Bu çalışma Ulusal Sağlık Enstitüleri ABD Savaş Gazileri İşleri Bakanlığı (Washington DC), ödenek 0.852.048, 0.731.201, 0.854.430 ve Ulusal Bilim Vakfı (Arlington, VA) gelen, ve hibe RO1 DK080897 (Bethesda, MD) tarafından desteklenmiştir . Sayın Khanna Bill &amp; Melinda Gates Vakfı (Seattle, WA) Sayın Edward Ross ve Dr Monica Moya cömert bir bağış destek aldı.

Burada protokol katmanlı aljinat mikro (Şekil 1) oluşturmak için üç aşamalı bir yordam açıklanır. İlk olarak, aljinat, mikro-küreler (Şekil 2A) oluşturulmaktadır. Bu prosedür, aşağıdaki bölümde 1 &#39;de tarif edilmektedir. Hücreleri ya da proteinler bir dağıtım sistemi olarak hareket etmek için, bu aşamada mikroboncukları eklenebilir. Bir sonraki adım mikroboncukları bir permselective tabakasının oluşturulmasını içerir ve 2. bölümünde tarif edilmiştir. Son adım ek bir aljinat tabakasının oluşturulmasını içerir ve bölüm 3&#39;te tarif edilmektedir. Boncuklar yüzeyi (Şekil 2B) dış kısmında, bu tabaka formları sistemi aşağıdaki nakli için hücresel tepkidir yönlendirmek için terapötik molekülleri (Şekil 2C) saklanması ve sağlamak için kullanılabilir. 1. Aljinat microbead Hazırlık <ol><li> 1 mL LVM aljinat 15 gram eritilmesi ile bir% 1.5 (w / v) LVM aljinat çözeltisi hazırlayıniç tabaka aljinat çözeltisi (pH 7.4 &#39;e ayarlanır DI su içinde 25 mM HEPES tampon, 118 mM NaCI, 5.6 mM KCI, 2.5 mM MgCl2,). Aljinat gücü tamamen açık, viskoz bir çözüm oluşturmak için çözünmüş kadar bir Vortexer üzerinde karıştırın. Not: Bu protokol aljinat mikrokapsüller içinde adacık encapsulation için en uygun koşulları açıklar 1 konsantrasyon ve aljinat mikroküreler kompozisyonu diğer uygulamalar (örneğin ilaç dağıtım, doku mühendisliği, vb) Not özelliklerini ayarlamak için değiştirilebilir. Adacık kapsülleme için adacıklar önceden microencapsulator olarak yüklenmesi için bu aşamada aljinat çözeltisine ilave edilebilir. </li><li> 100 mM CaCI2 ve DI su içinde 10 mM HEPES tampon çözücü ve pH 7.4 &#39;e ayarlayarak çapraz bağlayıcı çözelti (DI su içinde 22 mM CaCl2) hazırlayın. Not: Br 2 gibi başka iki değerlikli katyon +, Sr 2 + + gibi, aljinat jelleşme d doğasına bağlı olarak yerine Ca 2 kullanılabilmektediresired. </li><li> Bir şırınga için 25-gauge iğne ekleyerek ve iğne hava yeleği merkezinde olduğundan emin olmak için her iki tarafta vanalar ayarlamak tarafından iki kanallı hava yeleği aljinat microencapsulator ayarlayın. Farklı mastar iğnelerin hedeflenen aljinat mikroboncuk boyutuna bağlı olarak, bu adım için kullanılabilir. Bu aşama, aynı zamanda, bir şırınga ile yapılabilir, bir microencapsulator mevcut olmamalıdır. Şırıngaya aljinat çözeltisi ekleyin ve istenilen mikroboncuk boyutuna göre bir iğne gösterge seçin. </li><li> Doğrudan iğne altında çapraz bağlayıcı çözelti 10 mL içeren bir şişeye yerleştirin. Çözelti içinde bir karıştırma çubuğu yerleştirin. </li><li> Mikro-küreler oluşturmak üzere aljinat çapraz bağ amacıyla CaCI2 çözeltisi içine doğrudan damlacıklar enjekte edilir. Sürekli karıştırılarak, en az beş dakika boyunca tedavi etmek çapraz bağlayıcı çözelti içinde boncuklar inkübe. </li><li> Bir 15 ml&#39;lik bir santrifüj tüpüne boncuklar transfer edin. , Artık çözelti çıkarınve iki dakika her biri için normal tuzlu su çözeltisi içinde% 0.2 CaCI2 ile üç yıkama gerçekleştirmek. </li></ol> 2. Poli-L-ornitin ile kaplama mikroboncukları <ol><li> Normal serum fizyolojik içinde poli-L-ornitin (FKÖ) bir% 0.1 (w / v) çözeltisi 3 mL hazırlayın. Tamamen açık bir çözeltisinin oluşturulması, eriyene kadar bir girdap üzerine çözeltisi yerleştirin. Poli-L-lisin (PLL) seçici geçirgenliğe benzer seviyeleri ile sonuçlanır Bu adımda, bunun yerine FKÖ&#39;nün kullanılabilir. </li><li> FKÖ çözüm içine aljinat mikroküreler aktarın. FKÖ yeterli zaman polikatyon-polianyon kompleks oluşturarak, aljinat ile etkileşim sağlamak için 30 dakika boyunca bir vorteks koyun. 