We thank the laboratory of Dr. Stephen Moss for providing embryonic rat brain tissues. M.D.T. designed the original protocol. This work was funded by National Institutes of Health P41 Tissue Engineering Resource Center Grant EB002520. K.C. was supported with Postdoctoral Fellowship from German Research Foundation (DFG) (CH 1400/2-1).

Beyin dokusu izolasyon protokolü Tufts Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından onaylanmış ve Bakımı ve Kullanımı Laboratuvar Hayvanları (Kurumsal Hayvan Bakımı ve Kullanımı Komitesi B2011-45) Sağlık Rehberi National Institutes ile uyumludur edildi. 1. İpek İskele Hazırlık <ol><li> Ipek böceği kozası ipek solüsyonunun hazırlanması, daha önce 19,20 açıklandığı gibi. <ol><li> Makas kullanılarak 8 eşit parçaya her koza kesin. Parçalanmış koza 5 g için yaklaşık 11 koza kullanın. (15 dakika) </li><li> 0.02 M Na 2 CO 3 çözeltisi 2 L hazırlayın ve sıcak bir plaka kullanılarak kaynatın getirmek. (15 dakika) </li><li> Parçalanmış kozaların 5 g tartın ve 30 dakika süre ile, Na 2 CO 3 çözelti içinde kaynatın. Bir spatula ile kaynama ipek dakika her çift karıştırın. Ayrıca de-Gumming adlandırılan bu adım, hidrofil proteinler, sericins ipek fibroin arındırır. (30 dakika) </li><li&gt; elle fibroin sıkın ve kalan sericin ve kimyasallar dışarı yıkamak için distile su ile en az üç kez durulayın. (5 dakika) </li><li> Petri ıslak fibroin yerleştirin ve akış kaput O / N fibroin kurutun. </li><li> Bir sonraki gün kuru fibroin kütle ağırlığında ve bir cam beher içinde fibroin yerleştirin. (15 dakika) </li><li> 4. kuru fibroin kütlesini çarpılarak LiBr 9.3 M LiBr solüsyonu (ml) Gerekli hacmi hesaplamak içinde fibroin eritmek için yavaşça ipek fibroin üzerinde LiBr çözüm dökün ve spatula ile tüm fibroin lifleri batırmayın. Buharlaşmasını önlemek ve fiber çözünmesi için 4 saat süre ile 60 ° C &#39;de yerleştirmek için beher örtün. (15 dakika) </li><li> Şırınga kullanarak, kaptan fibroin çözümü toplamak ve MWCO 3500 diyaliz kasetleri içine yerleştirin. 48 saat süre ile destillenmiş suya karşı diyaliz gerçekleştirin. Suya her iki saatte değiştirin. </li><li> Şırınga kullanılarak, gelen Fibroin çözümü toplamak20 dakika boyunca 4 ° C&#39;de 9000 rpm&#39;de (12700 x g ~), iki kez 50 ml konik tüp ve santrifüj içine kasetleri. Her santrifüj aşamasından sonra yeni bir tüp içine süpernatant dökün ve pelet atın. (40 dakika) </li><li> Kuru ağırlığı tahminine göre fibroin konsantrasyonunu ölçün. Bir tartmak tekne içine ipek solüsyonu 1 ml dökün. 2 saat süre ile 60 ° C &#39;de fırında örnek kurutun. Kuru ipek fibroin tartılır ve 100 ile ipek çözeltisi beklenen konsantrasyon elde edilen ağırlık çarpılarak ipek fibroin çözeltisi konsantrasyonunu hesaplamak 6-9% (a / h). </li><li> Damıtılmış su içinde seyreltilmesi ile (ağırlık / hacim)% 6 ipek konsantrasyonu ayarlayın. Durdurma noktası: Sıvı ipek fibroin kapalı bir kapta en fazla bir ay süreyle 4 ° C&#39;de muhafaza edilebilir. </li></ol></li><li> Ipek çözeltisinden gözenekli iskelelerinin hazırlanması. <ol><li> Granüller daha sonra aşamalarda kullanılacak 500-600 um büyüklüğünde ayrı zerre NaCI Elek. Granül atıns 500 mikron altında ve 600 mikron üzerinde büyüklükte. (15 dakika) </li><li> Politetrafloroetilen (PTFE) kalıp (Şekil 1) içine 6% ipek çözeltisi 30 ml dökün. Yavaşça ipek üzerine elekten NaCl 60 gr dağılım. Tuz biçimli bir tabakasını elde etmek kabı dokunun. Ipek polimerize etmek için oda sıcaklığında 48 saat inkübe edilir. </li><li> Polimerizasyonu sonlandırmak ve kalan herhangi bir sıvı