Bu çalışma R21 AI113000 (ATM) ve R01 AI123392 (YF) Ulusal Sağlık Enstitüleri tarafından kısmen hibe desteklenmiştir.

Deneylerde kullanılan tüm hayvanlar Allegheny Sağlık Ağı / Allegheny Şarkıcı Araştırma Enstitüsü Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından incelenen ve onaylanan protokol çerçevesinde Allegheny-Singer Araştırma Enstitüsü&#39;nde hayvan tesisine yerleştirilmiştir bulundu. 1. kollagenaz Timüsü özümsemek <ol><li> Hasat timüs bezleri. <ol><li> Timus bezleri hasat için 3-5 haftalık C57BL6 / J karbondioksit fareler (CO 2) odasına euthanize. Ayrıntılı olarak, 3-5 dakika boyunca CO2 indüksiyonu altında bölme fareler yerleştirin ve kalp ya da solunum doğrulayarak ölüm teyit etmektedir. </li><li> Sırtında karkas yatırın ve% 70 etanol ile vücut ıslak. deri yoluyla karın üzerinde keskin, düz diseksiyon makas ile küçük bir yatay bir kesi yapmak. onun tersyüz böylece her iki ucunda (baş ve bacaklar) deriyi çekin. </li><li> diseksiyon makas hemen altında yatay bir kesim olundüşük kaburga ve yanlara diyafram kesti. Bir forseps ile kılıç şeklinde sürecini tutun ve her iki tarafta yukarı geçmeden kaburga orta kesti. timus açıkça görülebilecek şekilde kaburga / sternum yukarı çekin. Not: Thymus, doğrudan kalbin üstüne yatıyor 2 lob oluşur ve beyaz saydam renk olduğunu. </li><li> yavaşça kepçe ve bağ dokusu ve yıkama çözeltisi içinde transfer kapalı timus lobları çekin kavisli bir forseps kullanarak (Malzeme listesi bakınız). </li></ol></li><li> 9 ml RPMI-1640 (Roswell Park Memorial Institute orta 1640), 0.025 mg / ml saflaştınlmış kolajenaz, 10 mM HEPES [4- (2-hidroksietil) -1-piperazinetansülfonik asit] ve 0.2 mg / ml karıştırılarak sindirim çözeltisi hazırlayın 50 ml&#39;lik bir santrifüj tüpü içinde DNase I. kullanılana kadar 37 ° C su banyosu içinde sindirim çözeltisi tutun. Not: hazırlanan sindirim solüsyonunun miktarı bir 5 ml polistiren yuvarlık tabanlı birlikte inkübe edilebilir en fazla beş yetişkin fare Thymi için yeterlidiralt tüp. </li><li> 5-6 ml RPMI-1640 ihtiva eden bir 60 mm doku kültürü çanağı timus aktarın. </li><li> Lenfositler dışarı akmasına izin vermek, küçük parçalar halinde 28 G insülin şırınga iğnesi (yaklaşık 1 mm 3 kare) ile timus teşrih. </li><li> cam Pasteur pipeti ile petri olan RPMI-1640 ile timik parçaları durulayın. çanak eğin ve timik fragmanları dibe edelim. Yavaş yavaş aspire ve bir cam pipet kullanarak RPMI-1640 süpernatant atın. Solüsyon daha az opak olana kadar cam pipet ile 4-5 kez 5-6 ml RPMI-1640 ile bu durulama adımı yineleyin. Not: Cam pipetler bu aşamada tavsiye edilir, timik fragmanlar timik dokuların aşırı kaybına neden plastik sopa eğilimindedir beri. Prosedürün geri kalanı için, plastik pipet kullanın. </li><li> Adım 1.5&#39;de tarif edildiği gibi son yıkamadan sonra, süpernatant atılır. 3 mi sindirim çözeltisi ekleyin ve 5 ml&#39;lik yuvarlak dipli bir p haline timus parçaları ve çözeltisiniolystyrene tüp. </li><li> tüp rotator Tüp 12 rpm&#39;lik bir dönme hızında 6 dakika için 37 ° C de inkübe edilir. </li><li> kuluçkadan sonra timik fragmanları yerçekimiyle tüpün dibine yerleşmek sağlar. Pasteur pipeti ile süpernatan toplamak ve yıkama solüsyonu 10 ml bir 50 ml&#39;lik santrifüj tüpüne aktarılır. 5 dakika boyunca 300 xg&#39;de santrifüj, supernatant atmak ve 10 ml yıkama solüsyonu hücreleri tekrar süspansiyon. buz üzerinde tutun. </li><li> Yineleyin yuvarlak alt polistiren tüp 5 ml timik parçaları iki kez 1.6-1.8 adımları. Aynı 50 ml tüp içinde süpernatant havuz. Not: Santrifüj, ikinci ve üçüncü yıkama için gerekli değildir. </li><li> 50 ml santrifüj tüpüne tüp ve transferi herhangi bir geri kalan parçaları parçalamak için son sindirimden sonra, yukarı pipetleyin ve 2 ml plastik pipet serolojik aşağı. </li><li> Santrifüj 5 dakika boyunca 300 x g&#39;de 50 ml tüp, çamaşır Buffe 10 ml süpernatan, tekrar süspansiyon aspire100 mikron hücre süzgecinden r ve filtre. buz üzerinde tutun. </li></ol> Kesintili Yoğunluk Gradyan ile TECS 2. zenginleştirilmesi <ol><li> 2.4 mi yoğunluk gradyan ortamı (su içinde iyodiksanol ağ / hac% 60) ve 9.6 ml yıkama solüsyonu karıştırılarak% 21 yoğunluk gradyan çözeltisi hazırlayın. </li><li> 2.5 ml yıkama tamponunda 5 dakika ve tekrar süspansiyon için 300 xg&#39;de adım 1.11 50 ml toplama tüpü ayrışmış timik hücreleri Santrifüj. </li><li> Hücre süspansiyonu 20 ml RPMI-1640 ortamı ekleyin. </li><li> elektronik bir pipet ile 10 ml&#39;lik bir serolojik pipet% 21 yoğunluk gradyan çözeltisi 12 ml doldurun. 