تلقى سونيا Schrepfer ، كونرادي لينارد وارني هانسن التمويل من (DFG ؛ SCHR992/3-1 ، SCHR992/4-1 ، CO 858/1-1 ، HA 3423/3-1) دويتشه Forschungsgemeinschaft. وأيد أيضا العمل من خلال المنح المقدمة من الاتحاد الأوروبي لEschenhagen توماس (EUGeneHeart وAngioscaff).

1. EHT انشاء الشروط والثقافة أنجز تصنيع الليفين المستندة EHT في مكان آخر كما هو موضح 4. وباختصار ، لتوليد الليفين المستندة EHT لأغراض الزرع ، التي أعدت مزيجا رئيسية تحتوي على خلايا معزولة من ROSA الولدان / luciferase - قلوب الفئران المعدلة وراثيا LEW ، الفيبرينوجين الأبقار وMatrigel. وأعدت Agarose قوالب الصب باستخدام الفواصل وضعت في تفلون الأطباق ثقافة 6 - جيدا. بعد تصلب agarose ، أزيلت الفواصل ووضعت رفوف آخر سيليكون على أطباق مع الثقافة المشاركات التوصل إلى قوالب. لتوليد EHT ، كانت مختلطة لفترة وجيزة mastermix مع كمية محسوبة من ثرومبين وpipetted في القالب. بعد بلمرة الفيبرينوجين ، أزيلت بلطف مع رفوف سيليكون EHTs الانضمام إلى وظيفة من قوالب الصب agarose ، ونقل إلى 6 جديدة رفاه أطباق زراعة الخلايا والحفاظ عليها في 37 درجة مئوية ، و 7 ٪ CO 2 خلية حاضنة الثقافة باستخدام مصنوعة خصيصا خلية ثقافة المتوسط. تم الاحتفاظ بها في ظل ظروف EHT ثقافة الخلية حتى لمدة عشرة أيام. خلال هذه الفترة الزمنية ، بدأ الولدان خلايا قلب فأر على التواصل ، ومحاذاة ممدود على طول خطوط القوة بين الوظائف في سيليكون. في وقت الزرع ، وقد لوحظت تقلصات متماسكة عفوية ، تنحرف عن المشاركات السيليكون. 2. EHT زراعة تم تنفيذ EHT في غرس إما مناعيا الفئران براون (الجبهة الوطنية) والنرويج ، أو الفئران عارية العوز المناعي للتحقيق في استجابة خيفي المناعي. وتم شراء 150 غراما من نهر تشارلز ألمانيا (7 Sandhofer خبراء المياه ، D - Sulzfeld 97633) ، ويضم في ظل الظروف التقليدية ، وتغذية الفئران القياسية طعام وماء libidum الإعلانية -- BN عارية الفئران وزنها 100. ويعيش العوز المناعي الفئران عقيمة في الفراش ، الأقفاص ، والمياه المعقمة المستخدمة. الألماني لجنة المراجعة الأخلاقية مراجعتها والموافقة على الإجراءات الحيوانية. يتم تعقيم جميع الأدوات الجراحية قبل الاستخدام. يتم تنفيذ الإجراء زرع على النحو التالي : وanesthesized الفئران مع isoflurane (2.5-3 ٪) باستخدام غرفة التعريفي. يمكن الحد من تعرض الإنسان لisoflurane من خلال العمل في الجدول اسفل أو عدم إعادة تدوير غطاء محرك السيارة. يتم الاحتفاظ درجة حرارة الجسم أثناء العملية الجراحية. