1. Cilt eksplantlarından fibroblast kültürlerinin oluşturulması <ol><li> 100 ünite / ml penisilin, 100 ug / ml streptomisin, ve 0.25 ug / ml Fungizone takviye insan önkol elde edilen 4 mm punch biyopsi MEM yerleştirilir. </li><li> Herhangi bir deri altı yağ Trim ve ince petri dibine bir dizi 24 Bistüri sallanan tarafından küçük parçalar halinde biyopsi doğrayın. </li><li> Şişenin yüzeyi kadar birincil fibroblast büyüme ortamı (MEM% 10 FCS, 100 ünite / ml penisilin, 100 ug / ml streptomisin, ve 25 ug / ml Fungizone ile desteklenmiş) ile 25 cm2 doku kültürü şişesi içinde yer doku bulamaç eklendi kapalı ama sıvı derinliği doku float için yetersizdir. </li><li> % 5 CO2 nemlendirilmiş bir atmosfer içinde 37 3 gün inkübasyondan ° C ardından, birincil fibroblast büyüme ortamı olarak 3 ml eklenir. </li><li> Taze primer fibroblast büyüme ortamı ile başka bir 3 gün inkübasyon değişimi medya takiben veya cel eğerls onlar taze ortama 01:04 bölünebileceği neredeyse konfluent vardır. (Fungizone Bu noktadan itibaren ihmal edilebilir). </li></ol> 2. Evre I - sıçan kuyrukları gelen kollajen I hazırlanması Not: Yaklaşık 12-14 ergen (taze veya dondurulmuş) sıçan kuyrukları için protokol <ol><li> % 70 etanol içinde yıkanarak sıçan kuyruğu hazırlayın ve aşağıdaki gibi tendonlar kaldırın: </li><li> Ile yukarıdan aşağıya doğru kuyruk ortasında dilimleme sıçan kuyruk deri çıkarın bir neşter ve kuyruk uzunluğu boyunca pealing. </li><li> Kuyruk proksimal bölgenin çekirdek tendonu ayırın. </li><li> Dişli forseps kullanarak kılıfı kaçınarak kuyruk distal bölgeye yönelik tendonu çıkarın. </li><li> 48 saat için 4 ° C sıcaklıkta karıştırıldı, tendonun 1 g / L 0.5 M asetik asit, 250 ml çıkarın. </li><li> 30 dakika ekstraktı (7,500 xg) santrifüjleyin ve pelet atın. </li><li> Süpernatant% 10 eşit bir hacim (ağırlık / hacim) NaCl ekleyin ve 30-60 dakika için karıştırın. </li><li 30 dakika için&gt; Santrifüj (10,000 xg), süpernatanın atmak ve 4 C&#39;de 24 saat karıştırıldı ~ 1:1 oranında, 0.25 M asetik asit içinde yeniden çözülür çökelti ° C. </li><li> 6L 6-8 değişikliklere karşı kolajen çözeltisi ~ 17.5 mM asetik asit (soğuk su litre başına 1 ml glasiyel asetik asit, değişim 2 x günlük) Dialyze. </li><li> 1.5 saat için 30.000 xg&#39;de diyaliz kollajen santrifüjleyin. </li><li> Steril şişeye süpernatant ve yer çıkarın. </li><li> ~ 2 mg / ml arasında bir konsantrasyonda 4, 0.5 mM asetik asit kullanılarak ° C&#39;ye ayarlayın kolajen </li></ol> 3. Evre II - gömülü fibroblastlar (8 gün için sözleşme için izin) ile 3D matris kurma. <ol><li> Önceden soğutulmuş bir şişe 4&#39;te Soğuk reaktifler kullanarak ° C karışımı birleştirin. Buz üzerinde kollajen I tutun. </li><li> Konfluent birincil fibroblastlar biri T75 şişesi için (~ hücre sayısı 1 x 10 6) kullanımı: <ul><li> 25 ml fare kuyruk kolajeni (yaklaşık kons 2mg/ml) </li><li> 10x 3 miMEM </li><li> 0.22 M NaOH: Birincisi, 2ml, kollajen turuncu yanana kadar düşmesi-bilge iken, karıştırarak ekleyin </li><li> fakat pembe (genellikle en fazla 3 ml) eklenmiştir. Yaklaşık p H = 7.2. </li><li> Not: Hatta hafif alkali koşullara maruz kalırsa fibroblastlar jel sözleşme olmayacak gibi orta / jel nötral veya hafif asidik kalmasını sağlamak için önemlidir. </li></ul></li><li> , Fibroblastlar Trypsinise 5 dakika boyunca 400 xg&#39;de spin ve süpernatant kaldırmak. </li><li> 5 dakika boyunca üzerinde sıkma: 12 x 35 mm plastik tabaklar hazırlayın - normal fibroblastlar / kollajen karışımı bir şişeye 12 yemekleri ulaşmak. </li><li> 3 ml FCS fibroblastların yeniden askıya ve hemen kollajen karışıma ekleyin ve karıştırın. </li><li> Levha yaklaşık 2,5 ml tabak başına kollajen / fibroblast mümkün olduğunca çabuk. Kabarcıklar ve kollajen ayarını önlemek için deneyin. </li><li> Kollajen 10 dakika için hava içinde% 5 CO2 nemlendirilmiş bir atmosfer içinde 37 ° C &#39;de geçsin. </li><li> Fibroblast 1ml büyümeye ekleTh ortamı (DMEM +% 10 FCS). </li><li> Bir pipet kullanarak yemeğin taraftan kollajen / fibroblast matriks ayırın. </li><li> Ertesi gün: fibroblast büyüme ortamı (DMEM +% 10 FCS) 1ml ekleyin. </li><li> Her gün medya değiştirin. Onlar 24-iyi çanak uyum kadar, yaklaşık 8 gün boyunca sözleşme kollajen / fibroblast matriks İzin (çapı 1.5 cm ~ 3.5 cm Daralma, Şekil 1&#39;e bakınız). </li></ol> Not: kolajen konsantrasyon uygulamasına uyacak şekilde ayarlanması gerekmektedir. Daha fazla kollajen çözüm sulandırmak, daha hızlı fibroblastlar yaptırılacaktır. Daralma oranı küçük farklılıklar, aynı konsantrasyonda kollajen farklı seri ile yaşanacaktır. Benzer şekilde, farklı fibroblast kültürleri farklı oranlarda kollajen jeller daralacağını, daha fibroblastlar sunmak, daha hızlı daralma oranı. Kollajen konsantrasyon ve fibroblast yoğunluğu dolayısıyla tarafından, daralma oranı değiştirmek için ayarlanabilir olabilirkollajen jel ve nihai yoğunluk. 