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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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><tbody><tr><td>Triton X-100&amp;nbsp;</td><td>Fisher Scientific</td><td>BP151-100</td><td>Use in fume hood with eye protection and gloves.</td></tr><tr><td>Sodium Deoxycholate</td><td>Sigma-Aldrich</td><td>D6750-100g</td><td>Use in hood with eye protection and gloves.</td></tr><tr><td>Magnesium Sulfate</td><td>Sigma-Aldrich</td><td>M7506-500g</td><td>None</td></tr><tr><td>Calcium Chloride</td><td>Sigma-Aldrich</td><td>C1016-500g</td><td>None</td></tr><tr><td>DNase</td><td>Sigma-Aldrich</td><td>D5025-150KU</td><td>None</td></tr><tr><td>HCl</td><td>Sigma-Aldrich</td><td>258148-500ML</td><td>Use with eye protection and gloves.</td></tr><tr><td>Pepsin</td><td>Sigma-Aldrich</td><td>P6887-5G</td><td>Use in fume hood with eye protection and gloves.</td></tr><tr><td>Sodium Hydroxide</td><td>Fisher Scientific</td><td>BP359-500</td><td>Use with eye protection and gloves.</td></tr><tr><td>Genipin</td><td>Wako Chemicals</td><td>078-03021</td><td>Use in fume hood with eye protection and gloves.</td></tr><tr><td>PBS 10x</td><td>Quality Biological</td><td>119-069-151</td><td>None</td></tr><tr><td>PBS</td><td>VWR</td><td>45000-448</td><td>None</td></tr><tr><td>Filter Paper</td><td>Whatman</td><td>8519</td><td>N/A</td></tr><tr><td>Hand pump</td><td>Fisher Scientific</td><td>10-239-1</td><td>N/A</td></tr><tr><td>Graduate Beaker</td><td>VitLab</td><td>445941</td><td>N/A</td></tr><tr><td>Cryomill</td><td>SPEX</td><td>6700</td><td>Use cryogloves and eye protection.</td></tr><tr><td>Lyophilizer</td><td>FTS FlexiDry</td><td></td><td>Use gloves.</td></tr><tr><td>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

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

11.6K 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

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

Dinamik Maske Projeksiyon Fotolitografi kullanarak Sinir Kültür Sistemleri Micropatterned Hidrojeller Fabrikasyon

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

Biyomühendislik

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

Protein bazlı Hydrogels mimarilerde kolayca idare hücre kültür uygulamaları için

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

Biyokimya

Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue geometry. The resulting 3D hydrogel-based cellular constructs have been introduced as an alternative to animal experiments for advanced biological studies, pharmacological assays and organ transplant applications. Although hydrogel-based particles and fibers can be easily fabricated, it is difficult to manipulate them for tissue reconstruction. In this video, we describe a fabrication method for micropatterned alginate hydrogel sheets, together with their assembly to form a macro-scale 3D cell culture system with a controlled cellular microenvironment. Using a mist form of the calcium gelling agent, thin hydrogel sheets are easily generated with a thickness in the range of 100 - 200 &#181;m, and with precise micropatterns. Cells can then be cultured with the geometric guidance of the hydrogel sheets in freestanding conditions. Furthermore, the hydrogel sheets can be readily manipulated using a micropipette with an end-cut tip, and can be assembled into multi-layered structures by stacking them using a patterned polydimethylsiloxane (PDMS) frame. These modular hydrogel sheets, which can be fabricated using a facile process, have potential applications of in vitro drug assays and biological studies, including functional studies of micro- and macrostructure and tissue reconstruction.

We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment.

Micropatterned Makro ölçekli 3D Hücresel Mimarlık Modüler Hidrojel Levhaların İnşaatı

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue ...

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening.
Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. 
Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types.

We describe a set of protocols that together provide a tissue-mimicking hydrogel bioink with which functional and viable 3-D tissue constructs can be bioprinted for use in in vitro screening applications.

Bioprinting cellularized dokuya özel Hidrojel Bioink kullanılarak dahil edilmesi

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

Enjekte edilebilir rejeneratif terapi ve yüksek-den geçerek uyuşturucu tarama için 3D Microtissues

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

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 ...

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 ...

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 ...

Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes and therapeutic responses. Accounting for matrix effects earlier in the drug development pipeline is expected to increase the likelihood of clinical success of novel therapeutics. Biomaterial-based strategies recapitulating specific tissue microenvironments in 3D cell culture exist but integrating these with the 2D culture methods primarily used for drug screening has been challenging. Thus, the protocol presented here details the development of methods for 3D culture within miniaturized biomaterial matrices in a multi-well plate format to facilitate integration with existing drug screening pipelines and conventional assays for cell viability. Since the matrix features critical for preserving clinically relevant phenotypes in cultured cells are expected to be highly tissue- and disease-specific, combinatorial screening of matrix parameters will be necessary to identify appropriate conditions for specific applications. The methods described here use a miniaturized culture format to assess cancer cell responses to orthogonal variation of matrix mechanics and ligand presentation. Specifically, this study demonstrates the use of this platform to investigate the effects of matrix parameters on the responses of patient-derived glioblastoma (GBM) cells to chemotherapy.

The present protocol describes an experimental platform to assess the effects of mechanical and biochemical cues on chemotherapeutic responses of patient-derived glioblastoma cells in 3D matrix-mimetic cultures using a custom-made UV illumination device facilitating high-throughput photocrosslinking of hydrogels with tunable mechanical features.

Hidrojel dizileri, 3D Tümör Modellerinde Matris Bileşenlerinin ve Terapötiklerin Tarama Etkileri için Artan Verim Sağlar

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes ...

ECM Sertleşmesine Hücresel Tepkileri Araştırmak için Fototize Edilebilir Biyobaskılı Hidrojeller

3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture platforms and dynamically triggering changes, such as increasing microenvironmental stiffness, enables researchers to model changes in the extracellular matrix (ECM) that occur during fibrotic disease progression. Herein, a method is presented for 3D bioprinting a phototunable hydrogel biomaterial capable of two sequential polymerization reactions within a gelatin support bath. The technique of Freeform Reversible Embedding of Suspended Hydrogels (FRESH) bioprinting was adapted by adjusting the pH of the support bath to facilitate a Michael addition reaction. First, the bioink containing poly(ethylene glycol)-alpha methacrylate (PEG&#945;MA) was reacted off-stoichiometry with a cell-degradable crosslinker to form soft hydrogels. These soft hydrogels were later exposed to photoinitator and light to induce the homopolymerization of unreacted groups and stiffen the hydrogel. This protocol covers hydrogel synthesis, 3D bioprinting, photostiffening, and endpoint characterizations to assess fibroblast activation within 3D structures. The method presented here enables researchers to 3D bioprint a variety of materials that undergo pH-catalyzed polymerization reactions and could be implemented to engineer various models of tissue homeostasis, disease, and repair.

Yazar Spotlight: 3D Baskılı Fototable Hidrojeller Kullanarak Kronik Akciğer Hastalıklarını Anlamak 

This article describes how to 3D bioprint phototunable hydrogels to study extracellular matrix stiffening and fibroblast activation.

Fibroblast Aktivasyonunu İncelemek için 3D Biyobaskı Fototable Hidrojeller

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture ...