Bu çalışma, Biyomedikal Malzemeler (BMM) enstitü araştırma programı uygulandı. BMM Ekonomik işler, Tarım ve İnovasyon Bakanlığı tarafından Hollanda cofunded edilir. Nederlandse Hartstichting mali katkısı minnetle kabul edilmektedir.

1. İnsan Vena Saphena Hücreleri Kültür <ol><li> Schnell ve arkadaşları tarafından protokole göre ikincil kullanım malzeme kurallarına uygun olarak bir donörden alınan vena safena magna,,. 9 hücreleri izole ve sıvı azot içinde bu saklayın. Altı plaka kültürüne 2 x 2 mm bir donör kesim parçalardan vena safena magna kısmından. Başına iyi 2 adet kullanın. Genel olarak yeterli hücreleri, sıvı nitrojen içinde 0.25 x 10 6 hücre ile yaklaşık 3 şişeleri doldurmak için elde edilebilir. HVSCs vimentin, desmin hiçbir ifade ve α düz kas aktini 10 ifade bir alt ifade göstererek, miyofibroblastlar olarak karakterize edilirler. Sonraki hücrelerinin sayısını arttırmak için sıvı azottan hücreleri çözülme için protokol başlar. </li><li> T75 doku kültürü şişesi içine bir tüpten hücreleri yer ve İleri DMEM, 50 ml cenin sığır serumu (FBS), 5 ml penisilin / oluşan büyüme ortamı (GM), eklemestreptomisin ve 5 ml L-glutamax. 2-3 günde bir orta değiştirin. </li><li> Hücreler yaklaşık 14 gün sonra birleşik büyür. Store 0,5 sıvı azot içinde bir cam şişe içinde x 10 6 hücre, geçit 1 olarak adlandırılır. </li></ol> Not: Hücrelerin dondurularak HVSCs hasat zaman gerekli değildir, fakat sadece depolama için kullanılır. <ol start="4"><li> Bir T175 doku kültürü şişesi içine bir tüpten HVSCs yerleştirin ve GM ekleyin. 2-3 günde bir orta değiştirin. Hücreler yaklaşık 7 gün sonra birleşik büyür. Mağaza 3 ve sıvı azot içinde bir cam şişe içinde x 10 6 hücre, geçit 2 olarak anılacaktır. </li><li> Bir rollerbottle içine bir tüpten hücreleri yer ve GM ekleyin. 2-3 günde bir orta değiştirin. Hücreler, yaklaşık 8 gün sonra konfluent büyür. Bir rollerbottle yaklaşık 20-30 x 10 6 hücre içerir. Geçit 6 Kültür hücreleri. Hücre fenotip değişebilir, çünkü geçit 9 daha yüksek bir geçiş hücreleri kullanmayın. </li></ol&gt; 2. Fibrin tabanlı Doku Yapıları Mühendislik <ol><li> Sertleştirici (10:1) ile elastomer karıştırılarak silikon yapıştırıcı hazırlayın. Velcro 7 x 3 mm dikdörtgen parçaları kesin. Bir çapraz oluşturmak için, ve cırt bant arasında 3 mm kare boşluk bırakmak esnek membran ile 6 kuyu kültür plaka içine tutkal Velcro,. </li></ol> Notlar: Sadece Velcro yumuşak tarafı kullanın ve bu yan yukarı yüz. Ne zaman yapıştırma Velcro, sadece silikon yapıştırıcı ile Velcro kapağı, iyi boyunca tutkal yayılmış yok. Kültür plakaları silikon membran dipleri olduğundan, plaka için kolay yapıştırma sağlamak için, esnek zarlar takviye için plaka altında bir şey kullanın. <ol start="2"><li> Tutkalın sertleştirme sağlamak için 60 ° C&#39;de bir fırın içerisinde gece boyunca silikon yapıştırıcı kurutun. 