buharlaştırılması için 1 saat boyunca 60 ° C&#39;de bir fırında iskelesi ihtiva eden PTFE kalıp yerleştirin. </li><li> Tuz süzülecektir 48 st için damıtılmış su 2 L ihtiva eden bir beher içinde PTFE kalıp içerik yerleştirin. Suya günde 2-3 kez değiştirin. . Tuz tamamen noktası durdurma dışarı süzülen zaman kalıplardan sünger iskeleleri kaldırın: süngerler dehidratasyon gelen iskeleleri önlemek için kapalı bir kapta 4 ° C&#39;de suya saklanabilir. </li><li> Hazır olduğunuzda, 5 mm çapında biyopsi yumruk iskeleleri kesip. Yüksekliği yaklaşık 2 mm ulaşmak için iskeleleri Önceden kesilmiş. Pu2 mm çapındaki bir biopsi darbe ucu (Şekil 2A) ile birlikte, skafoldun merkezini NCH. (1 saat) </li><li> . Noktası durdurma (ıslak döngüsü 121 ° C, 20 dakika) ile sterilize edilmeleri için su içine batırılır iskelesi Otoklav: süngerler dehidratasyon iskeleleri önlemek için kapalı bir kapta, 4 ° C sıcaklıkta su içinde daldırılır saklanabilir. </li><li> Planlanan hücre ekim öncesi steril 0.1 mg / ml poli-D-lisin (PDL) çözelti içinde iskeleleri bırakın. 37 ° C &#39;de 1 saat süreyle inkübe edin. </li><li> Iskeleleri bağlı olmayan PDL çıkarılması için fosfat tamponlu salin (PBS) ile 3 kez yıkanır. (30 dakika) </li></ol></li></ol> Rat Kortikal nöronlar 2. izolasyonu <ol><li> Onaylı hayvan protokolü elde edildikten sonra daha önce Pacifici ve Peruzzi 27 tarafından açıklandığı gibi embriyonik günden itibaren 18 (E18) Sprague-Dawley sıçanları korteks parçalara ayır. (2 saat) </li><li> 3, 20 dakika boyunca (sığır pankreasından)% 0.3 DNase I ile% 0.25 tripsin 5 ml 10 korteksleri inkübe7 ° C. </li><li> 1 mg / ml soya fasulyesi proteinin eşit bir hacminin eklenmesiyle tripsin inaktive. </li><li> Tek bir hücre süspansiyonu oluşturulur kadar pipetleme 10 ml pipet kullanarak Pasteur korteks ve aşağı 20 kez Karışım. Nazik olun ve hava kabarcığı oluşumunu önlemek. (10 dakika) </li><li> 5 dakika boyunca 127 x g&#39;de hücre süspansiyonu santrifüjleyin. </li><li> Yeniden askıya kültür ortamında 10 ml hücre pelletini (Neuro temelli vasat, 1 x B27 takviyesi, 1x Glutamax,% 1 penisilin / streptomisin). Hücreleri saymak. Beklenen hücre konsantrasyonu yaklaşık 2x10 7 / ml&#39;dir. (20 dakika) </li></ol> 3. Meclis ve Kültür Construct <ol><li> Hücrelerle İskele tohumlama. <ol><li> Bir hücre kültürü kaput içinde steril iskeleleri ve gerekli tüm eşyaları taşıyın. Kullanma steril forseps bir 96-yuvalı hücre kültürü plakasında iskeleleri de başına bir iskele tahsis yerleştirin. (10 dakika) </li><li> Bunları önce, tohum hücre dengelenmeye hücre kültür ortamı içinde iskelesi bırakıning. 37 ° C &#39;de 1 saat süreyle inkübe edin. (10 dakika) </li><li> Iskeleler ortamın aşırı aspire. </li><li> Hücre süspansiyonu / iskele 100 ul uygulayın. (10 dakika) </li><li> Iskele hücre bağlanmasına olanak sağlamak için 37 ° de CO / N hücreleri inkübe edin. </li><li> Ertesi sabah olmayan bağlanmış hücreler aspire 200 ul / oyuk taze kültür ortamı uygulanır. (10 dakika) </li></ol></li><li> Kollajen matrisi ile gömme Temel Yapı. (2 saat) <ol><li> Ben buz üzerinde kollajen 10x PBS, su, 1 N NaOH ve sıçan kuyruğu yerleştirin. Üreticinin talimatlarına göre kollajen çalışma çözeltisi hazırlayın. Hücre-numaralı seribaşı yapıları (1 saat kadar) hazır olana kadar buz üzerinde koruyun. </li><li> Inkübatör hücre numaralı seribaşı ipek yapıları çıkarın ve ortamın aşırı aspire. </li><li> Kullanma steril forseps oyuklu bir plaka üzerinde boş oyuklara iskeleleri transferi ve 3 mg / ml kolajen çözeltisi 100 ul her bir iskele bırakın. Geri doku