Name: an in vitro organ culture model of the murine intervertebral disc
Uploaded: 2017-04-11T14:00:00.000+00:00
Duration: 8 min 3 s
Description: Watch this Scientific Journal Video about An In Vitro Organ Culture Model of the Murine Intervertebral Disc at JoVE.com

Bu çalışma Washington Üniversitesi Kas İskelet Araştırma Merkezi (NIH P30 AR057235), Moleküler Görüntüleme Merkezi (NIH P50 CA094056), mechanobiology Eğitim Grant (NIH 5T32EB018266), NIH R21AR069804 ve NIH K01AR069116 tarafından desteklenmiştir. Yazarlar veri toplamada katkılarından dolayı Patrick Wong teşekkür etmek istiyorum.

Tüm hayvan deneyleri Louis Hayvan Araştırmaları Komitesi Washington Üniversitesi ile uyumlu olarak gerçekleştirildi. 1. Hayvanlar <ol><li> (N = 6, BALB-E, BALB / cAnNTac) ve 10 haftalık nükleer faktör kappa-B-lusiferaz raportör hayvanları (NF-κβ-luc) bir yetiştirilen 10 haftalık BABL / c farelerinin iki soy elde edilir BALB / c arka plan (n = 6, BALB / c-Tg (Relà-luc) 31Xen). </li><li> Diseksiyon önce, zaman yaşayan ek bir 2 dakika, ardından 5 dakika boyunca 2.5-3 L / dk&#39;lık bir akış hızında CO2, aşırı dozda hayvanların euthanize. </li><li> Diseksiyon önce 2 dakika süreyle% 70 etanol banyosu içinde banyo ile hayvanın dış bölge dezenfekte. </li></ol> 2. Diseksiyon <ol><li> Vücut boşluğuna maruz küçük diseksiyon makas kullanılarak fare dorsal yüzeyi üzerinde bir uzunlamasına dikey kesim yapın. </li><li> başlayarak hayvanın omurganın her iki tarafında iki boyuna dikey kesimler yapınİlk omurga kaudal vertebra (L1) (C8). </li><li> neşter, ince forseps ve ince diseksiyon makas kullanarak dikkatli bir şekilde hayvanın vücut boşluğundan L1-C8 omurgaya çıkarın. </li><li> kazımak veya ventral tarafında IVD zarar değil sağlarken dikkatlice, omurga çevreleyen, yumuşak dokuları çıkarın. </li><li> Bundan başka, L1 / L2, L3 / L4, L5 / L6, C3 / C4, C5 / C6 ve C7 / C8 disklerinde vertebra-disk omurlar fonksiyonel omurga birimi (FSUs) içine omurga incelemek. </li><li> % 1 2 dakika boyunca penisilin-streptomisin ile takviye edilmiş Hank dengeli tuz çözeltisi içinde FSUs durulayın. </li><li> kültürlenmemiş ve işlenmemiş FSUs (Taze), kültürlenmiş ama tedavi edilmemiş FSUs (Kontrol) ve kültüre edildiği ve levha tedavi FSUs (Stab): rasgele üç gruba FSUs atar. </li><li> Ek bileşen hemen -20 ° C dondurucu içinde diseksiyon ve depo edilmesinden sonra sıvı azot ile taze ESB örnekleri dondurma. </li></ol> 3. Organ Kültür Koşulları<ol><li> steril kültür ortamı 500 mL Hazırlama 1: 1 Dulbecco&#39;nun modifiye edilmiş Eagle ortamı: Nutrient Mixture F12:% 20 fetal sığır serumu (FBS) ve% 1 penisilin-streptomisin ile takviye edilmiş (DMEM F12). </li><li> 24 kuyucuklu bir kültür plakasının her bir oyuğuna 10 ml serolojik pipetler, pipet kültür ortamı 2 ml kullanılması. </li><li> diseksiyon sonra ve durulama, ortam ile doldurulmuş, 24 oyuklu bir plaka içerisinde, bir birey haline her FSU yerleştirin. </li><li> 37 ° C,% 5 CO2,% 20 O2 ve&gt;% 90 nem muhafaza eden bir steril bir