50 ml santrifüj borusu içeren hücrelerin alt serolojik pipet ucu yerleştirin ve yoğunluk gradyan çözeltisi yerçekimi ile tüpün dibine drenaj sağlar. <ol><li> Yavaşça farklı yoğunluktaki iki kat üreten bitirmek için pipetlemeyin yoğunluk gradyan çözüm sınırdışı. </li></ol></li><li> 600 x g&#39;de santrifüj foR bir sallanan kepçeli rotor oda sıcaklığında 20 dakika. yavaş düzeyde yavaşlama hızını ayarlayın. </li><li> Yeni 50 ml tüp üst tabaka ve arayüz aktarın. Not: TECS çoğu arayüzü tutulur. Lenfositler Santrifüj işleminden sonra tüpün dibine çöker. Bu hücreler, başka amaçlar için kullanılmak üzere ise, üç kez 20 ml yıkama solüsyonu ile pelet yıkayın. Yıkama tamponu, 10 ml tekrar süspansiyon hücreleri. </li><li> 4 ° C&#39;de 6 dakika boyunca 400 xg&#39;de adım 2.6 ve santrifüj tüpe 40 ml yıkama solüsyonu ekleyin. Bir pipet ile süpernatant, aspire kaldırmak için. </li><li> Adım 2.7 açıklandığı gibi yıkama ve aspirasyon 2 kez daha tekrarlayın. </li><li> yıkama çözeltisi, 2 ml ve yeniden süspanse hemasitometre ile hücre sayımı. </li></ol> FACS ile 3. Yalıtımlı Tecs <ol><li> Yıkama solüsyonu ve transfer 1x10 6 hücre / 100 ul konsantrasyonunda adım 2.9 hücreleri ayarlayın(Akış sıralayıcı tarafından tavsiye edilen şekilde polipropilen veya polistirenin,) 5 mL&#39;lik yuvarlak dipli bir tüp hücre. </li><li> 1x10 6 hücre başına 2 ul anti-Fc reseptörü IgG ekleyin ve 10 dakika boyunca 4 ° C&#39;de inkübe edin. </li><li> (: 150 seyrelti, örnek başına 10 ul 1) ve anti-EpCAM, anti-CD45 ekleyin (1: 100 seyrelti, örnek başına 10 ul) antikorları ve en fazla 20 dakika süreyle 4 ° C&#39;de inkübe edin. </li><li> 4 ° C&#39;de 6 dakika boyunca 400 x g&#39;de 2 ml yıkama çözeltisi ve santrifüj ile hücreler yıkanır. Bir pipet ile süpernatant aspire. </li><li> 1 mi 1 x fosfat tamponlu tuzlu su (PBS) içinde tekrar süspansiyon hücreleri. </li><li> 100 mikron süzgeç süzülür. </li><li> sıralama enstrüman hücreleri uygulayın. Yan dağınık ışık (SSC) / ileri-dağınık ışık (FSC) paneli (ölü hücreler), 13 sıralamak için CD45- EpCAM + için seçilen nüfus ardından kapı üzerinde küçük hücreler hariç lenfosit ve TEC nüfusu seçin. </li></ol> 4. GTEC / EAK Agregalarının eneration Not: reaktif hazırlama ve laminer kaputun altında karıştırma hücreyi içeren adımları uygulayın. <ol><li> steril bir 1.5 ml mikrosantrifüj tüpü içinde, anti-His-tag IgG 4 ul anti-EpCAM IgG 8 ul ve PA / G 2.6 ul karıştırılarak pA / G Adaptör kompleksleri hazırlamak, ve 5 dakika boyunca oda sıcaklığında inkübe edilir. </li><li> Yıkama çözeltisi ve pipet düşük buharlaşmalı, 96 gözlü yuvarlak tabanlı doku kültürü plakası, bir oyuk 100 ul 5x10 4 hücre / ml aşama 3,7 kriteri Tecs ayarlayın. hücrelere 14.6 ul pA / G Adaptör kompleksleri ekleyin. 4 ° C&#39;de 20 dakika süreyle sallanan bir platform üzerinde plaka koyun. </li><li> 200 ul yıkama çözeltisi ile bir kez yıkanır. 6 dakika boyunca 400 x g&#39;de plaka santrifüj. Bir mikropipet ile süpernatant atın. </li><li> 200 ul% 10 sukroz çözeltisi ile bir kez yıkanır. 6 dakika boyunca 400 x g&#39;de santrifüjleyin. Bir mikropipet ile süpernatant atın. </li><li> 30 ul 10% s yeniden süspanse hücrelerioyuk başına terile sukroz çözeltisi. </li><li> EAKIIH6 10 ul (7.5 mg / ml) ilave edin ve oda sıcaklığında 5 dakika inkübe edilir. </li><li> TEC karışımına EAK16-II &#39;nin 20 ul (10 mg / ml) eklenir. </li><li> jelleşme başlatmak için sisteme tam ortam 100 ul ekle. Oda sıcaklığında 15-20 dakika süre ile bir çalkalama platformunda plaka koyun. </li><li> 37 ° C&#39;de inkübatör içinde plaka koyun ve% 5 CO2. 2-4 saat sonra, tam bir ortamda bir 100 ul ilave edin. </li><li> kullanılana kadar her gün orta değiştirin. <ol><li> Aspire bir mikropipet kullanarak ve yavaşça kuyunun duvarına pipet koyarak önceden ısıtılmış orta 100 ul eklemek jel bozmadan yüzeyden orta 100 ul. </li></ol></li></ol> TEC / EAK Agregalarının 5. Karakterizasyonu <ol><li> In vivo TEC / EAK agrega fonksiyonunu incelemek için, bir atimik çıplak böbrek kapsülü altında O / N kültür ve nakli sonrasında TEC / EAK hidrojel toplamakFare, daha önce prosedür 11,14 tarif kullanarak. </li><li> 4-6 hafta anti-CD4 bir kokteyli ile nakli ve lekeyi yayınlamak çıplak farelerde, -CD8, -CD45 EDTA içeren, kan toplama tüpüne 50-100 ul kan örnekleri hasadı çevre T hücrelerinin gelişimini izlemek ve -CD3 12 antikorları. </li><li> Tek hücre analiz yazılımı 12 kullanılarak akış sitometrisi ile çevresel kan lökositleri (FCM) T-lenfositlerin oranı analiz edin. </li></ol>