kültürü plakasınıinkübatörde 30 dakika kollajen polimerizasyonunun izin vermek için. </li><li> De / önceden ısıtılmış hücre kültür ortamında 100 ul uygulanır. Kültür bir hafta orta sadece yarısı hacmini değiştirerek her gün orta değiştirmek için yapıları. </li></ol></li></ol> 4. Mikroskopi Analizi <ol><li> Canlı / ölü boyama (1 saat) <ol><li> Propidium iyodür (PI) damıtılmış su içinde 1 mg / ml (1,5 mM) stok çözelti hazırlayın. </li><li> Aseton floresandiasetat (FDA) 2 mg / ml stok çözelti hazırlayın. </li><li> PBS içinde seyreltilmiş 10 uM PI ve 0.15 uM FDA içeren çalışma çözeltisi hazırlayın. Bir su banyosu içinde 37 ° C&#39;ye kadar çözüm önceden ısıtın. </li><li> Serum esterazlar kaldırmak için önceden ısıtılmış hücrelerin PBS ile yıkayın. PBS aspire. </li><li> 2 dakika için hücreler ile ilgili önceden ısıtılmış çalışma çözeltisi uygulanır ve PBS ile yıkayın. </li><li> FDA eski λ = 490 n; görüntüye epifluorescent mikroskop kullanın hücreleri (PI ex λ = 490 nm, em λ = 570 nmm, em λ) 514 nm =. Leke 40 dakika boyunca sabit kalır. Uzun süreli boyama PI hücreleri tarafından alınması ve hücrelerinde PI / FDA eş-lokalizasyonu kaynaklanan spesifik olmayan boyama ile sonuçlanabilir. </li></ol></li><li> Immün <ol><li> Kültür Arzu edilen zaman noktasında oda sıcaklığında 30 dakika süreyle% 4 paraformaldehid (PFA) ile hücrelerin tespit. Nedeniyle PFA toksisitesine sabitleme adımının kimyasal çeker ocak içinde yapılmalıdır dumanı. </li><li> . 3x PBS durdurma noktasına sahip iskeleleri yıkayın: PFA sabit yapılar daha başka aşamalar ile devam etmeden önce, birkaç gün 4 ° C&#39;de PBS içinde saklanabilir. </li><li> 4 ° C &#39;de primer antikor O / N içeren PBS içinde% 0.2 Triton,% 0.25 sığır serum albümini (BSA) iskeleleri inkübe edin. </li><li> Ertesi gün, boyama çözeltisi atılır ve bağlanmamış birinci antikorları ayıklamak için PBS ile iskeleler 3 x 30 dakika yıkama. </li><li> 2 PBS içinde% 0.2 Triton içinde seyreltilmiş sekonder antikor,% 0.25 BSA ile inkübe edinoda sıcaklığında sa. </li><li> Boyama çözeltisini çıkarın ve Bağlanmamış ikinci antikorları ayıklamak için PBS ile iskeleler 3 x 30 dakika yıkama. </li><li> Görüntü 1 mikron adım numune 100 mikron z-bölümleri alarak 20X amacı ile bir konfokal mikroskop kullanılarak iskeleler. Görüntü çözünürlüğü en az 1024 x 1024 piksel olmalıdır ince nevritlerle görselleştirmek için. </li></ol></li><li> RNA, DNA ve protein izolasyonu Not: RNA, DNA ve protein izolasyonu üretici talimatlarına uygun olarak bir ticari AllPrep DNA / RNA / Protein Mini Kit kullanılarak gerçekleştirilir. <ol><li> Kullanımı steril forseps 2 ml steril tüpe kültür plaka dışına iskeleleri aktarın. Tüp başına tek bir iskele yerleştirin. </li><li> Her tüpe RLT tamponu 200 ul ekle. </li><li> Steril microscissors ile parçalamadan tarafından yapı bozar. </li><li> Kesintiye doku homojenize, spin kolona tüpün içeriğini aktarmak. Bir mikrosantrifüj tam hızda 2 dakika süreyle Spin.Bu spin kolon boyunca geçerken lizat homojenize edilir. </li><li> Aracılığıyla akışını toplamak ve RNA, imalatçı protokolü aşağıdaki DNA ve protein izole etmek için hemen kullanabilirsiniz. </li><li> Nükleik asitlerin verim uyumlu herhangi bir yöntem (örneğin, NanoDrop) ölçmek. </li><li> Üreticinin talimatlarına uygun olarak BCA protein deneyi kullanılarak protein verimi ölçmek. Dalga boyu 562 nm mikroplaka okuyucu kullanarak tahlil sonuçlarını ölçün. Not: nükleik asit ve hücre yoğunluğuna göre bir yapı için protein beklenen verim Şekil 4 &#39;de gösterilmiştir. </li></ol></li></ol>

