Biz sağlam domuz akciğer dokusu bağış için Smithfield çiftlikleri teşekkür etmek istiyorum. Biz de bize kendi ekipman kullanmak için izin Dr. Hu Yang, Dr. Christina Tang ve VCU Plastik Cerrahi Bölümü&#39;ne teşekkür etmek istiyorum. Hidrojel ve doku örnekleri finansman formu NIH-NCI Kanser Merkezi Destek Grant, kısmen, NIH-NINDS Merkezi Çekirdek Grant 5 P30 NS047463 dan fon tarafından, kısmen desteklenen Anatomi ve Nörobiyoloji Mikroskopi Tesisi VCU Bölümü&#39;nde SEM için hazırlandı P30 CA016059. VCU Nanoteknoloji Çekirdek Karakterizasyonu Tesisi (KKK) numunelerin SEM görüntüleme. Bu çalışma, Ulusal Bilim Vakfı, CMMI 1351162 tarafından finanse edildi.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Çözüm </td><td> Steril Filtresi </td><td> Yol </td></tr><tr><td> Dihidro 2 O </td><td> Evet </td><td> Dihidro 2 O; Steril süzüldü </td></tr><tr><td> % 0.1 Triton X-100 Çözüm </td><td> Evet </td><td> Davlumbaz altında dihidroksi 2 O 100 ml, 100 ul, Triton-X 100 çözüm ekleyin ve çözülene kadar ajite; steril filtre. </td></tr><tr><td> % 2 Deoksikolat Çözelti </td><td> Evet </td><td> davlumbaz altında 2 gr Sodyum Deoksikolat çözüm ekleyin 100 mi dihidro 2 O ve eriyinceye kadar ajitasyon başı; steril filtre </td></tr><tr><td> 1 M NaCl </td><td> Evet </td><td> Dihidro 2 O 1 L 58.44 g NaCI ekleyin; çözülene kadar ajite; steril filtre </td></tr><tr><td> DNaz çözümü </td><td> Evet </td><td> 12,000 eklebirimleri DNaz 1 L DIH 2 O; 0.156 gr MgSO 4 ekleme (Susuz) ve 0.222 g CaCl2; çözülene kadar ajite; steril filtre </td></tr><tr><td> PBS </td><td> Evet </td><td> , 27.2 g Na 2 HPO 4 · 7H 2 O (dibazik heptahidrat) birleştirin 80 g NaCI ve 10 L dihidroksi 2 O 2 g KCI; çözülene kadar ajite; 7.4 pH ayarlamak; steril filtre </td></tr></tbody></table> Tablo 1: Doku Decellularization için gerekli çözümler. decellularization işlemi için yukarıdaki çözümler olun. 4 ° C&#39;de saklayın. Yaklaşık 2.5 L bir domuz akciğer için gerekli olacaktır. 1. Hidrojel Oluşumu <ol><li> (Referanslar 9,10 uyarlanmıştır) Domuz Akciğer Decellularization <ol><li> Kalp ve Vasc ile, bir mezbahadan, bir bloğun en normal bir domuz akciğer eldeulature bozulmamış. </li><li> kalp mümkün olduğunca yakın damarsal kesim, kalbi uzak incelemek için doku makas veya neşter kullanın, kalan damar perfüzyon için daha sonraki adımlarda kullanılacaktır. </li><li> Dikkatli bir neşter veya makas kullanarak trakea, bronşlar ve damarsal çevreleyen bağ dokusu uzak teşrih. NOT: decellularization sürecinin etkinliğini engelleyebilir büyük kesikler veya delikler plevra yoluyla ve atelektazi veya damar tıkanıklığına büyük miktarlarda olmadan bir akciğer seçerek prosedürün geri kalanı için kullanılacak en iyi akciğer belirleyin. </li><li> (Atılabilir kalp, bağ dokusu ve kaldırılan akciğer lobları) mümkün olduğunca trakea bağlı olduğu kadar bronşu bırakarak bırakıyorsa akciğer kesip. tek akciğer trakea ve damarsal bağlı kalan olmalıdır. NOT: İsterseniz histoloji için bölümleri saklayın. 9 </li><li> p kelepçeler veya sütür ile bağlantısız bronşlar kapatınRevent fazla geri tepme. </li></ol><ol start="6"><li> (Tablo 1), gerekli olana kadar, 4 ° C de decellularization çözümleri ve buzdolabında hazırlayın. </li><li> , Pulmoner arter uyum pulmoner arter ve trakea hem aracılığıyla akciğer dokusunda DI suyla 3 kez serpmek için kanüle bir el pompası kullanılarak. ilk her seferinde damarsal serpmek. damar sistemine yaklaşık 1 L ve her perfüzyon için trakea içine 1.5 L ile başlayın, ama hücresel enkaz kaldırıldıktan olarak daha likit sistemi ile perfüze edilebilir. </li><li> Triton X-100 çözeltisi, her iki damar ve trakea serpmek. </li><li> 4 ° C&#39;de 24 saat boyunca, Triton X-100 çözeltisi akciğer dokusu daldırın. </li><li> DI su ile perfuse damarsal ve trakea 3 kez durulayın. </li><li> deoksikolat çözeltisi ile iki damar ve trakea serpmek. </li><li> 4 ° C&#39;de 24 saat boyunca deoksikolat doku bölümleri daldırın. </li><li> DI su ile perfuse damarsal ve trakea 3 kez durulayın. </li><li>NaCl çözeltisi ile iki damar ve trakea serpmek. </li><li> 4 ° C&#39;de 1 saat boyunca, filtre NaCI çözeltisi doku daldırın. </li><li> DI su ile perfuse damarsal ve trakea 3 kez durulayın. </li><li> DNaz çözeltisi ile iki damar ve trakea serpmek. </li><li> 4 ° C&#39;de 1 saat boyunca, filtre DNase solüsyon içerisinde dokuyu daldırın. </li><li> damar ve trakea 5 kez PBS ile iki serpmek. </li><li> fark kıkırdaklı dokuyu, trakea ve sadece solunum bölgeleri (öncelikle periferal alanlar) bırakarak, solunum yollarını yapmasına (öncelikle hilusun ve akciğerlerin medial kısımları çevresinde bulunan) çapında tüm tübüller 2 mm veya daha büyük parçalara ayır. </li><li> 1 inç bölümleri veya daha küçük içine doku teşrih. doku Oryantasyon Bu aşama için önemli değil. </li><li> 50 ml konik tüpler aşırı sıvı ve yer dokusu dökün. -80 ° C&#39;de doku dondurun. Not: histolojik hücre ve hücre debri çıkarılmasını sağlamak için bölümler saklayıns, arzu edildiği takdirde. Biz H &amp; E kullanımı, α-galaktosidaz, Picogreen, hidroksiprolin, ninhidrin, alcian blue, SDS-PAGE ve kütle spektrometresi karakterize etmek. 