ويدعم تريسي C. Grikscheit، إريك R. بارثيل، وفريدريك سالا G. من معهد كاليفورنيا للطب التجديدي (CIRM)، وأرقام المنح RN2-00946-1 (TCG)، وTG2 01168-(إرب، FGS). أليسون L. سبير هو جمعية علماء جامعة Ethicon جراحي. ويتم تمويل Yashuhiro Torashima من قبل مستشفى الأطفال في لوس أنجلوس معهد بحوث سابان زمالة التطوير الوظيفي.

1. عضي وحدات التحضير <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> يجب تعقيم الأدوات المناسبة لتشريح الفأر (مقص وملقط) بواسطة الأوتوكلاف. </li><li style=";text-align:right;direction:rtl"> الموت ببطء إنسانية الماوس المانحة وفقا لبروتوكولات IACUC المحلية. تأكد من أن الحيوان قد مات قبل الشروع. </li><li style=";text-align:right;direction:rtl"> إجراء شق خط الوسط للوصول إلى التجويف البريتوني. يمكن أن تنعكس اللوحات الجلد حسب الحاجة لتحسين التعرض. </li><li style=";text-align:right;direction:rtl"> سلب الأمعاء الدقيقة وتقسيمها القاصي عادل للرباط ترايتس. فصل الأمعاء الدقيقة من مساريق وذلك باستخدام تشريح حادة حادة ورقيقة. تحديد تقاطع اللفائفية وتقسيم الأمعاء الصغيرة 5 مم الأقرب إلى ذلك. </li><li style=";text-align:right;direction:rtl"> باستخدام مقص، وفتح الأمعاء بالطول على طول الحدود مقابل المساريق في طبق بتري مع 10 مل 4 ° C، ملحي معقم هانكس &quot;مخزنة (HBSS، إينفيتروجن، كارلسباد CA) / 1X للمضادات الحيوية المضادة للفطريات (المضادة للمكافحة، إينفيتروجن) الحل. مسح البراز من الأمعاء مع فتحالتحريض لطيف ثم نقل إلى الأمعاء فتح أنبوب الطرد المركزي مع 10 مل 15ml من 4 ° C، HBSS العقيمة / 1X المضادة للسامية. </li><li style=";text-align:right;direction:rtl"> غسل الأمعاء فتح ثلاث مرات في 10 مل من 4 ° C، HBSS العقيمة / 1X المضادة للسامية في أنبوب اختبار. لا يمكن أن يؤديها كل غسيل مع اهتزاز خفيف من الأنبوب 15 مل لمدة 30 ثانية. بعد اهتزاز، والأنسجة المعوية المصارف إلى أسفل الأنبوب. تجاهل المواد العائمة، والتي هي الوسيطة الحطام. إزالة الحل الغسيل بعناية مع الماصة. </li><li style=";text-align:right;direction:rtl"> اللحم المفروم الأمعاء غسلها في طبق بتري مع 10 مل من 4 ° C، HBSS العقيمة / 1X مكافحة المضادة للقطع أقل من 1 ملم مربع باستخدام المقص. جمع المواد المفروم حتى مع الماصة التلقائي ووضعه في أنبوب الاختبار. </li><li style=";text-align:right;direction:rtl"> أجهزة الطرد المركزي في أنبوب في 500 دورة في الدقيقة لمدة 8 دقائق. تجاهل طاف، التي تحتوي على الدهون واللحمة المتوسطة. </li><li style=";text-align:right;direction:rtl"> هضم المفروم، المواد غسلها مع 10 مل من HBSS العقيمة / 1X المضادة للسامية بالإضافة إلى 0،125 ملغ / مل dispase (إينفيتروجن) وحدات 800 / مL كولاجيناز نوع 1 (ورثينجتون، ليكوود NJ). لإعداد 40 مل من محلول الهضم، وتزن من 5 ملغ من dispase، 142 ملغ من كولاجيناز، وإضافة HBSS العقيمة تصل إلى حجم 40 مل. إعداد هذا الحل الطازجة تعد كل وحدة عضي الوقت، والحفاظ في 4 درجات مئوية حتى جاهزة للاستخدام. إضافة الحل الهضم مباشرة إلى بيليه من الخطوة 1.8. </li><li style=";text-align:right;direction:rtl"> احتضان أنبوب اختبار يحتوي على المفروم، المواد غسلها مع الحل في الهضم C ° 37 لمدة 20 دقيقة. </li><li style=";text-align:right;direction:rtl"> استرداد أنبوب اختبار وتعطيل المزيد من الأنسجة هضمها من قبل سحن مع الماصة 10 مل. تكرار ما بين 20 إلى 50 مرة حتى يتم الحصول على مظهر موحد. </li><li style=";text-align:right;direction:rtl"> أجهزة الطرد المركزي في أنبوب الاختبار لمدة 5 دقائق في 800 دورة في الدقيقة. تجاهل طاف، التي تحتوي على خلايا واحد. </li><li style=";text-align:right;direction:rtl"> وقف رد فعل الهضم مع 10 مل من 4 ° C، عقيمة في Dulbecco معدلة النسر متوسطة (DMEM، إينفيتروجن)، بالإضافة إلى 10٪ V / V للحرارة المعطل مصل بقري جنيني (FBS-مرحبا، إينفيتروجن). إعادة تعليق على بيلير ويهز الأنبوب. </li><li style=";text-align:right;direction:rtl"> أجهزة الطرد المركزي في أنبوب الاختبار لمدة 5 دقائق في 800 دورة في الدقيقة. إزالة بعناية طاف مع الماصة التلقائي حتى قطرات القليلة الماضية. استخدام الماصة البلاستيكية للقطرات القليلة الماضية لتجنب resuspending بيليه. </li></ol> 2. التحميل من حمض Polyglycolic سقالة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> نموذج 2-مم، 5 مم القطر الخارجي السقالات أسطواني من حمض polyglycolic محبوكة (2-مم سمك ورقة، 60 ملغ الكثافة الظاهرية-3 سم؛ المسامية&gt; 95٪، ألياف كونكورديا، كوفنتري RI) كما هو موضح في المرجع. 