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J., Park, J., Kim, H. J., Wada, M., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+aqueous-derived+biomaterial+scaffolds+from+silk+fibroin.">Three-dimensional aqueous-derived biomaterial scaffolds from silk fibroin.</a> Biomaterials. 26 (15), 2775-2785 (2005).</li><li>Gil, E. S., Mandal, B. B., Park, S. H., Marchant, J. K., Omentetto, F. G., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicoidal+multi-lamellar+features+of+RGD-functionalized+silk+biomaterials+for+corneal+tissue+engineering.">Helicoidal multi-lamellar features of RGD-functionalized silk biomaterials for corneal tissue engineering.</a> Biomaterials. 31 (34), 8953-8963 (2010).</li><li>Hopkins, A. 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=Silk+hydrogels+as+soft+substrates+for+neural+tissue+engineering.">Silk hydrogels as soft substrates for neural tissue engineering.</a> Adv Funct Mater. 23 (41), 5140-5149 (2013).</li><li>Pacifici, M., Peruzzi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+rat+embryonic+neural+cells:+A+quick+protocol.">Isolation and culture of rat embryonic neural cells: A quick protocol.</a> Vis. Exp. (63), e3965 (2012).</li></ol>

Bu video-makale yayınlanması Corning tarafından desteklenmektedir.

Here we described the guidelines to assemble a compartmentalized 3D brain-like tissue model. The model is characterized by dense polarized culture of neurons resulting in the development of two morphologically different compartments: one containing densely packed cell bodies, second containing pure axonal networks. Overall, the scaffold architecture and growth pattern mimic brain cortical tissue including the six laminar layers and white matter tracts21.
The donut-shaped silk protein-based tissue model allows for modifications and tuning of mechanical properties, versatile structural forms, hydrogel fillers and cellular components. Thus, this tissue model establishes a base platform for a wide range of studies. Silk is a favorable candidate for biomaterial platforms due to its biocompatibility, aqueous-based processing, and robust mechanical and degradation properties22. The silk matrix also serves as a stable anchor to reduce collagen gel contraction over time in culture. The properties such as silk concentration and porosity of the scaffold used in this model have been previously adjusted to achieve optimal cell growth and mechanical properties resulting in brain-like tissue elasticity18,23,24. We suggest to keep these parameters constant to ensure the successful outcome and reproducibility of the experiments.
The 3D neuronal network formation was achieved by combining two types of biomaterials with different mechanical properties: a stiff scaffold to provide neuronal anchoring and a softer gel matrix to permit axon penetration and connectivity in 3D. Selective material preferences of the silk scaffold and soft hydrogel provide the underlying principle for compartmentalizing the neurons from the axonal connections. Due to the inert nature of the silk scaffold, functionalization with poly-D-lysine is required for neuronal attachment. However, other cell adhesion promoting factors can be applied such as RGD25 or fibronectin26. To achieve the 3D network formation the silk scaffold needs to be filled with hydrogel in a timely manner as soon as neurons are attached to the scaffold. Likewise, the collagen matrix filler can be replaced by other hydrogels, thus allowing the study of the influence of 3D extracellular environments on axonal network formation to serve as a platform to evaluate novel hydrogels in terms of neuronal compatibility. Additionally, apart from rat cortical neurons other neuronal sources such as hippocampal neurons or induced pluripotent cell (iPSc)-derived neurons can be utilized. Moreover, the tissue model can be used to study heterocellular interactions by including glial cells along with neurons and to build more complex brain-like tissue.
As shown previously, our model can be utilized to evaluate neuronal functionality in 3D microenvironment with a variety of assays such as cell viability, gene expression, LC-MS/MS and electrophysiology, thus demonstrating physiological relevance of the model18. Other methods, which are frequently utilized to evaluate 3D tissue-engineered constructs, such as immunostaining and microscopy8,9,11 can also be used to assess cell distribution and extent of axonal network formation. However, it has to be noted, that due to the high density of the collagen matrix, the penetration of antibodies and depth of the imaging is limited to few hundred micrometers. Moreover, the signal to noise ratio may be affected by nonspecific background fluorescence. This can be overcome by using lipophilic cell tracers and genetically expressed fluorescent proteins which diminish the need for immunostaining11. Alternatively, the imaging can be performed with 2-photon microscopy instead of usual one-photon technique, which may reduce the signal loss, photobleaching, and can be extended to several hundred micrometers of depth.
Summarizing, the silk and collagen-based brain-like tissue offers a robust platform to study neuronal networks in 3D and is a starting point for the development of more advanced models of neurological disorders in the future. Independent from the mode of evaluation, the relative simplicity of this tissue model supports its utility, success and reproducibility.