9 </li></ol></li><li> akciğer İşleme <ol><li> Decellularized akciğer dokusu içeren dondurulmuş tüplerin kapaklarını çıkarın. </li><li> Tüp deliğinin üzerine filtre kağıdı yerleştirin ve lastik bant ile sabitleyin. NOT: Tüp ve içeriği hala donmuş kadar aksi -80 ° C yerleştirmek dondurulmuş olmalıdır. </li><li> Tüm aşırı sıvı üretici talimatlarına göre bir dondurma kurutma kullanılarak, bitene kadar doku liyofilize. değirmen hazır olana kadar -80 ° C&#39;de saklayın. </li><li> çalışma koşullarına yalıtımı ve iç bileşenlerin soğutulması için dondurucu değirmene sıvı azot eklemek başlamadan önce. </li><li> değirmen tüpten manyetik değirmen çubuğunu çıkarın ve alt kapak doku ekleyin. </li><li> değirmen çubuğunu değiştirin ve gevşek paketlenmiş doku ekleyin. değirmen bar hala serbestçe hareket etmelidir. </li><li> değirmen tüpü ve pla kapatındondurucu değirmende ce. </li><li> maksimum doldurma çizgisine kadar sıvı azot doldurun. </li><li> Dondurucu değirmen ince toz haline tüm doku (yaklaşık 5 dakika, ~ 600 dakika çarpma hızı -1) bir paslanmaz çelik bir çarpma polikarbonat silindir hem de üretici talimatlarına göre dondurucu değirmen kullanılarak, paslanmaz çelik uç fişler. Kullanıma hazır olana kadar -80 ° C&#39;de saklayın. </li></ol></li><li> Mikro-gözenekli bir jel formasyonu (8 mg / ml) (referans uyarlanmıştır 9,11) <ol><li> % 1 ekleyin (a / h) hücresizleştirilmiş tozu ve% 0.1 (ağırlık / hacim), 0.01 M HCI pepsin sürekli ajitasyon altında, oda sıcaklığında, (sıvı üst düzeyde akış görmek gerekir) 48 saat. NOT: Toz statik toz tüp duvarlardan aşırı yıkamak için fırsat tanıyor sonra çok HCl ekleyerek tahsil edilir. </li><li> 48 saat boyunca sindirimden sonra, 5 dakika boyunca buz üzerinde solüsyonu ve reaktifleri. </li><li> Sıcaklık 10 olarak% (h / h), 0.1 M NaOH, birD 11.11% (h / h) 10 x PBS, 7.4 fizyolojik pH sindirilmiş protein çözeltisi getirmek (konsantrasyon 1x tüm çözüm getirmek için). NOT: Çözelti hemen bir haftaya kadar 4 ° C &#39;de muhafaza edilebilir. Istenirse reometre 9 kullanarak jelleşme kinetik gerçekleştirin. </li></ol></li></ol> 2. Mekanik Gücü Artırmak İçin hidrojellerin Çapraz bağlama <ol><li> 1,% 0.1 ve% v genipin çapraz bağlama çözeltisi w /% 0.01 solüsyonları,% 10 DMSO içinde genipin tozun gerekli miktarda çözülmesiyle hazırlanmıştır. </li><li> 1 saat çözüme her 15 dakika karıştırın. </li><li> Daha önce kısmen 1.3.4 monte edilmiştir ECM hidrojeller (96 çukurlu bir levhanın her bölmesine 100 ul) kapsayacak şekilde genipin solüsyonu ekleyin. </li><li> 37 ° C&#39;de 24 saat boyunca çapraz bağlanması için her ECM hidrojel bırakın. </li><li> yıkama solüsyonu artık mavi olana kadar ECM hidrojel PBS ile 3 kez durulayın. çapraz hidrojeller daha sonra karakterizasyon ve ce için hazır olacakll kültürü çalışmaları. </li></ol> Mikro Jel ile 3. Hücre Kültürü <ol><li> İki boyutlu kaplama <ol><li> 1.3.3 solüsyonun kullanılarak, 96 çukurlu bir muamele edilmemiş doku kültür plakasının her bir oyuğuna PREGEL çözeltisinin 20 ul ekle. </li><li> 4 ° C&#39;de bir gece buzdolabında protein adsorpsiyon plaka izin vermek. </li><li> Aspire Pregel çözümü ve PBS ile durulayın. </li><li> Geçiş hücre, 9. </li><li> Kuyulara 10.000 hücre / cm 2 ekleyin. </li><li> oyuk başına 100 ul ortam artırmak ve 37 ° C&#39;de inkübe edin. </li></ol></li><li> Üç boyutlu hücre kültürü 9 <ol><li> 1.3.3 solüsyonun kullanarak, PREGEL çözeltisi 1.000.000 hücre / ml &#39;lik bir konsantrasyona kadar pelet hücreleri yeniden süspanse edin. </li><li> Hızlı 3D hücre kültürü için ~ 500 um kalınlığında bir hidrojel oluşturmak üzere, 96 oyuklu bir plakanın her bir oyuğuna PREGEL hücre süspansiyonu 16 ul dağıtmak. </li><li> 30 dakika süre ile 37 ° C&#39;de inkübe edinjel dakika oluşturulması için. </li><li> oluşturulan hidroj en medya 100 ul ilave edin ve 37 ° C&#39;de inkübe edin. </li></ol></li><li> Değişken sertlik jeller Üç boyutlu hücre kültürü <ol><li> 96 oyuklu bir plakanın her bir oyuğuna pipetle, 1.3.3 gelen PREGEL çözeltisi 100 ul çözelti kullanılması. </li><li> Jel oluşturmak için 30 dakika boyunca 37 ° C&#39;de inkübe edin. Not: Burada kullanılan kısmı 2 adım Çapraz bağlama. </li><li> Geçiş hücre, 9. </li><li> Kuyulara 10.000 hücre / cm 2 ekleyin. </li><li> oyuk başına 100 ul ortam artırmak ve 37 ° C&#39;de inkübe edin. </li></ol></li></ol>

