Bu çalışma Yumurtalık Kanseri Araştırma tarafından desteklenen Fonu [hibe LT/UIC/01.2011], Klinik ve Translasyonel Bilim, UIC Kanser Merkezi ve Sağlık hibe C06RR15482 National Institutes of UIC Merkezi. UIC Hayvan Bakımı ve Kullanımı Komitesi tarafından onaylanmış hayvan bakım protokolleri altında AAALAC tarafından belirlenen kurallara ve yönetmeliklere uygun olarak hayvanlar üzerinde deneyler yapıldı. Bu proje için Animals of Illinois at Chicago Üniversitesi Biyolojik Kaynaklar Laboratuvarı (BRL) bariyer odalar yerleştirilmiştir. BRL, günde en az iki kez hayvanlar izler ve hayvan bakımı konusunda danışmanlık sağlayan bir tam veteriner bir kadroya sahiptir.

1. Yumurtalık diseksiyon ve doku yalıtımı <ol><li> % 0.5 &#39;lik (w / v) sodyum aljinat çözeltisi hazırlayın, protokol değiştirilmiş bir versiyonu hazırlamak steril PBS ve 37 ısı ° C kullanarak, daha önce 5 açıklanan Inversiyon veya sallanan karıştırın, ancak girdap yok. 25 G iğne ile 1 ml şırınganın içine aljinat çizin ve 37 ° korumak ° C </li><li> Crosslinking aljinat doku kapsülleme üzerine ve 37 sıcak ° C 10mM steril CaCl 2 çözüm hazırlayın </li><li> 1 ml kültür vasatı (αMEM + 5 adet penisilin ve 5 mg / ml streptomisin) başına her bir deney için 24 plaka hazırlayın. Kültür ortamında çeşitli ilaçlar, peptidler, ya da virüsler ekleyin. Organı kapsülleme önce transfeksiyon için, 37 ° C&#39;de dört saat için Lipofectamine 2000 ve plazmid DNA doku inkübe </li><li> Hayvan organları teşrih, herhangi bir yağ ya da kalıntı doku kaldırmak, yumurtalık çevresindeki bursa membran yavaşça çekin ve organ bisecting dört adet yumurtalık veya yumurtalık kesmek. Hayvanlar servikal dislokasyon CO 2 boğulma tarafından ötenazi edildi. Boruların için, yavaşça bağlantı membranlar ayrı çekme ve zıt yönlerde kesmeden önce forseps kullanarak tüp biter uzanan doku çözmek. </li><li> Steril bir örgü lif üzerine tek bir aljinat damlacık (~ 1-3 mcL) yerleştirin. </li><li> Steril forseps kullanarak, organ aljinat damlacık ve merkezi forseps veya steril bir iğne kullanarak damlacık içine bir parça taşımak. </li><li> Steril forseps ile örgü lif açgözlü ve yerçekimi asılı damla aljinat zorlamasına izin ters çevirerek organ parçası (organoid) içeren damlacık haricindekileri. </li><li> Forseps 10cm petri açılan çözüm denk gelecek şekilde 10mM CaCl 2 çözüm içeren bir kenarına dokunun. </li><li> 2 dakika boyunca inkübe, kültür ortamı içine ekstra 10mM CaCl 2, ve transfer serbest bırakmak için gözenekleri içeren steril kaşıkla çıkartın. 24 plaka kültür ortamı içeren tek bir kuyunun içine dört aljinat jeller transfer edilebilir. Kültür belirli bir süre için. </li></ol> 2. Aljinat ve toplama hücrelerin bozunması <ol><li> Aljinat jel çözünürleştirme için kültürler, 37 ° C&#39;de 30 dakika kadar veya jel tamamen eriyene Leibovitz L-15 medya çözünmüş aljinat liaz (3.4 mg / ml) kuluçkaya yatmaktadır. </li><li> Aljinat liaz doku çıkarın. </li><li> OSE toplanması için, 500 adet 1 saat 8 Leibovitz L-15 medya kollajenaz organoid