Bu proje, İsviçre Ulusal Bilim Vakfı (SNF # 310030-127586/1) tarafından desteklenmiştir.

1. İntervertebral Disk Hasat <ol><li> Tüm uzunluğu sığır kuyruğu, cilt varlığı kirlenme şansını artırır (Şekil 2), deri olmadan, mümkünse yerel bir mezbahaya elde edilir. </li><li> Büyük kesme tahtası hazırlayın ve bir kesme tahtası (Şekil 2) üstüne steril çalışma istasyonu ve aletleri hazırlamak. </li><li> 55mm sodyum sitrat içeren% 0.9 sodyum klorür ile nemlendirilmiş steril laminer akış kaputu steril gazlı bez altında hazırlanın ve 6-plaka her bir kuyunun içine koymak. </li><li> Bir havza hazırlayın ve musluk suyu ile 1:100 Betadine çözeltinin. </li><li> 5 dakika boyunca% 1&#39;lik Betadine çözeltisi içeren bir havzada tüm sığır kuyruğu bırakın. </li><li> Kısaca kuru steril gazlı bez ile kuyruk ve steril bir iş istasyonu üzerinde yerleştirin. </li><li> # 22 neşter bıçağı ile dikkatlice kuyruk etrafındaki kas çıkarın. </li><li> Gerekli değildir kuyruk parçaları, nispeten büyük ve çok küçük içeren kuyruk genellikle iki ucunda, uzak doğrayınIVD&#39;ler (Şekil 3). </li><li> IVD etrafında neşter bıçak kullanarak # 10 daha fazla kas ve tendonlar kesin. Disklerin dış anulus kesmek için dikkat edin. </li><li> Mark cerrahi cilt markeri IVD anterior (Bu adım isteğe bağlıdır, bizim biyoreaktör aksiyel rotasyon için merkezi belirlemenize yardımcı olur). </li><li> IVD ve vertebra bağlantı noktası kuyruk hafifçe hareket ettirerek bulun. Donuk bir neşter bıçak yüzü IVD ve kemik arasındaki sınır hissedin. Yarma site vertebra doğru IVD 1-2 mm olmalıdır. Yeri özel yarma sitesi (Şekil 4) endüstriyel bıçak tutucu. </li><li> Cleave IVD ve vertebra ısmarlama endüstriyel bıçak tutucu (Şekil 3) üstüne çekiç ile. </li><li> IVD-vertebranın bağlantısı, diğer tarafta tekrarlayın. </li><li> Wrap 55mm sodyum sitrat içeren% 0.9 sodyum klorür ile ıslatılmış steril gazlı bez ile IVD izole. </li><li> Istenen numune sayısı (genellikle 6 dolaşıp IVD&#39;ler izolasyonu Devam10-20 mm arasında çapı ile hasat edilebilir). </li><li> Jet steril Ringer solüsyonu ile lavaj sistemi (Zimmer, inc.) Bağlayın, 5L çanta (alternatif steril PBS veya% 0.9 salin solüsyonu) kullanışlı bir boyutta. </li><li> IVD Zimmer Pulsavac jet-lavaj sistemi (Şekil 1C) kullanarak forseps ve uç plakası yüzeyinin jet-lavajı her iki tarafında tutun. Jet tabanca 30 arasında bir açıyla uç plakası yüzeyinde çekilmiş olmalıdır - 60 ° her tarafında 30s hakkında. (Şekil 1 ve 7) </li><li> Jet başka bir disk örneği yıkarken IVD&#39;ler ile 6-plaka ve şal geri koyun gazlı bezle temizleyin. </li><li> IVD&#39;ler organ kültür ve aşağı akım uygulamaları (örneğin, hücre canlılığı, mekanik yüklenme, organ kültürü) (Şekil 1D) için hazırız. </li></ol> 2. Temsilcisi Sonuçlar Başarılı bir intervertebral disk hazırlık yargılamak için Sonuç ölçümleri difüzyon deneyi olabilir küçük bir molekül ağırlığı (örneğin procion kırmızı floresan boya)PBS 2% 1&#39;lik bir çözüm olarak hazırlanır. IVD sonra en az 100 mL boya çözüm ve boya, daha sonra ücretsiz şişme altında 24 saat için IVD pasif diffüz izin verilir