Merkezi sinir sistemi (CNS), yapısal, işlevsel, bulaşıcı veya dejeneratif damar, ilgili muhtelif rahatsızlıkların etkilenebilir. Tahminen 6,8 milyon kişinin 1 dünya çapında büyüyen bir sosyoekonomik yük temsil nörolojik bozukluklar sonucu olarak her yıl ölmektedir. Ancak, hastalıkların sadece birkaç kullanılabilir tedaviler var. Bu nedenle, nörolojik bozukluklar muzdarip hastalar için yeni terapötik stratejiler için ciddi bir ihtiyaç vardır. Ne yazık ki, pek çok CNS hedefli terapötik nedeniyle fizyolojik ilgili işlevsel okumalarla akut ve kronik etkilerinin değerlendirilmesine izin vermez yetersiz klinik öncesi araştırma modelleri, kullanımına kısmen klinik denemeler sırasında başarısız. Geçmiş yıllarda önemli araştırma çabalarına rağmen, CNS yapısı ve fonksiyonu hakkında bilinmeyen büyük bir miktardır. Daha fazla bilgi edinmek için, hayvan modelleri sık sık bize olaned özellikle pre-klinik çalışmalarda, bu tür travmatik beyin hasarı (TBI) veya demans gibi patolojik durumları, model. Bununla birlikte, hayvanlar insan hem de merkezi sinir sisteminin anatomisinde, hem de fonksiyonu, gen ekspresyonu ve metabolizma 2-4&#39;te önemli ölçüde farklıdır. Öte yandan, 2 boyutlu, in vitro kültürler, hücre biyolojisi araştırmak için yaygın yöntem ve rutin olarak ilaç keşfi için kullanılmıştır. Bununla birlikte, 2B hücre kültürleri karmaşıklığı ve insan beyin 5-7 ile karşılaştırıldığında fizyolojik önemi yoksundur. Düşük maliyet ve basitlik 2D hücre kültürü çalışmaları ya da hayvan modellerinde tarafından sağlanan karmaşıklığı için yerini hiçbir şey tutamaz iken, 3D doku mühendisliği vitro ve in vivo yaklaşımları 2D arasında mevcut boşluğu kapatmak için geliştirilmiş araştırma modellerini oluşturabilir. 3D doku mühendisliği 3D hücre-hücre etkileşimleri ve biyomalzeme iskelelerde tarafından sağlanan dışı ipuçları elde daha fizyolojik ilgili deneysel koşullar sağlar. Desp3D kültürlerin değeri arkasında önemli deliller ite, orada bu tür kök hücre türevli organoid kültürler 8-10 gibi birkaç 3D CNS doku modelleri, 11 neurospheroids anda ve hidrojel kültürleri 12,13 dağılmış. Çok katmanlı litografi 14, ve 3 boyutlu baskı 15 dahil olmak üzere gelişmiş teknik yöntemler akciğer, karaciğer ve böbrek dokusunu incelemek için kullanılmaktadır. Ancak, bu tür kortikal mimarisi ve biyoloji taklit olarak bölümlere nöronal büyüme için izin 3D CNS modellerin eksikliği vardır. Nöronal hücre gövdeleri nevritlerin Ayrılmış büyüme önceden akson yolu izleme, kalsiyum akını, ağ mimarisi ve faaliyetlerin çalışma izin mikroüretim 16,17 ile 2D kültürlerde ortaya konmuştur. Bu fikir hücre gövdeleri ve aksonal yolları beynin 18 katmanlı mimarisi taklit farklı bölümlerinde bulunan 3D polarize sinir dokusu geliştirmek için bize ilham </sup&gt;. Yaklaşımımız kapalı bir hacim içindeki hücreleri göz yüksek yoğunluk uygundur ve kolajen jele yoğun aksonal ağ aşırı büyümesini sağlayan benzersiz ipek skafold tasarım kullanımına dayanmaktadır. Burada iskele imalat ve nöronal kültürü gibi beyin gibi doku tam montaj işlemini göstermektedir.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Na2CO3</td>
			<td>Sigma-Aldrich</td>
			<td>223530</td>
			<td>-</td>
		</tr>
		<tr>
			<td>LiBr</td>
			<td>Sigma-Aldrich</td>
			<td>213225</td>
			<td>-</td>
		</tr>
		<tr>
			<td>MWCO 3500 dialysis cassettes&#160;</td>
			<td>Thermo Fisher</td>
			<td>66110</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Heating plate</td>
			<td>Fisher Scientific</td>
			<td>Isotemp</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Centrifuge 5804 R</td>
