Yazarlar Milli Eğitim Bakanlığı AcRF Tier 1 (RG41) fon kabul ve Nanyang Teknoloji Üniversitesi tarafından hibe başlatmak istiyorum. Alçak ve Tjin Nanyang Teknoloji Üniversitesi, Singapur Araştırma Öğrenci Bursu (RSS) tarafından finanse edilmektedir.

AECM Proteinler için rekombinant kodlayan plazmidlerin yapılması Vektör 1. Klonlama <ol><li> Fonksyonel (örneğin, hücre bağlama alanı ve elastin-benzeri tekrarlar) amino asit dizisini tasarımı. Fonksiyonel etki uçlarını yan Tasarım kısıtlama siteleri alt klonlama yazılımı talimatlarına göre özgür yazılım kullanarak (örn http://biologylabs.utah.edu/jorgensen/wayned/ape/) kolaylaştırmak için. Burada, planlanan bölgelere sindirimi sınırlandırmak için işlevsel bir etki mevcut değildir tek bir sınırlandırma bölgesine seçin. Çoklu klonlama yerinin ana vektörün (MCS) görünür kısıtlama bölgelerini seç (örn, pET22b (+)) alt-klonlama için. </li><li> Yazılım talimatlarına göre ücretsiz web sitelerini kullanarak nükleotid dizisi içine amino asit dizisini çevirmek Ters. (Örneğin, http://www.bioinformatics.org/sms2/rev_trans.html). Emin olun kodonları E. için optimize edilmiştir E. coli ana. </li><li> Ilgi Gen purchas olabilired, ticari olarak, tek sarmal kollu oligonükleotitlere, ve DNA tavlama yerine (oligonükleotidler &lt;100 baz çifti (bp) ise, yani,) (adım 1.4) maliyetini azaltmak için. Aksi takdirde, 100 bp daha büyük genler için, onlar ticari şirketler aracılığıyla satın alınabilir. </li><li> Oligonükleotidlerin DNA tavlama <ol><li> Istenen gen sekansı elde etmek için, DNA oligomerleri yeniden birleşir. 1 ug / ul nihai konsantrasyona (süzüldü, 2-amino-2- (hidroksimetil) -propan-1,3-diol, pH 8.0, 10 mM Tris), bir DNA oligomeri tamponu DNA oligonükleotidleri çözülür. </li><li> Toplam 40 ul karışım elde etmek için, DNA bağlanma tamponu 32 ul (10 mM Tris, 100 mM NaCI, 100 nM MgCl2), her bir oligomerin 4 ul ekleyin. </li><li> Bir ocak gözünü kullanılarak suyun beher kaynatılır ve 5 dakika, 95 ° C&#39;de karışım bırakın. Beher çıkarın ve yavaş yavaş strafor kutu O / N kurmak bütününü serin. oligomerleri tavlanmış ve sindirim hazır edilmiştir. </li></ol></li&gt; <li> Tavlanmış oligomerleri (örneğin, insert olarak anılacaktır) ve bir ev sahibi vektör ayrıca (yani pET22b (+)) sindirimi karşılık gelen restriksiyon enzimleri ekleyin. 37 ° C&#39;de 3-4 saat boyunca aşağıdaki tarifi (DNA 1-2 ug her bir sınırlama enzimi 2 ul, toplam 50 ul karışımına 10x sınırlayıcı enzim tamponu ve su yukarı 5 ul) kullanın. Not: pET22b (+) plazmid vektörü ampisilin antibiyotiklere dirençli ve C-termininde bir 6x-His etiketi içerir. PET22b (+) mevcut 6x-His etiketi 7. adımda western blotting ile hedef proteini tanımlamak için His-etiketli antikorlar kullanmamızı sağlar. </li><li> Her sindirim karışımı 6x yükleme boyası ekleyin. Ayrıca 100 V Visualize bir UV ışık ışığı ile% 1.2 DNA, agaroz jel, 1 saat boyunca bir UV floresan DNA leke ihtiva eden% 1.2 agaroz jelleri üzerinde, DNA, bir DNA merdiveni de dahil olmak üzere sindirim karışımları çalıştırın. </li><li> Ticari jel arıtma kitleri kullanılarak sindirilmiş DNA ürünleri ayıklamak için jel dilimleyin. Elute 50-100 ng / ul arasında en az bir DNA konsantrasyonu elde etmek için sütun için minimum hacim kullanılarak gerçekleştirilmiştir. </li><li> Sindirilmiş DNA inserti bağlanarak İlgi sırasıyla geni birleştirin (örneğin, DNA, tavlama ile elde elastin tekrarlar ya da hücre bağlama alanları), aşağıdaki tarifi kullanarak, T4 ligaz kullanılarak plazmid vektörüne (vektör 2 ul T4 ligaz 1 ul, T4 ligasyon tampon maddesi, x, ul ilavenin, 1.5 ul, toplam 15 ul karışım 10.5-X ul su). 