inkübatörde inkübe edin. </li><li> 24 saatlik bir başlangıç kültür döneminden sonra, mekanik olarak, Şekil 1A &#39;de gösterildiği gibi, steril bir 27 gauge iğne ile annulus fibrosus iğne delinmesi ile delinmeye grubu örnekleri zarar. </li><li> Yeni kullanılarak steril cımbız eski plakadan yeni bir ortam ile doldurulmuş 24 oyuklu bir kültür plakası hazırlanarak Ortam 48 saatte bir değiştirme ve aktarım örnekleri. Eski medya ve dispos aspireBir biyolojik tehlike atık kutusuna eski kültür plakasının e. </li><li> kültürün son gününde, 24 saat süre ile 0.75 mg / ml nitro mavi tetrazolyum klorür ihtiva eden ortam içinde tüm örnekleri inkübe edin. </li><li> Daha sonra, dondurarak tüm FSUs mekanik test veya histoloji (Şekil 1B) için hazır olana kadar -20 ° C dondurucu içinde sıvı azot ve mağaza ile. </li></ol> 4. Uzunlamasına Ölçümler NF-KB <ol><li> Görüntü 1 de NF-κβ-lusiferaz hayvanlardan FSUs, 5, 13 ve 19 gün NF-KB ifadesini değerlendirmek için. </li><li> 10 dakika boyunca 37 ° C&#39;de steril inkübatörde her 1 mg / ml lusiferin çözeltisinin 10 uL de ekleyin ve inkübe edilir. </li><li> Görüntü bir 1 dk maruziyet süresi ve 2 bin ayarıyla görüntüleme sistemi kullanılarak biyolüminesans için örnekler. </li><li> Eşzamanlı olarak, bir fotoğrafını çekip IVD içinde lüminesans anatomik yerini belirlemek için biyolüminesans görüntüsü ile bindirmek için makineyi kullanın. </li></ol><pclass = &quot;jove_title&quot;&gt; 5. Mekanik Değerlendirme <ol><li> Deplasman kontrollü dinamik sıkıştırma 9 kullanarak IVD&#39;ler mekanik davranışını belirler. </li><li> sağlam ve IVD&#39;nin bağlı kıkırdak uç plaka tutarken bir diseksiyon mikroskobu, neşter, ve cımbız kullanarak, her bir numuneden, eski Sovyetler kemik omur gövdelerinin çıkarın. </li><li> siyanoakrilat yapıştırıcı ile, bir 1 cm x 1 cm x 0,2 cm alüminyum levhaya izole IVD&#39;ler takın. </li><li> bir lazer mikrometre kullanılarak her diskin uzunlamasına ekseni üzerinde üç ölçümün ortalaması alınarak disk yüksekliğini ve genişliğini ölçün. maksimum disk genişliği ortalama disk yüksekliğine bölerek disk yükseklik oranını hesaplar. </li><li> mekanik test kullanılan girdi gerilme değerlerini hesaplamak için ölçülen disk yüksekliğini kullanın. Ortalama diski yüksekliği yaklaşık 690 um ± 39 um olduğu not edin. </li><li> compressi altında, bir fosfat tamponlu tuzlu su içinde numunelerMakinede 0.02 N&#39;ye disk önceden yüklenmesi </li><li> Siklik 3 denemeleri için 20 1% tür düzeyinde döngüleri ve% 5 suşu seviyesi için 1 Hz, her bir sinüs dalga formu kullanılarak disk sıkıştırmak ve IVD&#39;nin Yük ve yer değiştirme değerlerini kaydedin. dinlenmek ve yaralanmaları önlemek için disk için çalışmalar arasındaki dinlenme zaman 10 dakika bekleyin. </li><li> Son çevrimin yükleme fazı ortalama sertlik hesaplayın ve Yük ve yer değiştirme verileri arasındaki faz açısı kayıp tanjantı hesaplar. </li></ol> 6. Proteoglikan ve Kolajen Niceleme <ol><li> mekanik test sonra, bir ön-darası alınmış santrifüj tüpüne izole