30 dakika boyunca inkübe kuyular ve% 70 EtOH eklenerek sterilize edin. PBS ile 3x durulayın ve wel tüm PBS kaldırmakls ve Velcro. 30 dakika UV altına ve kullanılıncaya kadar steril tutmak. </li><li> Doku mühendisliği GM oluşan orta (TM) ve L-askorbik asit 2-fosfat ve 130 mg hazırlayın. </li><li> TM, 1 mg / ml &#39;ε-amino kaproik asit (ACA) ekleyin. ACA kırıcı 11 fibrin önlemek için kültürün ilk 7 gün için kullanılır. Alternatif olarak, aprotinin kullanılabilir. </li><li> Yığın oluşumunu önlemek için, fibrinojen açmadan önce oda sıcaklığına gelmesini bekleyin. Kümeleri fibrinojen olmadan daha kolay eriyecektir. ACA ile takviye edilmiş 10 mg gerçek protein / ml TM 7 arasında bir konsantrasyona fibrinojen çözülür. Steril filtre fibrinojen çözüm, birden çok filtre gerekebilir. Kullanılana kadar buz üzerinde muhafaza edin. </li></ol> Not: Çok fazla köpük oluşumunu önlemek için yavaşça fibrinojen karışımı çözülür için. <ol start="6"><li> ACA ile takviye edilmiş 10 IU / ml &#39;TM 7 arasında bir konsantrasyona trombin çözülür. Kullanılana kadar buz üzerinde muhafaza edin. </li> &lt;/ Ol&gt; Not: Buz Depolama trombin ve fibrinojen erken jelleşme önlemek için gereklidir. <ol start="7"><li> GM ve sayısında tekrar süspansiyon hücreleri, Trypsinize. </li><li> , Fibrin tabanlı doku yapıları (doku mühendisliği kalp kapakçıkları 7 metotlara göre konsantrasyon) tohum için 15 x 10 6 hücre / ml &#39;kullanın. Tek bir jel 100 ul GOF El ve böylece 1.5 x 10 6 hücre oluşur. Yapılacaktır jellerin sayısı kadar santrifüj tüpleri ile sonuçlanan bir santrifüj tüpü içinde 1.5 x 10 6 hücre koyun. </li><li> 7 dakika 350 x g&#39;de hücreleri Santrifüj ve süpernatant atın. 50 ul trombin hücrelerin tekrar süspansiyon. Bu süspansiyon mavi floresan polistiren mikro 4 ul ekleyin. Bir şişe içinde, fibrinojen 50 ul koyun. Hücreleri ile 50 ul trombin almak için bir pipet kullanın. 100 ul pipet hacmi artırın. Trombin çözeltisi ile 50 ul Pipetfibrinojen ile şişenin içine hücreleri trombin ve fibrinojen karıştırın ve 100 ul karışımı almak. </li></ol> Not: floresan polistiren mikro görüntü analizi için dahili referans işaretleri kullanılmaktadır. Karıştırma trombin ve fibrinojen, dikkatli bir karışım, pipetleme hava kabarcıklarının oluşumunu önlemek zaman. Hava kabarcıkları, fibrin jel delik neden olur. <ol start="10"><li> Içine ve Velcro şeritleri arasında jel karışımı pipetle. Fibrin jel için tipik jelleşme süresi, bir kez bileşenler karıştırılır, 20 sn &#39;dir. </li></ol> Notlar: karışım plaka pipetle önce jelleşme önlemek için mümkün olduğunca çabuk yapın. Hücreleri ve boncuklar kullanmadan önce uygulama. <ol start="11"><li> ACA ile kültür TM eklemeden önce jelleşme izin vermek için, nemlendirilmiş bir% 95 hava /% 5 CO2 kuluçka makinesinde 37 ° C&#39;de 30 dakika boyunca inkübe süspansiyonlar. </li><li> Ilk 7 gün ACA 2-3 gün her ile TM değiştirin. Bir hafta ACA olmadan kültive edilecek sonra jeller yeterince stabildir. İlk haftadan sonra 2-3 günde TM değiştirin. Başına iyi TM 6 ml ekleyin. Günde 12 hücreleri görünüm için yeterli kolajen doğurmuştur. </li></ol> 3. Gerilme ve Kısıtlamalar içinde uygulamak Gerilme ve indükleyen değişiklikler <ol><li> Statik yük hücresi çekiş ve sıkıştırma 12 nedeniyle, doğrudan hücre tarafından uygulanır. Kollajen yeniden ikna etmek için, doku tek yönde iki cırt bant gevşek inşa kesti. Tek yönlü kısıtlamaları (Şekil 1A) ile sonuçlanır. Yapı daha sonra istikrarlı yeterli ve kollajen tevdi beri, 12. günde bu gerçekleştirin. </li><li> Flexcell FX4000T sisteminin kullanımı ile, esnek bir membran ile kültür plakalarının altına bir vakum uygulanarak siklik gerilme uygulanır. Yükleme mesajların üst (ki inci bir parçası olan üzerinde, bir kaide üzerine tabak koyunE döngüsel yükleme cihazı). Pompa, membran üzerine bir vakum uygulanır ve böylece altında yatan dikdörtgen sonrası üzerinde membran çeker. Doku haç biçimli ekleri olarak, membranın (Velcro ile) oluşturur ve dikdörtgen sonrası uygulanan siklik suşu (Şekil 1B) tek eksenli. </li><li> Başlangıçta, döngüsel gerginlik uygulama gün 6 başlamadan önce, ilk mekanik bütünlük elde etmek için 5 gün boyunca statik kültür kullanın. Vakum pompası için kumanda içine programı döngüsel bir leke protokolü. Örneğin tek eksenli yön 13 ile, daha önce kurulmuş aralıklı döngüsel gerginlik protokolünü kullanır. Bu, 3 saat dinlenme süreleri ile dönüşümlü olarak 3 saat dönemleri boyunca 0 dan% 5 suşu için süzme 1 Hz ile bir sinüs dalgası, bir aralıklı gerginlik oluşur. Bir uyumlu kollajen organizasyon uyararak, 7 gün boyunca bu döngüsel gerginlik protokol yapın. </li><li> Tipik olarak 12 gün sonra, bir HVSCs hizalanmış kolajen organ ürettilern. Hizalanmış bir kolajen kuruluş ulaşıldıktan sonra, orijinal gerilme yönüne dik olduğu, tek eksenli siklik suşu yönünü değiştirmek. 90 ° dikdörtgen mesajları çevirerek yapın. </li></ol> 4. Görselleştirme Hücreler ve Kollajen <ol><li> Hücre canlılığı veya kollajen oluşumu ile karışmaz probları ile aktif, gerçek zamanlı kollajen yeniden, etiket örnekleri görselleştirmek için. Floresan hücre sitoplazma ve kollajen leke prob kullanın. </li><li> Konfokal lazer tarama mikroskobu kullanarak Alternatif olarak ikinci harmonik üretimi 780 nm ve 500-550 nm arasında algılama de uyarma ile otofloresansı 14, kullanarak kollajen yapıları görselleştirmek için kullanılabilir. </li><li> 3 saat dinlenme süresi boyunca periyodik olarak gergin örnekler için, kurulum ve inkübatör kültür plakaları çıkarın ve hücreleri ve kollajen görselleştirmek için bir konfokal lazer tarama mikroskobu taşırlar. </li></ol>

