Tracy C. Grikscheit, Erik R. Barthel ve Frédéric G. Sala California Rejeneratif Tıp Enstitüsü (CIRM), hibe numaraları RN2-00946-1 (TCG) ve TG2-01168 (ERB, FSG) tarafından desteklenmektedir. Allison L. Speer Üniversitesi Cerrahlar Ethicon bilgin bir Derneği&#39;dir. Yashuhiro Torashima Bir Çocuk Hastanesi Los Angeles Saban Enstitüsü Araştırma Kariyer Geliştirme Bursu tarafından finanse edilmektedir.

1. Organoid Üniteleri Hazırlık <ol><li> Fare diseksiyon (makas ve forseps) için uygun Araçlar otoklav ile sterilize edilmelidir. </li><li> Insanca yerel IACUC protokollere göre donör fare ötenazi. Hayvan geçmeden önce ölü olduğundan emin olun. </li><li> Periton boşluğuna erişmek için bir orta hat kesi olun. Pozlama geliştirmek için gerektiği gibi cilt flepleri yansıtılabilir. </li><li> Ince bağırsak canı çıksın ve Treitz ligamanı hemen distalinde bölün. Keskin ve yumuşak künt diseksiyon kullanarak mezenterden ince bağırsak ayırın. Ileoçekal bileşke belirlemek ve bu proksimal ince barsak 5 mm bölün. </li><li> Makas kullanılarak, (Invitrogen, anti-anti) 10 ml 4 ° C, steril Hanks &#39;tamponlu salin (HBSS, Invitrogen, Carlsbad, CA) / 1X-antifungal antibiyotik olan bir Petri tabağına antimezenterik sınır boyunca uzunlamasına çözelti bağırsak açın. Ile açılmış bağırsak dışkı TemizleYumuşak ajitasyon sonra, 10 ml 4 ° C, steril HBSS / 1X anti-anti ile bir 15 ml santrifüj tüpüne açılmış bağırsak aktarın. </li><li> 4 10 ml açılmış bağırsak üç kez yıkayın ° C arasında, bir test tüpü içinde anti-anti steril HBSS / 1X. Her yıkama 30 sn için 15 ml tüp hafif sallama ile gerçekleştirilebilir. Çalkaladıktan sonra, bağırsak doku tüpün altına düşer. Mezenkimal debris yüzen madde, atın. Bir pipet yardımıyla dikkatlice yıkanması çözüm çıkarın. </li><li> 4 10 ml bir Petri kabındaki yıkanmış bağırsak Kıyma ° C, steril HBSS / makas kullanılarak en az 1 mm kare parçalara 1X anti-anti. Otomatik bir pipet yardımıyla kıyma malzeme toplamak ve bir test tüpüne yerleştirin. </li><li> 8 dakika boyunca 500 rpm&#39;de tüp santrifüjleyin. Yağ ve mezenşimi içeren süpernatant, atın. </li><li> Steril HBSS / 1X anti-anti artı 0.125 mg / ml dispase (Invitrogen) ve 800 birim / m ile 10 ml kıyılmış ve yıkanmış malzeme, Digestl kollajenaz tip 1 (Worthington, Lakewood NJ). Sindirim çözelti, 40 ml hazırlamak için, dispase, kollajenaz 142 mg 5 mg tartmak, ve 40 ml bir hacme kadar steril HBSS kadar ekleyin. Her defasında taze organoid birimleri hazırlanan bu çözüm hazırlayın ve kullanmak için 4 ° C kadar hazır tutun. Adım 1.8 &#39;den direkt olarak pelet sindirim solüsyonu eklenir. </li><li> 20 dk için 37 ° C &#39;de sindirim çözelti ile kıyılmış ve yıkanmış malzeme içeren test tüpü inkübe edin. </li><li> Test tüpü ayrıca alma ve 10 ml lik bir pipet ile tritürasyon ile sindirilmiş doku bozar. Düzgün bir görüntü elde edilinceye kadar 20 ila 50 kez arasında tekrarlayın. </li><li> 800 rpm&#39;de 5 dakika süreyle test tüpü santrifüjleyin. Tek hücre içeren süpernatant, atın. </li><li> 4 ° C, steril Dulbecco Modifiye Eagle Ortamı (DMEM, Invitrogen) artı% 10 v / v ısı ile inaktive edilmiş fetal bovin serumu (HI-FBS, Invitrogen) 10 ml sindirim reaksiyonu durdurun. Pelle süspanset ve tüp sallayın. </li><li> 800 