2 saat boyunca oda sıcaklığında Ligasyon karışımı inkübe edin. Not: ekleme vektörün molar konsantrasyonu ligasyonu verimliliğini optimize etmek üzere, çeşitlendirilebilir gerekir. Tarifte X olarak adlandırılan parçanın hacmi adım 1.7 taşınan bir DNA konsantrasyonuna bağlıdır. </li><li> Çözülme E. E. coli DH5α kimyasal olarak kompetan hücreler (veya herhangi bir klonlama suşu) buz üzerinde. Sıcak eden 2xYT agar plakaları ampisilin (25 ug / ml) ihtiva eden (Tablo 1) 37 ° C arasındadır. </li><li> Kullanılarak hücrelerini dönüştürmekısı şoku: <ol><li> Kısım, temiz, önceden soğutulmuş mikro santrifüj tüplerine yetkin hücre 50 ul. Pipet 5 ul hücre içine ligasyon karışımının (100 ug ng ila 100 arası), pipetleme hafifçe yukarı ve aşağı karıştırın. 20 dakika boyunca buz karışımı bırakın. </li><li> 2 dakika için 42 ° C su banyosu içinde hücre karışımı ihtiva eden mikro santrifüj tüpü daldırın ve 2 dakika süreyle buz üzerinde döndü. Hücrelere ısı hasarı en aza indirmek için daldırma süresini zaman. </li><li> Mikro santrifüj tüpüne SOC ortamı (Tablo 1) 500 ul ilave edin ve 1 saat boyunca çalkalanarak 37 ° C&#39;de inkübe edin. </li><li> Ampisilin (25 ug / ml) ihtiva eden, oda sıcaklığına önceden ısıtılmış olan bir 2xYT agar plakası üzerine hücre / ligasyon karışımı 50 500 ul yayın ve plakalar 37 ° CO de baş aşağı inkübe / N (12-16 saat) . </li></ol></li><li> Ertesi gün, temiz pipet uçları kullanılarak agar plakasından DNA kolonileri seçin. (Tablo 2YT ortam 5 ml koloniler büyütün1) ihtiva eden ampisilin (25 ug / ml), O / N) çalkalanarak (225 rpm, 37 ° CO / N- (12-16 saat). </li><li> Ertesi gün, üreticinin protokolüne uygun olarak aldı her koloninin için DNA plazmidleri elde etmek için bir plazmid izolasyon kiti kullanmak. Su, 50 ul DNA Zehir. </li><li> 37 ° C&#39;de 2 saat boyunca inkübe edin (5 ul DNA, her biri, sınır enzimi 0.2 ul, toplam 10 ul karışım için 1 ul 10x sınırlayıcı enzim tamponu ve üst kadar su): Bir test, aşağıdaki tarifi kullanarak sınır enzimleri ile sindirmek gerçekleştirme büyük olasılıkla adım 1.6 olarak% 1.2 DNA agaroz jel üzerinde insert ve koşmak ihtiva sömürgeler için ekrana. NOT: sindirimi üzerine başarılı bir ligasyon gereken iki grup, vektör ve bunların ilgili molekül ağırlığına karşılık gelen insert (Şekil 1) için bir tane, her sonuçlanır. </li><li> Ileri T7 promotör kullanılarak DNA dizileme için olası koloniler Gönder ve ticari sequencer için primerler ters. </li></ol><ol><li> Sıralama sonucu itibaren başarıyla bağlandı edilmiş bir koloni seçin ve bir E. dönüşüm için DNA plazmid kullanmak E. coli ekspresyon konakçı. </li><li> Çözülme E. E. coli sentezleme türü buz üzerinde (BL21 (DE3) pLysS). Bu sırada, oda sıcaklığına kadar, ampisilin (25 ug / ml) ve kloramfenikol (34 ug / ml) ihtiva eden sıcak bir agar plakaları. Not: Bakteri içinde mevcut pLysS plasmidi BL21 (DE3) pLysS, bir