This work was funded by a Natural Sciences and Engineering Research Council (NSERC) Canada grant to MGM. JR is a NSERC-Vanier student fellow.

The authors have nothing to disclose.

الخطوة الأكثر أهمية في أي نظام تجربة إدراج ثقافة مشتركة يسكن فعلا في اختيار إدراج المناسب لاستخدامها. يجب أن تؤخذ حجم المسام والمواد غشاء بعين الاعتبار شامل، دون أن ننسى أن تنظر في نوع من الخلايا التي سيتم المصنف، والغرض من هذه التجربة. على سبيل المثال، قد المقايسات c...

دراسة الأنسجة والأعضاء أو الأنظمة في المختبر هي محاولة لتبسيط العلاقات المعقدة جدا القائمة بين عدة أنواع فرعية الخلوية التي تضم الكائنات متعددة الخلايا. في الواقع، في الدراسات المختبرية تجعل من الممكن الحصول على فهم مفصل للسكان خلية واحدة. هناك نوعان من المزايا الرئيسية لإجراء التجارب في المختبر: 1) انخفاض التفاعلات الخلوية، و2) القدرة على التعامل بسهولة البيئة الخلوية. العلماء ومن هنا، سمحت هذه المزايا اثنين للتنبؤ السلوك من أنواع معينة من الخلايا في الجسم الحي، مما أدى إلى القدرة على تنظيم النتائج من التأثيرات خارجي في الكائنات كلها. في هذا المعنى، في المختبر زراعة الخلايا غالبا ما يعمل كجسر يربط علوم الحياة الأساسية والتطبيقية. ومع ذلك، هناك أيضا العديد من عيوب العمل في المختبر، واحد أهمها أن تحفظ معين قد يسكن في فيزيولوجيآل أهمية الظواهر المرصودة. في الواقع، عندما يزرع نوع خلية واحدة في وعاء، تفقد الثقافة، إلى حد مختلف، وصلات خلية خلية مع أنواع الخلايا الأخرى، ومساهمتها في البيئة السائلة من الأنسجة والكائنات الأصلية، والمراسي داخل النسيج الذي مكنها من التمسك هيكل ثلاثي الأبعاد خاص حاسما في بعض الأحيان من أجل وظيفة الخلية. وقد تم تناول مسألة العلاقات خلية خلية من خلال تطوير تقنيات زراعة المختلطة. في هذه الطريقة، وتزرع اثنين أو أكثر من السكان الخلية معا في سفينة الثقافة نفسها. ومع ذلك، هذه الثقافات مختلطة تحمل المضايقات الهامة. من جهة، وبعض أنواع فرعية الخلية لا تتفاعل جسديا مع بعضها البعض في الأنسجة المنشأ والاعتماد فقط على الاتصالات نظير الصماوي التي لحقت العوامل القابلة للذوبان يفرز ومستقبلات المجاورة. هذا هو الحال لعدة عمليات الالتهابات التي تعتمد على إشارات خلوى القريبة. في ج مختلطةultures، والتفاعلات المادية التي لا يمكن تجنبها وتجعل من المستحيل لدراسة الاتصالات نظير الصماوي في غياب الاتصالات خلية خلية التي يمكن أن تسفر عن نتائج المتغيرة. من ناحية أخرى، وتحقيق التفسيرات خلايا محددة من داخل الخليط السكاني يصبح غير مجد دون استخدام تقنيات الفصل القاسية التي يمكن أن تؤثر بشكل كبير على النتائج. لحل هذه القضايا الهامة، وقد تم تطوير استخدام وسائل الإعلام مشروطة كأسلوب السماح للثقافات مجزأة ودراسة الإشارات نظير الصماوي. يتطلب هذا الأسلوب نقل طاف من نوع خلية واحدة، وبالتالي اسمه مكيفة المتوسطة، إلى الآبار التي تحتوي على مجموعة أخرى من الخلايا. ومع ذلك، ولكن هناك عائقا مهما هو أن جزيئات قصيرة الأجل لا البقاء على قيد الحياة لفترة كافية في المتوسط ​​مكيفة على أن يتم تحويلها إلى آبار السكان الثاني من الخلايا. سيتم حتى جزيئات طويلة العمر تضعف إلى حد كبير مع مرور الوقت بسبب نشرها. وعلاوة على ذلك، سواء خليةالسكان يشارك فقط في مجال الاتصالات نظير الصماوي أحادي الاتجاه وليس في اتصال ثنائي الاتجاه النشط. وهذا يقود إلى عدم وجود إشارات ردود الفعل التي أمر حيوي في إعادة العلاقات متعددة الخلايا دقيقة لأنها موجودة في الجسم الحي. ونتيجة لذلك وبدافع الحاجة لمحاكاة أفضل الأصلي في ظروف المجراة في المختبر في البيئة الخلوية، وقد تم تحقيق العديد من التطورات في تقنيات زراعة الخلايا على مر السنين. كان واحدا من التقدم الأكثر أهمية استخدام دعامات قابلة للاختراق مع الأغشية الصغيرة التي يسهل اختراقها لcompartmentalizing مزارع الخلايا، وتستخدم لأول مرة من قبل Grobstein في عام 1953 1. وقد جرى تصميم هذه الدعامات منفذة على مر السنين لاستيعاب العديد من أنواع الخلايا واستخدامها في العديد من التطبيقات المختلفة. في الوقت الحاضر، وهذه تدعم وجود تدرج كما جوفاء التي تم تصميمها للراحة في الآبار من لوحة زراعة الأنسجة multiwell أو في التعأطباق لار. في نظام الثقافة المشتركة، وإدراج يحتوي على نوع من الخلايا واحدة في حين البئر أو طبق يحتوي على السكان الخلوية الأخرى، مما يسمح لدراسة مساهمة