rpm&#39;de 5 dakika süreyle test tüpü santrifüjleyin. Son birkaç damla kadar otomatik bir pipet yardımıyla dikkatlice Süpernatantı. Pelet resuspending önlemek için son birkaç damla için tek kullanımlık plastik pipet kullanın. </li></ol> 2. Poliglikolik Asit İskele yüklenmesi <ol><li> Gibi Ref tarif; nonwoven poliglikolik asit (porozite&gt;% 95, Concordia Elyaf, Coventry RI 2 mm sac kalınlığı, 60 mg cm-3 yığın yoğunluğu) gelen Form 2-mm uzunluğunda, 5 mm dış çaplı silindirik iskelelerinin. 4. </li><li> Distile su içinde% 70 etanol ile hazırlanmış makasla Öneri atılabilir 1.000 mikrolitre pipeti distal 2 mm kesin. </li><li> 4 kuyu kültür plaka içine iskele yerleştirin. Birinci lümen içine ve daha sonra dış yüzey üzerine, 1.000 mikrolitre pipeti ile iskele üzerine organoid birimleri yerleştirin. Lümeninin kaplama sağlamak için bir forseps kullanın. Bozabilir veya polimer silindir şeklinde açık bozmayın. </li&gt; </ol> 3. Makine Fare içine İmplantasyonu <ol><li> Donör varsa aynı arka plan üzerine bir singenik ana farenizi kullanın. Aksi takdirde, bir immün obez olmayan diyabetik (Jackson Laboratuvarları, Sacramento CA) hayvan / ağır kombine immün yetmezliği veya NOD / SCID kullanır. </li><li> Izofluran ile genel anestezi øndükle. , Hazırlık Tıraş ve fare karın asmak. </li><li> Periton boşluğuna giriş kazanmak için 5 mm&#39;lik bir orta hat kesi olun. Belirleyin ve dikkatlice omentum bağırsaklarını. Omentum üzerine yüklenen polimer yerleştirin ve doku ile sarın. Omentum yırtmayın. </li><li> 5-0 monocryl sütür ile omentuma polimer sabitleyin. Yavaşça kendi anatomik pozisyonuna sarılmış polimer ile omentum değiştirin. </li><li> 4-0 vikril sütür kullanarak kat insizyon kapatın. Kas kapatma çalıştırın ve kesi altında abdominal organları kesmemeye dikkat edin. Cilt için suturlarla kullanın. </li><li> Kesi bitişik bir subkutan kabarıklık olarak steril su içinde 2 mg / kg ketoprofen (Ketofen, Fort Dodge Hayvan Sağlığı) ile postoperatif analjezi Yönet. Hayvanlar günlük olarak değerlendirilmelidir ve hayvan ağrı veya acı belirtileri gösteren ise, ketoprofen, ek bir doz ameliyat sonrası 2. günde tatbik edilebilir. Ameliyat sonrası üçüncü gününde, hayvan tam ağrı veya sıkıntı olmaksızın iyileşti edilmelidir. Ağrı veya sıkıntı post operatif 3. gün devam ederse, bu anormal kabul edilir ve IACUC ve hayvan bakım tesisi protokoller çerçevesinde ele alınmalıdır. </li><li> Iyileşmek için fare ve doku mühendisliği bağırsak dört hafta boyunca büyümeye izin verin. Kemirgen chow (Lab Diyet 5001, PMI Beslenme, St Louis MO) ve 5 ml (Hi-Tech Pharmacol, Amityville NY) 1:100 dilüsyon başına Septra 200 mg / 40 mg ile su hayvan ad libitum erişimi verin. </li></ol> 4. Hasat <ol><li> Humanely dört hafta implantasyonu sonrası ana hayvan ötenazi. </li><li> Orijinal kesi yeniden açın ve cilt yansıtan periton boşluğuna ulaşımı kolaylaştırmak için sefalad. </li><li> Kas tabakası açın ve doku bir küre olarak doku mühendisliği yapı tanımlar. </li><li> Keskin diseksiyon kullanarak intraabdominal organlar gelen yapı yapıştıkları aşağı atın. </li><li> Montaj sonra parafin için formalin yapı Fix, ya da real-time PCR veya protein izolasyonu gibi biyokimyasal testler için taze doku kullanın. </li></ol>