30 dakika sonunda, aljinat, mikro-küreler etrafında belirgin bir görünür beyaz kaplama olması gerekir. </li><li> FKÖ çözüm çıkarın ve iki dakika her biri için normal salin% 0.2 CaCl2 ile üç yıkar gerçekleştirin. </li></ol> 3. Dış Aljinatta oluşturma Layer <ol><li> Dış katman oluşturmak için kullanılacak olan istenilen konsantrasyonu bir aljinat çözeltisi hazırlayın. (1,1) &#39;da anlatıldığı gibi solüsyonu hazırlanır. Dış tabakasının boyutları (Şekil 3) kullanılan aljinat bileşimi ve konsantrasyonu tarafından etkilenir. </li><li> Bir hücre-süzgeç için aljinat mikroküreler aktarın. Mikroküreler mümkün olduğunca kurutmak için fazla çözüm emmek için bir Kimwipe kullanın. </li><li> Bir parafilm yüzeye mikro aktarın. Arzu edilirse, cam ya da plastik Petri tabağı gibi diğer düz yüzeyler, parafilm için ikame edilebilir. </li><li> Aljinat mikroküreler üzerine (3.1) hazırlanan aljinat çözüm aktarın. Aljinat çözeltisi hacmi tamamen aljinat mikroküreler kapsamalıdır edilebilir. Mikro-küreler 45 dakika boyunca aljinat çözelti içinde kalmasını sağlar. </li><li> Mikroküreler bağlı olmayan aşırı aljinat çözüm çıkarmak için bir pipet kullanın. </li><li> Içinde mikro-küreler aktarmak22mm CaCI2 içindeki bir çözeltisine. Bu, farklı dış aljinat tabakasının oluşumu ile sonuçlanarak, mikro-küreler etrafında aljinat çözeltisi çapraz bağlar. </li><li> Iki dakika her biri için serum fizyolojik CaCI2 22 mM üç yıkama yapın. Mikroskop altında mikro görüntüleniyor çekirdek microbead etrafında oluşan görülen ayrı bir dış aljinat kat için izin vermelidir. </li></ol> 4. Temsilcisi Sonuçlar <img src="/files/ftp_upload/3388/3388fig1.jpg" alt="Şekil 1" /> Şekil 1. Çok katmanlı aljinat, mikro-küreler oluşturmaya yönelik prosedürün şematik. Khanna ve ark izniyle. J Biomed Mater Res A. Kasım; 95 (2), 632-40 (2010). <img src="/files/ftp_upload/3388/3388fig2.jpg" alt="Şekil 2" /> Şekil 2,. (A) ve (B) aljinat mikroboncuk faz kontrast görüntülerdir. (A) &#39;a microbead gösterirsonra, Sentez, aşama (1,7), (B) adım (3,7) tamamlanmasından sonra, mevcut farklı bir dış tabaka aljinat gösterir iken. (C) dış tabaka içinde kapsüllenmektedir floresan-etiketli BSA bir protein FITC görüntüsüdür. Khanna ve ark izniyle. J Biomed Mater Res A. Kasım; 95 (2), 632-40 (2010). <img src="/files/ftp_upload/3388/3388fig3.jpg" alt="Şekil 3" /> Şekil 3. Dış aljinat tabakasının boyutları kullanılan aljinat bileşimi ve konsantrasyonuna dayalı çeşitlendirilebilir. Bizim sonuçlarımız LVM ve LVG aljinat her ikisi için, artan aljinat konsantrasyonu ile dış tabaka boyutu artar ve bu LVG aljinat eşit konsantrasyonlarda LVM aljinat daha kalın dış katmanları verir, göstermektedir. Khanna ve ark izniyle. J Biomed Mater Res A. Kasım; 95 (2), 632-40 (2010). <img src="/files/ftp_upload/3388/3388fig4.jpg" alt="Şekil 4"/&gt; Şekil 4. FGF-1 salınması, LVM ve LVG aljinat kullanılan bir konsantrasyon göre değiştiği dış tabakadan aljinat, bir büyüme faktörü anjiyojenik proteini. (A) ve (B) göstermektedir oranında salınma ve (C) ve (D) farklı bir dış tabaka formülasyonlar için zaman karşısında FGF-1, karşılık gelen yığın yayın temsil eder. Bir patlama ilk 5 h (A ve C) içindeki tüm koşulları için sergilenen bırakma ve 30 güne kadar düşük doz sürekli sürüm (B ve D) vardır. Khanna ve ark izniyle. J Biomed Mater Res A. Kasım;. 95 (2), 632-40 (2010) <a href="http://www.jove.com/files/ftp_upload/3388/3388fig4large.jpg" target="_blank">büyük rakamı görmek için buraya tıklayın</a> .