IVD yerleştirilmesi ve bir analitik terazi kullanılarak ağırlığının ölçülmesi disk ıslak ağırlık ölçer. </li><li> 250 uL papain sindirim çözeltisi izole IVD&#39;ler Digest (0,1 M sodyum asetat, 0.01 M EDTA, 0.005 M sistein HCI, pH 6.4) bir blok ısıtıcı kullanılarak bir gece boyunca 65 ° C&#39;de karıştırıldı. </li><li> 10 dakika süre ile santrifüj örnekleri2.000 x g&#39;de yüzeydeki sıvı toplamak ve. </li><li> kondroitin sülfat standartlarına dimetilmetilen mavisi (DMMB) analizi kullanılarak IVD proteoglikan içeriği ölçülür. </li><li> DMMB çözeltisi (21 mg, DMMB, 5 ml mutlak etanol, 1 L GKD 2 O 2 g sodyum format) hazırlayın. </li><li> Pipet her yuva içinde, DMMB çözeltisi 250 uL ile birlikte 96 oyuklu bir plaka içerisinde, üç kopya halinde 30 standartları uL ve örnekler. </li><li> Bir spektrofotometre kullanılarak 525 nm&#39;de plakanın emilmesini okuyun. </li><li> Üretici talimatlarına uygun olarak bir hidroksiprolin tahlili kiti kullanılarak kolajen içeriği ölçün. </li></ol> 7. Histoloji <ol><li> , 24 saat süresince% 4 paraformaldehit içinde numunelerinin bir alt saptamak 48 saat,% 5 formik asit içinde de-kalsifiye, etanol ile kurutmak, ve parafin içine yerleştirin. </li><li> sajital 10 um kalınlığında bölüm, numune bir mikrotom kullanılarak ve cam slaytlara bölümleri uygulanır. Safranin-O / Hızlı Gr kullanılarak bölümleri Lekeeen ve DAPI. </li><li> Bir ışık mikroskobu kullanılarak, görüntü 10X büyütmede kayar. </li></ol> 8. Kontrastlı microComputed Tomografi (mikroBT) <ol><li> 7 günlük bir zaman noktasında boylamasına iyoversol kontrastlı 10 ve terminal fosfomolibdik asit (PMA) kontrast iyileştirme sistemi kullanılarak, 0, 2, 5 ve 7 günlük zaman noktalarında numuneler bir alt tarama. </li><li> mikroBT tarama süresi öncesinde 4 saat, 4, 37 ° C&#39;de steril bir inkübatörde 350 mg / yaklaşık 50 mg / mL iyot içeren iyoversol solüsyonunun, nihai konsantrasyonu için kültür ortamına ml iyodin iyoversol çözeltisi 300 uL ekleyebilir ve inkübe h. </li><li> İnkübasyondan sonra, 1 mL mikrosantrifüj tüpü içinde, steril bir gazlı bez ve yerine örnek sarın. </li><li> mikroBT sisteminde yer örnekler ve 45 keVp, 177 uA, 10.5 um voksel boyutu, 300 ms, entegrasyon tarama. </li><li> İhracat DICOM dosyaları olarak mikroBT gelen veriler ve yazılımlar kullanılarak görselleştirmek ( <em&gt; örneğin OsiriX). </li><li> 7 günlük bir zaman noktasında, 3 gün süre ile% 5 PMA çözeltisi, ardından 24 saat boyunca bir% 4 paraformaldehid çözeltisi kullanılarak örnekleri saptamak ve adım 8.4 kullanılanlar ile aynı ayarları kullanarak tarama. </li></ol> 9. Üç Boyutlu Sonlu Eleman Model <ol><li> Segment manuel eşikleme (örneğin OsiriX) ile yazılımı kullanarak IVD&#39;ler DICOM dosyaları. </li><li> Meshlab kullanarak voksel tabanlı sonlu elemanlar kafes içine IVD NP ve AF hacimleri dönüştürün. </li><li> Tam bir IVD oluşturacak ve sonlu elemanlar yazılımında deneysel olarak belirlenen sınır koşulları kullanılarak NP ve AF mikro yapılarını birleştirin. </li></ol>