4. Evre II - matris üstüne faiz Kaplama hücreleri Not: Kullanmadan önce etanol ile tüm forseps ve ekipmanları sterilize edin. <ol><li> Künt forseps kullanarak, hafifçe 24-iyi çanak sözleşmeli matris taşıyın. Bunu kat değil emin olun. </li><li> Matris üstüne yaklaşık 4 x 10 4 / ml ve plaka 1ml (tripsin kurtulmak trypsinising, spin hücreler sonra) ilgi hücrelerinin süspansiyonu hazırlayın. Ihtiyaç duyulan hücre tam sayısı kullanılan hücre tipine bağlı olarak değişebilir. Hücre ortamı ilgi hücrelerin normal büyüme ortamı olmalıdır. </li><li> Hücrelerin matrisin üst, yaklaşık 3-5 gün izdiham büyümeye izin verin. </li></ol> 5. Evre III - işgali için bir ızgara (yaklaşık 0-21 gün) matris aktarılması <ol><li> Bir tripod oluşturmak ve (Bakınız Şekil 2) kullanmadan önce otoklav paslanmaz çelik ızgaralar kesin. </li><li> Steril ızgara yerleştirin6 cm çanak, ızgara üzerinde bir seviyede (yaklaşık 10.5ml) büyüme ortamı ekleyin. Yer ızgara üzerinde matris ve matris alt ortam maddesi ile temas halinde olduğu, ancak daldırılmayan kadar yavaşça ortam aspire. Bu istila teşvik eden bir eğim oluşturur hava / sıvı arayüz olarak adlandırılır. Her orta iki gün (Bakınız Şekil 2) değiştirin. </li><li> Canlı hücre floresan hücreleri kullanarak içeriksel görüntüleme bu aşamada veya daha erken de görüntülenebilir. Sözleşmeli veya fibriler kollajen ben ikinci harmonik üretimi (SHG, Şekil 3&#39;e bakınız) kullanılarak görüntülenebilir. Biz sitoplazmik GFP ifade hücreleri en az 200 mikron derin imaged olabilir oysa SHG, matriks içine en az 100 mikron yansıması bulabilirsiniz geniş alan algılama ile birlikte çoklu foton uyarma kullanma. </li></ol> Not: istilası ölçümü ile ilgili olarak, Matris ızgara üzerine edildiği gün 0. Gün tanımlar. Izgaraların üzerine Yerleştirme işgali int teşvik hücre kültürü ortamı bir degrade üretir<html> matris o. Örnekleri sonraki 1 üzerinde yansıması olabilir - 21 gün (veya daha uzun) gibi invazyon, çoğalma, hayatta kalma ya da farklılaşma (referans listesine bakınız) gibi biyolojik süreçleri değerlendirmek için. 6. Evre IV - Fiksasyon <ol><li> Falcon bir tüp içinde% 4 PFA 5 ml ilave edilir. </li><li> Bir düz yüzey üzerine matris aktarın. Taze temiz neşter (Eğer kesit boyama olacak gibi) ile yarıda matris Kes, gece boyunca düzeltmek, neşter ve% 4 PFA transferi ile matris kaldırın. </li><li> Organotipik matrisler artık antikor ile leke / (Bakınız Şekil 3) seçim leke hazırız. </li></ol> 7. Temsilcisi sonuçları: <img alt="Şekil 1" src="/files/ftp_upload/3089/3089fig1.jpg" /> Şekil fibroblast ve petri kopuk ve 6-8 gün içinde sözleşme izin sıçan kuyruk kollajen fibroblast sözleşmeli kollajen I. Karışım 1 Örnek. Şekil 2 &quot;src =&quot; / files/ftp_upload/3089/3089fig2.jpg &quot;/&gt; 2 Organotipik tahlil ilerlemesi Şekil. Organotipik kurulum ve progresyon A, şematik. B, paslanmaz çelik, hava / sıvı arayüzü oluşturmak için steril ızgara üstüne kollajen / fibroblast matris örneği. <img alt="Şekil 3" src="/files/ftp_upload/3089/3089fig3.jpg" /> Organotipik assay 3 Uygulamalar Şekil. A, B non-invaziv ve invaziv pankreatik duktal adenokarsinom (PDAC) hücreleri organotipik matris ile zamanla istila eder. C, fibroblast sözleşmeli kollajenöz dermal bileşeni katmanlı epidermis gösteren derisi eşdeğeri yaşıyor. D, C8161 melanom hücrelerinin işgalci bir fibroblast için kollajenöz Dermal eşdeğer. E organotipik matriks içinde etkileşim ve fibroblast (kırmızı) ile işgalci invaziv PDAC hücreler (yeşil)-daralmıştır. F </strong&gt;, ekstrasellüler matriks (mor) çevreleyen aşağılayıcı etkileşim invaziv PDAC hücreler (yeşil) multiphoton tabanlı ikinci harmonik üretimi kullanarak görüntülendi.

