باستخدام pipettor الروبوتية. في التصميم من الشكل 3، يمكن لكل لوحة 384 جيدا تحمل 3 عينات المريض مع 7 الأدوية المختلفة، أو عينة واحدة المرضى اختبار مع 21 المخدرات. يتم تمثيل كل دواء بنسبة 5 تركيزات، والمصنف كل شرط في التكرارات. والمصنف 4 آبار المراقبة (مضاف المخدرات) لكل مجموعة من 7 المخدرات (على سبيل المثال، الآبار 36-39، 126-129 و200-203). لضمان فاعلية من الأدوية المستخدمة، خط خلية الورم النخاعي الإنسان (على سبيل المثال، H929 أو MM1.S) هو أيضا المصنف، وتخديره في التكرارات على أعلى تركيز كل من العقاقير المستخدمة (الآبار 76-89). مراقبة خط الخلية إيجابية يضمن أن العقاقير المستخدمة لديها قوة كافية، منذ منحنيات الاستجابة للجرعة هي المتردد مقارنةتجارب روس. هي المصنفة بئرين مع خط خلية الورم النقوي عن السيطرة للظروف البيئية، وبعبارة أخرى، للكشف عن المشاكل المحتملة في مقاعد البدلاء كبار حاضنة أثناء التصوير (الآبار 75 و 90). تعداد الآبار يتبع نمطا متعرج من أجل تقليل المسافة التي مرحلة الآلية المجهر، مما يقلل من اكتساب الوقت ويقلل من التآكل من المعدات المشمولة.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>EVOS FL AUTO</td>
			<td>AMG</td>
			<td>AMAFD1000 + AMC1000</td>
			<td>Digital microscope equipped with motorized stage and bench top incubator.</td>
		</tr>
		<tr>
			<td>384-well plate CellBind</td>
			<td>CORNING</td>
			<td>CLS3683 SIGMA</td>
			<td>Corning CellBIND 384 well plates 
			384 well plate, polystyrene, CellBIND surface, sterile, clear flat bottom, black, w/lid</td>
		</tr>
		<tr>
			<td>1,536-well plate CellBind</td>
			<td>CORNING</td>
			<td>CLS3832 ALDRICH</td>
			<td>Corning 1536 well plates, low base, surface treatment CellBIND, sterile</td>
		</tr>
		<tr>
			<td>Ficoll-Paque Plus&#160;</td>
			<td>GE Healthcare</td>
			<td>GE17-1440-02 SIGMA</td>
			<td>For in vitro isolation of lymphocytes from human peripheral blood.</td>
		</tr>
		<tr>
			<td>CD138 magnetic beads</td>
			<td>Miltenyi&#160;</td>
			<td>130-051-301</td>
			<td>Antibody conjugated magnetic beads for selection of CD138+ cells.</td>
		</tr>
		<tr>
			<td>LS columns</td>
			<td>Miltenyi&#160;</td>
			<td>&#160;130-042-401</td>
			<td>Column for separation of cells.</td>
		</tr>
		<tr>
			<td>MidiMACS Separator</td>
			<td>Miltenyi&#160;</td>
			<td>130-042-302</td>
			<td>Magnet for separation column.</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Sigma-Aldrich</td>
			<td>E6909 SIGMA</td>
			<td>ECM Gel from Engelbreth-Holm-Swarm murine sarcoma</td>
		</tr>
		<tr>
			<td>Getrex</td>
			<td>Life Technologies</td>
			<td>A1413201</td>
			<td>LDEV-Free Reduced Growth Factor Basement Membrane Matrix</td>
		</tr>
		<tr>
			<td>HUVEC</td>
			<td>Life Technologies</td>
			<td>C-003-25P-B</td>
			<td>Human Umbilical Vein Endothelial Cells (HUVEC )</td>
		</tr>
		<tr>
			<td>RPMI-1640 media</td>
			<td>GIBCO</td>
			<td>11875-093</td>
			<td>RPMI 1640 Medium + L-Glutamine + Phenol Red</td>
		</tr>
		<tr>
			<td>FBS Heat inactivated</td>
			<td>GIBCO</td>
			<td>10082-147</td>
			<td>Fetal Bovine Serum, certified, heat inactivated, US origin</td>
		</tr>
		<tr>
			<td>3.1mg/mL Bovine collagen type I</td>
			<td>Advanced Biomatrix</td>
			<td>5005-B</td>
			<td>Bovine Collagen Solution, Type I, 3 mg/ml, 100 ml</td>
		</tr>
		<tr>
			<td>Precision XS</td>
			<td>BIOTEK</td>
			<td>PRC3841</td>
			<td>Robotic pipettor system</td>
		</tr>
	</tbody>
</table>

an organotypic high throughput system for characterization of drug sensitivity of primary multiple myeloma cells