kuluçkaya yatmaktadır. Vortex, 1 dakika için 1 saniye bakliyat (1 saniye vorteks ve 1 saniye geri kalanı) takip 1 dakika orta güç 2 saniye bakliyat (2 saniye girdap ve 2 saniye dinlenin),. Organı adet taze, steril mikrosantrifüj tüpe mikrosantrifüj tüp ve pipet süpernatant altına düşebilir. Pelet over yüzey epitel 3000 rpm&#39;de 3 dakika santrifüjleyin. </li><li> Tubal epitel toplanması için, 5 adet penisilin ve 5 mg / ml streptomisin ve% 4 (v / v) FBS DMEM doku kültürü plastik uzunlamasına tüp kesme ve yer. 3-5 gün içinde, hücreleri tubal stroma &quot;tarama&quot; ve doku kültürü plastic9 üzerinde büyümek. </li></ol> 3. Kesitler için aljinat kapsüllü organ kültürleri fiksasyonu <ol><li> Seçilen bir zaman noktasında kültür ortamı boncuk kapsüllü aljinat toplayın. </li><li> Iki dakika süreyle steril 10mM CaCl 2 çözüm kültür bırakarak jel pekiştirmek için aljinat Recrosslink. </li><li> Jel sağlam kalır ve sabitleme sırasında tamamen doku kapsüller sağlamak için% 2 paraformaldehid 50 mM sukroz, 10 mM CaCl 2 ve 50 mM sodyum cacodylate içeren aljinat kapsüllü organı sabitleyin. </li><li> Takiben, doku işleme, parafin ve 5 mikron bölümleri oluşturmak için bir mikrotom bölüm içine tüm aljinat-kapsüllü organ kültürü parçası gömün. </li><li> Aljinat çözünmüş ve ısı tabanlı antijen alımı nedeniyle immünohistokimya performans halinde slayt kaldırılır olacaktır. Aljinat hematoksilen ve eozin boyama ayakta altında kalır ve eozin leke. </li></ol> 4. Temsilcisi Sonuçlar: Yumurtalık ve yumurtalık üç boyutlu organ kültürünün bir örneği Şekil 1&#39;de gösterilmiştir. Yumurtalıklar farklı boyutta parçalar halinde kesilir ve OSE çoğalırlar ve bistüri ile kesi yaralandı yüzey tamir göç. Bu süreçte, temel stroma göre OSE doğru yönde yumurtalık encapsulating OSE ile korunur. Şekil 2&#39;de görüldüğü gibi, çeyrek veya sekizde oyulmuş yumurtalıklara karşılaştırıldığında yarıya yumurtalıkların OSE daha yumurtalık tamamen kapsülleyen. Over yara onarımıyüzey tüm yumurtalık bağlamında incelenebilir ve yumurtlama ve yüzey tamir over yüzey dönüşüm indükler nasıl ışık tutabilir. Deneyin son nokta, gerekli bilgileri bağlıdır. Mimari dokusu, hücresel morfoloji ve spesifik hücresel büyüme oranları kesitler kullanılarak kolayca takip edilmektedir. Örneğin, 7 gün boyunca 5 mg / ml insülin Ayrıca over yüzey epiteli ve çok katmanlı oluşumu hiperproliferasyonu indükler, tek bir over yüzey epitel tabakası 7 gün (Şekil 3A), serum serbest αMEM kültür sonra görülmektedir hücreleri (Şekil 3B). Fiksasyonu için 10 mcM 24 saat önce son bir konsantrasyonda kültürlere bromodeoxyuridine ekleyerek, hücre çoğalması (inset 3A ve 3B) ölçülebilir. Bakımı, serum serbest αMEM kültürler için, granüloza hücrelerinin daha büyük bir oranda çoğalırlar ise OSE yaklaşık% 3-5, 24 saat etiketleme dönemi sırasında (İçerlek 3A) yayılması tabi. Kültür ortamı insülin eklenmesi, toplam OSE yaklaşık% 30 (İçerlek 3B) OSE prolifere yüzdesi artar. Yeşil floresan proteininin (GFP) immünofloresan cDNA transfeksiyon (Şekil 