Yazarlar, &quot;uyarılmış pluripotent kök hücre davranışlarını kontrol için doku mühendisliği iskeleleri&quot; NSERC Discovery Hibe 402.462 tanımak istiyoruz.

Protokolü başlamadan önce Notlar: Fibrinojen bir kan proteini elde edilen ve bu nedenle uygun güvenlik eğitimi dokunmadan önce tamamlanması gerekir. Bu protokol, istenilen kök kültürlerin uygun onlar ekimi için hazır olduğundan emin olmak için zaman çok 2 gün tamamlamak için gerektirir. Ne kadar fibrinojen, tris üç 35 mm petri dışarı ağırlığında hesaplanması bakımından 400 ihtiva eden 1 24 kuyulu plakanın üretmek için yeterli olacak TBS 3 mL içinde çözülmüş fibrinojen 110-130 mg (TBS, pH 7.4) tamponlu salin Her iyi ul fibrin iskeleleri. 1. Gün: fibrinojen çözümleri ve geceleme diyaliz yapma <ol><li> Buzdolabının liyofilize fibrinojen çıkartın ve oda sıcaklığına gelmesini sağlamak için 20 dakika bekletin. </li><li> Tris ile ekle 3 mL her 35 mm petri için tuz (TBS) tamponlu. 12 ila 20 arasında değişen konsantrasyonlarda fibrinojenin fibrine çözeltisi verimleri her plakası 2-3 mLmg / mL ve fibrinojen gerekli toplam miktarının bu yaklaştırmalar göre hesaplanabilir. </li><li> Yaklaşık 100-130 mg fibrinojen (FG) dışarı tartılır ve her yemekte TBS mevcut yüzeyine serpin. Çözüm girmeye başlamak için fibrinojen için 5 dakika bekleyin. Kapağı ile petri örtün. </li><li> 37 inkübe petri ° C&#39;de 2 saat fibrinojen tam TBS çözümü gitmek için izin vermek. </li><li> TBS ile Islak diyaliz tüp (7000 MW kesilmiş). Hortum ve kelepçe sonu Fold Alt uç bir diyaliz kelepçe ile kapattı. </li><li> Diyaliz boru içine yemeklerinden fibrinojen çözümleri Pipeti. Üst ucuna katlayın ve bir diyaliz kelepçe ile kapattı kelepçe. </li><li> Yer diyaliz tüp TBS&#39;nin 4 litrelik kaba fibrinojen solüsyon içeren. Düşük hızda (100 rpm) ayarlanır heyecan plaka kullanarak Mix çözüm. </li><li> En az 12 saat için tabak karıştırın üzerine gecede çözüm Dialyze. TBS Çözüm bu dönem boyunca değiştirilmesine gerek yoktur. </li></ol&gt; 2. 2. Gün: iskelelerinin içine fibrin iskeleleri ve kök hücre ekim Polimerizasyonu Bu iskelesi kök hücre kültürleri ile ekildi edileceği gibi, bu işlem için açıklanan tüm adımları steril bir doku kültürü davlumbaz yapılmalıdır. <ol><li> Uygun büyüklükte konik tüp (15 ml veya 50 ml ya) içine diyaliz boruları ve yerden fibrinojen solüsyonu çıkarın. </li><li> Çözümü büyük kirleri ortadan kaldırmak için bir 5.0 mikron şırınga filtresi Filtre fibrinojen çözüm. Çözeltisinin hacmine bağlı olarak, çoklu filtrelerin kullanımının tüm çözüm işlemek için gerekli olabilir. </li><li> Bir 50mL konik tüp içine filtre 0.22 mikron ile Steril filtre fibrinojen çözüm. Uygun boyutta bir pipet kullanarak, hesaplamalar yaparken daha sonra kullanmak üzere filtre fibrinojen solüsyonun hacmi belirler. Çözeltisinin hacmine bağlı olarak, çoklu filtrelerin kullanımının ent işlemek için gerekli olabilirire çözüm. </li><li> Spektrofotometre ile 280 nm fibrinojen çözüm absorbansı ölçülür. 50 arasında bir seyreltme faktörü 1 uyarınca bir absorbans elde etmek için genellikle gerekir. </li><li> Aşağıdaki eşitliği kullanarak fibrine çözelti içinde mevcut proteini konsantrasyonu hesaplanır: [mg / mL &#39;de fibrinojen] = (A 280 x seyreltme faktörü) 1.55 insan fibrinojen 13 için bir 280 azından bir sönüm katsayısı ÷ 1.55. Sonraki 11.1 mg / başlangıçtaki hacminin dayalı fibrinojen mL çözelti konsantrasyonu sulandırmak için gerekli TBS miktarını hesaplar. Fibrin iskelesi protein son konsantrasyon polimerizasyon sonra 10 mg / ml olacak. </li><li> Steril trombin ve kalsiyum klorür çözümleri edinin. Birincisi, 15 uL trombin solüsyonu (konsantrasyon: 40 U / mL - iskele nihai konsantrasyonu 