tutulur. IVD sonra, sıvı nitrojen içinde flash dondurulmuş ve oda sıcaklığı ve aseton transferlerin bir dizi (ilk -80 olarak ° C, -20 ° C, daha sonra 4 ° C ve sonunda oda sıcaklığına kadar önceden soğutulmuş susuz geri getirdi. .) Disk sonra poli-metil-akrilat (PMMA) gömülü ve 100 mikron kalınlığında kesitler üretmek için keskin bir bıçak ile kesilir. Bu bölümler daha sonra herhangi bir standart aydınlık mikroskop ama tercihen procion kırmızı kırmızı floresan yayar (Şekil 5-6) bu yana floresan mikroskopi kullanımı ile görülebilir. Kemik yüzeyi trabeculi jet lavaging adım (Şekil 7) sonra çok temiz görünür. Başarılı bir organ kültürü parametreleri, kirlenme, hücre canlılığı muhafaza tüm yokluğu (Şekil 8-9), disk yüksekliği ilk vehistolojik kesitler ve safranin O / hızlı yeşil leke veya Meyer Hematoxilin 3 ile ölçülen genel &quot;disk bütünlüğü&quot;. Biz canlı disk dokusu leke ve başka bir konfokal lazer tarama mikroskobu ve canlı / ölü boyama kiti (Moleküler Probları, Invitrogen, Basel, İsviçre) kullanılarak 3 boyutlu hücre canlılığı tarama 4,5 yayınlanan protokolleri ve makrolar geliştirdi. Biz 3D doku veya 4-6 NIH ImageJ platformu kullanarak 3D taşıyıcılar hücre canlılığı tahmin etmek için otomatik hücre sayımı gerçekleştirmek için bir makro ve rutin daha geliştirdi. Alternatif protokolleri disk hücreleri hücre canlılığı pronase ve kollajenaz tip kullanarak ekstraselüler matriks sıralı sindirim kullanarak ilk sindirim belirlemek için 2 ve daha sonra hücre süspansiyonu 7 leke ileri sürülmüştür. Protokolün bir önceki sürümü PBS önce diskler hasat organ cu sonra 10 dakika süreyle on kat daha konsantre penisilin / streptomisin konsantrasyon içeren bir inkübasyon adım söz konusulture. Bu adımı kültür amaçları için yararlı gibi görünüyor bu yana jet lavaj adım ile bu adımı geçersiz olur. <img alt="Şekil 1" src="/files/ftp_upload/3490/3490fig1.jpg" /> Şekil 1 metodolojik adımları bakış, sağlam omurun ve kemik ince bir tabaka halinde (1-2mm) ile sığır coccygeal İntervertebral Diskler hazırlamak için . A) intervertebral disk (IVD) ve besin yollar şematik görünümü. B) Usulü ile IVD bıçak tutucu, özelleştirilmiş ve endüstriyel bir keskin bıçak kesmek için. C) Jet lavaj adım yüksek hücre canlılığı sağlamak ve uç plakaları ve her iki tarafta bağlı kemik 1-2 mm tutmak besin alışverişini sağlamak için. D) Son olarak IVD biyoreaktör numune odasında kültüre olabilir veya herhangi aşağı akım bir uygulama için kullanılmalıdır. <img alt="Şekil 2" src="/files/ftp_upload/3490/3490fig2.jpg" /> Şekil 2 TOP: Tam boy taze sığır kuyruğu (ideal obta2-3 saat içinde incelendiği yüksek hücre canlılığı sağlamak için post-mortem. Alt: kemik omurun (EP) ve vertebra gövdelerinin (VB) arasında büyüme plaklarının (GP), ikincil kemikleşme merkezi, varlığını gösteren yaklaşık 6 ay yaşlı bir sığır kuyruğu X-ray görüntü. <img alt="Şekil 3" src="/files/ftp_upload/3490/3490fig3.jpg" /> Şekil 3. A) Araçlar, steril koşullar altında sığır kuyruğu incelemek . B) Özelleştirilmiş bıçak tutucu, intervertebral disk kesim sırasında eylem sığır kuyruğu hareket