في هذا العمل وصفنا نهج الرواية التي تجمع بين خارج الجسم الحي المقايسات حساسية العقاقير وتحليل الصور الرقمية لتقدير chemosensitivity وتجانس المايلوما المتعددة (MM) الخلايا المشتقة من المريض. هذا النهج يتمثل في بذر خلايا MM الأولية المستخرجة حديثا من شفاطات نخاع العظم في غرف ميكروفلويديك تنفيذها في لوحات متعددة جيدا، يتألف كل منها من إعادة بناء المكروية نخاع العظم، بما في ذلك المصفوفة خارج الخلية (الكولاجين أو الغشاء القاعدي المصفوفة) وسدى (patient- الخلايا المشتقة الجذعية الوسيطة) أو الخلايا البطانية المستمدة البشرية (HUVECs). يتم تخديره غرف مع وكلاء وتركيزات مختلفة، ويتم تصوير متتالي لمدة 96 ساعة من خلال مشرق المجهري الميدان، في المجهر الآلية مزودة بكاميرا رقمية. الرقمي صورة برامج التحليل بالكشف عن الخلايا الحية والميتة من وجود أو عدم وجود حركة الغشاء، ويولد منحنيات التغير في جدوى بوصفها وظيفة سو تركيز الدواء ومدة التعرض. نستخدم النموذج الحسابي لتحديد معالم chemosensitivity من السكان الورم إلى كل دواء، فضلا عن عدد من السكان الفرعي الحالي كمقياس للورم عدم التجانس. ويمكن بعد هذه النماذج مصممة خصيصا المريض استخدامها لمحاكاة الأنظمة العلاجية وتقدير الاستجابة السريرية.

يوصف تدفق التجربة لفترة وجيزة في الشكل 1 إذا تم الانتهاء من جميع الخطوات بنجاح، يجب أن الصور التي حصلت عليها المجهر معادلا إلى الشكل 2: يعيش خلايا MM ينبغي النظر بوضوح الأقراص كما مشرق وBMSCs أو HUVECs ينبغي أن يكون بالكاد مرئية وتوزع بشكل جيد في الخلفية. كم...

Watch this Scientific Journal Video about An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells at JoVE.com

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.

نظام عضوي إنتاجية عالية لتوصيف الدواء حساسية خلايا المايلوما المتعددة الابتدائية

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and ...

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8.5K Views.  H. Lee Moffitt Cancer Center and Research Institute. We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.

Video: An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7.
Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8.
For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.

The organotypic brain slice coculture with carcinoma cells enables visualizing morphological changes by fluorescence as well as bright field (video) microscopy during the process of carcinoma cell invasion of brain tissue. This model system also allows for cell exchange and replenishment approaches and offers a wide variety of manipulations and analyses.

نظام Coculture مع عضوي النمط شريحة الدماغ و 3D كروي من خلايا سرطان

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in ...

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). 
Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. 
Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system.
Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. 
This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

عالية الإنتاجية توصيف الخلايا الجذعية البالغة المهندسة لتسليم العوامل العلاجية لاستراتيجيات اعصاب

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

إنشاء طعم أجنبي نموذج المايلوما المتعددة الإنسان في الدجاج لدراسة نمو الورم والغزو والأوعية الدموية

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

ورم في المخ / شريحة عضوي النمط المشارك ثقافة النظام لدراسة ورم المكروية والمستهدفة المخدرات العلاجات

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 &#181;m, C8 100 &#197;; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

أسلوب LC-MS/MS الأمثل لتحليل العينات السريرية إيفاكافتور، والايضات الرئيسية، ولوماكافتور في السوائل البيولوجية لمرضى التليف الكيسي الفائق

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

تحسين أسلوب لإنشاء في المختبر حاجز الدم – المخ نموذج استناداً إلى خلايا المخ الخنزيري بطانية

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

عزل خلايا Decidual البشرية الأولية من الأغشية الجنينية لمصطلح مترافق

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...

High-Throughput Analysis of Optical Mapping Data Using ElectroMap

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we present, in a step-by-step guide, the use of ElectroMap for analysis, quantification, and mapping of high-resolution voltage and calcium datasets acquired by optical mapping. ElectroMap analysis options cover a wide variety of key electrophysiological parameters, and the graphical user interface allows straightforward modification of pre-processing and parameter definitions, making ElectroMap applicable to a wide range of experimental models. We show how built-in pacing frequency detection and signal segmentation allows high-throughput analysis of entire experimental recordings, acute responses, and single beat-to-beat variability. Additionally, ElectroMap incorporates automated multi-beat averaging to improve signal quality of noisy datasets, and here we demonstrate how this feature can help elucidate electrophysiological changes that might otherwise go undetected when using single beat analysis. Custom modules are included within the software for detailed investigation of conduction, single file analysis, and alternans, as demonstrated here. This software platform can be used to enable and accelerate the processing, analysis, and mapping of complex cardiac electrophysiology.

