Bu çalışma NIH (HL118532, HL120142, CA187692), David ve Lucile Packard Vakfı, Camille ve Henry Dreyfus Vakfı ve Burroughs hibe tarafından kısmen desteklenmiştir Fonu hoş geldiniz. ASP Charlotte Elizabeth Procter yüceltici Kardeşliği tarafından kısmen desteklenmiştir.

Çözümler 1. hazırlanması <ol><li> Insülin, 5 mg / ml çözelti hazırlamak (çözücü, 100 ml, 500 mg insülin) O dH 2 5 mM, hidroklorik asit (HCI) ile toz haline getirilmiş insülin stokları sulandırmak. Damıtılmış su, 100 ml konsantre HCI, 50 ul ekleyerek çözücü 100 ml (DH 2 O) hazırlayın. </li><li> PBS 1x solüsyon yapmak için, steril koşullar altında DH 2 O 1x, 10x, fosfat tamponlu tuz (PBS) stok çözeltisi seyreltilir. </li><li> şöyle polidimetilsiloksan (PDMS) bir elastomer çözeltisi hazırlayın: 1 (ağ: ağ) oranı, bir 10 ısıyla sertleştirme maddesi ile bir araya PDMS prepolimeri karıştırın. </li><li> aşağıda hücre kültür ortamı hazırlayın: Hazır Dulbecco Modifiye Eagle Ortamı 500 ml: Besin Karışımı, F-12 (DMEM / F-12), 10, steril fetal sığır serumu ml (FBS), gentamisin reaktif 500 ul ve ek 5 mg / ml insülin, 500 ul. </li><li> (w% 1 ekleyin, 1 x PBS çözeltisi içinde% 1 sığır serum albümini (BSA) hazırlamak için: BSA tozu v) çözeltisi PBS 1x ve iyice karıştırın. </li><li> ekleyin I kolajen nötralize sığır tipi bir 4 mg / ml çözelti hazırlamak için, 50 ul 10x Hank dengeli tuz çözeltisi (HBSS) tampon maddesi, 30 ul 0.1 N sodyum hidroksit (NaOH), 30 ul hücre kültür ortamı ve hazır kollajen 400 ul soğutulmuş 1.5 ml mikrosantrifüj tüpüne. Yukarı ve aşağı pipetleme yavaş yavaş karıştırın; kabarcıkları almaktan kaçınması. Herhangi bir kabarcıklarını çıkarmak için, kısaca 4 ° C&#39;de karışım santrifüj. </li><li> 4 (v: v) 1 x PBS içinde% 16 paraformaldehit 1 seyrelterek bir sabitleyici çözelti hazırlayın. </li><li> 1,000 (v: v) stok çekirdekleri etiketleme çözümü 1 seyreltilmesi ile bir çekirdek etiketleme çözümü hazırlayın 1x PBS içinde. </li><li> Deterjan:% 0.3 (v v) ile 1X PBS karıştırılarak% 0.3 fosfat tamponlu tuzlu su ve deterjan (PBST) çözeltisi hazırlayın. </li><li> % 0.3 PBST: keçi serumu 01:10 (v v) seyreltilmesi ile engelleme tampon hazırlayın. </li><li> PRI seyrelterek, ilgilenilen özel bir protein için bir birincil antikor çözeltisi hazırlayın(Örneğin, tavşan anti-fokal yapışma kinaz (FAK)) Mary antikoru 1: 200 (hacim: hacim) blokaj tamponunda. </li><li> (V v) blokaj tamponunda 1.000 (örneğin, keçi anti-tavşan) 1 floresan prob ve konjüge olmamış antikor seyrelterek bir ikincil antikor çözeltisi hazırlayın. </li></ol> 3D micropatterning Elastomerik Pullar 2. Hazırlık Not: Elastomerik pulları PDMS ile yapılır. <ol><li> Adım 1.3 açıklandığı gibi aynı oranda bir PDMS çözüm yapmak ve iyice karıştırın. Karıştırma işlemi sırasında oluşan olan tüm hava kabarcıklarını çıkarmak için 15-30 dakika süreyle bir vakumlu odasında karışımın yerleştirin. </li><li> tekne ya da lithographically istenen geometri özellikleri ile desenli bir silikon ustası içeren Petri kabı tartın bir plastik içine Tolüen dökün. En iyi sonuçları elde etmek için, 12 saat ~ 60 ° C&#39;de PDMS çözeltisi tedavi. Not: silikon ustaları