<ol>
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Yazarlar hiçbir rakip mali çıkarlarının olmadığını beyan ederim.

Hücre kalabalık fibrin yapıların tarif model sistem hücre ve kollajen çalışma için büyük bir potansiyele sahiptir (yeniden) organizasyonu (de Jonge ve ark. 15), doku mühendisliği amaçlar için de kullanılabilir örneğin. Ilk hücre taşıyıcı olarak fibrin kullanarak, fibrin bozulduktan sonra, bir doku hücreleri ve endojen matriste ile oluşturulur. Bu şekilde, hücre sınır sertlik 12 algılama veya gerginlik kaçınma gösteren, kasılma güçleri 16,17...

Kardiyovasküler dokular önemli bir taşıyıcı fonksiyonu var. Hücre dışı matriks kollajen liflerinin belirli bir içerik ve organizasyonda yük taşıyan özellikleri katkı ve genel doku gücü 1 hakim. Doku mühendisliği yapı mekanik klima kullanılır - genellikle (döngüsel) süzme rejimlerinin oluşan - doku organizasyonu ve mekanik özellikleri 2,3 artırmak için. Önceden tanımlanmış kollajen mimarisi ile doku oluşturmak için karmaşık doku geometrilerde gerginlik kaynaklı kollajen organizasyonu tam bir anlayış henüz elde edilmiştir değil. Bu gelişmekte olan dokularda kollajen yeniden bizim sınırlı bilgi kaynaklanmaktadır. Mevcut modeller ağırlıklı olarak statik zorlanma 4-6 kullanımı ile kollajen yeniden nihai net sonucu hakkında bilgi vermek. Burada etkisi altında, 3D, gerçek zamanlı bir şekilde kolajen (yeniden) örgütün çalışma sağlayan son derece ayarlanabilir model sistem sağlarStatik veya siklik suşunun. Doku yapıları yapı tüm kollajen endojen sağlamak, fibrin tabanlı. Yapıları hücre ve kollajen organizasyon görüntülenmiştir ve bir iç referans sistemi bize zaman atlamalı analizi için hücreleri ve kollajen yapı taşınmaya sağlar. Bu hücreler dayalı olarak gelişmiş hücre dışı matriks üretim ve mühendislik kardiyovasküler dokularda 7 matris ve kurulan kullanım bozulmasina yeteneği, tanınırlar beri bu protokolde, İnsan Vena Saphena Hücreleri (HVSCs) için model sisteminin kullanımı anlatacağım de Jonge ve ark. 8 çalışmalarına

<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Culture plastic</td>
			<td>Greiner</td>
			<td></td>
			<td>Includes culture flasks and pipettes</td>
		</tr>
		<tr>
			<td>Advanced DMEM</td>
			<td>Gibco</td>
			<td>12491</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Greiner</td>
			<td>758075</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Gibco</td>
			<td>10378016</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMax</td>
			<td>Gibco</td>
			<td>35050-079</td>
			<td></td>
		</tr>
		<tr>
			<td>Elastomer and curing agent</td>
			<td>Dow Corning Corporation</td>
			<td>3097358-1004</td>
			<td>Silastic MDX 4-4210#</td>
		</tr>
		<tr>
			<td>Velcro</td>
			<td>Regular store</td>
			<td></td>
			<td>You can buy this at a regular store, only use the soft side</td>
		</tr>
		<tr>
			<td>Bioflex culture plates</td>
			<td>Flexcell Int</td>
			<td>BF-3001U</td>
			<td>Untreated</td>
		</tr>
		<tr>
			<td>L-Ascorbic Acid 2-phosphatase</td>
			<td>Sigma</td>
			<td>A8960</td>
			<td></td>
		</tr>
		<tr>
			<td>&#949;-Amino Caproic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>D7754</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine thrombin</td>
			<td>Sigma</td>
			<td>T4648</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine fibrinogen</td>
			<td>Sigma</td>
			<td>F8630</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 syringe filter</td>
			<td>Whatmann (Schleicher and Scheul)</td>
			<td>10462100</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene microspheres</td>
			<td>Invitrogen</td>
			<td>F-8829</td>
			<td>Blue fluorescent, 10 &#956;m diameter</td>
		</tr>
		<tr>
			<td>Flexcell FX-4000T</td>
			<td>Flexcell Int</td>
			<td></td>
			<td>Includes rectangular loading posts</td>
		</tr>
		<tr>
			<td>Cell Tracker Orange</td>
			<td>Invitrogen Molecular Probes</td>
			<td>C2927</td>
			<td></td>
		</tr>
		<tr>
			<td>CNA35-OG488</td>
			<td></td>
			<td></td>
			<td>Cordially provided by the Laboratory for Macromolecular and Organic Chemistry, Department of Biomedical Engineering, Eindhoven University of Technology</td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>LSM 510 Meta laser scanning microscope and Two-Photon-LSM mode</td>
		</tr>
		<tr>
			<td>Amphotericin</td>
			<td>Gibco</td>
			<td>15290-018</td>
			<td>Needed for cell isolation</td>
		</tr>
	</tbody>
</table>

engineering fibrin-based tissue constructs from myofibroblasts and application of constraints and strain to induce cell and collagen reorganization

Kolajen içerik ve kollajen doku gelişmekte olan organizasyon lokal doku suşları ve doku kısıtlaması tarafından etkilenebilir. Doku mühendisleri önceden tanımlanmış kollajen mimarileri ile dokuları oluşturmak için bu ilkeleri kullanmayı hedefliyoruz. Son mimari dokusu kontrol etmek için kollajen yeniden tam yatan süreçlerin tam bir anlayış, ancak eksik olduğunu. Özellikle, küçük bir doku mekanik yükleme koşullarındaki değişikliklere yanıt olarak kollajen liflerinin (yeniden) yönlendirme hakkında bilinmektedir. Biz daha kollajen aydınlatmak için, iki taraflı-kısıtlı miyofibroblast numaralı seribaşı fibrin yapıları oluşan, in vitro model sistem bir geliştirilen i yanıt olarak (yeniden) yönlendirme) tek eksenli statik yükleme koşulları ve ii iki eksenli geri alma) Çift yönlü-kısıtlı siklik tek eksenli yükleme Flexcell FX4000T yükleme cihazı kullanımı ile, yön değişikliği yükleme öncesi ve sonrası yapıları. Time-lapse konfokal görüntüleme vi için kullanılırbir tahribatsız şekilde kolajen (yeniden) yönlendirme sualize. Yapıları hücre ve kollajen organizasyon gerçek zamanlı olarak görüntülenmiştir olabilir, ve bir iç referans sistemi bize zaman atlamalı analizi için hücreleri ve kollajen yapı taşınmaya sağlar. Model sisteminin çeşitli yönleri hücre kaynağı ya da sağlıklı ve hastalıklı hücrelerin kullanımı gibi, ayarlanabilir. Katkı maddeleri örneğin MMP ekleyerek veya integrinler tarafından bloke edilmesi için, daha altta yatan mekanizmalar kollajen yeniden aydınlatmak için kullanılabilir. Bu yapı şekli ve büyüklüğü kolayca hücresi ve kollajen (yeniden) organizasyonu incelemek için bir çok ayarlanabilir modeli sistemi ile sonuçlanan, özel ihtiyaçlarına göre adapte edilebilir.

Bu model sistem kültür miyofibroblast numaralı seribaşı fibrin jeller için izin verir. Şekil 1A statik iki eksenli kısıtlar altında ilk kültüre bir doku gösterir. Doku kısıtlamaları tek eksenli statik kısıtlamaları oluşturmak için, iki kısıtlamaları fibrin jel kesim tarafından yayımlanan ve doku kompakt ve daha sonra remodels (Şekil 1A) vardır. Siklik bir suş için, doku hem de statik eksenli kısıtlamalar altında kültürlenir. 5 gün sonra, siklik tek ek...

Watch this Scientific Journal Video about Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization at JoVE.co

Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

Bu model sistem bir tahribatsız şekilde endojen kolajen (yeniden) organizasyonu gerçek zamanlı çalışma için kullanılabilecek bir miyofibroblast nüfuslu fibrin jel başlar. Bu model sistemi, farklı hücre kaynakları, orta katkı maddeleri ile birlikte kullanılabilir gibi çok ayarlanabilir, ve özel ihtiyaçlarına göre kolay bir şekilde adapte edilebilir.