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R., Panoskaltsis-Mortari, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+decellularized+lung+bioreactor+system+for+bioengineering+the+lung:+the+matrix+reloaded.">Development of a decellularized lung bioreactor system for bioengineering the lung: the matrix reloaded.</a> Tissue Eng. Part A. 16 (8), 2581-2591 (2010).</li><li>Freytes, D. O., Martin, J., Velankar, S. S., Lee, A. S., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+rheological+characterization+of+a+gel+form+of+the+porcine+urinary+bladder+matrix.">Preparation and rheological characterization of a gel form of the porcine urinary bladder matrix.</a> Biomaterials. 29 (11), 1630-1637 (2008).</li><li>Tilghman, R. 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Yazarlar ifşa hiçbir şey yok.

biyoloji ayrılmaz yönlerinden biri belirli bir görevi gerçekleştirmek hiyerarşik yapıların içine moleküllerin öz-örgütlenme olduğunu. Laboratuarda 13, kendinden montaj gibi tuz konsantrasyonu, pH ve sindirim süresi gibi pek çok faktöre bağlıdır. Gösterildiği gibi, bir kendi kendini düzenleyen hidrojel çözünür proteinler fizyolojik sıcaklığında tekrar. Oluşan hidrojel in vitro hücresel yapışma ve canlılık geliştirme kapasitesine sahiptir. <p class="jove_conten...