<ol>
	<li>Darrabie, M. D., Kendall, W. F., Opara, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+Poly-L-+Ornithine-coated+alginate+microcapsules.">Characteristics of Poly-L- Ornithine-coated alginate microcapsules.</a> Biomaterials. 26, 6846-6852 (2005).</li><li>Yamagiwa, K., Kozawa, T., Ohkawa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+alginate+composition+and+gelling+conditions+on+diffusional+and+mechanical+properties+of+calcium-alginate+gel+beads.">Effects of alginate composition and gelling conditions on diffusional and mechanical properties of calcium-alginate gel beads.</a> J. Chem. Eng. Jpn. 28, 462-467 (1995).</li><li>Amsden, B., Turner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+characteristics+of+calcium+alginate+gels.">Diffusion characteristics of calcium alginate gels.</a> Biotechnol. Bioeng. 65, 605-610 (1999).</li><li>Uriel, S., Brey, E. M., Greisler, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+low+levels+of+fibroblast+growth+factor-1+promote+persistent+microvascular+network+formation.">Sustained low levels of fibroblast growth factor-1 promote persistent microvascular network formation.</a> Am. J. Surg. 192, 604-609 (2006).</li><li>Moya, M. L., Lucas, S., Francis-Sedlak, M., Liu, X., Garfinkel, M. R., Huang, J. J., Cheng, M. H., Opara, E. C., Brey, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+delivery+of+FGF-1+increases+vascular+density+in+comparison+to+bolus+administration.">Sustained delivery of FGF-1 increases vascular density in comparison to bolus administration.</a> Microvasc. Res. 78, 142-147 (2009).</li><li>Moya, M. L., Cheng, M. H., Huang, J. J., Francis-Sedlak, M. E., Kao, S. W., Opara, E. C., Brey, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+FGF-1+loaded+alginate+microbeads+on+neovascularization+and+adipogenesis+in+a+vascular+pedicle+model+of+adipose+tissue+engineering.">The effect of FGF-1 loaded alginate microbeads on neovascularization and adipogenesis in a vascular pedicle model of adipose tissue engineering.</a> Biomaterials. 31, 2816-2826 (2010).</li><li>Khanna, O., Moya, M. L., Greisler, H. P., Opara, E. C., Brey, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilayered+Microcapsules+for+the+Sustained-Release+of+Angiogenic+Proteins+From+Encapsulated+Cells.">Multilayered Microcapsules for the Sustained-Release of Angiogenic Proteins From Encapsulated Cells.</a> Am. J. Surg. 200, 655-658 (2010).</li><li>Khanna, O., Moya, M. L., Opara, E. C., Brey, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+multilayered+alginate+microcapsules+for+the+sustained-release+of+fibroblast+growth+factor-1.">Synthesis of multilayered alginate microcapsules for the sustained-release of fibroblast growth factor-1.</a> J. Biomed. Mater. Res. A. 95, 632-640 (2010).</li></ol>

Çıkar çatışması ilan etti.