<ol>
	<li>Yamada, K. M., Cukierman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+tissue+morphogenesis+and+cancer+in+3D.">Modeling tissue morphogenesis and cancer in 3D.</a> Cell. 130, 601-610 (2007).</li><li>Edward, M., Gillan, C., Micha, D., Tammi, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour+regulation+of+fibroblast+hyaluronan+expression:+a+mechanism+to+facilitate+tumour+growth+and+invasion.">Tumour regulation of fibroblast hyaluronan expression: a mechanism to facilitate tumour growth and invasion.</a> Carcinogenesis. 26, 1215-1223 (2005).</li><li>Patsialou, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+of+human+breast+cancer+cells+in+vivo+requires+both+paracrine+and+autocrine+loops+involving+the+colony-stimulating+factor-1+receptor.">Invasion of human breast cancer cells in vivo requires both paracrine and autocrine loops involving the colony-stimulating factor-1 receptor.</a> Cancer Res. 69, 9498-9506 (2009).</li><li>Serrels, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+study+of+E-cadherin+and+membrane+dynamics+in+living+animals:+implications+for+disease+modeling+and+drug+development.">Real-time study of E-cadherin and membrane dynamics in living animals: implications for disease modeling and drug development.</a> Cancer Res. 69, 2714-2719 (2009).</li><li>Timpson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+Regulation+of+RhoA+Activity+during+Pancreatic+Cancer+Cell+Invasion+Driven+by+Mutant+p53.">Spatial Regulation of RhoA Activity during Pancreatic Cancer Cell Invasion Driven by Mutant p53.</a> Cancer Res. 71, 747-757 (2011).</li><li>Edward, M., Quinn, J. A., Pasonen-Seppanen, S. M., McCann, B. A., Tammi, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=4-Methylumbelliferone+inhibits+tumour+cell+growth+and+the+activation+of+stromal+hyaluronan+synthesis+by+melanoma+cell-derived+factors.">4-Methylumbelliferone inhibits tumour cell growth and the activation of stromal hyaluronan synthesis by melanoma cell-derived factors.</a> Br. J. Dermatol. 162, 1224-1232 (2010).</li><li>Fusenig, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+differentiation+characteristics+of+transformed+keratinocytes+from+mouse+and+human+skin+in+vitro+and+in+vivo.">Growth and differentiation characteristics of transformed keratinocytes from mouse and human skin in vitro and in vivo.</a> J. Invest. Dermatol. 81, 168s-175s (1983).</li><li>Nystrom, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+quantitative+method+to+analyse+tumour+cell+invasion+in+organotypic+culture.">Development of a quantitative method to analyse tumour cell invasion in organotypic culture.</a> J. Pathol. 205, 468-475 (2005).</li><li>Sabeh, F., Shimizu-Hirota, R., Weiss, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protease-dependent+versus+-independent+cancer+cell+invasion+programs:+three-dimensional+amoeboid+movement+revisited.">Protease-dependent versus -independent cancer cell invasion programs: three-dimensional amoeboid movement revisited.</a> J. Cell. Biol. 185, 11-19 (2009).</li><li>Amjad, S. B., Carachi, R., Edward, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keratinocyte+regulation+of+TGF-beta+and+connective+tissue+growth+factor+expression:+a+role+in+suppression+of+scar+tissue+formation.">Keratinocyte regulation of TGF-beta and connective tissue growth factor expression: a role in suppression of scar tissue formation.</a> Wound Repair Regen. 15, 748-755 (2007).</li><li>Gaggioli, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast-led+collective+invasion+of+carcinoma+cells+with+differing+roles+for+RhoGTPases+in+leading+and+following+cells.">Fibroblast-led collective invasion of carcinoma cells with differing roles for RhoGTPases in leading and following cells.</a> Nat. Cell. Biol. 9, 1392-1400 (2007).</li></ol>