Translating in vitro results to the clinic is one of the most challenging issues facing biomedical researchers. In vitro research on tissue culture plastic is easier, more convenient, and maintains high cell viability.1 This approach is a reasonable starting point, but the results have limited clinical translation. Increasingly, laboratories are incorporating three-dimensional constructs to replace the traditional two-dimensional methods. Reviews are available for many three-dimensional environments, from biological scaffolds to polymeric scaffolds.2,3
Biological frameworks can mimic characteristics of in vivo environments as they contain many of the protein and glycosaminoglycan components of the native matrix and provide familiar binding sites for cells to attach to and recognize. Extracellular matrix (ECM) derived materials have been shown to be capable scaffolds for cell attachment and proliferation.4 One challenge that limits the application of ECM hydrogel platforms stems from their inherently weak mechanical properties following gelation. Native tissue often has mechanical properties that are magnitudes higher than hydrogels. Non-toxic crosslinking agents can increase the mechanical properties of hydrogels to better mimic the native tissue environment. Genipin is a non-toxic, natural crosslinker derived from Gardenia plants with the ability to closely tailor mechanical properties of ECM with changes in genipin concentration5,6. 
Nearly all cells in the body exist in, and organize on, ECM that they either produce or maintain. New focus on the universal importance of ECM in the organization, condition, and function in every organ or system has sparked the production of matrix based platforms for in vitro investigation. Porcine small intestine submucosa is the most extensively studied naturally-derived scaffold, and it has been used to regenerate tendons, ligaments, skeletal muscle4, and even bone7. Matrices from other organs and donor species have also demonstrated good tissue regeneration potential. The use of foreign ECM components causes minimal issues with immunomodulation. After elimination of host cellular matter, the remaining ECM will be similar in amino acid content and organization to all other mammalian species8. There is a growing line of thinking that the best way to examine cell-ECM interactions in vitro is to utilize organ-specific ECM scaffolds. Each organ provides a unique composition of proteins and proteoglycans to create cellular niches. Niches provide structural, functional and even the enzymatic breakdown of the extracellular matrix contributing to biophysical signaling. To attain an in vitro microenvironment most similar to the in vivo microenvironment, use of tissue specific ECM would optimize the cellular niches for research. 
The goal of this protocol is to provide a method for establishing a hydrogel scaffold unique to the lung ECM. This method provides a platform for in vitro research on lung cell-ECM interactions.