Aljinat yosunu çıkarılan ve 1,4 &#39;-β-D-mannuronic asit (M) ve α-L-guluronic asit (G) 2,3 ünitelerinden oluşan bir doğal, asidik polisakkarittir. Basit jelleşme oluşur Ca 2 gibi iki değerlikli katyonlar, +, Sr 2 + ve Ba 2 + bitişik aljinat zincirleri arasındaki iyonik köprü oluşturan G-monomerler ile etkileşim. Aljinat mikroküreler kondrosit, hepatosit dahil fibroblast büyüme faktörü-1 (FGF-1), sinir büyüme faktörü, lösemi inhibitör fak...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td> Pronova Ultrapure LVG aljinat </td><td> Nova-Matrix </td><td> 4200006 </td><td> Aljinat formülasyonlar çeşitli mevcuttur. Aljinat tercih büyüklüğü de dahil olmak üzere mikroboncuk uç özellikleri, mekanik özelliklere, ve nakil etkiler. Kullanılan bileşimi verilen bir uygulama için optimize edilmesi gerekmektedir. </td></tr><tr><td> Pronova Ultrapure LVM aljinat </td><td> Nova-Matrix </td><td> 4200206 </td><td> Aljinat formülasyonlar çeşitli mevcuttur. Aljinat tercih büyüklüğü de dahil olmak üzere mikroboncuk uç özellikleri, mekanik özelliklere, ve nakil etkiler. Kullanılan bileşimi verilen bir uygulama için optimize edilmesi gerekmektedir. </td></tr><tr><td> Poli-L-ornitin hidroklorür </td><td> Sigma-Aldrich </td><td> P2533 </td><td></td></tr></tbody></table>

generation of alginate microspheres for biomedical applications

Aljinat esaslı malzemeler nedeniyle hidrofilik doğa, biyouyumluluk ve fiziksel mimari biyomedikal uygulamalar için oldukça dikkat çekmektedir. Uygulamalar hücre kapsülleme, ilaç dağıtım, kök hücre kültürü ve doku mühendisliği iskeleleri bulunmaktadır. Aslında, klinik çalışmalarda şu anda adacık tip I diyabet bir tedavi olarak FKÖ kaplı aljinat mikro kapsüllü hangi yapılmaktadır. Bununla birlikte, adacıklar, çok sayıda sağkalım aşağıdaki transplantasyonundan dolayı etkinliği için gereklidir. Yerel kapsüllü hücrelerinin etrafında mikrovasküler ağ oluşumunu teşvik yeteneği oksijen, glikoz ve diğer hayati besin gelişmiş ulaşım yoluyla canlılığı arttırabilir. Fibroblast büyüme faktörü-1 (FGF-1) kan damarlarının oluşumunu teşvik ve iskemik dokularda oksijen seviyelerini artırmak için mümkün olan bir doğal olarak meydana gelen büyüme faktörüdür. Bir sust olarak temin edildiğinde FGF-1 etkinliği arttırılıryerine tek bir büyük-bolus daha ained moda. Adacık kapsülleme sistemlerinden büyüme faktörlerinin yerel uzun vadeli sürüm potansiyel fonksiyonel greft sonuçlarının iyileştirilmesi, nakledilen hücrelerin doğrudan katkıda kan damarlarının büyümesini teşvik edebilir. Bu yazıda, biyomedikal uygulamalarda kullanım için aljinat mikroküreler hazırlanması için prosedürleri özetlemektedir. Buna ek olarak, biz çok katmanlı aljinat, mikro-küreler üretmek için geliştirilmiş bir yöntem açıklanmaktadır. Hücreler dış tabakadaki aljinat iç çekirdek aljinat, ve anjiyojenik proteinleri içinde kapsüllenmektedir edilebilir. Bu dış katmandaki proteinlerin serbest nakledilen adacıklar yönelik doğrudan yerel mikrovasküler ağların oluşumunu teşvik etmektedir.

Watch this Scientific Journal Video about Generation of Alginate Microspheres for Biomedical Applications at JoVE.com

Generation of Alginate Microspheres for Biomedical Applications

Aşağıdaki bölümlerde, biyomedikal uygulamalarda kullanım için aljinat mikroküreler hazırlanması için prosedürleri özetlemektedir. Biz özellikle tip 1 diyabet için potansiyel bir tedavi olarak hücre ve protein encapsulation çift amaçlı çok katmanlı aljinat mikroküreler oluşturmak için bir teknik göstermektedir.

Biyomedikal Uygulamalar için Aljinatta Mikrokürelerin Üretimi

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and ...

generation-of-alginate-microspheres-for-biomedical-applications

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Medicine

20.6K Görüntüleme.  Illinois Institute of Technology. Aşağıdaki bölümlerde, biyomedikal uygulamalarda kullanım için aljinat mikroküreler hazırlanması için prosedürleri özetlemektedir. Biz özellikle tip 1 diyabet için potansiyel bir tedavi olarak hücre ve protein encapsulation çift amaçlı çok katmanlı aljinat mikroküreler oluşturmak için bir teknik göstermektedir.