4. </li><li style=";text-align:right;direction:rtl"> تقليم القاصي 2 مم من الماصة المتاح ميكروليتر 1000 ترجيح مع مقص أعدت مع الايثانول 70٪ في الماء المقطر. </li><li style=";text-align:right;direction:rtl"> وضع سقالة في لوحة الثقافة 4-أيضا. تحميل وحدات عضي على السقالة مع الماصة ميكروليتر 1،000، لأول مرة في التجويف ومن ثم إلى السطح الخارجي. استخدام ملقط لضمان طلاء من التجويف. لا تعطيل أو كسر مفتوح على شكل أسطواني من البوليمر. </لى&gt; </ol> 3. غرس في الماوس المضيف <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> استخدام الماوس المضيف مسانج على خلفية نفس الجهة المانحة إذا كانت متوفرة. خلاف ذلك، وتوظيف والسكري nonobese المناعة / شديدة العوز المناعي مجتمعة أو NOD / SCID الحيوانية (جاكسون المختبرات، سكرامنتو CA). </li><li style=";text-align:right;direction:rtl"> حمل التخدير العام مع isoflurane. حلاقة والإعدادية وثنى البطن الفأر. </li><li style=";text-align:right;direction:rtl"> إجراء شق خط الوسط 5 مم للحصول على مدخل التجويف البريتوني. تحديد وسلب بعناية أكبر الثرب. وضع البوليمر تحميلها على الثرب والتفاف عليه مع الأنسجة. لا المسيل للدموع الثرب. </li><li style=";text-align:right;direction:rtl"> تأمين البوليمر إلى الثرب مع خياطة monocryl 5-0. استبدال بلطف الثرب مع البوليمر ملفوفة في موقفها التشريحية. </li><li style=";text-align:right;direction:rtl"> إغلاق شق البطن في طبقات باستخدام الخيوط الجراحية vicryl 4-0. إغلاق تشغيل العضلات والحرص على عدم إصابة الأحشاء في البطن دون شق. استخدام الخيوط الجراحية توقف للبشرة. </li><li style=";text-align:right;direction:rtl"> إدارة تسكين بعد العملية الجراحية مع كيتوبروفين ملغ / كغ 2 (Ketofen، وفورت دودج صحة الحيوان) في الماء المعقم الانتفاخ تحت الجلد باعتبارها المتاخمة للشق. وينبغي تقييم الحيوانات اليومية وإذا كان الحيوان مما يدل علامات الألم أو الضيق، يمكن إعطاء جرعة إضافية من كيتوبروفين في يوم آخر المنطوق 2. بعد يوم ما بعد الجراحة الثالثة، ينبغي الحيوان تعافى تماما دون وجود أدلة من الألم أو الضيق. إذا كان الألم أو الضيق يستمر في يوم آخر المنطوق 3، ويعتبر هذا غير طبيعي وينبغي أن تعالج وفقا لبروتوكولات مرفق الرعاية IACUC والحيوانية. </li><li style=";text-align:right;direction:rtl"> السماح الماوس لفترة نقاهة والأمعاء الأنسجة المهندسة في النمو لمدة أربعة أسابيع. إعطاء libitum الإعلانية وصول الحيوانات إلى طعام القوارض (معمل حمية 5001، PMI التغذية، وسانت لويس MO) والمياه مع 200 ملغ ملغ / 40 مل لكل 5 Septra (مرحبا التكنولوجيا صيدلي، NY Amityville) التخفيف 1:100 في. </li></ol> 4. حصاد <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> هوماالموت ببطء nely الحيوان المضيف أربعة أسابيع بعد الزرع. </li><li style=";text-align:right;direction:rtl"> رأسيا إعادة فتح شق الأصلي وتعكس الجلد لتسهيل الوصول إلى التجويف البريتوني. </li><li style=";text-align:right;direction:rtl"> فتح طبقة العضلات والأنسجة تحديد بناء الهندسة العالم باعتبارها من الأنسجة. </li><li style=";text-align:right;direction:rtl"> إنزال الالتصاقات إلى بناء الأحشاء داخل البطن باستخدام من تشريح حادة. </li><li style=";text-align:right;direction:rtl"> إصلاح بناء في الفورمالين لالبارافين في وقت لاحق المتصاعدة، أو استخدام الأنسجة جديدة للفحوصات البيوكيميائية مثل في الوقت الحقيقي PCR أو العزلة البروتين. </li></ol>