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	<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>

Çıkar çatışması ilan etti.

Biz bir organoid üniteleri-on-iskele yaklaşım kullanarak fare doku mühendisliği bağırsak üretmek için bir protokol mevcut. En kritik adımlar organoid birimlerin hazırlanması olanlardır. Bakım yeterince temiz ve mekanik doku işleme alınmalıdır, ancak sindirim gerçekleştirildikten sonra eşit bakım (adım 1.11) organoid birimleri overdigest ya overtriturate için değil alınmalıdır. Eğer bu yapılırsa, organoid birimleri adım 1.12 süpernatant içinde kaybolmuş olabilir tek hücreler, azaltılm...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td> HBSS </td><td> Gibco </td><td> 114170-112 </td><td></td></tr><tr><td> Antibiyotik-Antimikotik 100X </td><td> Invitrogen </td><td> 15240-062 </td><td></td></tr><tr><td> Dispase </td><td> Gibco </td><td> 17105-041 </td><td></td></tr><tr><td> Kollajenaz Tip 1 </td><td> Worthington </td><td> LS004194 </td><td></td></tr><tr><td> DMEM Yüksek Glikoz 1X </td><td> Gibco </td><td> 11995-065 </td><td></td></tr><tr><td> Isı inaktif FBS </td><td> Invitrogen </td><td> 16140-071 </td><td></td></tr><tr><td> Biofelt 100% PGA </td><td> Concordia Tıp </td><td> FELT01-1005 </td><td> Referans olarak polimer hazırlanması için.4 </td></tr><tr><td> Poli-L-laktik asit </td><td> Durect </td><td> B6002-1 </td><td> Referans olarak polimer hazırlanması için. 4 </td></tr><tr><td> I Kollajen, sıçan kuyruğu yazın </td><td> Sigma-Aldrich </td><td> C3867-1VL </td><td> Referans olarak polimer hazırlanması için. 4 </td></tr><tr><td> Ketoprofen 100 mg / ml </td><td> Fort Dodge Hayvan Sağlığı </td><td> 71-KETOI-100-50 </td><td></td></tr><tr><td> LabDiet 5001 kemirgen chow </td><td> LabDiet </td><td> 5001 </td><td></td></tr><tr><td> 5 ml, USP Septra başına 200 mg / 40 mg </td><td> Hi-Tech Pharmacol </td><td> 50383-824-16 </td><td></td></tr><tr><td> İzofluran, USP </td><td> Phoenix Pharmaceuticals </td><td> 57319-507-06 </td><td></td></tr></tbody></table>