Video: Generation of Alginate Microspheres for Biomedical Applications

Generation of Comprehensive Thoracic Oncology Database - Tool for Translational Research

The Thoracic Oncology Program Database Project was created to serve as a comprehensive, verified, and accessible repository for well-annotated cancer specimens and clinical data to be available to researchers within the Thoracic Oncology Research Program. This database also captures a large volume of genomic and proteomic data obtained from various tumor tissue studies. A team of clinical and basic science researchers, a biostatistician, and a bioinformatics expert was convened to design the database. Variables of interest were clearly defined and their descriptions were written within a standard operating manual to ensure consistency of data annotation. Using a protocol for prospective tissue banking and another protocol for retrospective banking, tumor and normal tissue samples from patients consented to these protocols were collected. Clinical information such as demographics, cancer characterization, and treatment plans for these patients were abstracted and entered into an Access database. Proteomic and genomic data have been included in the database and have been linked to clinical information for patients described within the database. The data from each table were linked using the relationships function in Microsoft Access to allow the database manager to connect clinical and laboratory information during a query. The queried data can then be exported for statistical analysis and hypothesis generation.

A thoracic oncology database was developed to serve as a comprehensive repository for clinical and laboratory data for the purposes of translational research. The database will serve translational cancer researchers within the Thoracic Oncology Research Program. This database is adaptable to other cancer models, as well as other human diseases.

Kapsamlı Göğüs Onkoloji Veritabanı Üretimi - Translasyonel Araştırma Aracı

The Thoracic Oncology Program Database Project was created to serve as a comprehensive, verified, and accessible repository for well-annotated cancer ...

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Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

Klinik ve Klinik Öncesi Araştırma Uygulamaları Tümör Hücre (CTC) Tahliller Sirkülasyon semiautomated adaptasyon

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

Yeni Nesil dizinleri için Toplama ve Tükrük DNA çıkarımı

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

İzolasyon, Kriyoprezervasyon ve Kültür Klinik Uygulamaları İnsan Amniyon Epitel Hücreleri

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Experimental Glaucoma Induced by Ocular Injection of Magnetic Microspheres

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present here a protocol for elevating intraocular pressure (IOP) in the rat, by injecting magnetic microspheres into the anterior chamber of the eye. The use of magnetic particles allows the user to manipulate the beads into the iridocorneal angle, thus providing a very effective blockade of fluid outflow from the trabecular meshwork. This leads to long-lasting IOP rises, and eventually neuronal death in the ganglion cell layer (GCL) as well as optic nerve pathology, as seen in patients with the disease. This method is simple to perform, as it does not require machinery, specialist surgical skills, or many hours of practice to perfect. Furthermore, the pressure elevations are very robust, and reinjection of the magnetic microspheres is not usually required unlike in some other models using plastic beads. Additionally, we believe this method is suitable for adaptation for the mouse eye.

We present a method for inducing elevated intraocular pressure (IOP), by injecting magnetic microspheres into the rat eye, to model glaucoma. This leads to strong pressure rises, and extensive neuronal death. This protocol is easy to perform, does not require repeat injections, and produces stable long-lasting IOP rises.

Deneysel Glokom Manyetik Mikrokürelerin Göz Enjeksiyon Bağlı

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

İnsan Hipofiz Adenom Hücreleri üç boyutlu Aljinat-boncuk Kültür

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

Kinomic Profil Yöntemi ve İlaç Tepki Testi için 3D İnsan BIOGEL Kültür Sisteminin Kullanılması Microtumors ve Hasta kaynaklı Glioblastom Hücreleri Üretimi

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Generation of Organ-conditioned Media and Applications for Studying Organ-specific Influences on Breast Cancer Metastatic Behavior

Breast cancer preferentially metastasizes to the lymph node, bone, lung, brain and liver in breast cancer patients. Previous research efforts have focused on identifying factors inherent to breast cancer cells that are responsible for this observed metastatic pattern (termed organ tropism), however much less is known about factors present within specific organs that contribute to this process. This is in part because of a lack of in vitro model systems that accurately recapitulate the organ microenvironment. To address this, an ex vivo model system has been established that allows for the study of soluble factors present within different organ microenvironments. This model consists of generating conditioned media from organs (lymph node, bone, lung, and brain) isolated from normal athymic nude mice. The model system has been validated by demonstrating that different breast cancer cell lines display cell-line specific and organ-specific malignant behavior in response to organ-conditioned media that corresponds to their in vivo metastatic potential. This model system can be used to identify and evaluate specific organ-derived soluble factors that may play a role in the metastatic behavior of breast and other types of cancer cells, including influences on growth, migration, stem-like behavior, and gene expression, as well as the identification of potential new therapeutic targets for cancer. This is the first ex vivo model system that can be used to study organ-specific metastatic behavior in detail and evaluate the role of specific organ-derived soluble factors in driving the process of cancer metastasis.