<ol>
	<li>Grikscheit, T. C., Siddique, A., Ochoa, E. R., 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=Tissue-engineered+small+intestine+improves+recovery+after+massive+small+bowel+resection.">Tissue-engineered small intestine improves recovery after massive small bowel resection.</a> Ann. Surg. 240, 748-754 (2004).</li><li>Wales, P. W., Christison-Lagay, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+bowel+syndrome:+epidemiology+and+etiology.">Short bowel syndrome: epidemiology and etiology.</a> Sem. Ped. Surg. 19, 3-9 (2010).</li><li>Evans, G. S., Flint, N., Somers, A. S., 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=The+development+of+a+method+for+the+preparation+of+rat+intestinal+epithelial+cell+primary+cultures.">The development of a method for the preparation of rat intestinal epithelial cell primary cultures.</a> J. Cell Sci. 101, 219-231 (1992).</li><li>Sala, F. G., Matthews, J. A., Speer, A. L., 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+multicellular+approach+forms+a+significant+amount+of+tissue-engineered+small+intestine+in+the+mouse.">A multicellular approach forms a significant amount of tissue-engineered small intestine in the mouse.</a> Tiss. Eng. Part A. 17, 1841-1850 (2011).</li><li>Speer, A. L., Sala, F. G., Matthews, J. A., Grikscheit, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+tissue-engineered+stomach+demonstrates+epithelial+differentiation.">Murine tissue-engineered stomach demonstrates epithelial differentiation.</a> J. Surg. Res. 171, 6-14 (2011).</li><li>Haxhija, E. Q., Yang, H., Spencer, A. U., 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=Intestinal+epithelial+cell+proliferation+is+dependent+on+the+site+of+massive+small+bowel+resection.">Intestinal epithelial cell proliferation is dependent on the site of massive small bowel resection.</a> Pediatr. Surg. Int. 23, 379-390 (2007).</li><li>Zhao, L., Cheng, Z., Dhall, D., 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+novel+corrective+pullthrough+surgery+in+a+mouse+model+of+Hirschsprung's+disease.">A novel corrective pullthrough surgery in a mouse model of Hirschsprung's disease.</a> J. Pediatr. Surg. 44, 759-766 (2009).</li><li>Petrosyan, M., Guner, Y. S., Williams, 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=Current+concepts+regarding+the+pathogenesis+of+necrotizing+enterocolitis.">Current concepts regarding the pathogenesis of necrotizing enterocolitis.</a> Ped. Surg. Int. 25, 309-318 (2009).</li><li>Shew, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+concerns+in+malrotation+and+midgut+volvulus.">Surgical concerns in malrotation and midgut volvulus.</a> Ped. Radiol. 39, S167-S171 (2009).</li><li>Sampietro, G. M., Corsi, F., Maconi, G., 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=Prospective+study+of+long-term+results+and+prognostic+factors+after+conservative+surgery+for+small+bowel+Crohn's+disease.">Prospective study of long-term results and prognostic factors after conservative surgery for small bowel Crohn's disease.</a> Clin. Gastroenterol. Hepatol. 7, 183-191 (2009).</li><li>Klempnauer, J., Grothues, F., Bektas, H., Pichlmayr, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+results+after+surgery+for+acute+mesenteric+ischemia.">Long-term results after surgery for acute mesenteric ischemia.</a> Surgery. , 121-239 (1997).</li><li>Fitzgibbons, S. C., Jones, B. A., Hull, M. A., 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=Relationship+between+biopsy-proven+parenteral+nutrition-associated+liver+fibrosis+and+biochemical+cholestasis+in+children+with+short+bowel+syndrome.">Relationship between biopsy-proven parenteral nutrition-associated liver fibrosis and biochemical cholestasis in children with short bowel syndrome.</a> J. Ped. Surg. 45, 95-99 (2010).</li><li>Spencer, A. U., Kovacevich, D., McKinney-Barnett, 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=Pediatric+short+bowel+syndrome:+the+cost+of+comprehensive+care.">Pediatric short bowel syndrome: the cost of comprehensive care.</a> Am. J. Clin. Nutr. 88, 1552-1559 (2008).</li><li>Kato, T., Tzakis, A. G., Selvaggi, G., 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=Intestinal+and+multivisceral+transplantation+in+children.">Intestinal and multivisceral transplantation in children.</a> Ann. Surg. 243, 756-766 (2006).</li><li>Reyes, J., Bueno, J., Kocoshis, S., 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=Current+status+of+intestinal+transplantation+in+children.">Current status of intestinal transplantation in children.</a> J. Ped. Surg. 33, 243-254 (1998).</li></ol>