Biz ifşa etmek başka bir şey var.

Burada invaziv hücre göçü 6-8 çalışmalar için uygun bir 3-boyutlu matrisi üretmek için bir yöntem sunmaktır. Matris birincil insan epidermal fibroblastlar tarafından birkaç gün bir süre içinde sözleşmeli kollajen tip 1 fibrillerinin oluşmaktadır. Kollajen poli-peptit ucunda tepkime özelliklerini koruyan ve tampon koşulları 9 nötrleştirme üzerine daha büyük agregalar halinde kollajen fibrillerin çapraz bağlanma teşvik asit ekstraksiyon, değil enzimatik sindirimi ile hazırlanır. Daha yakından in vivo kolajen özelliklerini taklit eder ve jel içinde kültür hücrelerinin invazif özellikleri için önemli sonuçlara sahiptir yeniden oluşturulmuş jel elde edilir. Kollajen çapraz bağlama genç hayvanlarda daha kararsızdır çünkü taze veya dondurulmuş başlangıç ​​malzemesi kullanılıp kullanılmadığını önemli, ama ergen sıçan kuyrukları kullanmak değildir önemlidir. Kollajen Ben genç hayvanlardan elde etmek bu nedenle kolay ve w yeniden oluştururITH yüksek sadakat. Kolajen matris sabittir ve lekeli istilacı tümör hücreleri ya da stromal fibroblastlann 2,10 ya karşı yönlendirilmiş antikorlar ile ve istila 5,6 inhibe ilaçların etkinliğini test etmek için son derece yararlıdır edilebilir. Bu protokol, birincil insan derisi fibroblast kullanımını göstermektedir, ancak, bu araştırma altında doku tipine bağlı olarak, diğer doku tipi, ikinci birincil fibroblast kullanmak mümkündür. Bu stabil bir şekilde floresan protein konjugatları ile transfekte edilmiş olan hücreler de dahil olmak üzere ölümsüzleştirilmiş, fibroblastlar, kültürleri kullanılarak oluşturulması da mümkündür. Bu şekilde stromal tümör her iki hücre invazyonu 11 sırasında hücre-hücre etkileşimleri görselleştirmek için etiketlenmiş olabilir.