3C) standart mikroskobu ile aljinat jel ile görüntülendi. Aljinat organ kültürü over yüzey epitel ve birincil foliküller büyüme ve analiz için sağlarken, olgun, ikincil foliküller bu kültürün sistemi (Şekil 3B) hayatta kalmak için başarısız olduğunu belirtmek gerekir. Başka bir yerde 10 bir aljinat hidrojel matriks içinde vade bireysel köklerinin Kültür tarif edilmiştir. Protein ya da RNA değişiklikler tüm organ veya özel hücre tiplerinden enzimatik bozulması (Şekil 4) kullanılarak analiz edilebilir. Bazı stromal hücreler izole olmasına rağmen (Şekil 4D) kollajenaz ile kültüre over organoids tedavisi, altta yatan stroma pelet tedavi sonrasında elde edilen hücre OSE zenginleşmesi için izin verirken (Şekil 4C) sağlam bırakır. <img alt="Şekil 1" src="/files/ftp_upload/2804/2804fig1.jpg" /> Şekil 1 yaralama sonra OSE yaymak için kullanılan Fare yumurtalık üç boyutlu kültür sistemi. A) Mikroskop altında, Bursa ve çevresindeki yağ yastığı çıkarıldı. B) Bozulmamış oviductal tüp yumurtalık (O) (T) ve yağ yastığı (F). C) Bursa ve yağ yastığıyla birlikte Yumurtalık kaldırıldı (solda) ve uncoiled yumurtalık (sağda). D) Yumurtalık veya oviductal organoids aljinat bir damlacık içine yerleştirildi. E) Aljinatta kaplı organoids CaCl 2 çözüm crosslinking bir jel formu için aljinat içine düştü. F) Aljinatta over organoid kapsüllü. G) Aljinatta oviductal organoid kapsüllü. H) Aljinatta kapsüllü organoids kültür ortamında inkübe edildi. <img alt="Şekil 2" src="/files/ftp_upload/2804/2804fig2.jpg" /> Şekil 2. Yüzey alanı sitokeratin 8 ile ölçülen over yüzey rejenere. Sekizde, çeyrek, yarım, BSA orta kültür ve sitokeratin 8 (CK8) antikoru ile immünohistokimyasal analizi ile ölçülen oyulmuş sonra OSE over organoids Encapsulation. <img alt="Şekil 3" src="/files/ftp_upload/2804/2804fig3.jpg" /> Şekil 3 Aljinatta kültür sistemi, geçiş ve büyüme faktörlerinin OSE içine cDNA transfeksiyon sağlar, ancak hızlı bir büyüme ve olgunlaşma köklerinin desteklemiyor . A) Yumurtalık organoid OSE işaretleri CK8 ifade analiz bazal ortamda 7 gün boyunca kültür. Ankastre, seri bölümü BrdU eklenmesi için boyandı. B) kültür ortamı insülin eklenmesi OSE hiperplazi neden olur. Ankastre, seri bölümü BrdU eklenmesi için boyandı. C) Yumurtalık organoid GFP ifade cDNA ile transfekte. D) Aljinatta kültür sistemi primordial foliküllerin (kırmızı ok ucu) büyümesini destekler, ama olgun değil ikincil foliküller (siyah ok). <img alt="Şekil 4" src="/files/ftp_upload/2804/2804fig4.jpg" /> Şekil 4 OSE analizi aşağıdaki kültürü için izole edilebilir. A) 2 haftaya kadar orta ve kültüre organoid. B) Encapsulated organoid organoid sağlam çıkmak için aljinat liaz ile tedavi edilir. Yumurtalık işareti OSE CK8 ile boyandı. C) organoid yatan stroma ayrı OSE kollajenaz ile tedavi edilir. Kalan doku parçası over yüzey kaldırma vurgulamak için CK8 ile boyandı. D) CK8 (kırmızı) boyanmış olarak tedavi sonrası Toplanan hücrelerin OSE zenginleştirilmiştir. Hücreler, çekirdekleri işaretlemek için DAPI ile zıt.