اثنين من مجموعات مختلفة من الخلايا في بيئة الخلطية منها (الشكل 1). ونتيجة لذلك، يتم الحفاظ على الاستقطاب الخلوي (basolateral مقابل إفراز قمي أو استقبال إشارة)، مما يمنح أنظمة إدراج ثقافة مشتركة ميزة مهمة ضد الثقافات المختلطة وتقنيات المتوسطة مكيفة. تتوفر عدة أنواع من المواد غشاء، وأكثرها شيوعا هو البوليستر (PET)، والبولي (PC) أو المغلفة الكولاجين تترافلوروإيثيلين (PTFE)، وكانت موجودة في أحجام المسام مختلفة تتراوح من 0.4 ميكرومتر إلى 12.0 ميكرون. هذه الأصناف من المواد وأحجام المسام تقدم تشكيلة واسعة من إدراج ممارسة ملامح متغيرة ذات الصلة إلى الخصائص البصرية، وسمك الغشاء والالتزام الخلية التي جعلها عملية على مختلف المستويات ليلي يستخدم سبيل المثال لا الحصر: -studyingتمايز الخلايا، التطور الجنيني، الانبثاث ورم وإصلاح الجرح بواسطة فحوصات chemotaxic من خلال نفاذية الأغشية. -evaluating تغلغل المخدرات عن طريق تقييم وسائل النقل من خلال الطبقات الوحيدة الظهارية أو البطانية مثقف على دعامات قابلة للاختراق، و. -إجراء خلية المشترك الثقافات لتحليل التحويرات سلوك الخلايا الناجم عن العوامل القابلة للذوبان يفرز في حالة عدم وجود خلية خلية الاتصال. والغرض من هذه المقالة هو وصف المبادئ التوجيهية المنهجية العامة لأداء وظيفة الثالثة المذكورة أعلاه، وهذا هو لتقييم التغيرات الخلوية بوساطة العوامل القابلة للذوبان يفرز في حالة عدم وجود خلية خلية الاتصال باستخدام نظام إدراج ثقافة مشتركة. العديد من المجالات المختلفة للبحث الاستفادة من أنظمة إدراج ثقافة مشتركة من أجل الإجابة على الأسئلة ذات الصلة لتأثير العوامل القابلة للذوبان يفرز على السكان من الخلايا. في الواقع، يشير نظير الصماوي الذي ينظم السلوك الخلوي على مختلف المستويات هي ذات الصلة في جميع الأنسجةوالنظم، مما يجعل الأنظمة شارك في ثقافة إدراج لا غنى عنها لضمان التقدم في هذه المجالات. على العكس من ذلك، استخدام إدراج ان اؤكد ان نقل الإشارة هو عن طريق الاتصال المباشر خلية خلية وليس العوامل يفرز. واحدة من أهم استخدامات تدرج في الدراسات التهاب 2-14 حيث يتم تقييم تأثير السيتوكينات يفرز في مختلف اللاعبين الخلوي للمناعة. على وجه الخصوص، وقد استفادت الدراسة من التهاب في الجهاز العصبي المركزي (CNS) كثيرا من الدراسات إدراج الثقافة المشتركة، التي سمحت لتحديد أفضل الأدوار نظير الصماوي متميزة من الخلايا العصبية والخلايا الدبقية الصغيرة في القيادة neuroinflammation 15-21. وقد وضعت هذه الأنظمة أيضا لدراسة إمكانية المضادة للالتهابات من الجزيئات التي تعتمد على قدرتها على تقليل أو منع إفراز العوامل المؤيدة للالتهابات 22-26. الأبحاث المتعلقة بسرطان 27-31، ولا سيما الآليات الكامنة وراء الأوعية الدموية 32-34 وinflammatiعلى 35-42 في تكون الأورام، يستفيد أيضا من أنظمة إدراج ثقافة مشتركة. وعلاوة على ذلك، والعوامل القابلة للذوبان هي ذات أهمية قصوى في العمليات التي تقود التمايز والعديد من الدراسات قد استخدمت تدرج للإجابة على الأسئلة في هذا المجال بشكل خاص 43-50. في الجهاز العصبي المركزي، كما نرى الأنسجة العصبية لديها امكانات تجديد محدودة للغاية، ودراسة neurotrophism والعصبية هي الأساسية، وقد ضمنت على نطاق واسع عن طريق استخدام الخلايا الجذعية في أنظمة شارك في ثقافة 51-56. وبالإضافة إلى ذلك، تستخدم تدرج أيضا في مجالات متنوعة مثل أمراض الكلى 57،58، والتفاعلات البطانية والأوعية الدموية 59-62، موت الخلايا المبرمج يشير 63-65، والتهاب في السمنة ومتلازمة الأيض 22،23،66-67، الأذن الداخلية حماية خلايا الشعر 68،69، وحتى في فطر الفوعة 70،71 و 72،73 الطفيليات. تقدم هذه المقالة المبادئ التوجيهية المنهجية العامة من أجل اقامة experimوالأنف والحنجرة في ضوء تقييم التغيرات الخلوية بوساطة العوامل القابلة للذوبان يفرز باستخدام نظام إدراج ثقافة مشتركة. على وجه الخصوص، سوف نركز اهتمامنا على الخلايا العصبية المشترك الثقافات واستخداماتها في دراسة عملية neuroinflammatory. وبالنظر إلى طيف واسع جدا من التجارب التي تدرج تجعل من الممكن التجريبية، فإنه أمر لا يطاق لتغطية كل من هذه التقنية زراعة الخلايا الجانب. وكمثال على ذلك، بروتوكول معين لقياس آثار السيتوكينات التي يفرزها lipopolysaccharide في (LPS) -activated الخلايا الدبقية الصغيرة N9 على الخلايا PC12 العصبية سيتم تفصيله، وتقديم فهم ملموس منهجية إدراج ثقافة مشتركة.