kloramfenikol direnç genini ihtiva süzün. pLysS plazmid konstitütif aECM proteininin sızıntılı ekspresyonunu sınırlamak için ifade edilen bir T7 represör geni içermektedir. Kloramfenikol kültür sırasında pLysS içeren bakteri hücreleri seçmek gereklidir. </li><li> Adımı yineleyin 1.10 E. dönüştürülmüştür olarak ulaşmak için Yapay proteinleri ifade hazırız coli hücreleri. Parafilm agar plakaları sarın ve 4 baş aşağı saklamak 6; bir ay kadar C. </li></ol> AECM Proteinlerin 3. Bakteriyel ekspresyon <ol><li> , Şekil 2, bir pipet ile transforme agar plakasından bir koloni çekme ve steril Terrific Broth (TB) ortam bir test tüpü içinde ampisilin ve kloramfenikol antibiyotik hem de ihtiva eden (Tablo 1) 10 ml inoküle bakınız. 225 rpm&#39;de çalkalanarak 37 ° de CO / N- (12-16 saat), bu başlatıcı kültür inkübe edin. </li><li> Transfer 3 L&#39;lik Erlenmeyen şişesi aynı antibiyotik ile takviye edilmiş 1 L&#39;lik, taze ve steril TBC ortamı içine başlatıcı kültür 10 mi. 2-3 saat boyunca 225 rpm&#39;de çalkalanarak 37 ° C&#39;de kültür inkübe ve kültür (OD 600), optik yoğunluğunu dikkate okuma için boş bir küvetin içine kültürün 1 ml pipetleme 0,6-0,8 ulaşmıştır. Önceden SDS-PAGE karakterizasyonu için indüksiyon kültürün 1 ml&#39;sinin kaydedin. <ol><li> Hücre kültürü OD 600 ölçmek için, bir küvet TB medya 1 flakon hazırlamakBoş bir ölçüm için. Ayrı olarak, bir yeni, boş bir küvet içinde kültür şişesi kültür 1 ml aktarın. 600 nm &#39;lik bir emişteki boş kontrol karşı bir spektrofotometre kullanılarak kültür optik yoğunluk ölçülür. </li><li> 2 dakika için 12,000 x g&#39;de bir 1.5 ml mikrosantrifüj tüpü, santrifüje kültür numune 1 ml aktararak (daha sonra, SDS-PAGE analizi için) örnek kaydedin ve süpernatant süzün. </li></ol></li><li> 1 mM&#39;lik nihai bir konsantrasyona kadar izopropil β-D-1-tiogalaktopiranosid (IPTG) ile kültür neden ve başka bir 4 saat boyunca 225 rpm&#39;de çalkalanarak 37 ° C&#39;de inkübe edin. SDS-PAGE karakterizasyonu için 4 saat sonra indüksiyon sonunda kültür 1 ml kaydedin. </li><li> 4 ° C&#39;de 30 dakika boyunca 12,000 xg&#39;de 1 L&#39;lik bir santrifüj şişeleri ve santrifüj kültürünün aktararak hücreleri hasat. Ug / ml 0.5 (pH = 8.0, 1 M Tris, 0.01 M EDTA, 0.1 M NaCl), süpernatant atılır TEN tamponu içinde hücre pelletleri ve tekrar süspansiyon tartın. </li></ol> Bakteriyel Kültürlerin 4. Liziz <ol><li> -80 ° CO / N de yeniden askıya hücre kültürü dondurun. Hücreleri lize etmek için, oda sıcaklığında veya buz üzerinde bir su banyosu içinde dondurulmuş hücre kültürü çözülme. Cozundururken 10 ug / ml Deoksiribonükleaz I (DNase I), 10 ug / ml ribonükleaz A (RNaz I) &#39;in ve 50 ug / ml fenilmetilsülfonil florid (PMSF) ilave edin ve yavaş karıştırma ile çözelti homojenleştirilir. </li><li> Tüm yeniden süspansiyon haline getirilmiş hücreler çözündürüldü sonra, su 12 içerisindeki protein çözünürlüğünü artırmak için, pH 9.0 çözeltisi ayarlayın. Homojen bir kıvam elde etmek için buz üzerinde karıştırılarak akıllıca 6 N NaOH damla ekleyin. 