2 U / ml olacak) 24 sıra plaka ilk satırdaki her iyi sağ tarafına. Daha sonra, 50 mM kalsiyum Chl 15 uL eklemeHer iyi sol tarafına oride çözüm. Son olarak, plaka fibrinojen çözeltinin 270 uL ekleyebilir. Taraftan çözeltilerinin karışmasını sağlamak için ön geri çalkalama takiben yan plakası çalkalanır. </li><li> Karışımları, oda sıcaklığında 5 dakika boyunca polimerize içeren satır edelim. Onlar polimerize olarak iskeleleri daha opak hale gelir. </li><li> Fibrin iskelelerinin istenilen sayıda polimerize edilmiş kadar adımları 2.6 ve 2.7 tekrarlayın. </li><li> Son 5 dakikalık inkübasyondan sonra, iskelelerinin tam polimerizasyonu için izin vermek için bir 37 ° C inkübatör içindeki plakaları yerleştirin. </li><li> Bir saat inkübe tamamlandıktan sonra, bir sonraki adım embriyoid organlarıyla tohumu iskelesi etmektir. Bir 20 uL pipettemen kullanılarak, fibrin iskele merkezinde bir birey embriyoid gövde ve bir yer seçmek. Her iyi tek bir embriyoid vücut içerdiğini mikroskop ile kontrol edin. </li><li> Ve 50 mM kalsiyum klorid ve 5 uL: trombin solüsyonu 5 uL (40 U / ml konsantrasyonda) ekleyinHer bir kuyuya fibrinojen solüsyonu 90 uL ardından her embriyoid vücut üstüne çözüm. Çözüm embriyoid organları özetleyen bir kabarcık oluşturmalıdır. </li><li> Polimerizasyon sağlamak için ek bir saat için geri 37 ° C inkübatöründe yer plaka. </li><li> Inkübasyondan sonra, 37 ° C inkübatör geri her iyi ve yerinde plakasına uygun Hücre kültür ortamına 1 ml. </li></ol> 3. Temsilcisi Sonuçlar Şekil 1 de bir embriyoid vücut. Şekil 2A ile tohumlanan 3D fibrin iskele kültür sistemi içeren bir bireyin yan görünümü şematik bir fare embriyonik fibroblast besleyici katmanlar kültüre olan fare indüklenen pluripotent kök hücrelerin temsilcisi görüntüleri gösterir gösterir. Bu hücreler daha sonra bir 8 gün retinoik asit tedavi protokolü (Şekil 2B) previousl olarak kullanarak embriyoid organları, sinir progenitörlerin içeren hücrelerin agrega oluşturmak için indüklenirŞekil 3, 3B fibrin iskelelerinin içinde kültür 3 gün sonra, bir iPS-türetilmiş embriyoid gövdesinin görünüşünü gösterir iken Y 14 tarif. Benzer sonuçlar fare embriyonik kök hücreleri 7 kullanarak daha önce elde edilmiştir. Ayrıca, bu yöntem, bu kültüre sisteminin yönlülüğü gösteren, retinoik asit ve purmorphamine 3D fibrin iskelelerinin 15 içinde yer aldığı, farklı bir protokol kullanılarak kültürü iPS-türetilmiş embriyoid gövdeleri için üretilen kullanılmıştır. <img alt="Şekil 1.." src="/files/ftp_upload/3641/3641fig1.jpg" /> Şekil 1. Iyi bir embriyoid gövdeli bir 3D fibrin iskele numaralı seribaşı içeren bir bireyin yan görünümü gösteren şematik. <img alt="Şekil 2." src="/files/ftp_upload/3641/3641fig2.jpg" /> Şekil 2. Fare uyarılmış pluripotent kök (iPS) hücre kültürü ve farklılaşma. Neur içine bu hücrelerin ayırt etmek içinal fenotipleri, iPS hücreleri embriyoid organları (EBS) denilen hücrelerin agrega üretmek için süspansiyon kültürlenmiştir. Bu EBS sonra nöral farklılaşma teşvik için retinoik asit ile tedavi edilir ve bu protokol daha önce embriyonik kök hücrelerinin sinir farklılaşma ikna etmek için kullanıldı. A) Farklılaşmamış fare iPS hücre kolonisi bir fare embriyonik fibroblast besleyici tabaka kültüre. B) süspansiyon kültüründe fare iPS hücreleri elde Embriyoid organları nöral farklılaşma teşvik için retinoik asit ile tedavi sonrasında 8. günde alınır. Ölçek çubuğu 100 mikron olduğunu. <img alt="Şekil 3." src="/files/ftp_upload/3641/3641fig3.jpg" /> Bir fare iPS Şekil 3. Örnek sonuçlar 3D fibrin iskele içinde kültür 3 gün sonra vücut embriyoid. Hücreler fibrin iskele iç göç ve ayırt etmeye başlamışlardır. Ölçek bar 500 mikron olduğunu.