This manuscript describes an ex vivo model system comprised of organ-conditioned media derived from the lymph node, bone, lung, and brain of mice. This model system can be used to identify and study organ-derived soluble factors and their effects on the organ tropism and metastatic behavior of cancer cells.

Meme Kanseri Metastatik Davran Organ spesifik Etkiler incelenmesi için Organ klimalı Medya ve Uygulamaları Üretimi

Breast cancer preferentially metastasizes to the lymph node, bone, lung, brain and liver in breast cancer patients. Previous research efforts have focused ...

Drug Repurposing Hypothesis Generation Using the "RE:fine Drugs" System

The promise of drug repurposing is that existing drugs may be used for new disease indications in order to curb the high costs and time for approval. The goal of computational methods for drug repurposing is to enable solutions for safer, cheaper and faster drug discovery. Towards this end, we developed a novel method that integrates genetic and clinical phenotype data from large-scale GWAS and PheWAS studies with detailed drug information on the concept of transitive Drug-Gene-Disease triads. We created "RE:fine Drugs," a freely available, interactive dashboard that automates gene, disease and drug-based searches to identify drug repurposing candidates. This web-based tool supports a user-friendly interface that includes an array of advanced search and export options. Results can be prioritized in a variety of ways, including but not limited to, biomedical literature support, strength and statistical significance of GWAS and/or PheWAS associations, disease indications and molecular drug targets. Here we provide a protocol that illustrates the functionalities available in the "RE:fine Drugs" system and explores the different advanced options through a case study.

Drug Repurposing Hypothesis Generation Using the &quot;RE:fine Drugs&quot; System

Here we describe a protocol using the web-based drug repurposing hypothesis generation tool: "RE:fine Drugs." This protocol can be modified to a user's preferences at the level of the query type (gene, drug or disease) and/or the range of available advanced options.

&quot;RE: İnce ilaçlar kullanılması&quot; İlaç repurposing Hipotez Üretim Sistemi

The promise of drug repurposing is that existing drugs may be used for new disease indications in order to curb the high costs and time for approval. The ...

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of genetically modified pig models, and the often-limited number of available animals of these models, establishment of (biobank) collections of adequately processed tissue samples suited for a broad spectrum of subsequent analyses methods, including analyses not specified at the time point of sampling, represent meaningful approaches to take full advantage of the translational value of the model. With respect to the peculiarities of porcine anatomy, comprehensive guidelines have recently been established for standardized generation of representative, high-quality samples from different porcine organs and tissues. These guidelines are essential prerequisites for the reproducibility of results and their comparability between different studies and investigators. The recording of basic data, such as organ weights and volumes, the determination of the sampling locations and of the numbers of tissue samples to be generated, as well as their orientation, size, processing and trimming directions, are relevant factors determining the generalizability and usability of the specimen for molecular, qualitative, and quantitative morphological analyses. Here, an illustrative, practical, step-by-step demonstration of the most important techniques for generation of representative, multi-purpose biobank specimen from porcine tissues is presented. The methods described here include determination of organ/tissue volumes and densities, the application of a volume-weighted systematic random sampling procedure for parenchymal organs by point-counting, determination of the extent of tissue shrinkage related to histological embedding of samples, and generation of randomly oriented samples for quantitative stereological analyses, such as isotropic uniform random (IUR) sections generated by the &#34;Orientator&#34; and &#34;Isector&#34; methods, and vertical uniform random (VUR) sections.

The practical application and performance of methods for generation of representative tissue samples of porcine animal models for a broad spectrum of downstream analyses in biobank projects are demonstrated, including volumetry, systematic random sampling, and differential processing of tissue samples for qualitative and quantitative morphologic and molecular analyses types.

Örnekleme stratejileri ve Biobank doku örnekleri domuz Biyomedikal modellerinden işlenmesi

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of ...