2 mm çapında yassı uç, 5 sn darbe kullanılarak buz üzerinde 20 dakika boyunca ultrasonik parçalama ile lizleyin. </li><li> 4 ° C&#39;de 30 dakika boyunca 12,000 x g&#39;de hücre çözümü santrifüjleyin. Daha sonra saflaştırma için 4 ° C sıcaklıkta, bir temiz, boş bir şişe ve mağaza süpernatant aktarın. </li><li> Bu arada, -80 ° C TEN tamponu ve yeniden dondurma ile tekrar hücre pelletini. Içinüç kez tam hücre parçalama, tekrar donma / çözülme ve sonication süreci kadar. SDS-PAGE karakterizasyonu için 20 ul hücre lizatı kaydedin. </li></ol> Ters Geçiş Bisiklet kullanarak aECM Proteinlerin 5. saflaştırılması <ol><li> Aşama 4.3 hücre lizatı harmanlayın ve elastin tabanlı proteinler 21 saflaştırılması için kullanılana benzer ITC kullanılarak aECM proteinlerini saflaştırmak için devam edin. Döngüleri 4 ° C olan, farklı sıcaklıklarda yapılabilir ve 37 ° C (&quot;soğuk&quot; olarak anılacaktır), sırasıyla devir (&quot;sıcak&quot; olarak anılacaktır). </li><li> 4 ° C&#39;de 2 saat süre ile 40.000 x g, hücre 50 ml&#39;lik santrifüj şişeler içine lizat ve santrifüj Böl. Hücre pelet görünümü koyu kahverengi olmalı ve akıntısı görünmelidir. </li><li> Pelet temiz bir ayrılık almak için pipetleme süpernatantı. Temiz santrifüj şişelerine supernatant toplamak ve (3 M maksimum) 1 M bir son konsantrasyona kadar NaCl ilave edin. Sıcak225 rpm&#39;de çalkalanarak, 2 saat boyunca 37 ° C&#39;ye kadar bir çözüm. NOT: NaCl ilavesi aECM neden agregasyon elastin bileşeninin geçişi tetikler. süpernatant bulanık dönecek. Protein konsantrasyonu yeterince yüksek olduğunda, beyaz köpüklü bir proteini santrifüj şişenin yanlarında görülebilir. </li><li> 37 ° C &#39;de 2 saat süre ile 40,000 x g de aşama 5.3 süpernatant santrifüjleyin. Süpernatant Durusu. Metal bir spatula kullanılarak pelet ezmek ve manyetik bir karıştırma çubuğu ve plaka kullanılarak 4 ° C&#39;de kuvvetli karıştırma O / N buz soğukluğunda otoklava damıtılmış su içinde pelet bitleri (50 mg / ml) yeniden süspanse edin. Pelet tamamen çözündürülmelidir. </li><li> Tekrarlayın protein yüksek saflıkta elde etmek için 5.2 başka 3-5 kez 5.4 adımları. </li><li> Saflaştırılmadan son çevriminden sonra, 4 ° C sıcaklıkta bulunan damıtılmış suyun karşı diyaliz protein çözeltisi desalt. Su, her 4 değişikliklerle 2-3 gün suya karşı protein çözeltisi Dialyzesaat ya da O / N 8 saat. SDS-PAGE için saflaştırılmış protein 20 ul kaydedin ve sonraki kullanıma kadar -80 ° C&#39;de saflaştırılmış protein ve mağaza geri kalanı liyofilize. </li></ol> SDS-PAGE elektroforezi kullanılarak aECM Proteinlerin 6. Karakterizasyonu <ol><li> (Moleküler ağırlığı büyük ise, daha iyi bir ayrılması için% 8 SDS-PAGE jellerinin kullanın), bir% 12 SDS-PAGE jeli hazırlayın. , Her bir örnek için 2 x SDS yükleme tamponu ekleyin ısı örnekleri 10 dakika boyunca 100 ° C &#39;de ve çalışma örnekleri jel üzerinde, 1 saat boyunca 100 V&#39;ta ya aECM proteininin molekül ağırlığına tekabül eden bir protein merdiveni orta ulaşıncaya kadar Jel. </li><li> Ayarlamak SDS-PAGE jelin almak ve bir Petri kabı içinde, 1 saat boyunca jel daldırın bir hacmi ile Coomassie Parlak Mavi leke (Tablo 2) ekleyin. Bir rocker 5 dakika boyunca bir destaining solüsyonu (Tablo 2) durulayın jel. Destaining çözümü değiştirin ve t arka kadar sallanan ile jel destain devamO Jel netleşiyor. Protein bantları açıkça görünür olmalıdır. </li><li> Hedef proteinin moleküler ağırlığı belirlemek için, protein merdiveni