segmentleri-out kesti. C) standart bir endüstriyel bıçak (Lutz, Almanya) ile özelleştirilmiş bıçak tutucu Yan görünümü. <img alt="Şekil 4" src="/files/ftp_upload/3490/3490fig4.jpg" /> Şekil 4 Çizim ve hareket segmentleri kesip yarma siteleri gösteren çevreleyen kas ve ligaman kaldırdıktan sonra sığır kuyruğu resmi. Kısmi vertebra wi &quot;çatlak&quot; olmalıdırısmarlama bıçak tutucu th ideal olarak 1-2 mm uzaklıkta, kıkırdak, omurun tam kemik yüzeyi sağlamak. <img alt="Şekil 5" src="/files/ftp_upload/3490/3490fig5.jpg" /> Şekil 5 önce jet-lavaj adım etkili temizlik Gösterme (A) ve sonrası (B). Püskürtme işlemi sonrasında intervertebral disk bölümleri temiz kemik yüzeylere dikkat edin. <img alt="Şekil 6" src="/files/ftp_upload/3490/3490fig6.jpg" /> Şekil 6 büyüme plağı tarafından blokaj etkisi gösteren, 24 saat boyunca% 1 procion kırmızı çözümü sol taze hazırlanmış eksize sığır kuyruğu intervertebral diskler (IVD&#39;ler) Serbest şişme difüzyon deneyi . Bir dijital X-ray sagital görünüm 2&#39;den sonra hala kıkırdak ve kemik uç plakası ile coccygeal sığır intervertebral disk eksplant büyüme plağı ve sagital kesilmiş kemik floresan mikroskopi görüntü ikinci bir tabaka içeren IVD4h serbest difüzyon. GP: büyüme plağı, 2ndry CO: ikincil kemikleşme merkezi. <img alt="Şekil 7" src="/files/ftp_upload/3490/3490fig7.jpg" /> Şekil 7, taze hazırlanmış, 24 saat% 1 procion kırmızı çözüm için sol eksize sığır kuyruğu intervertebral diskler (IVD&#39;ler) Serbest şişme deneyi, jet lavaj tedavi temizlik etkisi gösteren. (~ 100 mm), tedavi (Sağ) jet-lavaj tedavi olmadan jet lavaj tedavisi (Sol) Kontrol tarafında yapıldı ~ 1.5-mmthick kemik uç plakaları ile eksize sığır IVD&#39;ler sagittal kalınlığında kesitler. Giriş sprey Zimmer yara debridman sistemi kullanılan desen, 24 saat ücretsiz difüzyon deneyleri,% 1 procion kırmızı sığır intervertebral disk eksplant. Gösterir. <img alt="Şekil 8" src="/files/ftp_upload/3490/3490fig8.jpg" /> Şekil 8 konfokal Görüntüleme yığınları projeksiyonlar Live / Dead Görüntüler (250μm) bovin alınane intervertebral disk bölümleri düzeyde ayırır Nükleus pulpozusun (NP), iç anulus fibrosus (IA) ve dış anulus fibrosus (OA) için sırasıyla 0 ve 14 gün için serbest şişme altında tutulması bu jet-lavaj yöntemi ile hazırlanmıştır. Ethidium homodimer-1 tarafından boyanmış = Calcein AM (asetil metilester) ile boyanan canlı hücreler, kırmızı hücrelerin ölü hücreleri çekirdeği Yeşil hücreleri. <img alt="Şekil 9" src="/files/ftp_upload/3490/3490fig9.jpg" /> Şekil 9, canlı doku ve 3D yığın tarama konfokal lazer tarama mikroskobu kullanarak intervertebral disk hücre Canlılık . Hücre canlılığı düzeyde ayırır Nükleus pulpozusun (NP) ve annulus Fibrozis (AF), serbest-şişme koşul altında 21-gün-kültür üzerinde. (Ortalama ± SEM, N = 6) İstatistiksel farklılıklar non-parametrik Kruskal-Wallis rank sum testi gruplar arasında imzalanan kullanılarak test edildi. Gün 21 ve NP diğer tüm zaman noktaları arasında önemli farklılıklar tespit edilmiştir. AF önemli farklılıklar gün 7 arasında iken,gün 0. (* P &lt;0.05, ** p &lt;0.01).