son derece özelleştirilebilir; Bu protokol 50 ölçülerinde dikdörtgen yapılar kullanırmm x 200 mm x 50 mm 200 mm aralıklı. Silikon Masters, standart fotolitografi teknikleri kullanılarak yapılabilir. Kısaca, bir epoksi-bazlı fotorezist silikon göbeğin üzerine spin-kaplanır. İstenilen damga özelliklerini ayrıntılı bir maske daha sonra UV ışığına maruz fotorezist kaplı gofret, üstüne yerleştirilir. maske tarafından bloke istenen özellikleri ışığa maruz kalır ve ışığa dirençli kaplamanın geri kalanı çözünür olduğu ve yıkanmasını edilebilir ise bu yerlerde fotorezist, çapraz bağlanmış olur. </li><li> PDMS silikon ana istenilen özelliklere etrafında tamamen donduktan sonra plastik bir kaba gelen PDMS ve master&#39;ı çıkarın. Dikkatle silikon gofret PDMS ayırmak ve temiz bir jilet kullanarak baskılı özellikleri çevresinde aşırı PDMS çıkarın. </li><li> Hemen bir jilet ile tek tek dikdörtgen pullar halinde kalıplanmış özelliklerine (~ 8 mm x 5 mm), desenli PDMS kesti. Bir cle bu özellik yan-up MağazaBir 100 mm çaplı Petri kabı. </li><li> Ayrıca, dikdörtgen pulları için destek yapmak için adım 1.3 açıklanan PDMS çözümü kullanın. </li><li> bir eğirme kaplayıcı kullanılarak, 100 mm çapında bir Petri tabağına eşit PDMS çözeltisi ~ 2-3 g yaymak ve adım 2.1 60 ° C&#39;de ~ 12 saat boyunca PDMS tedavi. Sonra, bir jilet kullanarak, dikdörtgenler içine PDMS ince bir tabaka (~ 5 mm x 2 mm) kesti. Not: İki destekler adım 2.3 yapılan her PDMS damga için gereklidir. destekleri PDMS pulları içeren 100 mm çapında Petri kabındaki saklanabilir. </li><li> PDMS pulları ve destekler hücre kültürü için kullanılmadan önce, bir biyogüvenlik kabini içinde bir aspiratör (hücre kültürü kaput) kullanılarak% 70 etanol ve kuru batırılarak sterilize edin. </li></ol> 3D Epitel Dokuların 3. hazırlanması <ol><li> Bir biyo-güvenlik kabini, her pul en PBS içinde% 1 BSA, yaklaşık 50 ul&#39;lik bir damla ekleyin. Bir mini 4 ° C&#39;de pul kaplı damlacık yerleştirin4 saat içinde anne BSA damgası yüzeyine adsorbe sağlamak. </li><li> PDMS pul aspire BSA çözüm. </li><li> Her yıkamadan sonra aspire, hücre kültür ortamı (pul her yıkama başına 50 ul yeterli olmalıdır) ile iki kez pul yüzeyleri yıkayın. </li><li> Kavisli paslanmaz çelik cımbız kullanarak sterilize PDMS pulları ve destekleri anlaştım. Her damgası PDMS için bir biyogüvenlik kabini, bir 35 mm çaplı doku kültürü çanak yatıyordu. her yemeğin, PDMS pul uzunluğu biraz daha az bir mesafe ile ayrılmış iki PDMS destekler uzandı. </li><li> cımbız kullanarak, birçok yuvarlak cam lamelleri sterilize (çapı 15 mm, # 1)% 70 etanol kullanarak PDMS pulları olduğu gibi. cımbız ile onları tutarken lamelleri fazla sıvıyı aspire. Ayrı bir 100 mm çapında Petri kabındaki yıkandı lamelleri saklayın. </li><li> ~ Kollajen karışımı 50 ul eşit kat, her yüzey damgası PDMS için dağıtın. </li><li> AlmakHer kollajen kaplı PDMS cımbızla damga ve onları hafifçe ters çevirin. PDMS üstüne ters pullar indirin 35 mm çaplı doku kültüründe ortaya koydu destekler kollajen mühür ve doku kültürü çanak alt arasında olacak şekilde bombeli. 