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Yazarlar ifşa hiçbir şey yok.

Yukarıda detaylı Bu protokol, fare embriyonik ve uyarılmış pluripotent kök hücreleri için özel olarak, pluripotent kök hücre kültürü için 3D fibrin iskelesi üretmek için bir yöntem sağlar. Bu 3D biyomalzemenin bazlı kültürü sistemi daha doğru bir in vivo olarak bulundu kök hücre hücresi taklit eder ve bunun sonucu olarak, bu kök hücre farklılaşması 6 üzerindeki etkilerinin belirlenmesi için biyolojik kuyrukları taranması için kullanılabilir. Gözlemlerimiz bu isk...

Gerekli ekipman: Analitik terazi pH metre Doku kültürü inkübatöründe (37 ° C&#39;de,% 5 CO2) Plaka Stir Spektrofotometre Steril doku kültürü kaput Tris (artı yeterince fibrinojen çözünme için 4 L Need) (pH 7.4) tamponlu salin 50 mM kalsiyum klorür çözeltisi Steril konik bir boru (15 veya 50 mL) 35 mm Petri Diyaliz boru (7.000 MW kesim noktası) Diyaliz klipler 5.0 mikron şırınga filtreleri Ayrı ayrı sarılmış steril 0.22 mikron şırınga filtreleri Şırınga 24 iyi doku kültürü plakaları <table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> Fibrinojen (insan) </td><td> Calbiockenar </td><td> 341578 </td><td></td></tr><tr><td> Trombin (insan) </td><td> Sigma </td><td> T7009 </td><td></td></tr></tbody></table>