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Bu protokol IVD biyolojik değişikliklerin izlenmesi üzerinde durularak fare FSU bir organdır kültürünü özetliyor. Bu kültürlerin başarılı bakım dikkatli steril teknikler gerektirir. Özellikle, diseksiyon 2.1-2.6 adımları ve Kültür 3.1-3.6&#39;da steril koşulların korunmasını sağlamak için özel dikkat gerektirir adım, ve bu aşamalar kirleticiler en aza indirmek için bir HEPA hava akışı ile izole edilmiş bir uygulama odasında tercihen yapılmalıdır. Dis...

Bel ağrısı (BA) işyerinde küresel sakatlık için önde gelen faktör ve kayıp verimlilik ve Amerikalılar yalnız LBP tedavisi 1 50 milyar doların üzerinde harcama. yaygın olmakla birlikte, bel ağrısı etiyolojisi kompleks ve çok kalır. Bununla birlikte, intervertebral disk (IVD) dejenerasyon LBP 2 için en önemli risk faktörleri arasında yer almaktadır. IVD üç mikroyapıların meydana gelir: dış halka fibroz (AF), iç çekirdeği pulposus (NP) ve proksimal ve distal olarak 3 bütün yapı sandviç iki kıkırdak omurun. Yaşlanma ve dejenerasyon ile IVD bileşenleri kompozisyonel yapısal değişiklik ve mekanik 4. Bu değişiklikler, NP proteoglikanlar ve hidrasyon kaybı gibi, disk yüksekliği azalması ve mekanik yetkinlik 5 bozulmuştur. Bu değişiklikler şunlardırgenellikle LBP semptomları 6 yol edilen bir olay grubuyla sonuçlanan eklem yeri boşluğuna bir enflamatuvar yanıtı ve aynı zamanda nötrofil ve dorsal kök ganglion girmesini teşvik sitokinler ile birlikte. bel ağrısı verilmeden önceki dejenerasyon nedenini ortadan kaldırmak için çoğu zaman mümkün değildir, çünkü IVD dejenerasyon mekanizmalarının incelenmesi insanlarda zordur. Bu nedenle, IVD organa aşağı deneysel sistem basitleştirilmesi indirgemeci yaklaşım nedensel değişkenlerin mekanik ince ayarlanmasına izin verir ve bunların bir aşağı akış etkisine 5 incelenmesi. Sistem böylece IVD dejenerasyonu dış uyaranların etkileri doğrudan yorumlanmasını sağlar, ancak doğal hücre nüfusu ve hücre-dışı matrisi çevreleyen, indirgenir. Ayrıca, daha az maliyet ve fare modellerinin ölçeklenebilirlik, aynı zamanda, genetik olarak tadil edilmiş hayvanlar 7 çok sayıda, t izinIVD dejeneratif mekanizmalarla ve potansiyel terapilerin o hızla hedefe yönelik tarama. Burada, IVD hücre ve doku stabilitesi IVD&#39;ler, homeostatik mekanik, yapısal ve inflamatuar desen verilen belirli bir odak ile, 21 gün boyunca muhafaza edildiği bir murin organ kültürü sistemi tarif eder. Bu yöntemi kullanarak, disk dejenerasyonu altında yatan mekanizmaları anlamak için bir bıçak kaynaklı yaralanma modelinde 8 IVD&#39;ler fonksiyonel değişiklikleri izler. Ayrıca, kontrastlı microCT görüntüleme ve IVD üç boyutlu yüksek çözünürlüklü modelleme dahil başka çalışmalar yapmak için bu organ kültürü yönteminin çeşitli uygulamaları açıklar.