الإعلان عن أي تضارب في المصالح.

نقدم بروتوكول لإنتاج أنسجة الأمعاء الهندسة في الماوس باستخدام وحدات عضي على اساس سقالة النهج. الخطوات الأكثر أهمية هي تلك الوحدات من إعداد عضوي. يجب توخي الحذر بشكل كاف لتنظيف ومعالجة الأنسجة ميكانيكيا، ولكن يجب توخي الحذر بعدم المساواة overdigest أو overtriturate الوحدات عضي ...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> كتالوج رقم </td><td> التعليقات (اختياري) </td></tr><tr><td> HBSS </td><td> Gibco </td><td> 114170-112 </td><td></td></tr><tr><td> للمضادات الحيوية درجة التأثير 100X </td><td> إينفيتروجن </td><td> 15240-062 </td><td></td></tr><tr><td> Dispase </td><td> Gibco </td><td> 17105-041 </td><td></td></tr><tr><td> كولاجيناز النوع 1 </td><td> ورثينجتون </td><td> LS004194 </td><td></td></tr><tr><td> DMEM ارتفاع نسبة الجلوكوز 1X </td><td> Gibco </td><td> 11995-065 </td><td></td></tr><tr><td> FBS المعطل الحرارة </td><td> إينفيتروجن </td><td> 16140-071 </td><td></td></tr><tr><td> Biofelt PGA 100٪ </td><td> كونكورديا الطبية </td><td> FELT01-1005 </td><td> لإعداد البوليمر كما في المرجع.4 </td></tr><tr><td> بولي-L-حمض اللبنيك </td><td> Durect </td><td> B6002-1 </td><td> لإعداد البوليمر كما في المرجع. 4 </td></tr><tr><td> النوع الأول الكولاجين ذيل فأر، </td><td> سيغما الدريخ </td><td> C3867-1VL </td><td> لإعداد البوليمر كما في المرجع. 4 </td></tr><tr><td> كيتوبروفين 100 ملغ / مل </td><td> فورت دودج الصحة الحيوانية </td><td> 71-KETOI-100-50 </td><td></td></tr><tr><td> LabDiet 5001 تشاو القوارض </td><td> LabDiet </td><td> 5001 </td><td></td></tr><tr><td> Septra 200 ملغ / 40 ملغ لكل 5 USP، مل </td><td> مرحبا التكنولوجيا صيدلي </td><td> 50383-824-16 </td><td></td></tr><tr><td> Isoflurane، USP </td><td> طائر الفينيق الصيدلة </td><td> 57319-507-06 </td><td></td></tr></tbody></table>