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	<li>Anderson, G., Jenkinson, E. J., Moore, N. C., Owen, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MHC+class+II-positive+epithelium+and+mesenchyme+cells+are+both+required+for+T-cell+development+in+the+thymus.">MHC class II-positive epithelium and mesenchyme cells are both required for T-cell development in the thymus.</a> Nature. 362, 70-73 (1993).</li><li>Fan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymus-specific+deletion+of+insulin+induces+autoimmune+diabetes.">Thymus-specific deletion of insulin induces autoimmune diabetes.</a> The EMBO J. 28, 2812-2824 (2009).</li><li>Mohtashami, M., Zuniga-Pflucker, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+architecture+of+the+thymus+is+required+to+maintain+delta-like+expression+necessary+for+inducing+T+cell+development.">Three-dimensional architecture of the thymus is required to maintain delta-like expression necessary for inducing T cell development.</a> J Immunol. 176, 730-734 (2006).</li><li>Pinto, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+organotypic+coculture+model+supporting+proliferation+and+differentiation+of+medullary+thymic+epithelial+cells+and+promiscuous+gene+expression.">An organotypic coculture model supporting proliferation and differentiation of medullary thymic epithelial cells and promiscuous gene expression.</a> J Immunol. 190, 1085-1093 (2013).</li><li>Fung, S. Y., Yang, H., Chen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+colloidal+suspension+of+hydrophobic+compounds+with+an+amphiphilic+self-assembling+peptide.">Formation of colloidal suspension of hydrophobic compounds with an amphiphilic self-assembling peptide.</a> Colloids Surf B Biointerfaces. 55, 200-211 (2007).</li><li>Jun, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembly+of+the+ionic+peptide+EAK16:+the+effect+of+charge+distributions+on+self-assembly.">Self-assembly of the ionic peptide EAK16: the effect of charge distributions on self-assembly.</a> Biophys J. 87, 1249-1259 (2004).</li><li>Saunders, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+fluorogen+activating+proteins+into+self-assembling+materials.">Engineering fluorogen activating proteins into self-assembling materials.</a> Bioconjug Chem. 24, 803-810 (2013).</li><li>Zhang, S., Holmes, T., Lockshin, C., Rich, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+assembly+of+a+self-complementary+oligopeptide+to+form+a+stable+macroscopic+membrane.">Spontaneous assembly of a self-complementary oligopeptide to form a stable macroscopic membrane.</a> Proc of the Natl Acad Sci U S A. 90, 3334-3338 (1993).</li><li>Wen, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coassembly+of+amphiphilic+peptide+EAK16-II+with+histidinylated+analogues+and+implications+for+functionalization+of+beta-sheet+fibrils+in+vivo.">Coassembly of amphiphilic peptide EAK16-II with histidinylated analogues and implications for functionalization of beta-sheet fibrils in vivo.</a> Biomaterials. 35, 5196-5205 (2014).</li><li>Zheng, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+peptide-based+material+platform+for+displaying+antibodies+to+engage+T+cells.">A peptide-based material platform for displaying antibodies to engage T cells.</a> Biomaterials. 32, 249-257 (2011).</li><li>Tajima, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioengineering+mini+functional+thymic+units+with+EAK16-II/EAKIIH6+self-assembling+hydrogel.">Bioengineering mini functional thymic units with EAK16-II/EAKIIH6 self-assembling hydrogel.</a> Clin Immunol. 160, 82-89 (2015).</li><li>Fan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioengineering+Thymus+Organoids+to+Restore+Thymic+Function+and+Induce+Donor-Specific+Immune+Tolerance+to+Allografts.">Bioengineering Thymus Organoids to Restore Thymic Function and Induce Donor-Specific Immune Tolerance to Allografts.</a> Mol Ther. 23, 1262-1277 (2015).</li><li>Fan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymus-specific+deletion+of+insulin+induces+autoimmune+diabetes.">Thymus-specific deletion of insulin induces autoimmune diabetes.</a> The EMBO J. 28, 2812-2824 (2009).</li><li>Fan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compromised+central+tolerance+of+ICA69+induces+multiple+organ+autoimmunity.">Compromised central tolerance of ICA69 induces multiple organ autoimmunity.</a> J Autoimmun. 53, 10-25 (2014).</li><li>Palumbo, M. O., Levi, D., Chentoufi, A. A., Polychronakos, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+proinsulin-producing+medullary+thymic+epithelial+cell+clones.">Isolation and characterization of proinsulin-producing medullary thymic epithelial cell clones.</a> Diabetes. 55, 2595-2601 (2006).</li><li>Williams, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+analysis+of+complex+thymus+stromal+cell+populations:+rapid+thymic+epithelia+preparation+characterizes+radiation+injury.">Single cell analysis of complex thymus stromal cell populations: rapid thymic epithelia preparation characterizes radiation injury.</a> Clin Transl Sci. 2, 279-285 (2009).</li><li>McLelland, B. T., Gravano, D., Castillo, J., Montoy, S., Manilay, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+isolation+of+adult+thymic+epithelial+cell+subsets+for+multiparameter+flow+cytometry+and+gene+expression+analysis.">Enhanced isolation of adult thymic epithelial cell subsets for multiparameter flow cytometry and gene expression analysis.</a> J Immunol Methods. 367, 85-94 (2011).</li><li>Seach, N., Wong, K., Hammett, M., Boyd, R. L., Chidgey, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purified+enzymes+improve+isolation+and+characterization+of+the+adult+thymic+epithelium.">Purified enzymes improve isolation and characterization of the adult thymic epithelium.</a> J Immunol methods. 385, 23-34 (2012).</li><li>Xing, Y., Hogquist, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+identification,+and+purification+of+murine+thymic+epithelial+cells.">Isolation, identification, and purification of murine thymic epithelial cells.</a> J Vis Exp. , e51780 (2014).</li><li>Graham, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+monocytes+from+whole+human+blood.">Separation of monocytes from whole human blood.</a> ScientificWorldJournal. 2, 1540-1543 (2002).</li><li>Li, X., Donowitz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fractionation+of+subcellular+membrane+vesicles+of+epithelial+and+nonepithelial+cells+by+OptiPrep+density+gradient+ultracentrifugation.">Fractionation of subcellular membrane vesicles of epithelial and nonepithelial cells by OptiPrep density gradient ultracentrifugation.</a> Methods Mol Biol. 440, 97-110 (2008).</li><li>Mita, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+method+using+iodixanol+(OptiPrep)-based+density+gradient+significantly+reduces+cytokine+chemokine+production+from+human+islet+preparations,+leading+to+prolonged+beta-cell+survival+during+pretransplantation+culture.">Purification method using iodixanol (OptiPrep)-based density gradient significantly reduces cytokine chemokine production from human islet preparations, leading to prolonged beta-cell survival during pretransplantation culture.</a> Transplant Proc. 41, 314-315 (2009).</li><li>Anderson, G., Takahama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymic+epithelial+cells:+working+class+heroes+for+T+cell+development+and+repertoire+selection.">Thymic epithelial cells: working class heroes for T cell development and repertoire selection.</a> Trends Immunol. 33, 256-263 (2012).</li><li>Kyewski, B., Klein, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+central+role+for+central+tolerance.">A central role for central tolerance.</a> Annu Rev Immunol. 24, 571-606 (2006).</li><li>St-Pierre, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+sequencing+of+neonatal+thymic+epithelial+cells.">Transcriptome sequencing of neonatal thymic epithelial cells.</a> Sci Rep. 3, 1860 (2013).</li></ol>