			<td>Eppendorf</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sieve</td>
			<td>Fisher Scientific</td>
			<td>-</td>
			<td>No. 270, No. 35, No. 30</td>
		</tr>
		<tr>
			<td>PTFE mold</td>
			<td>-</td>
			<td>-</td>
			<td>made in house (Figure 1) 10 cm diameter, 2 cm height</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>71382</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Biopsy Punch 5mm</td>
			<td>World Precision Instrument</td>
			<td>501909</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Biopsy Punch 2mm</td>
			<td>World Precision Instrument</td>
			<td>501908</td>
			<td>-</td>
		</tr>
		<tr>
			<td>poly-D-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P6407-5MG</td>
			<td>final concentration 10 ug/ml, dissolved in water</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma-Aldrich</td>
			<td>D1283-500ML</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma-Aldrich</td>
			<td>T4049-500ML</td>
			<td>-</td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>Roche</td>
			<td>10104159001</td>
			<td>final concentration 0.3%</td>
		</tr>
		<tr>
			<td>Soybean protein</td>
			<td>Sigma-Aldrich</td>
			<td>T6522-100MG</td>
			<td>final concentration 1 mg/ml</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>warm up to 37&#176;C before use</td>
		</tr>
		<tr>
			<td>B27 supplement 50x</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Penicilin Streptomycin</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Collagen I, rat tail, 100 mg</td>
			<td>Corning</td>
			<td>354236</td>
			<td>final concentration 3 mg/ml</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma-Aldrich</td>
			<td>S2770</td>
			<td>corrosive</td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Sigma-Aldrich</td>
			<td>P4170-10MG</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>Fluorescein Diacetate</td>
			<td>Sigma-Aldrich</td>
			<td>F7378-5G</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Olympus MVX10</td>
			<td>Olympus</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>toxic, final concentration 4%</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7906-10G</td>
			<td>-</td>
		</tr>
		<tr>
			<td>anti-&#946;IIITubulin antibody&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T2200</td>
			<td>rabbit 1:500</td>
		</tr>
		<tr>
			<td>anti-rabbit Alexa-546</td>
			<td>Molecular Probes</td>
			<td>A11010</td>
			<td>goat 1:250</td>
		</tr>
		<tr>
			<td>goat serum</td>
			<td>Sigma-Aldrich</td>
			<td>G9023-5ML</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Leica SP2 confocal microscope</td>
			<td>Leica</td>
			<td>-</td>
			<td>objective 20x</td>
		</tr>
		<tr>
			<td>QIAshredder</td>
			<td>Qiagen</td>
			<td>79654</td>
			<td>-</td>
		</tr>
		<tr>
			<td>AllPrep DNA/RNA/Protein Mini Kit</td>
			<td>Qiagen</td>
			<td>80004</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Ethyl alcohol, Pure</td>
			<td>Sigma-Aldrich</td>
			<td>E7023&#160;</td>
			<td>-</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>63689</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 2000c/2000 UV-Vis Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>BCA protein assay</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Plate reader Spectramax M2</td>
			<td>Molecular Devices</td>
			<td>-</td>
			<td>absorbance 562 nm</td>
		</tr>
		<tr>
			<td>Micro Scissors, Economy, Vannas-type</td>
			<td>TedPella</td>
			<td>1346</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