<table border="0" cellpadding="0" cellspacing="0" width="841">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100&#160;</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">BP151-100</td>
			<td style="width:371px;">Use in fume hood with eye protection and gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Deoxycholate</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">D6750-100g</td>
			<td>Use with eye protection and gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium Sulfate</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">M7506-500g</td>
			<td>None</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium Chloride</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">C1016-500g</td>
			<td>None</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td>D5025-150KU</td>
			<td>None</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HCl</td>
			<td>Sigma-Aldrich</td>
			<td>258148-500ML</td>
			<td>Use with eye protection and gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pepsin</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td>P6887-5G</td>
			<td>Use in fume hood with eye protection and gloves.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sodium Hydroxide</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">BP359-500</td>
			<td>Use with eye protection and gloves.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Genipin</td>
			<td style="width:135px;">Wako Chemicals</td>
			<td style="width:119px;">078-03021</td>
			<td>Use in fume hood with eye protection and gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS 10x</td>
			<td style="width:135px;">Quality Biological</td>
			<td>119-069-151</td>
			<td>None</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td style="width:135px;">VWR</td>
			<td>45000-448</td>
			<td>None</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Filter Paper</td>
			<td>Whatman</td>
			<td align="right">8519</td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hand pump</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td>10-239-1</td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Graduate Beaker</td>
			<td style="width:135px;">VitLab</td>
			<td align="right" style="width:119px;">445941</td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryomill</td>
			<td>SPEX</td>
			<td>6700</td>
			<td>Use cryogloves and eye protection.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lyophilizer</td>
			<td>FTS FlexiDry</td>
			<td></td>
			<td>Use gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rheometer</td>
			<td>Discovery</td>
			<td>HR-2</td>
			<td>Use gloves and eye protection.</td>
		</tr>
	</tbody>
</table>

tunable hydrogels from pulmonary extracellular matrix for 3d cell culture

Burada, in vitro akciğer hücre kültürü için çok bileşenli hücre kültürü hidrojeller oluşturulması için bir yöntem sunulmaktadır. Domuz, sıçan veya fare bloğun akciğer dokusunda en sağlıklı ile başlayarak, doku perfüze selüler artığın ayrılması için, daha sonraki kimyasal deterjanlar içinde kalmasını sağlar. işlem öncesi ve sonrası dokunun histolojik karşılaştırma çift sarmallı DNA ve alfa galaktosidaz boyama% 95&#39;in üzerinde kaldırılmasını teyit hücresel enkaz çoğunluğu kaldırılır göstermektedir. Decellularization sonra, doku liyofılize edildi ve sonra toz haline cryomilled olup. Matris toz, bir asidik pepsin sindirim çözeltisi içinde 48 saat boyunca sindirilir ve daha sonra PREGEL çözelti oluşturmak için nötralize edilir. PREGEL çözeltisinin jelasyon, 37 ° C&#39;de inkübe edilerek uyarılabilir ve hemen ardından nötralizasyon kullanılabilir veya iki haftaya kadar 4 ° C &#39;de muhafaza edilebilir. Kaplama C için muamele edilmemiş plaka PREGEL çözeltisi kullanılarak oluşturulabilirarşın eki. Hücreler, hücreleri iskele boyunca göç veya kaplamalar üzerinde kaplama sağlayan bir oluşturulmuş jelin yüzeyi üzerine kaplanmış, bir 3D kültür elde etmek için kendiliğinden montaj öncesinde PREGEL, içinde süspanse edilebilir. stratejiye değişiklikler jelasyon sıcaklığının, kuvvet ya da protein parçası boyutları etkileyebilir sunulmaktadır. hidrojel oluşumu ötesinde, hidrojel sertlik genipin kullanılarak arttırılabilir.

Bu yöntem kullanılarak, normal domuz, sıçan ve fare akciğer (Şekil 1) gelen hidrojeller üretmişlerdir. İşlenmiş akciğerler bir sırası ile, 5 mg, 40 mg ve ECM tozun 10 g tahmin sağlar. Işlemin bir genel görünüşü Şekil 2 &#39;de gösterilmiştir. sürecinde kilit görselleştirme şunlardır: deoksikolat durulama sonra akciğerlerin beyaz bir görünüm; PREGEL oluşumundan sonra, çözelti, opak olmalı ve 4 ° C&#39;de muhafaza edil...