<table><tbody><tr><td>96 well plate</td><td>Midwest Scientific</td><td>TP92096</td><td>Used for biochemical assays</td></tr><tr><td>24 well plate</td><td>Midwest Scientific</td><td>TP92024</td><td>Used for organ culture</td></tr><tr><td>25 mL pipettes</td><td>Midwest Scientific</td><td>TP94024</td><td>Used for organ culture</td></tr><tr><td>10 mL pipettes</td><td>Midwest Scientific</td><td>TP94010</td><td>Used for organ culture</td></tr><tr><td>5 mL pipettes</td><td>Midwest Scientific</td><td>TP94005</td><td>Used for organ culture</td></tr><tr><td>500 mL bottle top filters, 22 &amp;micro;m</td><td>Midwest Scientific</td><td>TP99505</td><td>Used for filter media</td></tr><tr><td>10 &amp;micro;L pipette tips</td><td>Midwest Scientific</td><td>NP89140098</td><td>Used for biochemical assays</td></tr><tr><td>200 &amp;micro;L pipette tips</td><td>Midwest Scientific</td><td>NP89140900</td><td>Used for biochemical assays</td></tr><tr><td>1,000 &amp;micro;L pipette tips</td><td>Midwest Scientific</td><td>NP89140920</td><td>Used for biochemical assays</td></tr><tr><td>DMEM/F-12</td><td>Invitrogen</td><td>11330032</td><td>Used for culture media</td></tr><tr><td>Optiray 350</td><td>Guebert</td><td>19133341</td><td>Used for contrast enhanced microCT</td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma</td><td>F2442</td><td>Used for culture media</td></tr><tr><td>Penicillin Streptomycin&amp;nbsp;</td><td>Sigma</td><td>P4333</td><td>Used for culture media</td></tr><tr><td>Tetrazolium Blue Chloride</td><td>Sigma</td><td>T4375</td><td>Used for biochemical assays</td></tr><tr><td>D-Luciferin</td><td>Sigma</td><td>L6152</td><td>Used for bioluminescence imaging</td></tr><tr><td>Chondroitin Sulfate</td><td>Sigma</td><td>C9819</td><td>Used for biochemical assays</td></tr><tr><td>10% Phosphomolybdic Acid Solution</td><td>Sigma</td><td>HT152</td><td>Used for contrast enhanced microCT</td></tr><tr><td>Safranin O</td><td>Sigma</td><td>S8884</td><td>diluted to .1% concentration (in water)</td></tr><tr><td>Fast Green FCF</td><td>Sigma</td><td>F7258</td><td>.001% concentration</td></tr><tr><td>Papain from papaya latex</td><td>Sigma&amp;nbsp;</td><td>P3125</td><td>Used for biochemical assays</td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9542</td><td>Nucleic acid staining</td></tr><tr><td>Cyanoacrylate Glue</td><td>Loctite</td><td>234790</td><td>Adhesive&amp;nbsp;</td></tr><tr><td>1.5 mL Microcentrifuge Tubes</td><td>Fischer Scientific</td><td>S348903</td><td>Used for biochemical assays</td></tr><tr><td>&lt;strong&gt;Big Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BioDent</td><td>ActiveLife</td><td></td><td>For mechanical testing</td></tr><tr><td>Cytation 5</td><td>Biotek</td><td></td><td>Spectrophotometer</td></tr><tr><td>AxioCam503</td><td>Carl Zeiss AG</td><td></td><td>Microscope</td></tr><tr><td>VivaCT40</td><td>Scanco</td><td></td><td>MicroCT</td></tr><tr><td>Analytical balance</td><td>Denver Instrument Company</td><td>A-200DS</td><td>Analytical balance</td></tr><tr><td>Incubator HERAcell 150i</td><td>Thermo Scientific</td><td></td><td>Organ Culture</td></tr><tr><td>Dissection Scope</td><td>VistaVision</td><td></td><td>Used during dissection</td></tr><tr><td>Laser Micrometer</td><td>Keyence</td><td>LK-081</td><td>Measuring disc height</td></tr><tr><td>Microcentrifuge 5810 R</td><td>Eppendorf</td><td></td><td>Used for biochemical assays</td></tr><tr><td>Microtome</td><td>Leica&amp;nbsp;</td><td>RM2255</td><td>Used for histology</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Prism 7</td><td>GraphPad</td><td></td><td>For statistics</td></tr><tr><td>MATLAB R2014a</td><td>Mathworks</td><td></td><td>For modeling</td></tr><tr><td>Osiri-LXIV</td><td>Pixmeo</td><td></td><td>Open Source</td></tr><tr><td>MeshLab v1.3.3</td><td>Visual Computing Lab - ISTI - CNR</td><td></td><td>Open Source</td></tr><tr><td>PreView/FEBio 2.3</td><td>Utah MRL &amp;amp; Columbia MBL</td><td></td><td>Open Source</td></tr><tr><td>ImageJ</td><td>NIH</td><td></td><td></td></tr><tr><td>Microsoft Excel</td><td>Windows</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Dissection Tools&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cohan-Vannas Spring Scissors&amp;nbsp;</td><td>Fine Science Tools&amp;nbsp;&amp;nbsp;</td><td>15000-02</td><td>Or any nice pair of spring scissors</td></tr><tr><td>Fine Scissors - Sharp&amp;nbsp; (small)</td><td>Fine Science Tools&amp;nbsp;&amp;nbsp;</td><td>14060-09</td><td></td></tr><tr><td>Fine Scissors - Sharp&amp;nbsp; (larger)</td><td>Fine Science Tools&amp;nbsp;&amp;nbsp;</td><td>14060-11</td><td></td></tr><tr><td>Dumont #5 Forceps</td><td>Fine Science Tools&amp;nbsp;&amp;nbsp;</td><td>11252-40</td><td>At least 2; can also use #3&amp;nbsp;</td></tr><tr><td>Extra Fine Graefe Forceps, serrated</td><td>Fine Science Tools&amp;nbsp;&amp;nbsp;</td><td>11150-10</td><td>At least 2</td></tr><tr><td>Micro-Adson Forceps, serrated</td><td>World Precision Instruments</td><td>503719-12</td><td></td></tr><tr><td>Micro-Adson Forceps, teeth</td><td>World Precision Instruments</td><td>501244</td><td></td></tr><tr><td>Scalpel Handle - #3</td><td>Fine Science Tools&amp;nbsp;&amp;nbsp;</td><td>10003-12</td><td></td></tr><tr><td>Scalpel Handle - #4</td><td>Fine Science Tools&amp;nbsp;&amp;nbsp;</td><td>10004-13</td><td></td></tr><tr><td>Scalpel Blades - #23</td><td>Fine Science Tools&amp;nbsp;&amp;nbsp;</td><td>10023-00</td><td></td></tr><tr><td>Insect Pins, size 000</td><td>Fine Science Tools</td><td>26000-25</td><td></td></tr><tr><td>27 G Needle</td><td>BD PrecisionGlide Needles</td><td>BD305109</td><td></td></tr></tbody></table>