Authors declare no conflict of interest.

TECS timus stroma baskın nüfus ve timüs bezlerinin yapısı ve fonksiyonu için temel rolleri oynarken, onlar toplam timik sellülarite sadece yaklaşık 0.1-0.5% temsil etmektedir. Bunlar aynı zamanda, hücre ölümü, yüksek yüzdeleri zaman, yani, tedavi hücre dışı matrisin (ECM) ile ilgili Tecs ayırmak için, kollajenaz sindirimi aşağıdaki koşullar kırılgan hücrelerdir. Onların nadir (~ 200.000 fare timus başına) ve kırılganlık o TECS izole etmek zor bir görev yaptı. TEC izole yüks...

Timus edinilmiş bağışıklık sisteminin işlevi için önemlidir patojen reaktif, kendine dayanıklı T hücrelerinin çeşitli nüfusun oluşturulması için primer lenfoid organıdır. Bu timik stroma sünger benzeri üç boyutlu (3-D) matris boyunca göç, gelişmekte olan timositleri, lenfosit progenitör hücreler olarak kemik iliğinden göç dinamik bir organdır, soy-şartname ve farklılaşma tabi, ve sonunda da göç olgun T hücreleri. bu iyi programlanmış sürecin başarısı büyük ölçüde göç timositlerde ve konut timik epitel hücreleri (Tecs) kurulması ve timus mikroçevresinin bütünlüğünü korumak için gerekli olan timik stroma baskın nüfus arasındaki çapraz konuşma bağlıdır. anatomik bir konumda ve eşsiz fonksiyon esas alınarak, TECS iki alt ayrılabilir: kortekste Tecs yanıtlayabiliyor olan (cTECs)Kendi kendine MHC (majör histokompatibilite kompleks) seçilmesi olan kişidir T hücreleri (pozitif seçim) ve otoreaktif T hücreleri (negatif seçim) 1,2 ortadan kaldırmak için gerekli olan medullada Tecs (mTECs) yazılmıştır. Birçok faktör (örneğin, yaşlanma, enfeksiyon, radyasyon, ilaç tedavileri) tehlikeye adaptif bağışıklık sonuçlanan timik epitele geri dönüşü olmayan hasarlara neden olabilir. Çok sayıda girişimlerine rağmen, timus fonksiyonu yeniden nedeniyle timik mikro çoğaltmak zorluk zorlu olmuştur. 2-B ortamında kültürlenmiştir Tecs hızlı thymopoiesis 3,4 kritik genlerin ekspresyonunu azaltmak ise Özellikle, timik üç boyutlu (3-D) konfigürasyonu, Tecs hayatta kalma ve işlevi için önemlidir. EAK16-II (AEAEKAKAEAEAKAK) ve C-ucu histidinylated analog EAKIIH6 (AEAEKAKAEAEAKAKHHHHHH), düşük molekül ağırlıklı, deiyonize Su sağlama içinde çözünür olan amfifilik oligopeptitlerdir20 mM NaCI (insan vücut sıvısı normal tuz konsantrasyonunun 154 mM) daha yüksek iyonik güce maruz kaldığı zaman R, β-fibriller oluşturur jelleşme ama geçer. Bu çevre duyarlı özellik 3D yapıları oluşturmak üzere onları çok yönlü yapı taşlarını yapar. kompleksleri, protein ilaçları ve diğer biyomoleküllerin demirlemek için bir adaptör olarak hizmet verebilir (: EAKII-H6 üzerinde His-tag takma mekanizması, hangi anti-His IgG&#39;ler / Fc bağlayıcı rekombinant protein A / G (pA / G αH6) sağlar örneğin hidrojel karma yapısını 5-8, antikorlar). Daha önce hidrojel üzerine bağlanan flüoresan etiketli IgG molekülleri kadar 13 gün 9 enjeksiyon yerinde muhafaza edilebileceğini göstermiştir. ΑH6 Dahası,: Anti-CD4 IgG&#39;lerin ile sabitlenen pA / G adaptörleri EAK16 II / EAKIIH6 (EAK) hidrojel ilave edildi, CD4 + T hücreleri, spesifik olarak 10 ele geçirildi. benzer bir teknik kullanılarak, yakın zamanda göstermiştir ki TECS C 3-D-çekiş(: PA / G: αEpCAM αH6) TEC-özel anti-EpCAM antikorları taşıyan adaptör kompleksleri ile EAK hidrojel terfi ould. Çıplak farelerin böbrek kapsülleri altına nakledildiklerinde, EAK hidrojel gömülü TEC kümeleri etkili in vivo 11,12 işlevsel T hücrelerinin gelişimini destekler. Burada hızlı ve etkin bir floresan aktive hücre (FACS) ile Tecs arındırmak ve EAK hidrojel sistemi ile 3-D TEC agrega üretmek için bizim yöntemini gösterir.