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		<tr height="21">
			<td height="21" style="height:21px;width:232px;">RPMI-1640 medium</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">R8755</td>
			<td style="width:328px;">Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:232px;">Dulbecco&#8217;s Modified Eagle&#8217;s Medium/Nutrient Mixture F-12 Ham</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">D6421</td>
			<td style="width:328px;">Warm in 37 &#176;C water bath before use, must be supplemented with 0.365 gm/L L-glutamine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Horse serum</td>
			<td style="width:71px;">ATCC</td>
			<td style="width:119px;">30-2040</td>
			<td style="width:328px;">Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Fetal bovine serum</td>
			<td style="width:71px;">MultiCell</td>
			<td style="width:119px;">80350</td>
			<td style="width:328px;">Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:232px;">Nerve Growth Factor-7S from murine submaxillary gland</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">N0513</td>
			<td style="width:328px;">Reconstitute the lyophilized powder in a solution of buffered saline or tissue culture medium containing 0.1&#8211;1.0% bovine serum albumin or 1-10% serum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Trypsin-EDTA solution</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">T3924</td>
			<td style="width:328px;">Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">Lipopolysaccharides from&#160;Escherichia coli&#160;055:B5</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">L2880</td>
			<td style="width:328px;">Toxic</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">Cell culture inserts for use with 24-well plates</td>
			<td style="width:71px;">BD Falcon</td>
			<td style="width:119px;">353095</td>
			<td style="width:328px;">0.4 &#956;m pores</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">24-well plates</td>
			<td style="width:71px;">TrueLine</td>
			<td style="width:119px;">TR5002</td>
			<td style="width:328px;">Coat with collagen before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Routine PC12 cell culture medium</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>Routine N9 cell culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">-&#160;&#160;&#160;&#160;&#160;&#160; 85% RPMI medium</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>-&#160;&#160;&#160;&#160;&#160;&#160; 90% DMEM-F12 medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">-&#160;&#160;&#160;&#160;&#160;&#160; 10% heat-inactivated horse serum</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>-&#160;&#160;&#160;&#160;&#160;&#160; 10% heat-inactivated horse serum</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">-&#160;&#160;&#160;&#160;&#160;&#160; 5% heat-inactivated fetal bovine serum</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PC12 differentiation medium</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>N9 treatment medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">-&#160;&#160;&#160;&#160;&#160;&#160;&#160; 99% RPMI medium</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>-&#160;&#160;&#160;&#160;&#160;&#160; 99% DMEM-F12 medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">-&#160;&#160;&#160;&#160;&#160;&#160;&#160; 1% heat-inactivated fetal bovine serum</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>-&#160;&#160;&#160;&#160;&#160;&#160; 1% heat-inactivated horse serum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">-&#160;&#160;&#160;&#160;&#160;&#160;&#160; 50 ng/mL nerve growth factor</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PC12 treatment medium</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">-&#160;&#160;&#160;&#160;&#160;&#160;&#160; 99% RPMI medium</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">-&#160;&#160;&#160;&#160;&#160;&#160;&#160; 1% heat-inactivated fetal bovine serum</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