an in vitro organ culture model of the murine intervertebral disc

İntervertebral disk (IVD) dejenerasyonu, bel ağrısı için önemli bir katkıda bulunmaktadır. IVD iletmek ve omurgasında yükleri azaltacak hizmet veren bir fibrokartilajinöz eklemdir. IVD proteoglikandan zengin nucleus (NP) ve kıkırdak uç plakaları arasında kalan bir kolajen zengin halka fibroz (AF) oluşur. Birlikte komşu vertebralar ile omurlar-IVD yapısı fonksiyonel omurga birimi (ESB) oluşturmaktadır. Bu mikro benzersiz hücre türleri yanı sıra özgün hücre dışı matrisler içerir. FSU Whole organ kültür doğal hücre-dışı matrisi, hücre farklılaşması fenotipleri ve hücresel-matriks ilişkilerini kontrol korur. Bu nedenle, organ kültürü teknikleri IVD karmaşık biyolojik mekanizmaları araştırmak için yararlıdır. Burada, İVG hastalık mekanizmalarını ve terapileri eğitimi için ideal bir platform sağlar bütün bel fare FSUs yetiştirmek için kullanılan bir yüksek verimli bir yaklaşım açıklar. Ayrıca, s tarifkontrastlı microCT görüntüleme ve IVD üç boyutlu yüksek çözünürlüklü sonlu elemanlar modelleme dahil başka çalışmalar yapmak için bu organ kültürü yöntemi kullanan everal uygulamalar.

Şekiller 2-3 kültürlenmiş fare IVD&#39;ler için proteoglikan dağılımı, NF-KB ekspresyonu, sertlik, viskozite, disk yüksekliği ve yaş ağırlık temsili sonuçlar göstermektedir. Düzgün kültürlendirilmiş, Denetim grubunun IVD parametreleri Taze grubundan önemli ölçüde farklı olmamalıdır. Kültür tehlikeye aksi enfekte edilmiş veya ise, kontrol grubu, özellikle NF-KB ekspresyonu ve proteoglikan dağıtımında, taze grubundan farklı olacaktır (sonuçlar gösterilmemiştir). I...

Watch this Scientific Journal Video about An In Vitro Organ Culture Model of the Murine Intervertebral Disc at JoVE.com

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

intervertebral disk (IVD) Whole organ kültür doğal hücre-dışı matrisi, hücre fenotipleri ve hücresel-matriks ilişkilerini kontrol korur. Burada fare bel ve bunların fonksiyonel spinal birimlerinde kaudal IVD&#39;ler ve bu sistemi kullanan birçok uygulamaları kullanarak bir IVD kültür sistemi açıklanmaktadır.

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 ...

an-in-vitro-organ-culture-model-of-the-murine-intervertebral-disc

Research

JoVE Journal

Bioengineering

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

9.4K Görüntüleme.  Washington University in St. Louis.  intervertebral disk (IVD) Whole organ kültür doğal hücre-dışı matrisi, hücre fenotipleri ve hücresel-matriks ilişkilerini kontrol korur. Burada fare bel ve bunların fonksiyonel spinal birimlerinde kaudal IVD'ler ve bu sistemi kullanan birçok uygulamaları kullanarak bir IVD kültür sistemi açıklanmaktadır. 

Video: An In Vitro Organ Culture Model of the Murine Intervertebral Disc

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 Intact Bovine Tail Intervertebral Discs for Organ Culture

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

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

Organ Kültür bozulmamış Sığır Kuyruğu İntervertebral Disklerin Hazırlanması

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

Tissue Engineering of the Intestine in a Murine Model

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to preoperative weights within 40 days.1 In humans, massive small bowel resection can result in short bowel syndrome, a functional malabsorptive state that confers significant morbidity, mortality, and healthcare costs including parenteral nutrition dependence, liver failure and cirrhosis, and the need for multivisceral organ transplantation.2 In this paper, we describe and document our protocol for creating tissue-engineered intestine in a mouse model with a multicellular organoid units-on-scaffold approach. Organoid units are multicellular aggregates derived from the intestine that contain both mucosal and mesenchymal elements,3 the relationship between which preserves the intestinal stem cell niche.4 In ongoing and future research, the transition of our technique into the mouse will allow for investigation of the processes involved during TESI formation by utilizing the transgenic tools available in this species.5The availability of immunocompromised mouse strains will also permit us to apply the technique to human intestinal tissue and optimize the formation of human TESI as a mouse xenograft before its transition into humans. Our method employs good manufacturing practice (GMP) reagents and materials that have already been approved for use in human patients, and therefore offers a significant advantage over approaches that rely upon decellularized animal tissues. The ultimate goal of this method is its translation to humans as a regenerative medicine therapeutic strategy for short bowel syndrome.