tissue engineering of the intestine in a murine model

Doku mühendisliği ince bağırsak (TESI) başarıyla 40 gün içinde ameliyat öncesi ağırlıklarına dönmek sonuçlanan masif ince barsak rezeksiyonu sonrası Lewis fareleri kurtarmak için kullanılır olmuştur. İnsanlarda 1, masif ince barsak rezeksiyonu kısa barsak sendromu, fonksiyonel malabsorbtif neden olabilir önemli morbidite, mortalite ve parenteral beslenme bağımlılığı, karaciğer yetmezliği ve siroz ve multivisceral organ nakli ihtiyacı. 2 Bu yazıda da dahil olmak üzere sağlık giderleri bahşeder devlet, bir fare modelinde doku mühendisliği bağırsak oluşturmak için bizim protokol tanımlamak ve belgelemek çok hücreli organoid üniteleri-on-iskele yaklaşım. Organoid adet mukozal ve mezenkimal hem elementleri içeren bağırsak türetilen çokhücreli agrega, 3 intestinal kök hücre niş korur aralarında ilişki vardır. Süregelen ve gelecekteki araştırmalarda 4, içine bizim tekniği geçişiFare bu türün mevcut transgenik araçları kullanarak TESI oluşumu sırasında söz süreçlerin incelenmesi için izin verecektir. immünkompromize fare suşları 5 durumu da bize insan intestinal doku tekniği uygulamak ve bir insan TESI oluşumunu optimize etmek için izin verir insanlar içine onun geçiş öncesi fare ksenogreft. Bizim metodumuz iyi üretim uygulamaları (GMP) reaktifleri ve zaten insan hastalarda kullanım için onaylanmıştır ve bu nedenle decellularized hayvansal dokular güvenmemeniz yaklaşımlar üzerinde önemli bir avantaj sunuyor oylandı malzemeler kullanmaktadır. Bu yöntemin nihai hedefi kısa barsak sendromu için bir rejeneratif tıp tedavi stratejisi olarak insanlar onun çevirisidir.

Şekil 1 burada belgelenen protokolü için genel bir şema göstermektedir. Bu protokolün sonuç aynı yapı görüntüleyen bir lümen, mukoza, submukoza ve muskularis. Şekil 2A çevreleyen bir başlangıç ​​polimer iskele. Şekil 2B karşılaştırıldığında tipik bir dünya gösterir ile doku mühendisliği murin bağırsağın bir dünya veya küresel bir yapıdır keskin onun lümen ortaya bivalved. Şekil 3 inkübasyondan 4 hafta sonra ...

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

Bu makalede ve beraberindeki video bir organoid üniteleri-on-iskele yaklaşımı kullanarak, fare doku mühendisliği bağırsak üretmek için bizim protokol sunuyoruz.

Bir fare modelinde Bağırsak Doku Mühendisliği

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 Görüntüleme.  Keck School of Medicine of the University of Southern California. Bu makalede ve beraberindeki video bir organoid üniteleri-on-iskele yaklaşımı kullanarak, fare doku mühendisliği bağırsak üretmek için bizim protokol sunuyoruz.

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.

Kalp Doku Mühendisliği Fibrin Hidrojel kardiyomiyositlerinin Kapsülleme

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

Biyomühendislik

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.

Doku Mühendisliği için elektrospinning Liflerin Postprodüksiyon İşleme

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.

Elektrik Stimülasyonu murin myoblast Progenitör Hücreler ve Uygulama Mühendislik İskelet Kası Dokular

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.

Doku Mühendisliği için iki eksenli Mekanik Yükleniyor Biyoreaktör tasarımı

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.

İnsan 3D Doku Mühendisliği<em&gt; In vitro</em&gt; Tümör Test Sistemi

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.

Üç-boyutlu Tam Kalınlığı Deri Üretimi Eşdeğer ve Otomatik Yaralama

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

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.

Vasküler Doku Rejenerasyon için Mühendislik 3D cellularized Kollajen jeller

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.

Bir ıntrinsic Vaskülarizasyon tarafından Doku Mühendisliği<em&gt; İn Vivo</em&gt; Doku Mühendisliği Odası

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.

Bir Küçük Hayvan Modelinde Arteriovenöz (AV) Döngü Anjiogenez ve Vaskülarize Doku Mühendisliği Eğitim için

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 ...

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been &#39;conditioned&#39; by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

We present a model of ligament tissue in which three-dimensional constructs are treated with the human exercise-conditioned serum and analyzed for collagen content, function, and cellular biochemistry.

Egzersiz Koşullandırılmış Serum İle Ligament Yapılarının Tedavisi: Bir Translasyonel Doku Mühendisliği Modeli

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, ...