<ol>
	<li>Lee, C. R., Iatridis, J. C., Poveda, L., Alini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+organ+culture+of+the+bovine+intervertebral+disc:+effects+of+vertebral+endplate+and+potential+for+mechanobiology+studies.">In vitro organ culture of the bovine intervertebral disc: effects of vertebral endplate and potential for mechanobiology studies.</a> Spine (Phila Pa 1976). 31, 515-522 (1976).</li><li>Chan, S. C. W., Gantenbein-Ritter, B., Leung, V. Y., Chan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreserved+intervertebral+disc+with+injected+bone+marrow-derived+stromal+cells:+a+feasibility+study+using+organ+culture.">Cryopreserved intervertebral disc with injected bone marrow-derived stromal cells: a feasibility study using organ culture.</a> Spine. J. 10 (6), 486-496 (2010).</li><li>Chan, S. C., Ferguson, S. J., Wuertz, K., Gantenbein-Ritter, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+Response+of+the+Intervertebral+Disc+to+Repetitive+Short+Term+Cyclic+Torsion.">Biological Response of the Intervertebral Disc to Repetitive Short Term Cyclic Torsion.</a> Spine (Phila Pa 1976). , (2011).</li><li>Gantenbein-Ritter, B., Sprecher, C. M., Chan, S., Illien-J&#252;nger, S., Grad, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+imaging+protocols+for+live/dead+staining+in+three-dimensional+carriers.">Confocal imaging protocols for live/dead staining in three-dimensional carriers.</a> Methods Mol. Biol. 740, 127-140 (2011).</li><li>Gantenbein-Ritter, B., Potier, E., Zeiter, S., van der Werf, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+of+three+techniques+to+determine+cell+viability+in+3D+tissues+or+scaffolds.">Accuracy of three techniques to determine cell viability in 3D tissues or scaffolds.</a> Tissue Engineering Part C Methods. 14, 353-358 (2008).</li><li>Rasband, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ImageJ. , (1997).</li><li>Haschtmann, D., Stoyanov, J. V., Ettinger, L., Nolte, L. P., Ferguson, 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=Establishment+of+a+novel+intervertebral+disc/endplate+culture+model:+analysis+of+an+ex+vivo+in+vitro+whole-organ+rabbit+culture+system.">Establishment of a novel intervertebral disc/endplate culture model: analysis of an ex vivo in vitro whole-organ rabbit culture system.</a> Spine. 31, 2918-2925 (2006).</li><li>Gantenbein, B., Gr&#252;nhagen, T., Lee, C. R., van Donkelaar, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+organ+culturing+system+for+intervertebral+disc+explants+with+vertebral+endplates:+a+feasibility+study+with+ovine+caudal+discs.">An in vitro organ culturing system for intervertebral disc explants with vertebral endplates: a feasibility study with ovine caudal discs.</a> Spine. 31, 2665-2673 (2006).</li><li>Korecki, C. L., Kuo, C. K., Tuan, R. S., Iatridis, 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=Intervertebral+disc+cell+response+to+dynamic+compression+is+age+and+frequency+dependent.">Intervertebral disc cell response to dynamic compression is age and frequency dependent.</a> J. Orthop. Res. 27, 800-806 (2009).</li><li>Korecki, C. L., MacLean, J. J., Iatridis, 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=Dynamic+compression+effects+on+intervertebral+disc+mechanics+and+biology.">Dynamic compression effects on intervertebral disc mechanics and biology.</a> Spine (Phila Pa 1976). 33, 1403-1409 (1976).</li><li>Jim, B., Steffen, T., Moir, J., Roughley, P., Haglund, 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+an+intact+intervertebral+disc+organ+culture+system+in+which+degeneration+can+be+induced+as+a+prelude+to+studying+repair+potential.">Development of an intact intervertebral disc organ culture system in which degeneration can be induced as a prelude to studying repair potential.</a> European Spine Journal. , 1-11 (2011).</li></ol>