This article and the accompanying video present our protocol for generating tissue-engineered intestine in the mouse, using an organoid units-on-scaffold approach.

Bir fare modelinde Bağırsak Doku Mühendisliği

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to model well differentiated human airway epithelium and heart muscle cells. This could be an invaluable tool to study the deleterious effects of toxic inhaled chemicals, such as chlorine, that can normally interact with the cell surfaces, and form various byproducts upon reacting with water, and limiting their effects in submerged cultures. Our model using well differentiated human airway epithelial cell cultures at air-liqiuid interface circumvents this limitation as well as provides an opportunity to evaluate critical mechanisms of toxicity of potential poisonous inhaled chemicals. We describe enhanced loss of membrane integrity, caspase release and death upon toxic inhaled chemical such as chlorine exposure. In this article, we propose methods to model chlorine exposure in mammalian heart and airway epithelial cells in culture and simple tests to evaluate its effect on these cell types.

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

This protocol is designed to demonstrate exposure method of cell cultures to inhaled toxic chemicals. Exposure of differentiated air-liquid&#160;interface (ALI) cultures of airway epithelial cells provides a unique model of airway exposure to toxic gases such as chlorine. In this manuscript we describe effect of chlorine exposure on air-liquid&#160;interface cultures of epithelial cells and submerged culture of cardiomyocytes. In vitro exposure systems allow important mechanistic studies to evaluate pathways that could then be utilized to develop novel therapeutic agents.

Zehirli İnhale Kimyasal Test in vitro Hücre Kültürü Modeli

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

Yeniden İnşa İnsan Kornea gibi Epitelyal (RHCE) Doku Modeli kullanılarak Göz tahriş Kimyasalların Tehlike Tanımlaması Göz tahrişi Testi (EIT)

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Ovine Lumbar Intervertebral Disc Degeneration Model Utilizing a Lateral Retroperitoneal Drill Bit Injury

Intervertebral disc degeneration is a significant contributor to the development of back pain and the leading cause of disability worldwide. Numerous animal models of intervertebral disc degeneration have been developed. The ideal animal model should closely mimic the human intervertebral disc with regard to morphology, biomechanical properties and the absence of notochordal cells. The sheep lumbar intervertebral disc model fulfils these criteria. We present an ovine model of intervertebral disc degeneration utilizing a drill bit injury through a lateral retroperitoneal approach. The lateral approach significantly reduces the incision and potential morbidity associated with the traditional anterior approach to the ovine spine. Utilization of a drill-bit method of injury affords the ability to produce a consistent and reproducible injury, of precise dimensions, that initiates a consistent degree of intervertebral disc degeneration. The focal nature of the annular and nucleus pulposus defect more closely mimics the clinical condition of focal intervertebral disc herniation. Sheep recover rapidly following this procedure and are typically mobile and eating within the hour. Intervertebral disc degeneration ensues and sheep undergo necropsy and subsequent analysis at periods from eight weeks. We believe that the drill bit injury model of intervertebral disc degeneration offers advantages over more conventional annular injury models.

Intervertebral disc degeneration is a significant contributor to back pain and a leading cause of disability worldwide. Numerous animal models of intervertebral disc degeneration exist. We demonstrate an ovine model of intervertebral disc degeneration, utilizing a drill bit, which achieves a consistent disc injury and reproducible level of disc degeneration.

Yanal Retroperitoneal Matkap Ucu Yaralanmasını Kullanan Koyun Lomber İntervertebral Disk Dejenerasyon Modeli

Intervertebral disc degeneration is a significant contributor to the development of back pain and the leading cause of disability worldwide. Numerous ...

Tıp

A Proinflammatory, Degenerative Organ Culture Model to Simulate Early-Stage Intervertebral Disc Disease.