development of an insert co-culture system of two cellular types in the absence of cell-cell contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

استخدام نظم شارك في ثقافة إدراج يرتدي أهمية خاصة في دراسة العمليات neuroinflammatory التي تظهر العلاقات نظير الصماوي بين اللاعبين الخلوية المختلفة للجهاز العصبي المركزي. ويتم إنجاز الحصانة في الجهاز العصبي المركزي بشكل رئيسي من قبل خلايا مقيمة تسمى الخلا?...

Watch this Scientific Journal Video about Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact at JoVE.com

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

تطوير نظام إدراج المشارك ثقافة نوعين الخلوية في غياب خلية خلية الاتصال

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...

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Biology

27.7K Views.  University of Québec. In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

Video: Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

والخلايا العصبية ونجمية المشارك الثقافة الفحص لتحليل محتوى عال من العصبية

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

The use of SC1 (Pluripotin) to Support mESC Self-renewal in the Absence of LIF

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1 However, LIF is expensive and activation of the LIF/JAK/STAT3 pathway is not absolutely required to maintain the self-renewal state.2 The SC1 small molecule may be an economical alternative to LIF. SC1 functions through dual inhibition of Ras-GAP and ERK1.3 Illustration of its mechanism of action makes it a useful tool to study the fundamental molecular mechanism of self-renewal. Here we demonstrate the procedure for culturing mouse ES cells in the presence of SC1 and show that they are able to maintain self-renewal in the absence of LIF. Cells cultured with SC1 showed similar morphology compared to cells maintained with LIF. Both exhibited typical mouse ES morphology after five passages. Expression of typical pluripotency markers (Oct4, Sox2, Nanog, and SSEA1) was observed after five passages in the presence of SC1. Furthermore, SC1 caused no overt toxicity on mouse ES cells.

The use of SC1 Pluripotin to Support mESC Self-renewal in the Absence of LIF

SC1 functions through dual inhibition of Ras- GAP and ERK1. We tested the function of SC1 in supporting mouse ES cell self-renewal in the absence of LIF and showed that SC1 is able to maintain self-renewal of mouse ES cell cultures.

استخدام SC1 (Pluripotin) لدعم MESC التجديد الذاتي في غياب LIF

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1 However, LIF is expensive and ...

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures 

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical research. The hypoxic chamber offers a simple system to control oxygenation in standard culture vessels, but lacks precise temporal and spatial control over the oxygen concentration at the cell surface, preventing its application in studying a variety of physiological phenomena. Other systems have improved upon the hypoxic chamber, but require specialized knowledge and equipment for their operation, making them intimidating for the average researcher. A microfabricated insert for multiwell plates has been developed to more effectively control the temporal and spatial oxygen concentration to better model physiological phenomena found in vivo. The platform consists of a polydimethylsiloxane insert that nests into a standard multiwell plate and serves as a passive microfluidic gas network with a gas-permeable membrane aimed to modulate oxygen delivery to adherent cells. The device is simple to use and is connected to gas cylinders that provide the pressure to introduce the desired oxygen concentration into the platform. Fabrication involves a combination of standard SU-8 photolithography, replica molding, and defined PDMS spinning on a silicon wafer. The components of the device are bonded after surface treatment using a hand-held plasma system. Validation is accomplished with a planar fluorescent oxygen sensor. Equilibration time is on the order of minutes and a wide variety of oxygen profiles can be attained based on the device design, such as the cyclic profile achieved in this study, and even oxygen gradients to mimic those found in vivo. The device can be sterilized for cell culture using common methods without loss of function. The device's applicability to studying the in vitro wound healing response will be demonstrated.