Biz ifşa etmek başka bir şey var.

Başarılı organ kültürü için ilk adım eksplant kontamine olmaması gerektiğini emin olun. Prosedüre başlamadan önce kuyruk tenli olmalıdır. Herhangi bir hayvan kılı steril bir laboratuvar haline getirdiği kirlenme açısından sorunlu olabilir. Sığır kuyruğu ideal (bu ilk hücre canlılığı etkiler) mümkün olduğunca taze olmalıdır. Ayrıca, betadin yıkama adım daha kontaminasyon riskini azaltmak için önerilmektedir. IVD vertebra gövdesi ayrı bir ölçüye göre bıçak tutucu ve bir çek...

<table border="1"><tbody><tr><td> Reaktifi Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td colspan="4"> Yerel mezbahasında (ideal saat sonra otopsi içinde ve cilt) Taze sığır sığır kuyrukları intervertebral disk dokusu,. </td></tr><tr><td> Pulsavac Ayrıca AC Sistemi </td><td> Zimmer inc, İsviçre </td><td> 00-5150-486-01 </td><td> Hip-sprey başlığı ile ve AC güç kaynağı ile en iyi performans (8 adet AA pil ile çalışır ancak daha az uygundur.) </td></tr><tr><td> Yüksek Kapasiteli Fan Sprey w / Splash Kalkan, 12.7cm uzunluk </td><td> Zimmer inc, İsviçre </td><td> 00-5150-175-00 </td><td> Çeşitli sprey başları vardır, başarılı bir test </td></tr><tr><td> Neşter bıçaklar # 22 # 10 </td><td> Swann-Mortİngiltere, </td><td> # 10: 0201 # 22: 0208 </td><td></td></tr><tr><td> Neşter bıçak tutucu # 3 ve # 4 </td><td> Hausmann, Almanya </td><td> # 3: 06.103.00 # 4: 06.104.00 </td><td></td></tr><tr><td> Lutz endüstriyel bıçak </td><td> Lutz, Almanya </td><td> 1022.0884 </td><td></td></tr><tr><td> Fosfat tamponlu salin (PBS) </td><td> Invitrogen, İsviçre </td><td> 10010-023 </td><td></td></tr><tr><td> Dulbecco&#39;nun Modifiye Kartal Medium (DMEM) </td><td> Gibco, İsviçre </td><td> 11960-044 </td><td></td></tr><tr><td> Ringer solüsyonu (glikoz) </td><td> Bichsel, İsviçre </td><td> 133 0002 </td><td></td></tr><tr><td> 6-çok-plaka </td><td> DYP, İsviçre </td><td> 92006 </td><td></td></tr><tr><td> Betadine çözüm </td><td> Mundipharma, İsviçre </td&gt; <td> 10055025 </td><td></td></tr><tr><td> Cerrahi cilt markeri </td><td> Porex Cerrahi, İsviçre </td><td> 9560 </td><td></td></tr><tr><td> Büyük kesme tahtası </td><td></td><td></td><td> Herhangi bir marka mümkündür </td></tr></tbody></table>