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Ifşa etmek için hiçbir şey yoktur.

Aljinat hidrojeller kullanan üç boyutlu organ kültürü sisteminin gelişimi organizmaların geniş bir yelpazede birçok farklı doku tipleri analiz etmek için çok yönlü bir yöntem temsil eder. 11 hücreleri büyümek için bir iskele sağlar 3D kültürlerin kullanımı, doku mühendisliği ve rejeneratif tıp alanlarında uzatılabilir . Şu anda, bizim laboratuar insan yumurtalık ve fallop tüpü doku kullanarak ilk çalışmalar geçiyor, ancak, insan ve insan dışı primat folikülleri k?...

<table border="1"><tbody><tr><th> Reaktifin Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar (isteğe bağlı) </th></tr><tr><td> Leibovitz L-15 orta </td><td> Gibco </td><td> 11415 </td><td></td></tr><tr><td> αMEM orta </td><td> Gibco </td><td> 12000-022 </td><td></td></tr><tr><td> Sodyum aljinat </td><td> Novamatrix </td><td></td><td></td></tr><tr><td> Sodyum cacodylate </td><td> Sigma Aldrich </td><td> C0250 </td><td></td></tr><tr><td> Polipropilen örgü lif </td><td> McMaster Carr </td><td></td><td> 45 &quot;geniş rulo; 0,0041&quot; açılış </td></tr><tr><td> Lipofectamine 2000 </td><td> Invitrogen </td><td> 11668 </td><td></td></tr><tr><td> Penisilin / streptomisin </td><td> Gibco </td><td> 15070-063 </td><td></td></tr><tr><td> Aljinat liyaz </td><td> Sigma Aldrich </td><td> A1603 </td><td></td></tr><tr><td> A1603 </td><td> Sigma Aldrich </td><td> C9697 </td><td> Clostridium histolyticum itibaren </td></tr><tr><td> Sığır serum albumini </td><td> MP Biyomedikal </td><td> 103700 </td><td></td></tr><tr><td> Fetal sığır serumu </td><td> Gibco </td><td> Gibco </td><td></td></tr><tr><td> Çözüm </td><td> Roche </td><td> 11074547001 </td><td> İnsülin / transferrin / sodyum selenit </td></tr><tr><td> Sitokeratin 8 antikor </td><td> Gelişim Çalışmaları Hibridoma Bankası </td><td> TROMA-1 </td><td> 1:200 az kullanın </td></tr><tr><td> DAPI </td><td> Vektör Labs </td><td> H1200 </td><td></td></tr><tr><td> Bromodeoxyuridine </td><td> Sigma </td><td> B50002-1G </td><td> Final konsantrasyon 10 mcM </td></tr></tbody></table>

alginate hydrogels for three-dimensional organ culture of ovaries and oviducts

Yumurtalık kanseri, kadınlarda kanser ölümlerinin beşinci önde gelen nedenidir ve Amerika Birleşik Devletleri 1% 63 ölüm oranına sahiptir . Over kanseri için menşe hücre tipi, söz konusu hala ya da over yüzey epitel (OSE) veya fallop tüpü fimbria 2,3 distal epitel olabilir. In vitro bir temel kültür olarak normal hücreleri Kültürleme, bilim adamları, böylece kesin olarak belirlenmesi, farklı epitel kökenli hücre tipi yumurtalık kanserine yol açabilir belirli değişiklikler modeline sağlayacaktır. Bu, bu hastalığın hedefleyen daha doğru biyobelirteçleri, doku spesifik gen değişiklikleri ile hayvan modellerinde ve daha iyi önleme stratejileri gelişimini sağlayacaktır. Aljinat hidrojeller normal hücreleri korumak, kendi üç boyutlu bağlamında hücrelerin in vitro kültürü kısa vadeli teşvik ve plazmid DNA, siRNA ve küçük moleküllerin giriş izin verir. Tek bir hayvan deney sayısını artırarak, tek bir organ oluşturulabilir bir neşter, çeşitli kültürler ile stratejik kesimler türetilmiştir adet kültür organları tarafından. Bu OSE çoğalması, yumurtalık kanseri oluşumu ile ilgili önde gelen yumurtlama modeli yönlerini keser. Hücre tipleri, plastik yüzeylerde iyi büyümek değil OSE olarak bu yöntemi kullanarak kültürlü ve normal hücresel süreçler veya 4 kanser oluşumu ilk olaylar soruşturma kolaylaştırmak olabilir. Aljinatta hidrojeller dokuların 5 birçok çeşit büyümeyi desteklemek için kullanılabilir. Aljinat zarar dokuların 6,7 nazik olmayan bir jelleşme eylem sonucunda, β-D-mannuronic asit ve kalsiyum iyonları ile çapraz bağlı olabilir α-L-guluronic asit birimleri tekrar oluşan lineer bir polisakkarit. Matrigel olarak diğer üç boyutlu hücre kültürü matrisler gibi, aljinat dokular için mekanik destek sağlar ancak, proteinler aljinat matris ile reaktif değildir ve bu nedenle hücre 5 sinyalizasyon başlatmak değildir sentetik ekstraselüler matriks olarak aljinat fonksiyonları. Aljinat hidrojel, standart hücre kültürü ortamında yüzer ve in vitro doku büyümesini mimarisini destekler . Hazırlama, ayırma, yumurtalık ve oviductal organ parçaları, iki hafta kadar kültür muhafaza edilebilir aljinat hidrojeller, içine yerleştirilmesi için bir yöntem sunulmuştur. Organ kültürü protein ve RNA örnekleri analiz için hücrelerin enzimatik sürümü de açıklanmıştır. Son olarak, birincil hücre tipleri büyüme, farelerin genetik ölümsüzleşme olmaksızın mümkün ve nakavt ve transgenik fareler kullanmak araştırmacılar izin verir.