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures

Fabrication and validation of an add-on platform that offers enhanced control over the spatial and temporal oxygenation in a 6-well plate. The device is adaptable to a number of culture systems and can be used to investigate the effects of oxygen on wound healing.

تصنيع وتشغيل إدخال الأوكسجين للثقافات الخلوية منتسب

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical ...

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility4-6. Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer7.
Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development8. Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages2,9. Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology5,6,10,11. While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression12, antibody staining13, and electron microscopy1. Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures14. Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail15. We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.

تصور انواع معينة ايليجانس</emالهياكل البشرة> استخدام الصبغة الحيوية محبة للدهون ، صلاح الغيدان

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

Two Types of Assays for Detecting Frog Sperm Chemoattraction

Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension and microscopically visualize sperm accumulation around the pipette1. Sperm chemoattraction in vertebrates such as frogs, rodents and humans is more difficult to detect and requires quantitative assays. Such assays are of two major types - assays that quantitate sperm movement to a source of chemoattractant, so-called sperm accumulation assays, and assays that actually track the swimming trajectories of individual sperm.
Sperm accumulation assays are relatively rapid allowing tens or hundreds of assays to be done in a single day, thereby allowing dose response curves and time courses to be carried out relatively rapidly. These types of assays have been used extensively to characterize many well established chemoattraction systems - for example, neutrophil chemotaxis to bacterial peptides and sperm chemotaxis to follicular fluid. Sperm tracking assays can be more labor intensive but offer additional data on how chemoattractancts actually alter the swimming paths that sperm take. This type of assay is needed to demonstrate the orientation of sperm movement relative to the chemoattrractant gradient axis and to visualize characteristic turns or changes in orientation that bring the sperm closer to the egg.
Here we describe methods used for each of these two types of assays. The sperm accumulation assay utilized is called a "two-chamber" assay. Amphibian sperm are placed in a tissue culture plate insert with a polycarbonate filter floor having 12 &mu;m diameter pores. Inserts with sperm are placed into tissue culture plate wells containing buffer and a chemoatttractant carefully pipetted into the bottom well where the floor meets the wall (see Fig. 1). After incubation, the top insert containing the sperm reservoir is carefully removed, and sperm in the bottom chamber that have passed through the membrane are removed, pelleted and then counted by hemocytometer or flow cytometer.
The sperm tracking assay utilizes a Zigmond chamber originally developed for observing neutrophil chemotaxis and modified for observation of sperm by Giojalas and coworkers2,3. The chamber consists of a thick glass slide into which two vertical troughs have been machined. These are separated by a 1 mm wide observation platform. After application of a cover glass, sperm are loaded into one trough, the chemoattractant agent into the other and movement of individual sperm visualized by video microscopy. Video footage is then analyzed using software to identify two-dimensional cell movements in the x-y plane as a function of time (xyt data sets) that form the trajectory of each sperm.

Eggs and the extracellular coatings around eggs frequently release peptides, proteins and small molecules that communicate with sperm to guide them to the egg thereby promoting fertilization. Using frog sperm we describe and compare two classes of assays used to detect sperm chemoattraction &#x2013; sperm accumulation assays and sperm tracking assays.

نوعان من فحوصات الكشف عن الضفدع Chemoattraction الحيوانات المنوية

Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

نظام ل<em&gt; خارج الجسم</em&gt; التثقيف البنكرياس الجنينية

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

أداة تحليلية يحدد مقدار التغييرات الخلوية الصرف من التقاط صور ثلاثية الأبعاد الإسفار

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

الفأر الجنينية التنمية في الجامعة نظام الثقافة الأجنة الخالية من المصل

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

و<em&gt; خارج الحي</em&gt; نظام الثقافة لدراسة التنمية الغدة الدرقية

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

Establishment of an Extracellular Acidic pH Culture System

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

إنشاء درجة الحموضة خارج الخلية الحمضية منظومة الثقافة

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...

تطوير نظام إدراج المشارك ثقافة نوعين الخلوية في غياب خلية خلية الاتصال

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