Watch this Scientific Journal Video about Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture at JoVE.com

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Bu akciğer hücre dışı matristen 3 boyutlu hücre kültürü iskele oluşturmak için bir yöntemdir. Bozulmamış akciğer üç boyutlu hücrelerin büyümesini destekleyen hidrojeller olarak işlenir.

3D Hücre Kültürü için Pulmoner Ekstrasellüler Matrix Ayarlanabilir Hidrojellerinin

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

tunable-hydrogels-from-pulmonary-extracellular-matrix-for-3d-cell

Research

JoVE Journal

Bioengineering

11.5K Görüntüleme.  Virginia Commonwealth University.  Bu akciğer hücre dışı matristen 3 boyutlu hücre kültürü iskele oluşturmak için bir yöntemdir. Bozulmamış akciğer üç boyutlu hücrelerin büyümesini destekleyen hidrojeller olarak işlenir. 

Video: Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Hücre Kültürü Uygulamaları için Hidrojeller Işık aracılı Oluşumu ve Desenlendirme

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Biyomühendislik

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

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

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

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

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

Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies of endothelial-epithelial bilayers have traditionally relied on permeable support models that enable bilayer culture, but permeable supports are limited in their ability to replicate the diversity of human basement membranes. In contrast, hydrogel models that require chemical synthesis are highly tunable and allow for modifications of both the material stiffness and the biochemical composition via incorporation of biomimetic peptides or proteins. However, traditional hydrogel models are limited in functionality because they lack pores for cell-cell contacts and functional in vitro migration studies. Additionally, due to the thickness of traditional hydrogels, incorporation of pores that span the entire thickness of hydrogels has been challenging. In the present study, we use poly-(ethylene-glycol) (PEG) hydrogels and a novel zinc oxide templating method to address the previous shortcomings of biomimetic hydrogels. As a result, we present an ultrathin, basement membrane-like hydrogel that permits the culture of confluent cellular bilayers on a customizable scaffold with variable pore architectures, mechanical properties, and biochemical composition.

Current bilayer culture models do not allow for functional in vitro studies that mimic in vivo microenvironments. Using polyethylene glycol and a zinc oxide templating method, this protocol describes the development of an ultrathin biomimetic basement membrane with tunable stiffness, porosity, and biochemical composition that closely mimics in vivo extracellular matrices.

Kültür Ultrathin Porated elastik Hydrogels bir Biomimetic membran için çift olarak hücre

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

Bir böbrek korteks ekstraselüler matrisi elde edilen hidrojel imalatı

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

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 ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. One way to address this challenge is to develop new materials that enable measurement of cell function within the material. Here, a method is presented for measurement of cellular matrix metalloproteinase (MMP) activity in 3D hydrogels in a 96-well format. In this system, a poly(ethylene glycol) (PEG) hydrogel is functionalized with a fluorogenic MMP cleavable sensor. Cellular MMP activity is proportional to fluorescence intensity and can be measured with a standard microplate reader. Miniaturization of this assay to a 96-well format reduced the time required for experimental set up by 50% and reagent usage by 80% per condition as compared to the previous 24-well version of the assay. This assay is also compatible with other measurements of cellular function. For example, a metabolic activity assay is demonstrated here, which can be conducted simultaneously with MMP activity measurements within the same hydrogel. The assay is demonstrated with human melanoma cells encapsulated across a range of cell seeding densities to determine the appropriate encapsulation density for the working range of the assay. After 24 h of cell encapsulation, MMP and metabolic activity readouts were proportional to cell seeding density. While the assay is demonstrated here with one fluorogenic degradable substrate, the assay and methodology could be adapted for a wide variety of hydrogel systems and other fluorescent sensors. Such an assay provides a practical, efficient and easily accessible 3D culturing platform for a wide variety of applications.

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

Küresel hücresel matriks metalloproteinaz ve 3D Hydrogels metabolik etkinliğini ölçme

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

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

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

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

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

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

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

Bir İnsan 3D Ekstrasellüler Matrix-Adiposit Kültür Modeli Matrix-Hücre Metabolik Crosstalk Çalışma için

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

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 ...

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

Canlı Mikroskopi Görüntüleme Altında 3D Hidrojellerin Kontrollü Suşu

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...