30 dakika boyunca 37 ° C &#39;de yemekler inkübe edin. </li><li> Dairesel lamelleri her birine ~ kalan kollajen karışımı 50 ul koyun ve 30 dakika boyunca 37 ° C&#39;de inkübe (daha sonra bu, tamamen kollajen bunları kapsüllemek için mühendislik dokularının üzerine yerleştirilir). </li><li> % 40 konfluansa ~ epitel hücreleri (ör. EpH4 fare meme epitelyal hücreleri) ihtiva eden bir 100 mm çaplı bir doku kültürü çanağı elde edilir. Hücre kültür ortamı aspire edildi ve PBS 1x 10 ml ile bir kez yıkanır. hücrelere tripsin 2 ml ilave edilir ve 5-10 dakika boyunca 37 ° C&#39;de inkübe edin. </li><li> doku kültürü çanağı, tripsinize edilmiş hücre ihtiva eden çanak kalan yapışık hücreler ayırma taze kültür ortamı 8 ml ekleyin. pipetleme ile yavaşça karıştırın ve 5 dakika boyunca 100 x g &#39;de 15 ml konik bir tüp ve santrifüj hücre karışımı hareket ettirin. </li><li> Konik tüpten aspire ve hücre kültür ortamı içinde hücre pelletini. hücreler bir hemasitometre kullanarak saymak süspansiyon konsantrasyonunu belirlemek için. 10 6 -10 7 hücre / ml ~ nihai bir konsantrasyon elde etmek için süspansiyon ses seviyesini ayarlayın. </li><li> inkübatör jölelenmişti kollajen örnekleri çıkarın. cımbız kullanarak, yavaşça kalıplı kollajen onları ayırmak ve onları atmak için düz yukarı PDMS pulları kaldırın. </li><li> istenen geometriye kalıplanmış boşluklar içeren her kollajen jel yüzeyi üzerine ~ konsantre hücre süspansiyonu 30 ul koyun. Hafifçe yemekler yan sallayarak boşlukları içinde yerleşen hücreyi teşvik yan ise 10X / 0.25 NA amacı ile bir aydınlık mikroskop altında hücreleri gözlemleyin. boşluklar ~ 5 dakika içinde doldurulması gerekmektedir. </li><li> rBoşlukların etrafında aşırı hücre birikimleri temizlenmelidir yan, her bir doku kültürü çanağı eğim ve yavaşça kollajen jel yüzeyi üzerine hücre kültürü ortamında ul 400 ~ dağıtın. Her yıkama arasında mikroskop altında kolajen jelleri kontrol sıvı aspire ve yıkama 1-2 kez daha tekrarlayın. </li><li> fazla hücreler kolajen kalıp hücre dolu boşlukların etrafında temizlendikten sonra, 15 dakika boyunca 37 ° C&#39;de bir hücre kültür inkübatörü içine doku kültür kaplarına yerleştirin. Ardından, cımbız kullanarak, yavaşça kollajen kaplı sınıf lamelleri ters ve lamelleri kollajen hücre dolu boşlukların üzerine bir kap oluşturduğu hücre dolu kollajen kalıpların üstüne koyun. 15 dakika boyunca 37 ° C&#39;de inkübe edin. </li><li> Kollajen kapaklar hücre dolu kollajen kalıba yapıştırılır sonra, jeller üstüne cam lamel üzerinde yavaşça ~ 2-2.5 ml hücre kültür ortamı dağıtmak. Kültür 1-3 gün boyunca 37 ° C &#39;de örnekler. Not:24 saat ilk ekimden sonra epitel dokular, epidermal büyüme faktörü (EGF) veya dal uyarılması için hepatosit büyüme faktörü (HGF) gibi büyüme faktörleri ile muamele edilir. </li></ol> 4. İmmünofloresan ve Görüntü Analizi <ol><li> Doku kültür kaplarından, hücre kültür ortamı aspire ve hücre-ihtiva eden jeller karşılamak için yeterli sabitleyici solüsyonu ekleyin. 