Biz Robin Nye (Tufts Tıp Merkezi), Tomoya Uchimura ve Dana Cairns (Tufts Üniversitesi), sinoviyal sıvı depolama ve santrifüj ile yardım sağlamak için teşekkür etmek istiyorum. Bu çalışma LZ (1R01AR059106-01A1) NIH tarafından finanse edildi

Sinoviyal sıvı, kültür, insan eklem kondrosit için bir 3D sistemi Bu çalışmada, modifiye edilmiş bir imalat önerilen kapsülleme protokolü (Lonza, ve 31) kullanılarak aljinat boncuk insan eklem kondrosit kapsüllü. Bu 3D yapıları kullanarak, çeşitli yüzdeler insan sinovyal sıvı içeren bir kültür ortamı kültür hücreleri için bir sistem geliştirdik ve kıkırdak gen ekspresyonu için bu 3 boyutlu yapıları değerlendirilir. 1. Üç boyutlu (3D) kapsülleme için insan eklem kondrosit hazırlayın (HAC) <ol><li> Insan eklem kondrosit (HAC) (Lonza) (Passage 2) 37 ° C su banyosunda 1 dakika için bir şişe (1 ml) Çözülme. </li><li> Kondrosit büyüme ortam (Lonza) hücreleri toplamak için 3-5 dakika süreyle 3000 rpm&#39;de santrifüj 1 ml ile karıştırın. Süpernatantı atın. </li><li> Kondrosit büyüme ortamı yeniden süspanse hücre pelletini (Lonza). </li><li> 10 cm doku Levha hücrelerikültür plakasına (BD Biosciences), kondrosit büyüme ortamı (Lonza) kültür ve hücrelerin konfluent. kadar. HACs 3 veya 4 den daha yüksek (P3 veya P4) bir geçiş sayısı kullanılmalıdır. </li><li> Aljinat boncuk kapsülleme hücreleri toplamak için trypsinzation takip 155mm NaCl yerine standart PBS, plaka üzerinde HACs yıkayın. Hücreleri müstakil, 5dakika 3000rpm hücreler aşağı doğru döndürün. Hücreler artık 3D kapsülleme için hazır. </li></ol> 2. 3D boncuk HACs kapsülleyen 8x10 5 hücre / ml yoğunluğu% 1.2 aljinat çözeltisi (Sigma) HACs tekrar Hücre sayıları kullanarak, standart bir cep sayacı önce kapsülleme belirlendi. Bu çok önemli bir boncuk hücrelerin eşit dağılımını sağlamak için iyice karıştırın. <ol><li> Pipet 22-gauge iğne (Tyco sağlık, Inc.) Bağlı 12 ml enjektöre HAC / Aljinatta çözüm karışımı. </li><li> Bu arada, 50 ml beher 102 m hazırlamak.Bir ses seviyesinde M CaCl 2 5 kez HAC / aginate çözüm. Yerleştirin ve bir kabın içinde karıştırın çubuğunda (yaklaşık 150 rpm&#39;de) 102 mM CaCl 2 çözümü yavaş yavaş karıştırın . </li><li> CaCl 2 çözüm yüzeyinden yaklaşık 6 inç şırınga ucu tutun ve CaCl 2 çözüm damla damla içine HAC / aljinat karışımı ekleyin. Genel olarak, aljinat boncuk yaklaşık 10 4 hücre / boncuk bir kapsülleme yoğunluğu ile çapı 2mm. Not: CaCl 2 çözüm üzerinde şırınga yüksekliği önemlidir. Kısa mesafe düzensiz mekanik bütünlüğü ve hücre kapsülleme neden, küresel şekilli boncuk yerine şeklindeki gözyaşı damla neden olacaktır. </li><li> Boncuklar ilave edin, 20-25 dakika süreyle CaCl 2 çözüm olsun . Diğer aljinat encapsulation protokoller 10dk çok daha kısa bir inkübasyon süresi işaret ederken, biz CaCl 2 çözümü ile inkübasyon süresi daha uzun olabilir bütünlüğünü gelişmiş bulundu.reklamlar. </li><li> CaCl 2 çözüm çıkarın, kondrosit farklılaşmasını orta (Lonza) bir kez daha sonra 2-3 defa 2-3 hacim NaCl boncuk yıkayın ve. Aljinat boncuk encapsulation protokoller yukarıdaki yıkama işlemleri için bir filtre kullanarak ilgili olsa da, biz genellikle böylece encapsulation etkinliğini azaltan aljinat boncuklar, filtre ve kuru bir çıkış tuzak haline bulundu. Biz en verimli boncuk atılmadan önce beher alt çözüm yerleşmek için izin bulundu. </li><li> Boncuk kültür kaplarına transferi için standart bir spatula kullanın. Biz genellikle kültür kondrosit büyüme ortamında 2 gün 3 cm başına 12 boncuk kondrosit sürecinden önce sinovyal sıvı ile kondrosit farklılaşmasını orta (Lonza) geçiş yetiştiği ortamda ayarlamak için izin vermek. </li></ol> 3. Sinoviyal sıvı kültür ortamında Kültür kondrosit <ol><li> Taze sinoviyal sıvı bir o elde edilebilirutpatient kliniği (sinoviyal sıvı Tufts Tıp Merkezi&#39;nden elde edilen). Sinovyal sıvı laboratuvarda 15ml şahin tüpler ve hemen 15 dakika için 3000 rpm&#39;de santrifüj hücre enkaz kaldırmak için olabilir. 