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.

Çörek şekli Katı ipek süngerler hazır kesilmiş sinir dokusu (Şekil 2A) benzeyen bir mimarisi bölümlere ulaşmak için benzersiz ve basit bir fikri temsil etmektedir. Küçük bir hacim içinde tohumlama ve yüksek hücre yoğunluğuna büyümesini sağlayan son derece yüksek bir yüzey alanı içinde yaklaşık 500 um sonuç gözenek boyutlu iskele oldukça gözenekli bir yapı (2x10 7 / ml) (Şekil 2B). Ayrıca, iskelenin yüksek gözeneklilik genişletilmiş zaman dilimlerinde üzerinde yoğun hücre kültürlerinin üstün canlılığı ile sonuçlanan besin ve atıkların sınırsız yayılması için izin verir. Nöronlar tohumlama sonra hızlı bir şekilde ve homojen bir skafold gözenek yüzeyine bağlanır. Hücre bağlanma tamamlandığında ve hücreler, ipek alt-tabaka üzerinde dengelenir sonra, sünger skafold aksonal ağı oluşumu (Şekil 3 için 3 boyutlu bir ortam sağlamak amacıyla yumuşak kolajen matriks ile doldurulur </strong&gt;). Nöronların sadece 2B yüzeyler (Şekil 3B) ile bulunduğu gibi karışık ağlarda elde edilen gözenek yüzeyi üzerinde uzantılar büyüdükçe kollajen matris yokluğunda akson ve hücre gövdeleri bölmelere mümkün değildir. Bunun aksine, kollajen jel nöronal hücre gövdeleri ipek iskele (Şekil 3C) bağlı kalır ise, 3D geniş aksonal dış gelişimini destekleyen bir ağ örgüsü içerir. Bu büyüme paterni hücre gövdeleri ve saf aksonal ağlarda beyinde korteks gri ve beyaz madde benzer (Şekil 3D), compartmentalization yol açar. Ayrı mikroskopisi, konstruktlar deney arkasındaki soruya bağlı olarak, diğer yöntemlerle 18 çeşitli değerlendirilebilir. Örneğin, konstruktların DNA, RNA ve protein izolasyonu kolayca ticari kitler (Şekil 4) kullanılarak gerçekleştirilmiştir olabilir. Nükleik asitler ve protein pe verimir yapı başlangıçta ipek iskelelerinin numaralı seribaşı hücrelerin sayısına bağlıdır. Daha fazla Hücreler DNA, RNA ve protein verimi artar. <img alt="figür 1" src="/files/ftp_upload/52970/52970fig1.jpg" /> Ipek sünger iskele hazırlanmasında kullanılan Şekil 1. PTFE kalıp ölçüleri:.. 10 cm çapında, 2 cm yüksekliğinde <a href="https://www.jove.com/files/ftp_upload/52970/52970fig1large.jpg" target="_blank">bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 2," src="/files/ftp_upload/52970/52970fig2.jpg" /> Şekil 2. 3B beyin gibi doku modeli. (A) Gözenekli ipek sünger iskele. Daha önce kolajen (yeşil-canlı hücreler, kırmızı ölü hücreleri) gömme ipek iskele üzerinde tohumlama üzerine günde 1 nöronların (B) Canlı / ölü boyama. Sağ tarafta Panelleri ma göstermekKırmızı çerçeveli yakalanan alanın gnification. <a href="https://www.jove.com/files/ftp_upload/52970/52970fig2large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 3," src="/files/ftp_upload/52970/52970fig3.jpg" /> Şekil 3. nöronal 3D beyin gibi doku modelinde bölümlere akıbet (A) İskele bölmeleri:. Kırmızı hücre gövdesi bölmesi, Mavi-aksonal bölmesi. (B) kollajen katıştırma önce tohumlama üzerine 1 gün de Nöronal büyüme paterni. (C). Hücre gövdesi bölmesinde tohumlama üzerine günde 7 nöronal akıbet. (D) akson bölmeye tohumlama üzerine 7. günde Nöronal sonucudur. Yeşil -. ΒIIITubulin <a href="https://www.jove.com/files/ftp_upload/52970/52970fig3large.jpg" target="_blank">bu büyük halini görmek için tıklayınız</a>rakam. <img alt="Şekil 4," src="/files/ftp_upload/52970/52970fig4.jpg" /> (A) RNA ve DNA ve iskele başına seribaşı hücrelerin miktarına bağlı olarak yapı başına (B) protein Şekil 4. Beklenen verim (± SD, n = 5). <a href="https://www.jove.com/files/ftp_upload/52970/52970fig4large.jpg" target="_blank">Bu rakamın büyük halini görmek için tıklayınız .</a>

Watch this Scientific Journal Video about Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue at JoVE.com

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

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.

Polarize Sinir Doku Engineered 3D İpek-kolajen bazlı Modeli

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 ...

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12.4K Görüntüleme.  Tufts University. 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.

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

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

2D ve 3D Ortamlarda Sinir Progenitör Hücreleri Elektrik Alanı kontrollü Yönetmen Göç

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

Biyomühendislik

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.

Tri-broşür Polimer Vanalar göreceli hidrodinamik Değerlendirme Protokolü

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

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 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.

Bir Fare Modeli Doku mühendisliği Kapların Üretimi ve Tırnak

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.