preparation of intact bovine tail intervertebral discs for organ culture

Intervertebral disk (IVD) vertebra vertebra için bağlayan omurga ortak. Bu, omurga yükleme aktarmak ve omurga esnekliği sağlamak için fonksiyonlar. Üç bölmeden oluşur: anulus fibrosus (AF) ve her iki tarafta vertebral gövde NP ve AF bağlayan iki kıkırdak omurun kapsayan içteki düzeyde ayırır Nükleus pulpozusun (NP). Muhtemelen dejeneratif intervertebral disk hastalığı (DDH) ve disk hernileri nedeniyle Diskojenik ağrısı, modern toplumda önemli bir sorun olarak tespit edilmiştir. IVD yozlaşma olası mekanizmaları incelemek için, canlı disk hücreleri ile yapılan in vitro organ kültürü sistemleri son derece çekici. Sağlam sığır coccygeal IVD&#39;ler in vitro kültürü, iyi kontrollü fizyolojik ve mekanik ortamda mekanik-biyolojik yönlerini çalışma sağlayan ilgili bir model sistem, ilerlemiştir . Sığır kuyruğu IVD&#39;ler yüksek sayıda nispeten kolay elde edilebilird insan lomber IVD&#39;ler hücre yoğunluğu, hücre popülasyonu ve boyutları bakımından çok benzer. Ancak, beslenme yollarının açıkça pıhtılaşmış kan tarafından bloke edildi yana, kıkırdak omurun ve kemik omurun istinat önceki sığır kaudal IVD hasat teknikleri, kültür 1-2 gün sonra başarısız oldu. IVD&#39;ler büyük avasküler organları, böylece NP hücrelere besin, sadece komşu vertebral gövde kılcal tomurcukları aracılığıyla difüzyon bağımlı. Son plağın yüzeylerde kemik enkaz ve pıhtılaşmış kan varlığı, disk ve uzlaşma hücre canlılığı merkezi haline besin difüzyon engel teşkil edebilir. Grubumuz için düşük kontaminasyon riski ile kuyruk IVD&#39;ler &quot;crack&quot; için görece hızlı bir protokoldür kurdu. Biz taze kesilmiş, kan pıhtılaşması ve kesim artıkları kaldırır ve son derece verimli beslenme difüzyon yolu yeniden açar bir cerrahi jet lavaj sistemi kullanarak kemik uç plakası yüzeyler permeabilize mümkünIVD merkezinde. Her iki tarafta vertebral kemik büyüme plaklarının varlığı kaçınılması gereken ve kültürüne önce kaldırılmalıdır. Bu video, hazırlanması sırasında çok önemli adımlar anahat ve yüksek hücre canlılığı sürdürmek için 14 gün ücretsiz şişme kültürün etkisi altında başarılı bir organ kültürü anahtarı göstermek. Uygun mekanik çevre mekanik yüklenme biyoreaktör kullanılarak korunabilir kültürü süresi uzatılabilir. Burada gösterilen tekniği, domuz, koyun ve tavşangillere kaudal ve lomber IVD izolasyon gibi diğer hayvan türlerinin uzatılabilir.

Watch this Scientific Journal Video about Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture at JoVE.com

Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

Bu protokol, organ kültürü için coccygeal sığır intervertebral diskler için hasat tekniği göstermektedir.<em&gt; In vitro</em&gt; Organ kültürü.

Organ Kültür bozulmamış Sığır Kuyruğu İntervertebral Disklerin Hazırlanması

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

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JoVE Journal

Bioengineering

14.4K Görüntüleme.  University of Bern. Bu protokol, organ kültürü için coccygeal sığır intervertebral diskler için hasat tekniği göstermektedir. In vitro Organ kültürü.