<table><tbody><tr><td>&lt;strong&gt;TEC Isolation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>70% Ethanol</td><td>Decon Laboratories</td><td>2701(1 Gallon)</td><td>Ethanol 200 Proof, deionized water</td></tr><tr><td>Dissecting scissors, straight</td><td>Fine Science Tools, Inc.</td><td>91460-11</td><td></td></tr><tr><td>Graefe forceps, straight</td><td>Fine Science Tools, Inc.</td><td>11053-10</td><td></td></tr><tr><td>Graefe forceps, curved</td><td>Fine Science Tools, Inc.</td><td>11052-10</td><td></td></tr><tr><td>Washing solution</td><td></td><td></td><td>1x PBS, 0.1% BSA, 2&amp;nbsp;mM EDTA</td></tr><tr><td>1x PBS (Phosphate buffered saline)</td><td>Gibco</td><td>10010-023</td><td></td></tr><tr><td>BSA (Bovine serum albumin)</td><td>Sigma</td><td>A1470-100G</td><td></td></tr><tr><td>EDTA (ethylenediaminetetraacetic acid)</td><td>Invitrogen</td><td>15575-038</td><td></td></tr><tr><td>Digestion solution</td><td></td><td></td><td>9 ml RPMI-1640, 0.025&amp;nbsp;mg/ml Liberase TM Research grade, 10&amp;nbsp;mM HEPES, 0.2&amp;nbsp;mg/ml DNaseI</td></tr><tr><td>RPMI-1640</td><td>Gibco</td><td>11879-020</td><td></td></tr><tr><td>Liberase TM Research Grade</td><td>Roche</td><td>05 401 127 001</td><td>referred as &quot;purified collagenase&quot;</td></tr><tr><td>1&amp;nbsp;M HEPES</td><td>Lonza</td><td>17-737E</td><td></td></tr><tr><td>Dnase I</td><td>Roche</td><td>10 104 159 001</td><td></td></tr><tr><td>50&amp;nbsp;ml Centrifuge tube</td><td>Corning</td><td>430290</td><td></td></tr><tr><td>60&amp;nbsp;mm tissue culture dish</td><td>Falcon</td><td>353002</td><td></td></tr><tr><td>1/2&amp;nbsp;cc U-100 Insulin syringe 28&amp;nbsp;1/2&amp;nbsp;G</td><td>Becton Dickinson</td><td>329461</td><td></td></tr><tr><td>5&amp;nbsp;ml Polystyrene round-bottom tube</td><td>Falcon</td><td>352058</td><td></td></tr><tr><td>5&amp;nbsp;ml glass pipet</td><td>Fisher Healthcare</td><td>13-678-27E</td><td>Use for rinsing the thymic fragments. Thymic fragments tend to stick to the wall with plastic pipets.</td></tr><tr><td>MACSmix tube rotator</td><td>Miltenyi</td><td>130-090-753</td><td></td></tr><tr><td>100&amp;nbsp;&amp;micro;m Cell strainer</td><td>Falcon</td><td>352360</td><td></td></tr><tr><td>Density gradient medium: OptiPrep</td><td>Axis-Shield</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Cell Sorting&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>5&amp;nbsp;ml Polypropylene round-bottom tube</td><td>Falcon</td><td>352063</td><td></td></tr><tr><td>Anti-mouse CD16/CD32 (Fc Block)</td><td>BD Biosciences</td><td>553142</td><td>Use as undiluted, 2&amp;nbsp;&amp;micro;l per sample</td></tr><tr><td>Anti-mouse CD45-PercpCy5.5</td><td>eBioscience</td><td>45-0451-80</td><td>Use at 1:150, 10&amp;nbsp;&amp;micro;l per sample</td></tr><tr><td>Anti-mouse CD326 (EpCAM)-PE</td><td>eBioscience</td><td>12-5791-82</td><td>Use at 1:100, 10&amp;nbsp;&amp;micro;l per sample</td></tr><tr><td>BD Influx</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>Single cell analysis software</td><td>FlowJo</td><td></td><td></td></tr><tr><td>&lt;strong&gt;EAK Gel Assembly&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anti-His-Tag</td><td>AnaSpec</td><td>29673</td><td>&quot;anti-His-Tag IgG&quot;</td></tr><tr><td>Purified anti-mouse CD326 (EpCAM)</td><td>BioLegend</td><td>118202</td><td>&quot;anti-EpCAM IgG&quot;</td></tr><tr><td>Recombinant protein A/G</td><td>Pierce Biotechnology</td><td></td><td></td></tr><tr><td>1.5&amp;nbsp;ml Safe-Lock Tubes, Biopur, Sterile</td><td>Fisher Healthcare</td><td>05-402-24B</td><td>referred as &quot;1.5&amp;nbsp;ml microcentrifuge tube&quot;</td></tr><tr><td>96-well, Tissue culture plate, Round-bottom with low evaporation lid</td><td>BD Falcon</td><td>353917</td><td></td></tr><tr><td>Rocking platform: Nutator Mixer no.1105</td><td>BD Clay Adams</td><td></td><td></td></tr><tr><td>10% sucrose</td><td>Sigma</td><td>S0389</td><td>Prepare with sterile distilled water</td></tr><tr><td>EAK16-II (AcNH-AEAEAKAKAEAEAKAK-CONH&lt;sub&gt;2&lt;/sub&gt;)</td><td>American Peptide Company&amp;nbsp;</td><td></td><td>custom synthesized, 10&amp;nbsp;mg/ml</td></tr><tr><td>EAKIIH6 (AcNH-AEAEAKAKAEAEAKAKHHHHHH-CONH&lt;sub&gt;2&lt;/sub&gt;)</td><td>American Peptide Company&amp;nbsp;</td><td></td><td>custom synthesized, 7.5&amp;nbsp;mg/ml</td></tr><tr><td>Complete medium</td><td></td><td></td><td>RPMI-1640, 10% FBS, 1% Pen/Strep, 1% L-glutamine, 1% NEAA, 5&amp;nbsp;mM HEPES, 50&amp;nbsp;&amp;micro;M 2-Mercaptoethanol</td></tr><tr><td>RPMI-1640</td><td>Gibco</td><td>11879-020</td><td></td></tr><tr><td>FBS (Fetal Bovine Serum)</td><td>Atlanta Biologicals</td><td>S11150</td><td>Heat inactivated before use</td></tr><tr><td>Pen/Strep</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>L-glutamine 200&amp;nbsp;mM (100x)</td><td>Gibco</td><td>25030-081</td><td></td></tr><tr><td>NEAA (non-essential amino acid) 100x</td><td>Gibco</td><td>11140-050</td><td></td></tr><tr><td>1&amp;nbsp;M HEPES</td><td>BioWhittaker</td><td>17-737E</td><td></td></tr><tr><td>2-Mercaptoethanol (100x)</td><td>Millipore</td><td>ES-007-E</td><td></td></tr><tr><td>Platform shaker: The Belly Dancer</td><td>Stovall Life Sciences Inc.</td><td>model: USBDbo</td><td></td></tr></tbody></table>

promoting 3-d aggregation of facs purified thymic epithelial cells with eak 16-ii/eakiih6 self-assembling hydrogel

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing thymus is crucial to maintain effective T cell-mediated acquired immune response against invading pathogens. However, thymus regeneration and revitalization proved to be challenging, largely due to the difficulties of reproducing the unique 3D microenvironment of the thymic stroma that is critical for the survival and function of thymic epithelial cells (TECs). We developed a novel hydrogel system to promote the formation of TEC aggregates, based on the self-assembling property of the amphiphilic EAK16-II oligopeptides and its histidinylated analogue EAKIIH6. TECs were enriched from isolated thymic cells with density-gradient, sorted with fluorescence-activated cell sorting (FACS), and labeled with anti-epithelial cell adhesion molecule (EpCAM) antibodies that were anchored, together with anti-His IgGs, on the protein A/G adaptor complexes. Formation of cell aggregates was promoted by incubating TECs with EAKIIH6 and EAK16-II oligopeptides, and then by increasing the ionic concentration of the medium to initiate gelation. TEC aggregates embedded in EAK hydrogel can effectively promote the development of functional T cells in vivo when transplanted into the athymic nude mice.