preparation of 3d fibrin scaffolds for stem cell culture applications

Kök hücreler doğal genellikle kök hücre hücresi 1 olarak adlandırılan in vivo, in 3D mikro meydana bulunur. 3D biyomalzeme iskelelerinin içinde Kültürleme kök hücreler polistiren yanı sıra, mühendislik yedek dokular 2 için bir yöntem kullanılarak geleneksel 2B kültür yöntemleri üzerinde bir avantaj sağlayarak, doğru bir şekilde bu mikro taklit etmek için bir yol sağlar. 2B doku kültürü polistren hücre kültür deneylerinde çoğunluğu için kullanılmış olsa da, 3D biyomalzeme iskelelerinin daha yakından ortamında hücre polarite daha doğru kurulmasını sağlayan ve yumuşak doku benzer biyokimyasal ve mekanik özelliklere sahip olan in vivo olarak bulundu mikroçevrelerde çoğaltabilirsiniz. 3 çeşitli doğal kaynaklı ve sentetik biyomalzeme iskelelerde kök hücre büyümesini desteklemek için 3D ortamlar olarak incelenmiştir. Sentetik iskelesi daha büyük bir r sahip sentezlenebilir ikenmekanik ve kimyasal özellikleri ange ve genellikle daha büyük bir tekrarlanabilirlik sahip, doğal biyomalzemeler genellikle extracelluar matris içinde ve hücre yapışması için bağlama bölgeleri içermesi ve kolaylıkla hücre kültürü destekleyen bir sonucu olarak bulundu proteinleri ve polisakkaritleri oluşmaktadır. Plazma elde edilen protein fibrinojen polimerize tarafından üretilen fibrin iskelesi, yaygın olarak in vitro ve in vivo hem de in 4 doku mühendisliği çeşitli uygulamalar için incelenmiştir. Böyle iskeleleri iyileştirici faktörler, 5 sunmak için kontrollü salım sistemlerini birleştirmek için çeşitli yöntemler kullanılarak değiştirilebilir. Önceki İş bu iskeleleri başarıyla kültür embriyonik kök hücreler için kullanılabilir ve bu iskele tabanlı kültür sistemi 6,7 içindeki ekilmiş kök hücrelerin farklılaşması üzerine çeşitli büyüme faktörlerinin etkilerini taramak için kullanılabilir göstermiştir. Bu protokol, ayrıntıları polymerizin işlemig fibrin trombinin enzim etkinliği kullanarak fibrine çözeltilerinden iskeleleri. Polimerizasyon süreci inhibe sitratlar kaldırmak için fibrinojen çözümü için bir gece diyaliz adım da dahil olmak üzere, tamamlamak için 2 gün sürer. Bu detaylı yöntemleri embriyonik ve uyarılmış pluripotent kök hücre kültürleri için optimal olarak belirlenmiştir fibrinojen güveniyor. Diğer gruplar daha fazla hücre tipleri ve geniş bir uygulama yelpazesi için fibrin iskeleleri araştırdık - Bu yaklaşımın 8-12 çok yönlülüğünü gösteren.

Watch this Scientific Journal Video about Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications at JoVE.com

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Bu çalışma detayları 3D fibrin hazırlanması kültür ve plutipotent kök hücrelerin ayrıştırılması için iskeleleri. Bu tür iskelesi sıra ilaç verme sistemleri içeren değiştirilmiş olarak kök hücre davranışı üzerindeki çeşitli biyolojik etkiler bileşiklerin taranması için kullanılabilir.

3D Fibrin hazırlanması Kök Hücre Kültürü Uygulamaları için iskeleleri

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

preparation-of-3d-fibrin-scaffolds-for-stem-cell-culture-applications

Research

JoVE Journal

Bioengineering

25.1K Görüntüleme.  University of Victoria. Bu çalışma detayları 3D fibrin hazırlanması kültür ve plutipotent kök hücrelerin ayrıştırılması için iskeleleri. Bu tür iskelesi sıra ilaç verme sistemleri içeren değiştirilmiş olarak kök hücre davranışı üzerindeki çeşitli biyolojik etkiler bileşiklerin taranması için kullanılabilir.

Video: Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

3-Boyutlu Hücre Kültürü için Self-raporlama iskeleleri

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Biyomühendislik

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

3D Hücre Kültürü Modelleri Kütle Spektrometre Görüntüleme Hazırlık Stratejileri Numune

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

Eklem Kondrosit Kültür ve Kıkırdak Üretimi için 3D hidrojel iskeleleri

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

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 ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

Biyomedikal Uygulamalar için Mekanik ayarlanabilir ve Biyoaktif Metal iskelelerinin Fabrikasyon

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D Etkileşimleri Çalışmaları 3D Kollajen jeller ve Mikrokanallarda Hazırlanması<em&gt; İn Vivo</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

Micropatterning ve 3D mikrodamarlar Meclisi

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

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 ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

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

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

3D Hücre Kültürü için Pulmoner Ekstrasellüler Matrix Ayarlanabilir Hidrojellerinin

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

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

3D Mekansal Hücre Kültürleri için hücre yapı iskeleleri olarak biyo-uyumlu sıvı kristal Elastomer Köpüklerin sentezi

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...