karşı hedef proteinin konumunu Karşılaştırması. </li><li> Bundan başka, hedef proteinin varlığını teyit etmek için, aşama 7&#39;deki gibi Batı blotting işleminin gerçekleştirilmesi. </li></ol> Western Blot kullanarak aECM Proteinlerin 7. Karakterizasyon <ol><li> Bir boyama yöntemi ile, Bölüm 6 &#39;daki gibi bir% 12 SDS-PAGE jeli üzerinde örnekleri çalıştırın. </li><li> SDS-PAGE jeli biraz daha büyük bir boyuta nitroselüloz zar kesilir. Aynı boyuta filtre kağıdı 4 adet kesin. </li><li> Western transfer tamponuna filtre kağıdı ıslak bir parça (% 20 h / h metanol, 25 mM Tris, 190 mM glisin, pH 8.3), filtre kağıdı bir parça ile, ardından filtre kağıdı üstüne SDS-PAGE jeli, yer daha sonra nitroselüloz zara aktarıldı ve filtre kağıdı nihai Diğer iki adettir. Emin olun, tüm set-up yeterli batı aktarım tamponu ile batık. Not: proteinler temas üzerine membrana bağlanan olabilir gibi jel ve membran zaman bu noktada temas etmemesi gerekmektedir. </li><li> Nitroselüloz zar, ardından bir Batı yarı kuru bir transfer ünitesinin içine iki filtre kağıtları yerleştirerek nitroselüloz zar üzerine transfer proteinleri ve dikkatli bir mesafede olan ve membran üzerinde SDS-PAGE jel yer son olarak son iki ıslak filtre kağıdı yer SDS-PAGE jeli. 45 mA, 30 dakika batı transferi çalıştırın. Voltaj daha yüksek 30 V ise tampon ekle Not: Tercihen bir girişimde membran üzerinde SDS-PAGE jeli yerleştirmek ve proteinler temas üzerine membrana bağlanan olabilir gibi zar üzerinde gereksiz jel çevrilmesini önlemek. </li><li> Al ve oda sıcaklığında 2 saat boyunca (filtre kağıdı ile filtre edildi, PBS pH 7.4 içinde% 5 yağsız süt) çözeltisi bloke ile nitroselüloz zarı bloke eder. Tampon engelleme atınız ve durulama PBS ekleyin. NOT: Yeni eldiven değiştirin istenmeyen prote ile membran kirletmesini önlemek içinins. </li><li> 1 PBS içinde seyreltme ile birincil anti-His antikoru inkübe: 1000, oda sıcaklığında 1 saat boyunca çalkalanarak. 1000 antikoru 1 ul (stok konsantrasyonu: 1 mg / ml) ekleyin: seyreltme oranı 1, örneğin, tüm membran daldırın olabilecek bir hacimde bir karışım hazırlayın, bir 1 ml toplam karışım için 999 ul PBS. </li><li> (% 0.1 Tween 20 ile PBS) PBST 5 ml zar yıkayın. </li><li> 1 PBS içinde seyreltilmesi ile bayırturpu peroksidaz (HRP) konjuge edilmiş ikincil antikor ile inkübe: 5000, oda sıcaklığında 1 saat boyunca çalkalanarak. 5000 antikoru 1 ul (stok konsantrasyonu: 1 mg / ml) ekleyin: seyreltme oranı 1, örneğin, tüm membran daldırın olabilecek bir hacimde bir karışım hazırlayın, 5 ml toplam karışım için 4999 ul PBS. </li><li> PBST 5 ml zar yıkanır ve iki kez tekrarlayın. </li><li> Membran kapak ve kullanılan alt tabaka için yönergeleri izleyerek inkübe hacmi zara kemilüminesan substrat uygulayın. NOT: Protein tespiti için alt tabakanın hassasiyeti değişebilir, biz sinyallerini artmaz antikor konsantrasyonunu arttırarak olay çok düşük sinyaller için azami hassasiyet ile bir alt tabaka öneririz. </li><li> Bir CCD kamera tabanlı görüntüleyici kullanarak chemiluminescent sinyalleri yakalayın. Sinyallerin zayıf ve non-spesifik olup olmadığını birincil ve ikincil antikor seyreltme oranını ayarlayın. Arka sinyal yüksek ise çözümü engelleme uzun kuluçkaya yatmaktadır. </li></ol>