3D Doku mühendisliği İnsan Özofagus Mukozal Modeli Üretimi, Karakterizasyonu ve Potansiyel Kullanımları

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

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific mutational background. The model is generated on a decellularized porcine scaffold that reproduces tissue-specific characteristics regarding extracellular matrix composition and architecture including the basement membrane. We standardized a protocol that allows artificial tumor tissue generation within 14 days including three days of drug treatment. Our article provides several detailed descriptions of 3D read-out screening techniques like the determination of the proliferation index Ki67 staining&#39;s, apoptosis from supernatants by M30-ELISA and assessment of epithelial to mesenchymal transition (EMT), which are helpful tools for evaluating the effectiveness of therapeutic compounds. We could show compared to 2D culture a reduction of proliferation in our 3D tumor model that is related to the clinical situation. Despite of this lower proliferation, the model predicted EGFR-targeted drug responses correctly according to the biomarker status as shown by comparison of the lung carcinoma cell lines HCC827 (EGFR -mutated, KRAS wild-type) and A549 (EGFR wild-type, KRAS-mutated) treated with the tyrosine-kinase inhibitor (TKI) gefitinib. To investigate drug responses of more advanced tumor cells, we induced EMT by long-term treatment with TGF-beta-1 as assessed by vimentin/pan-cytokeratin immunofluorescence staining. A flow-bioreactor was employed to adjust culture to physiological conditions, which improved tissue generation. Furthermore, we show the integration of drug responses upon gefitinib treatment or TGF-beta-1 stimulation - apoptosis, proliferation index and EMT - into a Boolean in silico model. Additionally, we explain how drug responses of tumor cells with a specific mutational background and counterstrategies against resistance can be predicted. We are confident that our 3D in vitro approach especially with its in silico expansion provides an additional value for preclinical drug testing in more realistic conditions than in 2D cell culture.

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

We present a three-dimensional (3D) lung cancer model based on a biological collagen scaffold to study sensitivity towards non-small-cell-lung-cancer-(NSCLC)-targeted therapies. We demonstrate different read-out techniques to determine the proliferation index, apoptosis and epithelial-mesenchymal transition (EMT) status. Collected data are integrated into an in silico model for prediction of drug sensitivity.

Kombine bir 3D doku mühendisliği<em&gt; İn Vitro</em&gt; /<em&gt; Siliko</emSpesifik mutasyonel Arka İlaç Etkinliği tahmin için&gt; Akciğer Tümör Modeli

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting kıkırdak ve cilt doku analogları bir roman pasif birimi teknik Bioink Precellularization için karıştırma kullanmak

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

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.

Tohum ve bir biyosentetik doku Mühendisliği trakeal greft bir fare modeli implantasyonu

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.

Sıçanlarda Çevresel Özofagus Rekonstrüksiyonu için Doku-Mühendislik Greft

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

3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also respond to neural injuries and act to protect the tissue from degenerative events. In vitro studies of astrocytes&#39; functionality are important to elucidate the mechanisms involved in such events and contribute to developing therapies to treat neurological disorders. This protocol describes a method to biofabricate a neural-like tissue structure rich in astrocytes by 3D bioprinting astrocytes-laden bioink. An extrusion-based 3D bioprinter was used in this work, and astrocytes were extracted from C57Bl/6 mice pups&#39; brain cortices. The bioink was prepared by mixing cortical astrocytes from up to passage 3 to a biomaterial solution composed of gelatin, gelatin-methacryloyl (GelMA), and fibrinogen, supplemented with laminin, which presented optimal bioprinting conditions. The 3D bioprinting conditions minimized cell stress, contributing to the high viability of the astrocytes during the process, in which 74.08% &#177; 1.33% of cells were viable right after bioprinting. After 1 week of incubation, the viability of astrocytes significantly increased to 83.54% &#177; 3.00%, indicating that the 3D construct represents a suitable microenvironment for cell growth. The biomaterial composition allowed cell attachment and stimulated astrocytic behavior, with cells expressing the specific astrocytes marker glial fibrillary acidic protein (GFAP) and possessing typical astrocytic morphology. This&#160;reproducible protocol provides a valuable method to biofabricate 3D neural-like tissue rich in astrocytes that resembles cells&#39; native microenvironment, useful to researchers that aim to understand astrocytes&#39; functionality and their relation to the mechanisms involved in neurological diseases.

Here we report a method of 3D bioprinting murine cortical astrocytes for biofabricating neural-like tissues to study the functionality of astrocytes in the central nervous system and the mechanisms involving glial cells in neurological diseases and treatments.

Mühendislik Nöral Benzeri Dokular için Murine Kortikal Astrositlerin 3D Biyobaskı

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also ...