Video: Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

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

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

Yumurtalıklar ve fallop borularında Üç Boyutlu Organ Kültür Aljinatta Hidrojeller

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

Biyomühendislik

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

3D Fibrin hazırlanması Kök Hücre Kültürü Uygulamaları için iskeleleri

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

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

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

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

3D Hücre Kültürü Modelleri Kütle Spektrometre Görüntüleme Hazırlık Stratejileri Numune

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

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

Korelatif Mikroskopisi kullanılarak sağlam hücrelerde Epidermal Büyüme Faktörü Alıcısının stokiyometrisinden incelenmesi

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

bir<em&gt; İn Vitro</emKemirgen İntervertebral Disk&gt; Organ Kültürü Modeli

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

İmalat ve Organ-on-chip sistemin doğrudan Transendothelial elektrik direncini ölçmek için entegre elektrotlar ile doğrulama

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

Fragility Assessment of Bovine Cortical Bone Using Scratch Tests

Bone is a complex hierarchical material with five distinct levels of organization. Factors like aging and diseases like osteoporosis increase the fragility of bone, making it fracture-prone. Owing to the large socio-economic impact of bone fracture in our society, there is a need for novel ways to assess the mechanical performance of each hierarchical level of bone. Although stiffness and strength can be probed at all scales &#8211; nano-, micro-, meso-, and macroscopic &#8211; fracture assessment has so far been confined to macroscopic testing. This limitation restricts our understanding of bone fracture and constrains the scope of laboratory and clinical studies. In this research, we investigate the fracture resistance of bone from the microscopic to the mesoscopic length scales using micro scratch tests combined with nonlinear fracture mechanics. The tests are performed in the short longitudinal orientation on bovine cortical bone specimens. A meticulous experimental protocol is developed and a large number (102) of tests are conducted to assess the fracture toughness of cortical bone specimens while accounting for the heterogeneity associated with bone microstructure.

This study assesses the fracture toughness of bovine cortical bone at the sub-meso levels using microscopic scratch tests. This is an original, objective, rigorous, and reproducible method proposed to probe fracture toughness below the macroscopic scale. Potential applications are studying changes in bone fragility due to diseases like osteoporosis.

Kazıma testi kullanarak sığır kemik kırılganlığını değerlendirilmesi

Bone is a complex hierarchical material with five distinct levels of organization. Factors like aging and diseases like osteoporosis increase the ...

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human patients. Human Organ-on-a-Chip (Organ Chip) microfluidic cell culture devices, which provide an experimental in vitro platform to assess efficacy, toxicity, and pharmacokinetic (PK) profiles in humans, may be better predictors of therapeutic efficacy and safety in the clinic compared to animal studies. These devices may be used to model the function of virtually any organ type and can be fluidically linked through common endothelium-lined microchannels to perform in vitro studies on human organ-level and whole body-level physiology without having to conduct experiments on people. These Organ Chips consist of two perfused microfluidic channels separated by a permeable elastomeric membrane with organ-specific parenchymal cells on one side and microvascular endothelium on the other, which can be cyclically stretched to provide organ-specific mechanical cues (e.g., breathing motions in lung). This protocol details the fabrication of flexible, dual channel, Organ Chips through casting of parts using 3D printed molds, enabling combination of multiple casting and post-processing steps. Porous poly (dimethyl siloxane) (PDMS) membranes are cast with micrometer sized through-holes using silicon pillar arrays under compression. Fabrication and assembly of Organ Chips involves equipment and steps that can be implemented outside of a traditional cleanroom. This protocol provides researchers with access to Organ Chip technology for in vitro organ- and body-level studies in drug discovery, safety and efficacy testing, as well as mechanistic studies of fundamental biological processes.

Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro.

Ölçeklenebilir gerilebilir, Çift kanal, mikrosıvısal Organ cips imalatı

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human ...