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M., Guan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular+Design+in+Natural+and+Biomimetic+Soft+Materials.">Modular Design in Natural and Biomimetic Soft Materials.</a> Angewandte Chemie International Edition. 50 (39), 9026-9057 (2011).</li><li>Gagner, J. E., Kim, W., Chaikof, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+protein-based+biomaterials+for+medical+applications.">Designing protein-based biomaterials for medical applications.</a> Acta Biomater. 10 (4), 1542-1557 (2014).</li><li>Gomes, S., Leonor, I. B., Mano, J. F., Reis, R. L., 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=Natural+and+genetically+engineered+proteins+for+tissue+engineering.">Natural and genetically engineered proteins for tissue engineering.</a> Prog Polym Sci. 37 (1), 1-17 (2012).</li><li>Werkmeister, J. A., Ramshaw, J. A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+protein+scaffolds+for+tissue+engineering.">Recombinant protein scaffolds for tissue engineering.</a> Biomedical Materials. 7 (1), 012002 (2012).</li><li>MacNeil, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterials+for+tissue+engineering+of+skin.">Biomaterials for tissue engineering of skin.</a> Materials Today. 11 (5), 26-35 (2008).</li><li>Fong, E., Tirrell, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+Cell+Migration+on+Artificial+Extracellular+Matrix+Proteins+Containing+Full-Length+Fibronectin+Domains.">Collective Cell Migration on Artificial Extracellular Matrix Proteins Containing Full-Length Fibronectin Domains.</a> Advanced Materials. 22 (46), 5271-5275 (2010).</li><li>Ratner, B. D., Bryant, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BIOMATERIALS:+Where+We+Have+Been+and+Where+We+are+Going.">BIOMATERIALS: Where We Have Been and Where We are Going.</a> Annual Review of Biomedical Engineering. 6 (1), 41-75 (2004).</li><li>Cai, L., Heilshorn, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+ECM-mimetic+materials+using+protein+engineering.">Designing ECM-mimetic materials using protein engineering.</a> Acta Biomater. 10 (4), 1751-1760 (2014).</li><li>Annabi, N., 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=Elastomeric+recombinant+protein-based+biomaterials.">Elastomeric recombinant protein-based biomaterials.</a> Biochemical Engineering Journal. 77, 110-118 (2013).</li><li>Heilshorn, S. C., DiZio, K. A., Welsh, E. R., Tirrell, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell+adhesion+to+the+fibronectin+CS5+domain+in+artificial+extracellular+matrix+proteins.">Endothelial cell adhesion to the fibronectin CS5 domain in artificial extracellular matrix proteins.</a> Biomaterials. 24 (23), 4245-4252 (2003).</li><li>Simnick, A. J., Lim, D. W., Chow, D., Chilkoti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomedical+and+Biotechnological+Applications+of+Elastin-Like+Polypeptides.">Biomedical and Biotechnological Applications of Elastin-Like Polypeptides.</a> Polymer Reviews. 47 (1), 121-154 (2007).</li><li>Tjin, M. S., Chua, A. W. C., Ma, D. R., Lee, S. T., Fong, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Epidermal+Keratinocyte+Cell+Response+on+Integrin-Specific+Artificial+Extracellular+Matrix+Proteins.">Human Epidermal Keratinocyte Cell Response on Integrin-Specific Artificial Extracellular Matrix Proteins.</a> Macromolecular Bioscience. 14 (8), 1125-1134 (2014).</li><li>MacNeil, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+opportunities+for+tissue-engineered+skin.">Progress and opportunities for tissue-engineered skin.</a> Nature. 445 (7130), 874-880 (2007).</li><li>Kariya, Y., 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=Differential+regulation+of+cellular+adhesion+and+migration+by+recombinant+laminin-5+forms+with+partial+deletion+or+mutation+within+the+G3+domain+of+&#945;3+chain.">Differential regulation of cellular adhesion and migration by recombinant laminin-5 forms with partial deletion or mutation within the G3 domain of &#945;3 chain.</a> J Cell Biochem. 88 (3), 506-520 (2003).</li><li>Shang, M., Koshikawa, N., Schenk, S., Quaranta, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+LG3+module+of+laminin-5+harbors+a+binding+site+for+integrin+&#945;3&#914;1+that+promotes+cell+adhesion,+spreading,+and+migration.">The LG3 module of laminin-5 harbors a binding site for integrin &#945;3&#914;1 that promotes cell adhesion, spreading, and migration.</a> J Biol Chem. 276 (35), 33045-33053 (2001).</li><li>Hassouneh, W., Christensen, T., Chilkoti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastin-like+polypeptides+as+a+purification+tag+for+recombinant+proteins.">Elastin-like polypeptides as a purification tag for recombinant proteins.</a> Current Protocols in Protein Science. , 6.11.1-6.11.16 (2010).</li><li>Keeley, F., Mecham, R., Keeley, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+4.">Ch. 4.</a> Evolution of Extracellular Matrix.Biology of Extracellular Matrix. , 73-119 (2013).</li><li>Le, D. H. T., 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=Self-Assembly+of+Elastin-Mimetic+Double+Hydrophobic+Polypeptides.">Self-Assembly of Elastin-Mimetic Double Hydrophobic Polypeptides.</a> Biomacromolecules. 14 (4), 1028-1034 (2013).</li><li>MacEwan, S. R., Hassouneh, W., Chilkoti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-chromatographic+Purification+of+Recombinant+Elastin-like+Polypeptides+and+their+Fusions+with+Peptides+and+Proteins+from+Escherichia+coli.">Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli.</a> Journal of visualized experiments: JoVE. (88), e51583 (2014).</li><li>Meyer, D. E., Chilkoti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+synthesis+of+protein-based+polymers+with+precisely+specified+molecular+weight+and+sequence+by+recursive+directional+ligation:+examples+from+the+elastin-like+polypeptide+system.">Genetically encoded synthesis of protein-based polymers with precisely specified molecular weight and sequence by recursive directional ligation: examples from the elastin-like polypeptide system.</a> Biomacromolecules. 3 (2), 357-367 (2002).</li></ol>