This protocol describes the setup and use of ElectroMap, a MATLAB-based open-source software platform for analysis of cardiac optical mapping data. ElectroMap provides a versatile high-throughput tool for analysis of optical mapping voltage and calcium datasets across a wide range of cardiac experimental models.

تحليل عالي الإنتاجية لبيانات الخرائط البصرية باستخدام الخريطة الكهربائية

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we ...

A High-Throughput Electrochemiluminescence 7-Plex Assay Simultaneously Screening for Type 1 Diabetes and Multiple Autoimmune Diseases

Islet autoantibodies (IAbs) are widely used in type 1 diabetes (T1D) diagnosis and prediction. Four major IAbs to insulin (IAA), glutamate decarboxylase-65 (GADA), insulinoma antigen-2 (IA-2A), and zinc transporter-8 (ZnT8A) are equally important in disease prediction. Presently, up to 40% of patients diagnosed with T1D go on to develop other autoimmune disorders. Unfortunately, current screening methods using a single autoantibody for measurement are laborious and inefficient for large scale screening studies. We recently developed a simple multiplexed electrochemiluminescence (ECL) assay to address these current issues. The assay combines all 7 autoantibody tests into one well. Each well includes three IAbs (IAA, GADA, and IA-2A), autoantibodies to thyroid peroxidase (TPOA) and thyroid globulin (ThGA) to detect autoimmune thyroid disease, autoantibodies to tissue transglutaminase (TGA) for celiac disease, and autoantibodies to interferon alpha (IFN&#945;A) for autoimmune polyglandular syndrome-1 (APS-1); all of which screen for T1D and other relevant autoimmune diseases, simultaneously. The multiplex ECL assay is based on the single ECL assay platform, but instead uses the multiplex plate combining multiple autoantibody assays, up to 10, into a single well. The main difference from the single ECL assay is that each antibody-antigen complex formed in the fluid-phase is restrained onto a specific spot on each well through a linker system on the multiplex plate. The 7-Plex ECL assay, in the present study, is validated against standard radio-binding assays (RBA) and single ECL assays, using a large cohort of newly diagnosed T1D patients and age-matched healthy controls, resulting in excellent assay sensitivity and specificity.

We model a simple multiplexed ECL assay that combines 7 autoantibody assays together. The assay is capable of screening for T1D and multiple other autoimmune diseases, simultaneously, including celiac disease, autoimmune thyroid disease, and autoimmune polyglandular syndrome 1.

فحص التلألؤ الكهروكيميائي عالي الإنتاجية 7-Plex في وقت واحد لمرض السكري من النوع 1 وأمراض المناعة الذاتية المتعددة

Islet autoantibodies (IAbs) are widely used in type 1 diabetes (T1D) diagnosis and prediction. Four major IAbs to insulin (IAA), glutamate ...

Characterization of a Novel Human Organotypic Retinal Culture Technique

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal pigment epithelium-choroid (RPE-choroid) and sclera, the integrity of the fragile retina can easily be compromised. The aim of this study was to develop a novel HORC model that contains the retina, RPE-choroid and sclera to maintain retinal integrity when culturing retinal explants.
After cutting circumferentially along the limbus to remove iris and lens, four deep incisions were made to flatten the eyecup. In contrast to previous HORC protocols, a trephine was used to cut through not only the retina but also the RPE-choroid and sclera. The resultant triple-layered explants were cultured for 72 h. Hematoxylin and Eosin staining (H&#38;E) was used to assess anatomical structures and retinal explants were further characterized by immunohistochemistry (IHC) for apoptosis, M&#252;ller cell integrity and retinal inflammation. To confirm the possibility of disease induction, explants were exposed to high glucose (HG) and pro-inflammatory cytokines (Cyt), to mimic diabetic retinopathy (DR). The Luminex magnetic bead assay was used to measure DR-related cytokines released into the culture medium.
H&#38;E staining revealed distinct retinal lamellae and compact nuclei in retinal explants with the underlying RPE-choroid and sclera, while retinas without the underlying structures exhibited reduced thickness and severe nuclei loss. IHC results indicated absence of apoptosis and retinal inflammation as well as preserved M&#252;ller cell integrity. The Luminex&#160;assays showed significantly increased secretion of DR-associated pro-inflammatory cytokines in retinal explants exposed to HG + Cyt relative to baseline levels at 24 h.
We successfully developed and characterized a novel HORC protocol in which retinal integrity was preserved without apoptosis or retinal inflammation. Moreover, the induced secretion of DR-associated pro-inflammatory biomarkers when exposing retinal explants to HG + Cyt suggests that this model could be used for clinically translatable retinal disease studies.

This study aims to develop a novel human organotypic retinal culture (HORC) model that prevents compromising retinal integrity during explant handling. This is achieved by culturing the retina with the overlying vitreous and the underlying retinal pigment&#160;epithelium-choroid (RPE-choroid) and sclera.

توصيف رواية الإنسان Organotypic تقنية ثقافة الشبكية

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal ...