Name: design and construction of artificial extracellular matrix (aecm) proteins from escherichia coli for skin tissue engineering
Uploaded: 2015-06-11T13:00:00.000+00:00
Duration: 10 min 30 s
Description: 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

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><tbody><tr><td>pET22b (+)</td><td>Novagen</td><td>69744</td><td>T7 expression vectors with resistance to ampicillin&amp;nbsp;</td></tr><tr><td>BL21(DE3)pLysS&amp;nbsp;</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>1 M 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&amp;trade; 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 ...

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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

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 ...

Biyomühendislik

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

A Tam Cilt Hata Modeli Biyomateryallerin vaskülarizasyonunu değerlendirin<em&gt; In Vivo</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

Acellular extracellular matrix preparations are useful for studying cell-matrix interactions and facilitate regenerative cell therapy applications. Several commercial extracellular matrix products are available as hydrogels or membranes, but these do not possess tissue-specific biological activity. Because perfusion decellularization is usually not possible with human heart tissue, we developed a 3-step immersion decellularization process. Human myocardial slices procured during surgery are first treated with detergent-free hyperosmolar lysis buffer, followed by incubation with the ionic detergent, sodium dodecyl sulfate, and the process is completed by exploiting the intrinsic DNase activity of fetal bovine serum. This technique results in cell-free sheets of cardiac extracellular matrix with largely preserved fibrous tissue architecture and biopolymer composition, which were shown to provide specific environmental cues to cardiac cell populations and pluripotent stem cells. Cardiac extracellular matrix sheets can then be further processed into a microparticle powder without further chemical modification, or, via short-term pepsin digestion, into a self-assembling cardiac extracellular matrix hydrogel with preserved bioactivity.

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

This protocol describes the complete decellularization of human myocardium while preserving its extracellular matrix components. Further processing of the extracellular matrix results in the production of microparticles and a cytoprotective self-assembling hydrogel.

İnsan kalp dokusu hücre dışı matriks hidrojel Vitro ve In Vivo uygulamaları için kendi kendine montaj doğru işlenmesi

Acellular extracellular matrix preparations are useful for studying cell-matrix interactions and facilitate regenerative cell therapy applications. ...

Gelişim Biyolojisi

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. ...

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

Geliştirilmesi 2D ve 3D Cilt<em&gt; İn Vitro</emMakromoleküler Dışlamasını Kullanma&gt; Modeller

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

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, ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

Elastin benzeri Protein Hydrogels üretimi için saklama ve Immunostaining hücre 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure and healing. Extraction of extracellular matrix from fibrogenic cell cultures in vitro has potential for generation of ECM from human- and potentially patient-specific cell lines, minimizing the presence of xenogeneic epitopes which has hindered the clinical success of some existing ECM products. A significant challenge in in vitro production of ECM suitable for implantation is that ECM production by cell culture is typically of relatively low yield. In this work, protocols are described for the production of ECM by cells cultured within sacrificial hollow fiber membrane scaffolds. Hollow fiber membranes are cultured with fibroblast cell lines in a conventional cell medium and dissolved after cell culture to yield continuous threads of ECM. The resulting ECM fibers produced by this method can be decellularized and lyophilized, rendering it suitable for storage and implantation.

The goal of this protocol is production of whole extracellular matrix fibers targeted for wound repair which are suitable for preclinical evaluation as part of a regenerative scaffold implant. These fibers are produced by the culture of fibroblasts on hollow fiber membranes and extracted by dissolution of the membranes.

Hücre dışı matriks liflerinin kurban Hollow Fiber membran hücre kültürü ile üretim

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure ...

Generation of Self-assembled Vascularized Human Skin Equivalents

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to study skin development, wound healing, and grafting techniques. Many HSEs continue to lack vasculature and are additionally analyzed through post-culture histological sectioning which limits volumetric assessment of the structure. Presented here is a straightforward protocol utilizing accessible materials to generate vascularized human skin equivalents (VHSE); further described are volumetric imaging and quantification techniques of these constructs. Briefly, VHSEs are constructed in 12 well culture inserts in which dermal and epidermal cells are seeded into rat tail collagen type I gel. The dermal compartment is made up of fibroblast and endothelial cells dispersed throughout collagen gel. The epidermal compartment is made up of keratinocytes (skin epithelial cells) that differentiate at the air-liquid interface. Importantly, these methods are customizable based on needs of the researcher, with results demonstrating VHSE generation with two different fibroblast cell types: human dermal fibroblasts (hDF) and human lung fibroblasts (IMR90s). VHSEs were developed, imaged through confocal microscopy, and volumetrically analyzed using computational software at 4- and 8-week timepoints. An optimized process to fix, stain, image, and clear VHSEs for volumetric examination is described. This comprehensive model, imaging, and analysis techniques are readily customizable to the specific research needs of individual labs with or without prior HSE experience.

The goal of this protocol is to describe the generation and volumetric analysis of vascularized human skin equivalents using accessible and simple techniques for long term culture. To the extent possible, the rationale for steps is described to allow researchers the ability to customize based on their research needs.

Kendi Kendine Monte Edilmiş Vaskülerleştirilmiş İnsan Derisi Eşdeğerlerinin Üretimi

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to ...

<meta charset="utf8"></head><body>Esnek Araştırma Uygulaması için Verimli 3D Cilt Modelleri

Building Up Skin Models for Numerous Applications - from Two-Dimensional (2D) Monoculture to Three-Dimensional (3D) Multiculture

Due to the complex structure and important functions of the skin, it is an interesting research model for the cosmetic, pharmaceutical, and medical industries. In the European Union, there has been a total ban on testing cosmetic products and their ingredients on animals. In the case of medicine and pharmaceuticals, this possibility is also constantly limited. In accordance with the 3Rs principle, it is becoming more and more common to test individual compounds as well as entire formulations on artificially created models. The cheapest and most widely used are the 2D models, which consist of a cell monolayer but do not reflect the real interactions between the cells in the tissue. Although the commercially available 3D models provide a better representation of the tissue, they are not used on a large scale. This is because they are expensive, the waiting time is quite long, and the available models are frequently limited to only those typically used. 
In order to move the conducted research to a higher level, we have optimized the procedures of various 3D skin model preparations. The described procedures are cheap and simple to prepare as they can be applied in numerous laboratories and by researchers with different experiences in cell culture.

<meta charset="utf8"></head><body>Yazar Spotlight: Uygun Maliyetli 3D Hücresel Modellerle Cilt Modeli Çeşitliliğini Artırma

Here, we present inexpensive and simple procedures to introduce various 3D skin models for routine research in a cell culture laboratory. Researchers can create models tailored to their needs without relying on commercially available models.

Çok Sayıda Uygulama için Cilt Modelleri Oluşturma - İki Boyutlu (2D) Monokültürden Üç Boyutlu (3D) Çoklu Kültüre

Due to the complex structure and important functions of the skin, it is an interesting research model for the cosmetic, pharmaceutical, and medical ...

Tasarım ve Yapay Ekstrasellüler Matrix İnşaatı (aECM) Proteinler gelen

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