Yazarlar hiçbir rakip mali çıkarlarını olduğunu beyan ederim.

Rekombinant protein mühendisliği aşağıdan yukarıya yaklaşımı kullanarak yeni protein malzemeleri oluşturmak için çok yönlü bir tekniktir. Protein bazlı malzemeler ilgi uygulamasına göre birden fazla işlevleri sahip olacak şekilde dizayn uygun olabilir. Nedeniyle klonlanması ve protein ifadesi teknolojileri artan ilerleme için, bir tekrar üretilebilir ve ölçeklenebilir şekilde yapay proteinler çeşitli oluşturmak için göreceli olarak basit (ve uygun maliyetli) olmuştur. elastin-benzeri domen...

For several decades, both synthetic and natural materials have been explored for use as scaffolds in tissue engineering1,2. While synthetic materials such as polymers offer excellent structural integrity and tunable mechanical properties, they often have insufficient bioactivity to promote growth and infiltration of tissues. On the other hand, natural materials such as extracellular matrix (ECM) proteins have excellent biological activity, but have limitations such as batch-to-batch variability, rapid degradation and immunogenicity issues. As such, recombinant proteins are desired, since they can be designed to mimic only the desirable properties of native proteins3,4.
Recombinant protein engineering has garnered widespread interests as a versatile platform for the design and production of novel artificial protein biopolymers. By controlling the genetic sequence, the functionalities of the artificial proteins can be tailored for a wide variety of applications5,6. In particular, artificial extracellular matrix (aECM) proteins can be tailored to have multiple functionalities for applications in tissue engineering, regeneration and wound repair2,7. More importantly, advances in cloning and purification technologies have increased scalability and reduced the cost of manufacturing recombinant proteins tremendously. It is possible to produce large quantities of recombinant proteins at low production costs which are economic for use in the clinic5. 
Artificial extracellular matrix proteins have been developed for tissue engineering applications8-11. For instance, Tirrell et al. designed a small diameter vascular graft using artificial proteins containing fibronectin CS5 sequence and elastin-like repeats (ELP-CS5). They showed that human umbilical vein endothelial cells (HUVECs) were able to adhere and grow on these materials12. Others have also incorporated short bioactive sequences taken from fibronectin, collagen, laminin, fibrinogen and vitronectin as well as structural domains that mimic elastin, spider silk and collagens to create a variety of fusion proteins10. Bulk cross-linked films made out of elastin-based aECM proteins also exhibited mechanical properties similar to that of native elastin (elastic moduli ranges between 0.3-0.6 MPa)13. Subsequently, aECM proteins containing longer fibronectin fragments were also reported to accelerate wound healing in vitro due to increased integrin binding affinities8. 
Recently, integrin-specific artificial ECM proteins have been developed by Tjin and coworkers14. Each aECM protein contains a bioactive cell-binding domain taken from ECM components of native human skin2,7,15, such as laminin-5, collagen-IV and fibronectin. For example, the integrin &#945;3&#914;1 has been shown to bind the PPFLMLLKGSTR sequence found in the laminin-5 alpha-3 chain globular domain 3 (LG3)16,17. In their report, they showed that primary human skin epidermal keratinocytes preferentially engage different integrins for binding to each of the aECM proteins, depending on the type of cell binding domain present.
The aECM proteins discussed in the work by Tjin et al. contain flanking elastin-like domains {(VPGIG)2VPGKG(VPGIG)2}8 that confer elasticity which mimics the mechanical properties of human skin. In addition, the incorporation of lysine residues within the elastin-like repeats also increases the overall protein solubility in aqueous solvents. In addition, the lysine residues also serve as crosslinking sites to facilitate the formation of crosslinked aECM films12. Inclusion of elastin-like repeats within the aECM protein sequence allow the proteins to be readily purified via Inverse Transition Cycling (ITC)14. Elastins undergo a sharp and reversible phase transition at a specific temperature known as the lower critical solution temperature (LCST) or the inverse transition temperature (Tt)18-20. Elastins and elastin-like repeats adopt hydrophilic random coil conformations below their LCST and become soluble in water, whereas above their LCST, elastins aggregate rapidly into micron-size particles. Such phase transitions are reversible and hence, can be exploited to allow elastin-based aECM proteins to be readily purified via the ITC technique21.
In this work, we report a generalized procedure to design, construct and purify artificial ECM proteins containing bioactive cell-binding domains, fused to elastin-like repeats. The process to design and clone the plasmids that encode for the amino acid sequences for the aECM proteins is described. The steps involved to purify the aECM proteins using ITC are outlined. Finally, the methods to determine the purity of the aECM proteins obtained using SDS-PAGE electrophoresis and Western Blotting are discussed.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>pET22b (+)</td>
			<td>Novagen</td>
			<td>69744</td>
			<td>T7 expression vectors with resistance to ampicillin&#160;</td>
		</tr>
		<tr>
			<td>BL21(DE3)pLysS&#160;</td>
			<td>Invitrogen</td>
			<td>C6060-03</td>
			<td>additional antibiotics - chloramphenicol</td>
		</tr>
		<tr>
			<td>Isopropyl-beta-D-thiogalactoside (IPTG)</td>
			<td>Gold Biotechnology</td>
			<td>I2481C</td>
			<td>1M stock solution with autoclaved water, make fresh prior to induction.</td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td>plasmid isolation kit</td>
		</tr>
		<tr>
			<td>T4 ligase</td>
			<td>New England Biolabs</td>
			<td>M0202S</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Affymetrix</td>
			<td>11259</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Affymetrix</td>
			<td>23660</td>
			<td></td>
		</tr>
		<tr>
			<td>Zymoclean&#8482; gel DNA recovery kit</td>
			<td>Zymo Research</td>
			<td>D4001</td>
			<td></td>
		</tr>
		<tr>
			<td>XL10-gold strain</td>
			<td>Agilent Technologies</td>
			<td>200315</td>
			<td></td>
		</tr>
	</tbody>
</table>