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> حلق منطقة البطن وتطبيق مرهم العين لمنع جفاف العين من خلال التخدير. </li><li style=";text-align:right;direction:rtl"> وضع فأر على ظهرها ووضع قناع على الأنف والفم والخمسين للحفاظ على التخدير. </li><li style=";text-align:right;direction:rtl"> تطهير منطقة البطن باستخدام Betadine ثم الايثانول 80 ٪. مسحة من نظيف لالقذرة ، من وسط منطقة حلق تتحرك إلى الخارج في اتجاه منطقة الشعر. </li><li style=";text-align:right;direction:rtl"> فحص ردود الفعل معسر القدمين الخلفيتين للتأكد من أن الفأر هو تخدير بما فيه الكفاية. </li><li style=";text-align:right;direction:rtl"> ثنى الحيوان وإجراء شق البطن خط الوسط التي تفصل بين الجلد والعضلات في خطوتين لفتح البطن. وتستخدم حبة تعقيم الساخن بين الجلد والعضلات الافتتاح. </li><li style=";text-align:right;direction:rtl"> وضع الامعاء في درجة حرارة المياه المالحة قفاز مسحوق رطب الحرة. طوى القفازات حول الامعاء لمنع فقدان الرطوبة. </li><li style=";text-align:right;direction:rtl"> إزالة الأنسجة الدهنية وفضح الطحال. </li><li style=";text-align:right;direction:rtl"> تصور وانتشار أكبر الثرب بالملقط. </li><li style=";text-align:right;direction:rtl"> قطع بعناية EHT من السيليكون وتعليق المشاركات هاربورفرونت الثرب أكبر. </li><li style=";text-align:right;direction:rtl"> التفاف حول أكبر omentus EHT والإصلاح باستخدام اثنين من الغرز واحدة 7-0 prolene (Ethicon ، Norderstedt ، المانيا) </li><li style=";text-align:right;direction:rtl"> الخطوة التالية في الأمعاء مرة أخرى في البطن. </li><li style=";text-align:right;direction:rtl"> مسح البطن مع المالحة prewarmed العقيمة. </li><li style=";text-align:right;direction:rtl"> إغلاق الطبقة العضلية للجدار البطن باستخدام 6-0 prolene الغرز تشغيل (Ethicon ، Norderstedt ، ألمانيا). </li><li style=";text-align:right;direction:rtl"> استخدام prolene 5-0 (Ethicon ، Norderstedt ، ألمانيا) تشغيل الغرز لإغلاق الجلد. </li><li style=";text-align:right;direction:rtl"> في حين أن الفئران لا يزال في التخدير وحقن 5 ملغ / كلغ كاربروفين تحت الجلد. </li><li style=";text-align:right;direction:rtl"> لتوفير ما يكفي من وعي لهذا النوع من الإجراء ، وهو يضاف مسكن (مثل Metamizol) لمياه الشرب (50 ملجم لكل 100 مل Metamizol) لعلاج الألم لمدة 3 أيام بعد الزرع. </li></ol> 3. تلألؤ بيولوجي من التصوير وبعد زرع EHT تم استخدام منصة bioimaging IVIS (Xenogen ، ألاميدا ، كاليفورنيا ، الولايات المتحدة الأمريكية) للتصوير تلألؤ بيولوجي غير الغازية في الجسم الحي ، ويتم تحليل النتائج باستخدام صورة حية IVIS (Xenogen) حزمة البرمجيات. يتم إجراء التصوير يوميا بدءا 2 ساعة بعد الجراحة. تخدير الفئران مع isoflurane (2.5 -- 3 ٪) باستخدام غرفة التعريفي. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تطهير منطقة البطن والصدر به على نطاق واسع ، بروفو اليود ، واستخدام الايثانول 80 ٪ المقبل ، كرر هذه الخطوة ثلاث مرات. </li><li style=";text-align:right;direction:rtl"> حقن د Luciferin (Xenogen ؛ 375 من وزن الجسم ملغ / كلغ) الملكية الفكرية </li><li style=";text-align:right;direction:rtl"> مكان الفئران مخدرة الى غرفة التصوير IVIS (ويمكن تحليل ما يصل إلى 4 الفئران في الوقت نفسه). </li><li style=";text-align:right;direction:rtl"> تقييم قوة إشارة لUC + الخلايا داخل EHT في وحدات ثانية / الفوتونات / سم 2 / steridianin المنطقة ذات الاهتمام (ROI) في الأساس (120 دقيقة بعد زرع) وفي أيام 1 ، 2 ، 3 ، 5 ، 7 ، 10 ، وفي الأسابيع 2 ، 3 و 4. لاحظ إشارة الذروة (يظهر حوالي 20 دقيقة بعد الحقن Luciferin د). </li></ol> إنشاء رسم بياني (اكسل) تظهر كثافة إشارة مرور الوقت. 4. الربط بين النتائج وBLI ELISPOT وكانت دراسة الخلوية داخل الجسم الحي الاستجابات المناعية في BN مناعيا والفئران عارية العوز المناعي بعد زرع EHT. وكان حصاد الطحال من الحيوانات المتلقية 5 أيام بعد زرع EHT لعزل splenocytes المتلقية. المقايسات Elispot باستخدام mitomycin - منعت EHTs (سيغما الدريتش ، وسانت لويس ، MO ، الولايات المتحدة الأمريكية) ، ومنشط و 5 splenocytes X10 المتلقي كما تم تنفيذ 6 خلايا المستجيب وفقا لبروتوكول الشركة المصنعة دينار بحريني (العلوم البيولوجية) باستخدام الإنترفيرون γ والمغلفة IL - 4 لوحات للتحقيق TH1 ، والاستجابة TH2 ، على التوالي. الإنترفيرون γ وIL - 4 نقاط كانت أعلى بشكل ملموس في النموذج ، مناعيا بالمقارنة مع النموذج العوز المناعي (IFN - γ : ع = 0.001 ؛ IL - 4 : ع = 0.023 ؛ الطالب اختبار t). 5. ممثل النتائج : <img src="/files/ftp_upload/2605/2605fig1.jpg" alt="الشكل 1" /> الشكل 1. الربط بين النتائج وBLI ELISPOT أ) ودرس في الاستجابات المناعية الخلوية المجراة في النرويج براون مناعيا والفئران عارية العوز المناعي بعد زرع EHT. كانت تحصد splenocytes المتلقي 5 أيام بعد غرس EHT. الإنترفيرون؟ وجرى تقييم IL - 4 التعبير المقايسات ELISpot mitomycin به ، كما منعت EHT التحفيز والتشجيع وsplenocytes المتلقي 5x106 كخلايا مستجيب. وTH1 والاستجابات TH2 أعلى بكثير في النموذج مناعيا بالمقارنة مع النموذج العوز المناعي كما يتضح من IFNγ و- IL - 4 الإنتاج على التوالي. ب) تم زرع لوك + EHTs في omentus أكبر من أي من الفئران أو الجرذان BN مناعيا عارية العوز المناعي. وأعقب طوليا بقاء الخلية BLI. وتقدم نسبة الحيوانات التي رفضت EHTs المزروعة. جميع الفئران BN رفض زرع EHT بسرعة ، في حين نجا الخلايا في الفئران الخلايا التائية ناقصة.