Mühendislik Fibrin tabanlı Doku miyofibroblastlar ve Kısıtlar Uygulamasından Yapıları ve Hücre ve Kollajen (Re) Organizasyon tetikleyebilecek süzün

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim ...

engineering-fibrin-based-tissue-constructs-from-myofibroblasts

Research

JoVE Journal

Bioengineering

10.8K Görüntüleme.  Eindhoven University of Technology.  Bu model sistem bir tahribatsız şekilde endojen kolajen (yeniden) organizasyonu gerçek zamanlı çalışma için kullanılabilecek bir miyofibroblast nüfuslu fibrin jel başlar. Bu model sistemi, farklı hücre kaynakları, orta katkı maddeleri ile birlikte kullanılabilir gibi çok ayarlanabilir, ve özel ihtiyaçlarına göre kolay bir şekilde adapte edilebilir. 

Video: Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do not have suitable autologous donor vessels due to disease progression or previous harvest. The aim of vascular tissue engineering is to develop a suitable alternative source for these bypass grafts. In addition, engineered vascular tissue may prove valuable as living vascular models to study cardiovascular diseases. Several promising approaches to engineering blood vessels have been explored, with many recent studies focusing on development and analysis of cell-based methods.2-5 Herein, we present a method to rapidly self-assemble cells into 3D tissue rings that can be used in vitro to model vascular tissues.
To do this, suspensions of smooth muscle cells are seeded into round-bottomed annular agarose wells. The non-adhesive properties of the agarose allow the cells to settle, aggregate and contract around a post at the center of the well to form a cohesive tissue ring.6,7 These rings can be cultured for several days prior to harvesting for mechanical, physiological, biochemical, or histological analysis. We have shown that these cell-derived tissue rings yield at 100-500 kPa ultimate tensile strength8 which exceeds the value reported for other tissue engineered vascular constructs cultured for similar durations (&lt;30 kPa).9,10 Our results demonstrate that robust cell-derived vascular tissue ring generation can be achieved within a short time period, and offers the opportunity for direct and quantitative assessment of the contributions of cells and cell-derived matrix (CDM) to vascular tissue structure and function.

This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.

Hücresel Self-Meclis Biyomekanik Analizi ve Doku Mühendisliği, Hücre-Türetilmiş Doku Yüzüklerin Üretiyor yönelik

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

Biyomühendislik

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

Raman Spektroskopisi kullanarak Hücreler ve Ekstrasellüler Matriks Temassız, Etiket ücretsiz izleme

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

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 ...

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

Kanser Hücre Invasion Uygulama ile Tümör Stromal mikroçevresinin Doku Mühendisliği

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

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. ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Sandviç gibi mikroçevrelerde Hücre / Malzeme Etkileşimleri Harness

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

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, ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

Rekombinant kolajen peptid Microcarriers hücre genişletme ve hücre teslim sistemi bir biyoreaktör olarak potansiyel kullanım için ben Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily identified and used. Currently, this gap is filled with tendon auto-, allo-, xeno-, or artificial grafts, but clinical methods to secure them are not necessarily translatable to animals because of the scale. In order to evaluate new biomaterials or study a tendon graft made up of collagen type 1, we have developed a modified suture technique to help maintain the engineered tendon in alignment with the tendon ends. Mechanical properties of these grafts are inferior to the native tendon. To incorporate engineered tendon into clinically relevant models of loaded repair, a strategy was adopted to offload the tissue engineered tendon graft and allow for the maturation and integration of the engineered tendon in vivo until a mechanically sound neo-tendon was formed. We describe this technique using incorporation of the collagen type 1 tissue engineered tendon construct.

In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue.

Büyük Tendon Kusurlarını Onarmak İçin Doku Mühendisliği Kollajen Greftli Yeni Bir Tenorhaphy Dikiş Tekniği

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily ...

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

Decellularized extracellular matrix (dECM) can provide suitable microenvironmental cues to recapitulate the inherent functions of target tissues in an engineered construct. This article elucidates the protocols for the decellularization of pancreatic tissue, evaluation of pancreatic tissue-derived dECM bioink, and generation of 3D pancreatic tissue constructs using a bioprinting technique.

3D Hücre Yüklü Pankreas Dokusu Yapıları Baskı için Pankreas Doku Türetilmiş Ekstrasellüler Matrix Bioink

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...