tissue engineering of the intestine in a murine model

وقد تم بنجاح الأنسجة المهندسة الأمعاء الدقيقة (TESI) تستخدم لانقاذ الفئران لويس بعد استئصال الأمعاء الدقيقة ضخمة، مما أدى إلى العودة إلى الأوزان قبل الجراحة في غضون 40 يوما. 1 في البشر، ضخمة استئصال الأمعاء الدقيقة يمكن أن يؤدي إلى متلازمة الأمعاء القصيرة، وmalabsorptive الوظيفية الدولة التي تمنح قسط كبير من المراضة والوفيات وتكاليف الرعاية الصحية بما في ذلك الاعتماد التغذية الوريدية، وفشل الكبد وتليف الكبد، والحاجة إلى زرع الأعضاء multivisceral. 2 في هذه الورقة، ونحن وصف وتوثيق بروتوكول لدينا لخلق نسيج الأمعاء الهندسة في نموذج الفأر مع نهج وحدات متعددة الخلايا على اساس عضوي سقالة. وحدات متعددة الخلايا عضي هي المجاميع المشتقة من الأمعاء التي تحتوي على عناصر كل من الغشاء المخاطي والوسيطة، 3 العلاقة بين الذي يحافظ على الخلايا الجذعية المعوية المتخصصة. 4 في البحوث الجارية والمستقبلية، والانتقال من أسلوب لدينا فيوسوف تسمح الماوس للتحقيق في العمليات التي ينطوي خلال تشكيل TESI من خلال الاستفادة من أدوات المعدلة وراثيا المتوفرة في هذا النوع. 5 وتوافر سلالات الفئران المناعة ستسمح لنا أيضا لتطبيق هذه التقنية في الأنسجة المعوية البشرية وتحسين تشكيل TESI الإنسان بوصفه طعم أجنبي الماوس قبل انتقاله إلى البشر. أسلوبنا توظف الممارسات التصنيعية الجيدة (GMP) الكواشف والمواد التي تمت الموافقة عليها بالفعل للاستخدام في المرضى من البشر، ولذا فهي توفر تفوقا كبيرا على النهج التي تعتمد على الأنسجة الحيوانية decellularized. الهدف النهائي لهذه الطريقة هو ترجمتها إلى البشر كاستراتيجية الطب التجديدي العلاجية لمتلازمة الأمعاء القصيرة.

ويظهر الشكل 1 مخطط العام لبروتوكول توثيقها هنا. النتيجة النهائية لهذا البروتوكول هو بنية كروية أو العالم من الأنسجة المهندسة الأمعاء الفئران مع التجويف، الغشاء المخاطي، مخاطية، والعضلية المحيطة. الشكل 2A يظهر العالم نموذجي بالمقارنة مع بداية 2...

Watch this Scientific Journal Video about Tissue Engineering of the Intestine in a Murine Model at JoVE.com

Tissue Engineering of the Intestine in a Murine Model

هذه المادة والفيديو المصاحبة لدينا هذا البروتوكول لتوليد أنسجة الأمعاء الهندسة في الماوس، وذلك باستخدام وحدات عضي على اساس سقالة النهج.

هندسة الأنسجة من الأمعاء في نموذج الفئران

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

tissue-engineering-of-the-intestine-in-a-murine-model

Research

JoVE Journal

Bioengineering

14.8K Views.  Keck School of Medicine of the University of Southern California. هذه المادة والفيديو المصاحبة لدينا هذا البروتوكول لتوليد أنسجة الأمعاء الهندسة في الماوس، وذلك باستخدام وحدات عضي على اساس سقالة النهج.

Video: Tissue Engineering of the Intestine in a Murine Model

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

تغليف الخلايا العضلية في هيدروجيل الفايبرين للهندسة الأنسجة القلبية

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

المعالجة بعد الإنتاج من الألياف Electrospun للهندسة الأنسجة

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

مناديل الهندسة العضلات الهيكلية من الفئران الخلايا الاصلية Myoblast وتطبيق تحفيز الكهربائية

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

تصميم مفاعل حيوي ذو محورين الميكانيكية جار للهندسة الأنسجة

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

هندسة الأنسجة ل3D الإنسان<em&gt; في المختبر</em&gt; نظام ورم اختبار

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

جيل من سمك ثلاثي الأبعاد الجلد كامل أي ما يعادل واصابة الآلي

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

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 ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

الهندسة 3D Cellularized الهلام الكولاجين لتجديد الأنسجة الوعائية

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

هندسة الأنسجة عن طريق الجوهرية الأوعية الدموية في<em&gt; في فيفو</em&gt; غرفة هندسة الأنسجة

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.