CD45- stromal hücreler ettirecek yoğunluk gradyan ayırma protokolü kullanılarak etkinliğini incelemek için, arayüz ve çökelen lenfosit pelet iki hasat hücreleri, anti-CD45 ve anti-EpCAM antikor ile boyandı. Hem anti-Ulex Europaeus Agglutinin 1 (UEA1) ve anti-MHC Sınıf II antikorlar yanı sıra diğer CTEC ve MTec alt kümeleri tespit etmek boyama kokteyli alındı. Şekil 1&#39;de gösterildiği gibi, rutin TECS 15-20 kat zenginleşme yakın yakalamayı ba?...

Watch this Scientific Journal Video about Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel at JoVE.com

Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel

This video demonstrates a protocol to enrich thymic epithelial cells (TECs) with density gradient for FACS isolation. It also shows the use of EAK16-II/EAKIIH6 peptides to promote the TEC aggregate formation. The microenvironments of EAK16-II/EAKIIH6 hydrogel provide the 3-D configuration necessary to maintain the survival and function of the TECs.

EAK 16-II ile FACS Arıtılmış Timik Epitel Hücreleri 3-D Toplama Teşvik / EAKIIH6 Kendi toplanan Hidrojel

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing ...

promoting-3-d-aggregation-facs-purified-thymic-epithelial-cells-with

Research

JoVE Journal

Immunology and Infection

6.5K Görüntüleme.  Allegheny Health Network. This video demonstrates a protocol to enrich thymic epithelial cells (TECs) with density gradient for FACS isolation. It also shows the use of EAK16-II/EAKIIH6 peptides to promote the TEC aggregate formation. The microenvironments of EAK16-II/EAKIIH6 hydrogel provide the 3-D configuration necessary to maintain the survival and function of the TECs.

Video: Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel

3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse this scaffold in a highly coordinated temporal and spatial order while sequentially passing obligatory check points, i.e., T cell lineage commitment, followed by T cell receptor repertoire generation and selection prior to their export into the periphery. The two major resident cell types forming this scaffold are cortical (cTECs) and medullary thymic epithelial cells (mTECs). A key feature of mTECs is the so-called promiscuous expression of numerous tissue-restricted antigens. These tissue-restricted antigens are presented to immature thymocytes directly or indirectly by mTECs or thymic dendritic cells, respectively resulting in self-tolerance.
Suitable in vitro models emulating the developmental pathways and functions of cTECs and mTECs are currently lacking. This lack of adequate experimental models has for instance hampered the analysis of promiscuous gene expression, which is still poorly understood at the cellular and molecular level. We adapted a 3D organotypic co-culture model to culture ex vivo isolated mTECs. This model was originally devised to cultivate keratinocytes in such a way as to generate a skin equivalent in vitro. The 3D model preserved key functional features of mTEC biology: (i) proliferation and terminal differentiation of CD80lo, Aire-negative into CD80hi, Aire-positive mTECs, (ii) responsiveness to RANKL, and (iii) sustained expression of FoxN1, Aire and tissue-restricted genes in CD80hi mTECs.

Studying medullary thymic epithelial cells in vitro has been largely unsuccessful, as current 2D culture systems do not mimic the in vivo scenario. The 3D culture system described herein - a modified skin organotypic culture model - has proven superior in recapitulating mTEC proliferation, differentiation and maintenance of promiscuous gene expression.

Medüller Timik epitel hücre proliferasyonu, farklılaşması ve Promiscuous Gen İfade Destekleme 3D Organotipik Co-kültür Modeli

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse ...

Gelişim Biyolojisi

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

3 boyutlu montaj Self-Peptid Hidrojel İnsan Sinir Progenitör Hücreler Yetiştiriciliği

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Biyomühendislik

Generation and Recovery of &beta;-cell Spheroids From Step-growth PEG-peptide Hydrogels

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional microenvironment with tissue-like elasticity and high permeability for culturing therapeutically relevant cells or tissues. Hydrogels prepared from poly(ethylene glycol) (PEG) derivatives are increasingly used for a variety of tissue engineering applications, in part due to their tunable and cytocompatible properties. In this protocol, we utilized thiol-ene step-growth photopolymerizations to fabricate PEG-peptide hydrogels for encapsulating pancreatic MIN6 b-cells. The gels were formed by 4-arm PEG-norbornene (PEG4NB) macromer and a chymotrypsin-sensitive peptide crosslinker (CGGYC). The hydrophilic and non-fouling nature of PEG offers a cytocompatible microenvironment for cell survival and proliferation in 3D, while the use of chymotrypsin-sensitive peptide sequence (CGGY&darr;C, arrow indicates enzyme cleavage site, while terminal cysteine residues were added for thiol-ene crosslinking) permits rapid recovery of cell constructs forming within the hydrogel. The following protocol elaborates techniques for: (1) Encapsulation of MIN6 &beta;-cells in thiol-ene hydrogels; (2) Qualitative and quantitative cell viability assays to determine cell survival and proliferation; (3) Recovery of cell spheroids using chymotrypsin-mediated gel erosion; and (4) Structural and functional analysis of the recovered spheroids.

Generation and Recovery of &#946;-cell Spheroids From Step-growth PEG-peptide Hydrogels

The following protocol provides techniques for encapsulating pancreatic &#x03B2;-cells in step-growth PEG-peptide hydrogels formed by thiol-ene photo-click reactions. This material platform not only offers a cytocompatible microenvironment for cell encapsulation, but also permits user-controlled rapid recovery of cell structures formed within the hydrogels.

Adım büyüme PEG-peptid Hidrojeller kaynaktan β-hücre sferoidlerin Üretimi ve Kurtarma

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional  microenvironment with tissue-like elasticity and high permeability for  ...