200 rpm&#39;de bir çalkalayıcıda 15 dakika oda sıcaklığında inkübe edin yemekler. </li><li> , Fiksatif aspire 1X PBS ile doku kültür kaplarına doldurun ve 200 rpm&#39;de bir çalkalama 15 dakika oda sıcaklığında inkübe edin. Üç toplam yıkar iki kez tekrarlayın. </li><li> Doku kültürü çanak aspire PBS ve Hoechst çözümü ile değiştirin: çekirdekleri etiketlemek için. 15-20 dakika boyunca oda sıcaklığında inkübe edilir. FAK veya antikorlar ile saptanabilir başka marker için leke, 4.5 adıma geçin. </li><li> nükleer etiketleme çözeltisi aspire ve hücre-ihtiva eden g yıkamakAşama 4.2 olarak 1 x PBS ile üç kez kavuşmaktadır. Lekeli örnekler sonraki kullanıma kadar 4 ° C &#39;de 1 x PBS içinde saklanabilir. </li><li> , PBS aspire doku kültür kaplarından% 0.3 PBST 300 ul ve 15 dakika için oda sıcaklığında inkübe edilir örnek: ilgi konusu bir protein için leke için. </li><li> PBS aspire Blokaj tamponu ile jeller kapak ve ~ 4 saat boyunca oda sıcaklığında bir çalkalama (200 rpm) üzerinde inkübe edin. </li><li> Aspire bloke tamponu, primer antikor çözeltisi ile jeller kapağı ve 4 ° C&#39;de bir gece 200 rpm&#39;de bir çalkalama üzerinde inkübe edin. </li><li> Primer antikor çözeltisi aspire% 0.3 PBST solüsyonu ekleyin ve 30 dakika için oda sıcaklığında 200 rpm&#39;de bir çalkalama üzerinde inkübe edin. PBST aspire ve 3-4 saat süreyle her 30 dakikada bir tekrarlayınız. </li><li> Tekrarlayın 4.7 ve 4.8, ikincil antikor çözeltisi ile inkübe bu kez yineleyin. İkincil antikor photobleaching önlemek için alüminyum folyo ile doku kültür tabaklarında sarın. fina sonraL yıkama, lekeli numuneler sonraki kullanıma kadar 4 ° C &#39;de 1 x PBS içinde saklanabilir. </li><li> Örnekleri görselleştirmek için, epitel dokuda midplane odaklanmış bir 10X / 0.30 NA objektif kullanın. </li><li> Hücre çekirdekleri, UV aydınlatma altında 10X / 0.30 NA objektif kullanarak bir nükleer işaretleyici ile etiketlenmiş görüntü sabit örnekleri görselleştirmek için. </li><li> Ters bir floresan mikroskopu üzerinde bir 10X / 0.30 NA objektif kullanılarak ilgi konusu bir protein, görüntü için boyandı örnekleri görselleştirmek için. </li><li> aynı ilk geometri birçok dokuda (tipik olarak 50 veya daha fazla) protein boyama frekans haritalar oluşturmak için, ilk ithalat standart görüntü analiz yazılımı kullanarak her görüntü (Malzeme) ve 8-bit gri tonlama onları dönüştürmek. </li><li> Bu görüntülerin eşik için, gri tonlama kesme noktası tanımlayarak ikili (yani, yarım ton / siyah ve beyaz) onları dönüştürmek; eşiğin altına gri tonlama değerleri siyah olur ve eşiğin üstünde o beyaz olur. Daha sonra, kombinetek bir görüntü yığını içine bireysel ikili görüntülerin her. </li><li> Sonraki yığılmış görüntüleri kayıt (yani hizalamak veya onları maç) analiz yazılımı. Birçok görüntü analiz yazılımı paketleri serbestçe kullanılabilir kayıt eklentisi ile geliyor. <ol><li> sonraki görüntüye uyum için bir şablon olarak bir yığın her görüntü kullanın, tüm yığın görüntüleri yayılması ile hizalanır şekilde. Son olarak, bindirme (proje) ortalama yoğunluğuna dayalı hizalanmış görüntü yığınında görüntüleri piksel frekansı harita ile tek bir görüntü oluşturmak için. Bu görüntü daha sonra renk kodlu seçim 10,11 görüntü düzenleme yazılımını kullanarak olabilir. </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