Symptomatic intervertebral disc (IVD) degeneration (IDD) is a major socioeconomic burden and is characterized by inflammation and tissue degradation. Due to the lack of causative therapies, there is an urgent need for innovative experimental organ culture models to study the mechanisms involved in the progression of the disease, find therapeutic targets, and reduce the need for animal models. We here present a novel, three-dimensional organ culture model protocol mimicking the proinflammatory and catabolic microenvironment, which is present during IDD. 
Initially, bovine caudal IVDs were dissected, cleaned, and cultured in the tissue culture medium. Dynamic physiologic or pathologic loading was applied in a custom-made bioreactor for 2 hours per day. IVDs were assigned to a control group (high glucose medium, physiological loading, phosphate-buffered saline injection) and a pathological group (low glucose medium, pathological loading, tumor necrosis factor-alpha injection) for four days. Gene expression analysis from collected nucleus pulposus cells of the IVDs and enzyme-linked immunosorbent assay of the conditioned organ culture media was performed. 
Our data revealed a higher expression of inflammatory markers and reduced disc heights after loading in the pathological group compared to the control group. This protocol is reliable to simulate IVD inflammation and degeneration and can be further expanded to broaden its application scope.

This protocol presents a novel experimental model of proinflammatory, degenerative bovine organ culture to simulate early-stage intervertebral disc degeneration.

Erken Evre Intervertebral Disk Hastalığını Simüle Etmek için Proinflamatuar, Dejeneratif Organ Kültürü Modeli.

Symptomatic intervertebral disc (IVD) degeneration (IDD) is a major socioeconomic burden and is characterized by inflammation and tissue degradation. Due ...

Optical Sectioning and Visualization of the Intervertebral Disc from Embryonic Development to Degeneration

Intervertebral disc (IVD) degeneration is a leading cause of low back pain and it entails a high degree of impairment for the affected individuals. To decode disc degeneration and to be able to develop regenerative approaches a thorough understanding of the cellular biology of the IVD is essential. One aspect of this biology that still remains unanswered is the question of how cells are spatially arranged in a physiological state and during degeneration. The biological properties of the IVD and its availability make this tissue difficult to analyze. The present study investigates spatial chondrocyte organization in the anulus fibrosus from early embryonic development to end-stage degeneration. An optical sectioning method (Apotome) is applied to perform high resolution staining analyses using bovine embryonic tissue as an animal model and human disc tissue obtained from patients undergoing spine surgery. From a very high chondrocyte density in the early embryonic bovine disc, the number of cells decreases during gestation, growth, and maturation. In human discs, an increase in cellular density accompanied the progression of tissue degeneration. As had already been demonstrated in articular cartilage, cluster formation represents a characteristic feature of advanced disc degeneration.

We present a method to investigate spatial chondrocyte organization in the anulus fibrosus of the intervertebral disc using an optical sectioning method.

Embriyonik Gelişimden Dejenerasyona Kadar Intervertebral Diskin Optik Kesitlenmesi ve Görselleştirilmesi

Intervertebral disc (IVD) degeneration is a leading cause of low back pain and it entails a high degree of impairment for the affected individuals. To ...

Biyoloji

Generating a Pro-Inflammatory Organ Culture Model to Simulate an Early Intervertebral Disc Disease

Source: Saravi, B., et al. <a href="https://app.jove.com/v/62100">A Proinflammatory, Degenerative Organ Culture Model to Simulate Early-Stage Intervertebral Disc Disease.</a> J. Vis. Exp. (2021).
This video demonstrates a technique for culturing bovine intervertebral discs in bioreactors to develop a three-dimensional organ culture model. Specific nutrient-deprived media and degenerative pressure conditions are applied to the discs, mimicking the pro-inflammatory and degenerative microenvironment observed during early-stage intervertebral disc disease. The disc heights are measured post-treatment to analyze the effects of nutrient deprivation and degenerative loading.

Erken İntervertebral Disk Hastalığını Simüle Etmek için Bir Proinflamatuar Organ Kültürü Modeli Oluşturma

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Dissection of the bovine ...

JoVE Encyclopedia of Experiments

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Generating an Organ Culture Model to Simulate Early-Stage Intervertebral Disc Disease

Source: Saravi, B. et al., <a href="https://app.jove.com/v/62100">A Proinflammatory, Degenerative Organ Culture Model to Simulate Early-Stage Intervertebral Disc Disease.</a>&#160;J. Vis. Exp. (2021).
This video demonstrates the development of a pro-inflammatory and degenerative organ culture model for early-stage intervertebral disc (IVD) disease using TNF-&#945;. It outlines the procedure for injecting TNF-&#945; into the intervertebral disc and explains the mechanisms through which TNF-&#945; induces inflammation and degradation of the IVD.

Source: Saravi, B. et al., A Proinflammatory, Degenerative Organ Culture Model to Simulate Early-Stage Intervertebral Disc Disease.&#160;J. Vis. Exp. ...

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