Anterior Segment Organ Culture Platform for Tracking Open Globe Injuries and Therapeutic Performance

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and medical intervention in rural environments and military medicine applications where ophthalmic care is not readily available. Untreated injuries are susceptible to infection after the eye has lost its watertight seal, as well as loss of tissue viability due to intraocular hypotension. Therapeutics to temporarily seal open globe injuries, if properly developed, may be able to restore intraocular pressure and prevent infection until proper ophthalmic care is possible. To facilitate product development, detailed here is the use of an anterior segment organ culture open globe injury platform for tracking therapeutic performance for at least 72 h post-injury. Porcine anterior segment tissue can be maintained in custom-designed organ culture dishes and held at physiological intraocular pressure. Puncture injuries can be created with a pneumatic-powered system capable of generating injury sizes up to 4.5 mm in diameter, similar to military-relevant injury sizes. Loss of intraocular pressure can be observed for 72 h post-injury confirming proper injury induction and loss of the eye&#39;s watertight seal. Therapeutic performance can be tracked by application to the eye after injury induction and then tracking intraocular pressure for multiple days. Further, the anterior segment injury model is applicable to widely used methods for functionally and biologically tracking anterior segment physiology, such as assessing transparency, ocular mechanics, corneal epithelium health, and tissue viability. Overall, the method described here is a necessary next step toward developing biomaterial therapeutics for temporarily sealing open globe injuries when ophthalmic care is not readily available.

Open globe eye injuries may go untreated for multiple days in rural or military-relevant scenarios, resulting in blindness. Therapeutics are needed to minimize loss of vision. Here, we detail an organ culture open globe injury model. With this model, potential therapeutics for stabilizing these injuries can be properly evaluated.

Açık Küre Yaralanmalarını ve Terapötik Performansı İzlemek için Ön Segment Organ Kültürü Platformu

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and ...

Reconstituting Cytoarchitecture and Function of Human Epithelial Tissues on an Open-Top Organ-Chip

Nearly all human organs are lined with epithelial tissues, comprising one or multiple layers of tightly connected cells organized into three-dimensional (3D) structures. One of the main functions of epithelia is the formation of barriers that protect the underlining tissues against physical and chemical insults and infectious agents. In addition, epithelia mediate the transport of nutrients, hormones, and other signaling molecules, often creating biochemical gradients that guide cell positioning and compartmentalization within the organ. Owing to their central role in determining organ-structure and function, epithelia are important therapeutic targets for many human diseases that are not always captured by animal models. Besides the obvious species-to-species differences, conducting research studies on barrier function and transport properties of epithelia in animals is further compounded by the difficulty of accessing these tissues in a living system. While two-dimensional (2D) human cell cultures are useful for answering basic scientific questions, they often yield poor in vivo predictions. To overcome these limitations, in the last decade, a plethora of micro-engineered biomimetic platforms, known as organs-on-a-chip, have emerged as a promising alternative to traditional in vitro and animal testing. Here, we describe an Open-Top Organ-Chip (or Open-Top Chip), a platform designed for modeling organ-specific epithelial tissues, including skin, lungs, and the intestines. This chip offers new opportunities for reconstituting the multicellular architecture and function of epithelial tissues, including the capability to recreate a 3D stromal component by incorporating tissue-specific fibroblasts and endothelial cells within a mechanically active system. This Open-Top Chip provides an unprecedented tool for studying epithelial/mesenchymal and vascular interactions at multiple scales of resolution, from single cells to multi-layer tissue constructs, thus allowing molecular dissection of the intercellular crosstalk of epithelialized organs in health and disease.

The present protocol describes the capabilities and the essential culture modalities of the Open-Top Organ-Chip for the successful establishment and maturation of full-thickness organ-on-chip cultures of primary tissues (skin, alveolus, airway, and intestine), providing the opportunity to investigate different functional aspects of the human epithelial/mesenchymal and vascular niche interface in vitro.

İnsan Epitel Dokularının Üstü Açık Bir Organ Çipi Üzerindeki Sitomimarisinin ve İşlevinin Yeniden Yapılandırılması

Nearly all human organs are lined with epithelial tissues, comprising one or multiple layers of tightly connected cells organized into three-dimensional ...