Watch this Scientific Journal Video about Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts at JoVE.com

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Üç boyutlu bağlamında normal hücreleri Kültür, hücresel dönüşüm ve tümörogenez için gerekli erken olayları incelemek için alternatif bir yöntem temsil eder. Bu yöntem, normal yumurtalık ve yumurtalık kanseri oluşumu erken olayları incelemek için oviductal hücreleri büyümek için kullanılır.

Yumurtalıklar ve fallop borularında Üç Boyutlu Organ Kültür Aljinatta Hidrojeller

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

alginate-hydrogels-for-three-dimensional-organ-culture-ovaries

Research

JoVE Journal

Bioengineering

21.9K Görüntüleme.  University of Illinois at Chicago. Üç boyutlu bağlamında normal hücreleri Kültür, hücresel dönüşüm ve tümörogenez için gerekli erken olayları incelemek için alternatif bir yöntem temsil eder. Bu yöntem, normal yumurtalık ve yumurtalık kanseri oluşumu erken olayları incelemek için oviductal hücreleri büyümek için kullanılır.

Video: Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

İletken Mürekkepler düzlemsel ve Üç Boyutlu Baskı

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Biyomühendislik

Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

The intervertebral disc (IVD) is the joint of the spine connecting vertebra to vertebra. It functions to transmit loading of the spine and give flexibility to the spine. It composes of three compartments: the innermost nucleus pulposus (NP) encompassing by the annulus fibrosus (AF), and two cartilaginous endplates connecting the NP and AF to the vertebral body on both sides. Discogenic pain possibly caused by degenerative intervertebral disc disease (DDD) and disc herniations has been identified as a major problem in our modern society. To study possible mechanisms of IVD degeneration, in vitro organ culture systems with live disc cells are highly appealing. The in vitro culture of intact bovine coccygeal IVDs has advanced to a relevant model system, which allows the study of mechano-biological aspects in a well-controlled physiological and mechanical environment. Bovine tail IVDs can be obtained relatively easy in higher numbers and are very similar to the human lumbar IVDs with respect to cell density, cell population and dimensions. However, previous bovine caudal IVD harvesting techniques retaining cartilaginous endplates and bony endplates failed after 1-2 days of culture since the nutrition pathways were obviously blocked by clotted blood. IVDs are the biggest avascular organs, thus, the nutrients to the cells in the NP are solely dependent on diffusion via the capillary buds from the adjacent vertebral body. Presence of bone debris and clotted blood on the endplate surfaces can hinder nutrient diffusion into the center of the disc and compromise cell viability. Our group established a relatively quick protocol to "crack"-out the IVDs from the tail with a low risk for contamination. We are able to permeabilize the freshly-cut bony endplate surfaces by using a surgical jet lavage system, which removes the blood clots and cutting debris and very efficiently reopens the nutrition diffusion pathway to the center of the IVD. The presence of growth plates on both sides of the vertebral bone has to be avoided and to be removed prior to culture. In this video, we outline the crucial steps during preparation and demonstrate the key to a successful organ culture maintaining high cell viability for 14 days under free swelling culture. The culture time could be extended when appropriate mechanical environment can be maintained by using mechanical loading bioreactor. The technique demonstrated here can be extended to other animal species such as porcine, ovine and leporine caudal and lumbar IVD isolation.