The protocol described above outlines a method to produce identical epithelial tissues of pre-defined shape, enabling spatial control of the mechanical stress experienced by cells in the tissue. An elastomeric mold is used to create cavities in type I collagen that are then filled with epithelial cells and covered with an additional collagen layer such that cells are completely encapsulated in a 3D collagen matrix environment. Further culture of these tissues and treatment with growth factors to induce branching from the...

morfojenezi dallanma olarak bilinen kollara ayrılmış epitelyal dokuların gelişimi, hücre-türevli, fiziksel ve çevresel faktörler ile düzenlenir. meme bezinde, morfolojilerinden dallanma toplu hücre göçü bir ağaç gibi mimari oluşturur güdümlü aracılığıyla tekrarlanan bir süreçtir. İlk adım şube başlatılması ve uzama 1,2 izledi süt kanallarından birincil tomurcuğu oluşumu vardır. Çevredeki stromaya dalları Invasion ergenlik steroid hormonların sistemik salınımı ile indüklenir. Yeni birincil tomurcukları sonra varolan şube uçlarından başlatabilir ve bu süreç bir epitel ağacı 3 yaratmaya devam ediyor. Birçok önemli biyokimyasal sinyaller tespit edilmiş olmasına rağmen, bu karmaşık gelişim süreci şu anda eksiktir rehberlik hücre biyolojik mekanizmaların kapsamlı bir anlayış. Ayrıca, belirli ipuçlarının etkilere mekanik çalışmalar deney gelen yapısızlaştırmak zorin vivo likte olarak hassas uzaysal tedirginlikler ve ölçümler çoğu zaman mümkün değildir. Böyle tüm organ kültürü, birincil organoids ve hücre kültür modelleri gibi üç boyutlu (3D) kültür teknikleri, sistematik doku morfolojilerinden 4-6 yatan mekanizmaları araştırmak için yararlı araçlardır. Bu hareketi, çoğalması ve farklılaşması dahil olmak üzere hücre davranışları çeşitli, bu gibi mekanik güçler ve biyokimyasal sinyallerin gibi, tek tek özel faktörlerin etkileri belirlemek için yararlı olabilir. 6 tasarlanan hücre kültürü modelleri, özellikle, hali hazırda pertürbasyon etkinleştirmek tek tek hücreler ile bunların mikro arasında. Böyle bir kültür modeli a ile uyarılan zaman tutarlı ve tekrarlanabilir toplu göç dalları oluşturan kontrollü 3D yapısı ile örnek meme epitel doku mühendisi bir mikroüretim tabanlı bir yaklaşım kullanırppropriate büyüme faktörleri. modelin en büyük avantajı tam işlemek ve bu tür yüksek istatistiksel güven ile mekanik stres desen, fiziksel ve biyokimyasal faktörlerin etkilerini ölçmek için yeteneğidir. Bu teknik, bir araya hesaplama modelleme ile, daha önce meme epitelyal dokusunda ve dallı epitel 7-11 normal gelişimin rehberliğinde fiziksel ve biyokimyasal sinyallerin nispi katkılarını belirlemek için kullanılmıştır. Burada sunulan kolaylıkla hücreler ve hücre-dışı matrisi (ECM) jellerin diğer tiplerinin uzatılabilir, bu model, dokular, bina için ayrıntılı bir protokoldür ve terapötiklerinin test edilmesi için potansiyel bir araç olarak hizmet eder.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Polydimethylsiloxane (PDMS)</td>
			<td>Ellsworth Adhesives</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr>
			<td>PDMS curing agent</td>
			<td>Ellsworth Adhesives</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithographically patterned silicon master</td>