Reaggregate Thymus Cultures

Stromal cells within lymphoid tissues are organized into three-dimensional structures that provide a scaffold that is thought to control the migration and development of haemopoeitic cells. Importantly, the maintenance of this three-dimensional organization appears to be critical for normal stromal cell function, with two-dimensional monolayer cultures often being shown to be capable of supporting only individual fragments of lymphoid tissue function. In the thymus, complex networks of cortical and medullary epithelial cells act as a framework that controls the recruitment, proliferation, differentiation and survival of lymphoid progenitors as they undergo the multi-stage process of intrathymic T-cell development. Understanding the functional role of individual stromal compartments in the thymus is essential in determining how the thymus imposes self/non-self discrimination. Here we describe a technique in which we exploit the plasticity of fetal tissues to re-associate into intact three-dimensional structures in vitro, following their enzymatic disaggregation. The dissociation of fetal thymus lobes into heterogeneous cellular mixtures, followed by their separation into individual cellular components, is then combined with the in vitro re-association of these desired cell types into three-dimensional reaggregate structures at defined ratios, thereby providing an opportunity to investigate particular aspects of T-cell development under defined cellular conditions. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

In this video the preparation of 2-dGuo-treated reaggregate thymus cultures is demonstrated.

Thymus Kültürler Reaggregate

Stromal cells within lymphoid tissues are organized into three-dimensional structures that provide a scaffold that is thought to control the migration and ...

Biyoloji

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

Doku Mühendisliği 3D Hidrojeller Hücresel Kapsülleme

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

Elastin benzeri Protein Hydrogels üretimi için saklama ve Immunostaining hücre 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

Initial 3D Cell Cluster Control in a Hybrid Gel Cube Device for Repeatable Pattern Formations

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its restrictions. Producing few repeatable results of pattern formation deteriorates the analysis of the mechanisms underlying the self-organization. Reducing variation in initial culture conditions, such as the cell density and distribution in the extracellular matrix (ECM), is crucial to enhance the repeatability of a 3D culture. In this article, we demonstrate a simple but robust procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain highly repeatable pattern formations. A micromold with a desired shape was fabricated by using photolithography or a machining process, and it formed a 3D pocket in the ECM contained in a hybrid gel cube (HGC). Highly concentrated cells were then injected in the pocket so that the cell cluster shape matched with the fabricated mold shape. The employed HGC allowed multi-directional scanning by its rotation, which enabled high-resolution imaging and the capture of the entire tissue structure even though a low-magnification lens was used. Normal human bronchial epithelial cells were used to demonstrate the methodology.&#160;

We present a procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain a repeatable pattern formation. A cubic device containing two different hydrogels is employed to achieve multi-directional imaging for tissue pattern formation.

İlk 3D hücre küme denetimi içinde a Hybrid jel küp aygıt için tekrarlanabilir model oluşumları

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its ...

<meta charset="utf8"></head><body>iPSC'den Türetilmiş Organoidlerle Timik Mikro Ortamın Yeniden Oluşturulması

Formation of Human Thymus Organoids in Three-Dimensional Fibrin Hydrogels

Generation of a functional and self-tolerant T cell repertoire is a complex process dependent on the thymic microenvironment and, primarily, on the properties of its extracellular matrix (ECM). Thymic epithelial cells (TECs) are crucial in thymopoiesis, nurturing and selecting developing T cells by filtering self-reactive clones. TECs have been empirically demonstrated to be particularly sensitive to physical and chemical clues supplied by the ECM and classical monolayer cell culture leads to a quick loss of functionality until their death. Because of this delicate maintenance combined with relative rarity, and despite the high stakes in modeling thymus biology in vitro, models able to faithfully mimic the TEC niche at scale and over time are still lacking. Here, we describe the formation of a multicellular human thymic organoid model, in which the TEC compartment is derived from human induced pluripotent stem cells (iPSC) and reaggregated with primary early thymocyte progenitors in a three-dimensional (3D) fibrin-based hydrogel. This model answers current needs for a scalable culture system that reproduces the thymic microenvironment ex vivo and demonstrates functionality, i.e., the ability to produce T cells and to support thymus organoid growth over several weeks. Thus, we propose a practical in vitro model of thymus functionality through iPSC-derived organoids that would benefit research on TEC biology and T cell generation ex vivo.

<meta charset="utf8"></head><body>Yazar Spotlight: İnsan Timik Organoidleri ile Timik Epitel Hücrelerini ve T-Hücresi Araştırmalarını İlerletmek

Here, we describe a protocol for the formation of human iPSC-derived thymus organoids grown in 3D fibrin hydrogels aiming to support thymic epithelial cell (TEC) maturation and prolonged maintenance, as well as thymopoiesis in vitro.

Üç Boyutlu Fibrin Hidrojellerinde İnsan Timus Organoidlerinin Oluşumu

Generation of a functional and self-tolerant T cell repertoire is a complex process dependent on the thymic microenvironment and, primarily, on the ...

EAK 16-II ile FACS Arıtılmış Timik Epitel Hücreleri 3-D Toplama Teşvik / EAKIIH6 Kendi toplanan Hidrojel

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Üç Boyutlu Fibrin Hidrojellerinde İnsan Timus Organoidlerinin Oluşumu

Üç Boyutlu Fibrin Hidrojellerinde İnsan Timus Organoidlerinin Oluşumu

Üç Boyutlu Fibrin Hidrojellerinde İnsan Timus Organoidlerinin Oluşumu