<table border="1"><tbody><tr><th> Reaktif Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar (isteğe bağlı) </th></tr><tr><td> 10x MEM </td><td> Gibco </td><td> 21430 </td><td> - </td></tr><tr><td> NaOH </td><td> Sigma </td><td> 367176-500G </td><td> Su içinde 0.22 M stok hazırlayın </td></tr><tr><td> FCS </td><td> PAA Laboratuvarlar </td><td> A15-101 </td><td> - </td></tr><tr><td> 35 mm yemekleri </td><td> Şahin </td><td> 353001 </td><td> Adım 3.4 için </td></tr><tr><td> 60 mm yemekleri </td><td> Şahin </td><td> 353004 </td><td> Adım 5.2 için </td></tr><tr><td> Bahar forseps künt </td><td> Samco </td><td> E003/02 </td><td> Düzgün değil, Dişli </td></tr><tr><td> % 16 paraformaldehit </td><td> Elektron Mikroskopi Hizmetleri </td><td> 15710 </td><td> Kullanımdan önce PBS içinde% 4 ile seyreltilir </td></tr><tr><td> Cd-1, boyutu 40 mesh için ekranlar </td><td> Sigma </td><td> S07707-5EA </td><td> Paslanmaz çelik ızgaralar, paket başına 5 </td></tr><tr><td> Diyaliz boru </td><td> Medicell Uluslararası </td><td> 7607 2295 </td><td> 12 - 14 kD </td></tr><tr><td> PBS </td><td> Oxoid </td><td> BR0014G </td><td> - </td></tr><tr><td> Asetik asit </td><td> Sigma </td><td> 242853 </td><td> - </td></tr><tr><td> 24 de çanak </td><td> Şahin </td><td> 353047 </td><td> Adım 4.1 için </td></tr><tr><td> Fungizone </td><td> Vitrogen In </td><td> 15290018 </td><td> - </td></tr></tbody></table>

organotypic collagen i assay: a malleable platform to assess cell behaviour in a 3-dimensional context

Hücre göçü gelişimi, yara iyileşmesi, bağışıklık sisteminin hücresel yanıtları, ve tümör hücrelerinin metastaz gibi biyolojinin birçok yönleri için esastır. Göç ışık mikroskobu ile incelenmesi için mükellef hücresel dinamikleri yapmak için cam lameller üzerine çalışılmıştır. Ancak, hücre göçü birçok açıdan sadakatle cam ve plastik 1 gibi sert iki boyutlu yüzeyler tarafından temsil edilmeyen, elastikiyet, protein kompozisyonu ve gözenek boyutu dahil yerel çevrenin özelliklerine bağlı olduğu açık hale gelmiştir. Ayrıca, stromal fibroblastlann 2 ve 3 bağışıklık hücreleri dahil olmak üzere diğer hücre tipleri ile etkileşimi kanser hücreleri istila teşvik edilmesinde önemli bir rol oynadığı gösterilmiştir. Moleküler düzeyde incelenmesi giderek karşılaştırıldığında aynı hücrelerin ilaç tedavisine yanıt dahil moleküler dinamik, önemli ölçüde farklı olduğunu göstermiştir <em&gt; In vitro ve in vivo 4. İdeal olarak, bu canlılar kendi doğal bağlamında hücre göçü incelemek için iyi olurdu, ancak bu her zaman mümkün değildir. Böyle hücre elde matrisi, Matrigel, organotipik kültürü (burada açıklanan) doku eksplant, organoids ve xenografts gibi Orta doku kültürü sistemleri, bu nedenle önemli deneysel ara vardır. Bu sistemler yaklaşık bazı in vivo ortamda yönlerini ancak böyle stably transfekte hücre hatları, ilaç tedavisi rejimleri, uzun vadeli ve yüksek çözünürlüklü görüntüleme kullanımı gibi deneysel manipülasyon için daha uygundurlar. Böyle ara sistemleri probları doğrulamak ve görüntü öncesinde vivo 5 görüntüleme üstlenen hücreleri ve floresan gazetecilere dinamik tepki için gerekli parametreleri kurmak Vaitoudden olarak özellikle yararlıdır. Bu nedenle, onlar livin üzerinde deney ihtiyacını azaltmada önemli bir rol hizmet edebilirg hayvanlar.