This protocol illustrates a harvesting technique for coccygeal bovine intervertebral discs for organ culture for in vitro organ culture.

Organ Kültür bozulmamış Sığır Kuyruğu İntervertebral Disklerin Hazırlanması

The intervertebral disc (IVD) is the joint of the spine connecting  vertebra to vertebra. It functions to transmit loading of the spine and give  ...

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

Tümör Hücre Invasion üzerine İnterstisyel Akışkan Etkilerinin Ölçülmesi için üç boyutlu Hücre Kültürü Modeli

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

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

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

Bir Üç boyutlu Kollajen Matrix içindeki Hücre Göç Analizi

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

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper composition, targeted modulus, and well-defined architectural features. Biomaterials that recapitulate the intrinsic architecture of in vivo tissue are vital for studying diseases as well as to facilitate the regeneration of lost and malformed soft tissue. A novel biofabrication technique was developed which combines state of the art imaging, three-dimensional (3D) printing, and selective enzymatic activity to create a new generation of biomaterials for research and clinical application. The developed material, Bovine Serum Albumin rubber, is reaction injected into a mold that upholds specific geometrical features. This sacrificial material allows the adequate transfer of architectural features to a natural scaffold material. The prototype consists of a 3D collagen scaffold with 4 and 3 mm channels that represent a branched architecture. This paper emphasizes the use of this biofabrication technique for the generation of natural constructs. This protocol utilizes a computer-aided software (CAD) to manufacture a solid mold which will be reaction injected with BSA rubber followed by the enzymatic digestion of the rubber, leaving its architectural features within the scaffold material.

An innovative biofabrication technique was developed to engineer three-dimensional constructs that resemble the architectural features, components, and mechanical properties of in vivo tissue. This technique features a newly developed sacrificial material, BSA rubber, which transfers detailed spatial features, reproducing the in vivo architectures of a wide variety of tissues.

Üç boyutlu Biyomimetik Teknoloji: Roman Biorubber Kollajen Hidrojeller içinde Mikro ve Makro ölçekli Mimarileri Tanımlı oluşturur

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

Ters Kolloidal Kristal Poli (etilen glikol) İskele imalatı: Karaciğer Doku Mühendisliği için Üç boyutlu Hücre Kültürü Platformu

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

Katmanlı Aljinat Yapıları: Heterojen hücre popülasyonlarının Co-kültür için bir platform

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

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 ...

Fragmenting Bulk Hydrogels and Processing into Granular Hydrogels for Biomedical Applications

Granular hydrogels are jammed assemblies of hydrogel microparticles (i.e., &#34;microgels&#34;). In the field of biomaterials, granular hydrogels have many advantageous properties, including injectability, microscale porosity, and tunability by mixing multiple microgel populations. Methods to fabricate microgels often rely on water-in-oil emulsions (e.g., microfluidics, batch emulsions, electrospraying) or photolithography, which may present high demands in terms of resources and costs, and may not be compatible with many hydrogels. This work details simple yet highly effective methods to fabricate microgels using extrusion fragmentation and to process them into granular hydrogels useful for biomedical applications (e.g., 3D printing inks). First, bulk hydrogels (using photocrosslinkable hyaluronic acid (HA) as an example) are extruded through a series of needles with sequentially smaller diameters to form fragmented microgels. This microgel fabrication technique is rapid, low-cost, and highly scalable. Methods to jam microgels into granular hydrogels by centrifugation and vacuum-driven filtration are described, with optional post-crosslinking for hydrogel stabilization. Lastly, granular hydrogels fabricated from fragmented microgels are demonstrated as extrusion printing inks. While the examples described herein use photocrosslinkable HA for 3D printing, the methods are easily adaptable for a wide variety of hydrogel types and biomedical applications.

This work describes straightforward, adaptable, and low-cost methods to fabricate microgels with extrusion fragmentation, process the microgels into injectable granular hydrogels, and apply the granular hydrogels as extrusion printing inks for biomedical applications.

Biyomedikal Uygulamalar için Dökme Hidrojellerin Parçalanması ve Granüler Hidrojellere İşlenmesi

Granular hydrogels are jammed assemblies of hydrogel microparticles (i.e., &#34;microgels&#34;). In the field of biomaterials, granular hydrogels have ...