<ol>
	<li>Eschenhagen, T., Zimmermann, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+myocardial+tissue.">Engineering myocardial tissue.</a> Circ Res. 97, 1220-1231 (2005).</li><li>van der Bogt, K. E., Schrepfer, S., Yu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+transplantation+of+adipose+tissue-+and+bone+marrow-derived+mesenchymal+stem+cells+in+the+infarcted+heart.">Comparison of transplantation of adipose tissue- and bone marrow-derived mesenchymal stem cells in the infarcted heart.</a> Transplantation. 87, 642-652 (2009).</li><li>Hakamata, Y., Murakami, T., Kobayashi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Firefly+rats"+as+an+organ/cellular+source+for+long-term+in+vivo+bioluminescent+imaging.">"Firefly rats" as an organ/cellular source for long-term in vivo bioluminescent imaging.</a> Transplantation. 81, 1179-1184 (2006).</li><li>Hansen, A., Eder, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Drug+Screening+Platform+Based+on+Engineered+Heart+Tissue.">Development of a Drug Screening Platform Based on Engineered Heart Tissue.</a> Circ Res. , (2010).</li></ol>

الإعلان عن أي تضارب في المصالح.

قبل التنفيذ السريرية التكنولوجيا EHT لإصلاح القلب ، والمسائل الأساسية مثل استمناع كل من المكونات الخلوية والمصفوفة أو البقاء على قيد الحياة بعد زرع الفساد تحتاج إلى تقييم في النماذج التجريبية. التصوير داخل الجسم الحي تلألؤ بيولوجي يمثل تقنية جديدة مفيدة للبقاء ...

<ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> LEW - تيراغرام (GT (روزا) 26Sor لوك) 11Jmsk الفئران (NBPR عدد الفئران 0299 ​​، http://www.anim.med.kyoto-u.ac.jp/NBR/) </li><li style=";text-align:right;direction:rtl"> البقري الفيبرينوجين (محلول المخزون : 200 ملغ / مل زائد أبروتينين 5 ميكروغرام / مل في الفيبرينوجين كلوريد الصوديوم 0.9 ٪ ، سيغما F4753) </li><li style=";text-align:right;direction:rtl"> Matrigel دينار بحريني (356235 العلوم البيولوجية) </li><li style=";text-align:right;direction:rtl"> DMEM (صباغ حيوي المنشأ F0415) </li><li style=";text-align:right;direction:rtl"> حصان المصل (Gibco 26050) </li><li style=";text-align:right;direction:rtl"> البنسلين / الستربتوميسين (Gibco 15140) </li><li style=";text-align:right;direction:rtl"> الانسولين (سيغما الدريخ I9278) </li><li style=";text-align:right;direction:rtl"> أبروتينين (سيغما الدريتش A1153) </li><li style=";text-align:right;direction:rtl"> د Luciferin (L - Biosynth 8220) </li><li style=";text-align:right;direction:rtl"> انزيم مرتبط المناعي لوحات بقعة مقايسة (ELISpot) (الحسا Laborbedarf ، S2EM004M99) </li></ul>

bioluminescence imaging for assessment of immune responses following implantation of engineered heart tissue (eht)

واتبعت أساليب مختلفة للهندسة الأنسجة القلبية في العقود الماضية بما في ذلك استراتيجيات السقالات باستخدام المواد الأصلية أو الحيوي الصناعي سقالة ، انحباس myocytes القلب في الهلاميات المائية مثل الليفين أو الكولاجين والتراص من monolayers العضلية 1. هذه المفاهيم تهدف إلى استعادة وظيفة القلب للخطر (على سبيل المثال بعد احتشاء عضلة القلب) أو نماذج تجريبية (مثل علم السموم التنبؤي وفحص مادة أو النمذجة المرض). الرصد الدقيق للبقاء على قيد الحياة بعد زرع خلايا أنسجة القلب هندسيا (EHT) أصبح من الممكن الآن استخدام التقنيات في الجسم الحي ، تلألؤ بيولوجي (BLI) التصوير 2. نحن هنا وصف جيل من الليفين المستندة EHT الفئران من سلالة معدلة وراثيا مع التعبير في كل مكان من luciferase يراعة (ROSA / تيراغرام luciferase - LEW ؛ 3). يتم تنفيذ غرس في أكبر سلالات الفئران الثرب مختلفة لتقييم الاستجابات المناعية للكائن المتلقي التالية زرع EHT. المقارنة بين النتائج التي تم إنشاؤها بواسطة BLI وانزيم مرتبط تقنيات سبوت المناعي (ELISPOT) تأكيد قابليتها للBLI لتقييم الاستجابات المناعية.

Watch this Scientific Journal Video about Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT at JoVE.com

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

هذا الفيديو يوضح استخدام<em&gt; في الجسم الحي</em&gt; التصوير تلألؤ بيولوجي لدراسة الاستجابات المناعية بعد زرع أنسجة القلب المهندسة (EHT) في الفئران.

التصوير تلألؤ بيولوجي لتقييم الاستجابات المناعية عقب زرع أنسجة القلب المهندسة (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or ...

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Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

9.6K Views.  University Heart Center Hamburg. هذا الفيديو يوضح استخدام في الجسم الحي التصوير تلألؤ بيولوجي لدراسة الاستجابات المناعية بعد زرع أنسجة القلب المهندسة (EHT) في الفئران.