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Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Bir yöntem olup kollajen tip I ve primer insan fibroblast oluşan bir 3-boyutlu matrisi hazırlanması için tarif edilmektedir. Bu doku stroma temel özelliklerini taklit eder ve mikroskopi birçok formları için müsait olduğundan bu organotipik jel invaziv hücre göçü değerlendirmek için yararlı bir substrat olarak hizmet vermektedir.

Organotipik Kollajen Ben Assay: 3-Boyutlu Bağlamında Hücre Davranışı değerlendirin A Dövülebilir Platformu

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

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22.3K Görüntüleme.  University of Glasgow. Bir yöntem olup kollajen tip I ve primer insan fibroblast oluşan bir 3-boyutlu matrisi hazırlanması için tarif edilmektedir. Bu doku stroma temel özelliklerini taklit eder ve mikroskopi birçok formları için müsait olduğundan bu organotipik jel invaziv hücre göçü değerlendirmek için yararlı bir substrat olarak hizmet vermektedir.

Video: Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

Proteolitik Degradable 3 Boyutlu Sistem Yumurtalık Folikül Encapsulation ve Kültür Bir Yöntem

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

Biyomühendislik

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Protein ve hücre Etrafının İki Boyutlu Desenli Yüzeyler Oluşturma

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

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 ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

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

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

3-Boyutlu Hücre Kültürü için Self-raporlama iskeleleri

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

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

Cilt Tip I Kolajen hazırlanması için geliştirilmiş bir yöntem

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

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 ...

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

Planar Gradient Difüzyon Sistemi 3D Kollajen Matrix Kemotaksis Araştırma

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

Sıkıştırılmış Kolajen kıyılmış Doku: Tek aşamalı Rekonstrüktif Onarım için Cep içeren Biotransplant

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

Rekombinant kolajen peptid Microcarriers hücre genişletme ve hücre teslim sistemi bir biyoreaktör olarak potansiyel kullanım için ben Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

Rodent Behavioral Testing to Assess Functional Deficits Caused by Microelectrode Implantation in the Rat Motor Cortex

Medical devices implanted in the brain hold tremendous potential. As part of a Brain Machine Interface (BMI) system, intracortical microelectrodes demonstrate the ability to record action potentials from individual or small groups of neurons. Such recorded signals have successfully been used to allow patients to interface with or control&#160;computers, robotic limbs, and their own limbs. However, previous animal studies have shown that a microelectrode implantation in the brain not only damages the surrounding tissue but can also result in functional deficits. Here, we discuss a series of behavioral tests to quantify potential motor impairments following the implantation of intracortical microelectrodes into the motor cortex of a rat. The methods for open field grid, ladder crossing, and grip strength testing provide valuable information regarding the potential complications resulting from a microelectrode implantation. The results of the behavioral testing are correlated with endpoint histology, providing additional information on the pathological outcomes and impacts of this procedure on the adjacent tissue.

We have shown that a microelectrode implantation in the motor cortex of rats causes immediate and lasting motor deficits. The methods proposed herein outline a microelectrode implantation surgery and three rodent behavioral tasks to elucidate potential changes in the fine or gross motor function due to implantation-caused damage to the motor cortex.