			<td>self-made</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic weigh boat</td>
			<td>Fisher Scientific</td>
			<td>08-732-115</td>
			<td></td>
		</tr>
		<tr>
			<td>100-mm-diameter Petri dishes</td>
			<td>BioExpress</td>
			<td>D-2550-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl Alcohol 200 Proof</td>
			<td>Pharmco-Aaper</td>
			<td>111000200</td>
			<td>Make a 70% EtOH (v:v) solution by mixing with dH2O</td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>American Safety Razor</td>
			<td>620179</td>
			<td></td>
		</tr>
		<tr>
			<td>1:1 Dulbecco&#8217;s Modified Eagle&#8217;s Medium : Ham&#8217;s F12 Nutrient Mixture (DMEM/F12) (1:1)</td>
			<td>Hyclone</td>
			<td>SH30023FS</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>S11150H</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Hank&#8217;s balanced salt solution (HBSS)</td>
			<td>Life Technologies</td>
			<td>14185-052</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma Aldrich</td>
			<td>I6634-500MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Life Technologies</td>
			<td>15750-060</td>
			<td></td>
		</tr>
		<tr>
			<td>10X Phosphate-buffered saline (PBS)</td>
			<td>Fisher Scientific</td>
			<td>BP399-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Sigma Aldrich</td>
			<td>221465-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine type I collagen (non-pepsinized)</td>
			<td>Koken</td>
			<td>IAC-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin from bovine serum (BSA)</td>
			<td>Sigma Aldrich</td>
			<td>A-7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved stainless steel tweezers</td>
			<td>Dumont</td>
			<td>7</td>
			<td></td>
		</tr>
		<tr>
			<td>35-mm-diameter tissue culture dishes</td>
			<td>BioExpress</td>
			<td>T-2881-6</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tubes</td>
			<td>BioExpress</td>
			<td>C-3394-2</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Eppendorf Safe-Lock Tube</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
			<td></td>
		</tr>
		<tr>
			<td>Circular #1 glass coverslips, 15-mm in diameter</td>
			<td>Bellco Glass Inc.</td>
			<td>Special order</td>
			<td></td>
		</tr>
		<tr>
			<td>0.05% 1X Trypsin-EDTA</td>
			<td>Life Technologies</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>VWR</td>
			<td>100503-916</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Perkin Elmer</td>
			<td>N9300260</td>
			<td>Detergent</td>
		</tr>
		<tr>
			<td>HGF</td>
			<td>Sigma Aldrich</td>
			<td>H 9661</td>
			<td>Resuspended in dH2O at 50 mg/mL</td>
		</tr>
		<tr>
			<td>Rabbit anti-mouse FAK antibody</td>
			<td>Life Technologies</td>
			<td>AMO0672</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit Alexa 488 antibody</td>
			<td>Life Technologies</td>
			<td>A-11034</td>
			<td></td>
		</tr>
		<tr>
			<td>Adobe Photoshop</td>
			<td>Adobe</td>
			<td>N/A</td>
			<td>Used for color-coding pixel frequency maps.</td>
		</tr>
		<tr>
			<td>FIJI (ImageJ)</td>
			<td>NIH</td>
			<td>N/A</td>
			<td>Free image analysis software used for thresholding, registering, and overlaying images to create a pixel frequency map. The StackReg plugin was used for registering binary images.</td>
		</tr>
	</tbody>
</table>