Video: Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

المغناطيسي منهجية Elastography الرنين لتقييم الأنسجة المهندسة النمو كونستركت

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage&#160;and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.

There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions.

بروتوكول لتقييم هيدرودينامي النسبية للثلاثي النشرة بوليمر صمامات

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

تقنية جراحية لزرع الأنسجة المهندسة الأوعية الدموية الطعوم واللاحقة<em&gt; في فيفو</em&gt; الرصد

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

جيل ومسمار سفن الهندسة الأنسجة في نموذج الفأر

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

إنتاج وتوصيف والمحتملة استخدامات الأنسجة المهندسة-3D الإنسان المريء المخاطي نموذج

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

استنادا الحرير الكولاجين المهندسة 3D نموذج الاستقطاب العصبية الأنسجة

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

تحليل الآلي تقلص الإنسان المهندسة القلب الأنسجة للفحص القلب سلامة الأدوية

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

Custom Engineered Tissue Culture Molds from Laser-etched Masters

As the field of tissue engineering has continued to mature, there has been increased interest in a wide range of tissue parameters, including tissue shape. Manipulating tissue shape on the micrometer to centimeter scale can direct cell alignment, alter effective mechanical properties, and address limitations related to nutrient diffusion. In addition, the vessel in which a tissue is prepared can impart mechanical constraints on the tissue, resulting in stress fields that can further influence both the cell and matrix structure. Shaped tissues with highly reproducible dimensions also have utility for in vitro assays in which sample dimensions are critical, such as whole tissue mechanical analysis.
This manuscript describes an alternative fabrication method utilizing negative master molds prepared from laser etched acrylic: these molds perform well with polydimethylsiloxane (PDMS), permit designs with dimensions on the centimeter scale and feature sizes smaller than 25 &#181;m, and can be rapidly designed and fabricated at a low cost and with minimal expertise. The minimal time and cost requirements allow for laser etched molds to be rapidly iterated upon until an optimal design is determined, and to be easily adapted to suit any assay of interest, including those beyond the field of tissue engineering.

Herein we present a rapid, facile, and low-cost method for fabricating custom polydimethylsiloxane molds that can be used for producing hydrogel-based engineered tissues with complex geometries. We additionally describe results from mechanical and histological assessments conducted on engineered cardiac tissues produced using this technique.

مخصص تصميم قوالب زراعة الأنسجة من سادة ليزر محفوراً

As the field of tissue engineering has continued to mature, there has been increased interest in a wide range of tissue parameters, including tissue ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

بذر وغرس الاختلاس الرغامى السكروز هندسة الأنسجة في طراز الماوس

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant technique with nutritional support. Recently, there have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in early epithelization in the special environment of peristalsis. Here, we developed an artificial esophagus that can improve the regeneration of the esophageal mucosa and muscle layers through a two-layered tubular scaffold, a mesenchymal stem cell-based bioreactor system, and a bypass feeding technique with modified gastrostomy. The scaffold is made of polyurethane (PU) nanofibers in a cylindrical shape with a three-dimensional (3D) printed polycaprolactone strand wrapped around the outer wall. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. We improved the graft survival rate by applying surgical anastomosis and covering the implanted prosthesis with a thyroid gland flap, followed by temporary nonoral gastrostomy feeding. These grafts were able to recapitulate the findings of initial epithelialization and muscle regeneration around the implanted sites, as demonstrated by histological analysis. In addition, increased elastin fibers and neovascularization were observed in the periphery of the graft. Therefore, this model presents a potential new technique for circumferential esophageal reconstruction.

Esophageal reconstruction is a challenging procedure, and development of a tissue-engineered esophagus that enables regeneration of esophageal mucosa and muscle and that can be implanted as an artificial graft is necessary. Here, we present our protocol to generate an artificial esophagus, including scaffold manufacturing, bioreactor cultivation, and various surgical techniques.

الترقيع المهندس بالأنسجة لإعادة بناء المريء المحيطي في الفئران

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...