design and construction of artificial extracellular matrix (aecm) proteins from escherichia coli for skin tissue engineering

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins that have incorporated functional domains derived from nature (or created de novo) have been reported. In particular, artificial extracellular matrix (aECM) proteins have been developed; these aECM proteins consist of biological domains taken from fibronectin, laminins and collagens and are combined with structural domains including elastin-like repeats, silk and collagen repeats. To date, aECM proteins have been widely investigated for applications in tissue engineering and wound repair. Recently, Tjin and coworkers developed integrin-specific aECM proteins designed for promoting human skin keratinocyte attachment and propagation. In their work, the aECM proteins incorporate cell binding domains taken from fibronectin, laminin-5 and collagen IV, as well as flanking elastin-like repeats. They demonstrated that the aECM proteins developed in their work were promising candidates for use as substrates in artificial skin. Here, we outline the design and construction of such aECM proteins as well as their purification process using the thermo-responsive characteristics of elastin.

Elastin benzeri tekrarları içeren füzyon proteinleri tasarımında, genel bir elastin içeriğini, füzyon proteininin 18 arasında yeterince büyük bir kısmını muhafaza etmek önemlidir. Bu füzyon proteini yapısı saflaştırılması için ITC kullanmak için, onun elastin gibi özelliklerini muhafaza sağlamaktır. aECM proteinleri tasarımı ve bu bölümde tarif edilen sekanslar, özel olarak Tjin ve ark., 14 tarafından gerçekleştirilen çalışmadan alınmıştır.</em...

Watch this Scientific Journal Video about Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering at JoVE.com

Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats.

Tasarım ve Yapay Ekstrasellüler Matrix İnşaatı (aECM) Proteinler gelen<em&gt; Escherichia coli</em&gt; Cilt Doku Mühendisliği

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins ...

design-construction-artificial-extracellular-matrix-aecm-proteins

Research

JoVE Journal

Bioengineering

Design and Construction of Artificial Extracellular Matrix (aECM) Proteins from Escherichia coli for Skin Tissue Engineering

8.8K Görüntüleme.  Nanyang Technological University. Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats. 

Video: Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering

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, ...

Biyomühendislik

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Kardiyak Doku Mühendisliği kılcal Kuvvetleri Litografi

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

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

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

Doku Mühendisliği için iki eksenli Mekanik Yükleniyor Biyoreaktör tasarımı

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

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

Doku Mühendisliği: Katmanlı Hücre Sheets teslimi için bir Multisellüler 3D İskele İnşaatı

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

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

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

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

Üç-boyutlu Tam Kalınlığı Deri Üretimi Eşdeğer ve Otomatik Yaralama

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

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

Epitel Hücre Repopulation ve Böbrek Doku Kalkınma Kemirgen Ekstrasellüler Matriks iskelelerinin hazırlanması

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

Ekstraselüler Matrix içinde Gömülü Mühendislik Üç boyutlu Epitel Dokular

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis.

We present a protocol for the application of Brillouin light scattering spectroscopy to elastin and trypsin-purified type I collagen fibers of the extracellular matrix to extract their full elastic properties.

Brillouin Spektroskopisi için Ekstraselüler Matriks Proteini Elyaf Hazırlama

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

3D Hücre Kültürü için Pulmoner Ekstrasellüler Matrix Ayarlanabilir Hidrojellerinin

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

Doku spesifik Hücre Kültürü ve Delivery Platform olarak Ekstrasellüler Matriks kaynaklı Köpükler ve mikro Fabrikasyon

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Tasarım ve Yapay Ekstrasellüler Matrix İnşaatı (aECM) Proteinler gelen

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