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.

Meme epitelyal dokusu mikroimalat genel şematik Deneysel çalışma akışını özetleyen mikroimalat prosedürünün genel şematik Şekil 1&#39;de gösterilmiştir. Sonuçta tamamen bir ECM jel içinde gömülü olan özdeş geometri ve aralık epitel dokuda bir dizidir. Temsili deney I, 4 mg / ml bir konsantrasyonda kolajen sığır türü bir jel içinde kültürlenmiş EpH4 fare meme epitelyal hücreleri kullanılmakta...

Watch this Scientific Journal Video about Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix at JoVE.com

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

Bu yazıda, üç boyutlu (3D) tek tip diziler ekstraselüler matriks çevrili tanımlanan geometri epitel doku mühendisi yumuşak litografi tabanlı teknik anlatılmaktadır. Bu yöntem, hücre tipleri ve deneysel bağlamlarda çeşitli için uygun olan ve aynı çoğaltır yüksek verimli tarama sağlar.

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, ...

engineering-three-dimensional-epithelial-tissues-embedded-within

Research

JoVE Journal

Bioengineering

7.5K Görüntüleme.  Princeton University.  Bu yazıda, üç boyutlu (3D) tek tip diziler ekstraselüler matriks çevrili tanımlanan geometri epitel doku mühendisi yumuşak litografi tabanlı teknik anlatılmaktadır. Bu yöntem, hücre tipleri ve deneysel bağlamlarda çeşitli için uygun olan ve aynı çoğaltır yüksek verimli tarama sağlar. 

Video: Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Üç boyutlu optik çözünürlük foto akustik Mikroskopi

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Biyomühendislik

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

Üç boyutlu Matrix floresan etiketli Proteinler ifade Geçiş Hücreler Canlı hücre Görüntüleme

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

Bir Üç boyutlu Kollajen Matrix içindeki Hücre Göç Analizi

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

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.

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 ...

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 ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

İçinde üç boyutlu Vitro hücre göç karakterize ortamlar yara

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

Üç boyutlu peptid iskele memeli hücrelerinde kültür

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

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 ...

Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the major glial cells of the PNS, play a vital role in promoting PNS regeneration by dedifferentiating into a regenerative cell phenotype following injury. However, the dedifferentiated state of SCs is challenging to maintain through the time-period needed for regeneration and is impacted by changes in the surrounding extracellular matrix (ECM). Therefore, determining the complex interplay between SCs and differing ECM to provide cues of regenerative potential of SCs is essential. To address this, a strategy was created where different ECM proteins were adsorbed onto a tunable polydimethylsiloxane (PDMS) substrate which provided a platform where stiffness and protein composition can be modulated. SCs were seeded onto the tunable substrates and critical cellular functions representing the dynamics of SC phenotype were measured. To illustrate the interplay between SC protein expression and cellular morphology, differing seeding densities of SCs in addition to individual microcontact printed cellular patterns were utilized and characterized by immunofluorescence staining and western blot. Results showed that cells with a smaller spreading area and higher extent of cellular elongation promoted higher levels of SC regenerative phenotypic markers. This methodology not only begins to unravel the significant relationship between the ECM and cellular function of SCs, but also provides guidelines for the future optimization of biomaterials in peripheral nerve repair.

This methodology aims to illustrate the mechanisms by which extracellular matrix cues such as substrate stiffness, protein composition and cell morphology regulate Schwann cell (SC) phenotype.

Schwann Hücre Fenotip Spesifikasyonunu Değerlendirmek Için Hücre Dışı Matriks Mikroortamlarının Hazırlanması

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the ...

Generation of a Simplified Three-Dimensional Skin-on-a-chip Model in a Micromachined Microfluidic Platform

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of concept, a simplified and undifferentiated human skin containing a dermal (stromal) and an epidermal (epithelial) compartment has been modelled. To accomplish this, a versatile and robust, vinyl-based device divided into two chambers has been developed, overcoming some of the drawbacks present in microfluidic devices based on polydimethylsiloxane (PDMS) for biomedical applications, such as the use of expensive and specialized equipment or the absorption of small, hydrophobic molecules and proteins. Moreover, a new method based on parallel flow was developed, enabling the in situ deposition of both the dermal and epidermal compartments. The skin construct consists of a fibrin matrix containing human primary fibroblasts and a monolayer of immortalized keratinocytes seeded on top, which is subsequently maintained under dynamic culture conditions. This new microfluidic platform opens the possibility to model human skin diseases and extrapolate the method to generate other complex tissues.

Here, we present a protocol to generate a three-dimensional simplified and undifferentiated skin model using a micromachined microfluidic platform. A parallel flow approach allows the in situ deposition of a dermal compartment for the seeding of epithelial cells on top, all controlled by syringe pumps.

Mikromakine Mikroakışkan Platformda Basitleştirilmiş Üç Boyutlu Çip Üzerinde Cilt Modeli Üretimi

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of ...