1,5 mL mikrosantrifüj tüpler içine kısım hücre ücretsiz sinovyal sıvı, donma-çözülme tekrarlanan önlemek için. Süpernatant -80 ° C kullanana kadar saklanabilir. </li><li> Önce kültür, sabit bir 100 mM askorbik asit konsantrasyonu ve 9 mM CaCl 2 (2 CaCl bu iz miktar aljinat bütünlüğünü sağlamak için, hacimsel oranları değişen kondrosit farklılaşması orta sinovyal sıvı karışımı (CDM, Lonza) boncuk gibi). </li><li> 37 ° C inkübatör içinde sallanan platform üzerinde plaka tutun (sallanan frekansı: yaklaşık 75 kez / dk) aljinat boncuklar içinde besin dağıtım yardımcı olmak ve boncuk topaklanma azaltmak için. Bu zaman uzun bir süre için (en fazla 4 hafta) kültürü bu kondrosit olarak özellikle önemlidir. NotKondrosit büyüme ve farklılaşma medya (Lonza) iki yaygın olarak kullanılan antibiyotik gentamisin ve amfoterisin içerirken kondrosit kültür için farklı yüzde sinovyal sıvı kullanıldığı zaman, herhangi bir antibiyotik takviyesi vermedi. No kirlenme hiç gözlendi. </li><li> Her 2-3 günde bir medya karışımlar değiştirin. Sinovyal sıvının viskozitesi, uzun vadeli kültürleri sırasında aljinat boncuk kapsüllü kültür kondrosit büyük bir engel olmuştur. Biz de aljinat boncuk bütünlüğünü güçlendirir 102mm CaCl 2 (% 50 V / V), sinoviyal sıvı takviye orta sulandırmak için en etkili bulundu. Bu şekilde, orta boncuklar zarar vermeden yavaş yavaş bir pipetlemeyin kaldırılır olabilir. </li></ol> 4. Gen ekspresyon analizi için aljinat boncuk Hasat HACs Gen ekspresyon analizi için aljinat boncuk HACs hasat zaman özel bir dikkat gerekir. <ol><li> Aljinat boncuk 3 ti yıkayınher zaman için yaklaşık 5 dakika 102 mM CaCl 2 mes. </li><li> 4-5 kez boncuk hacmi 55 mM NaCitrate ekleyerek kondrosit al. Aljinat boncuk NaCitrate çözümü tamamen dalmış olduğu önemlidir. 20-30 dakika sallayın veya kaya. </li><li> Hücrelerin Spin ve süpernatantı atın. </li><li> RT-PCR analizi için, hücre, hücre lizis tamponunda pelet (Qiagen RNA izolasyon kiti) ve RNA arıtma ile devam tekrar süspansiyon. </li></ol> 5. Histolojik analiz için aljinat boncuk HACs Fix Özel bakım, histolojik analiz için 3 boyutlu boncuk HACs hasat alınmalıdır. <ol><li> 5 dakika hakkında her zaman için 102 mM CaCl 2 aljinat boncuk, 3 kere yıkayın . Viskoz sinovyal sıvı tamamen uzak yıkamak için, biz, 37 ° C inkübatör (sallanan frekans de sallanan bir gecede kondrosit farklılaşmasını orta (Lonza) ile yıkamak için en etkili bulundu: yaklaşık 75 kezs / dak). Kondrosit büyüme ortamı, bu uzun süreli yıkama kondrosit hayatta kalmasını sağlamak için ve sabitleme ajanı maksimum penetrasyon sağlamak için kullanılır oldu. </li><li> Bir gecede% 70 etanol boncuk Fix ve histolojik analizi ile devam edin. DAPI boyama gerçekleştirmek için, nazik sallanan altında 1hr DAPI çözüm (500ng/ml) ile aljinat boncuk inkübasyon ve 102mm CaCl 2 15 dakika için her zaman 3 kere yıkayın. DAPI görüntüler daha sonra bir floresan mikroskop altında görülebilir. Boncuklar da alcian mavi ve H &amp; E boyama kesitli olabilir. </li></ol> 6. Temsilcisi Sonuçlar Sinovyal sıvıda yüksek oranda insan kondrosit 3D kültür yöntemi şematik diyagramı Şekil 1&#39;de gösterildiği gibi tasvir edilmiştir. Insan kondrosit aljinat boncuk kapsüllü sonra, onlar, sinovyal sıvı oranları değişen ile takviye ortamda büyümeye izin verildi. Çünkü sinovyal sıvının viskozitesi, esastırkültür kondrosit sürekli sallanan koşullar altında, kıkırdak yapıları topaklanma önlemek ve hatta besinlerin dağılımını sağlamak için. Bu sabitleme veya lizis tamponu boncuklar nüfuz edebilir (Şekil 1), kondrosit almadan önce yoğun aljinat boncuk yıkamak için de çok önemlidir. Aljinat boncuk kondrosit Parlak alan (BF) görüntüleri Şekil 2&#39;de gösterilmiştir. DAPI boyama, boncuk içinde hücrelerin usniform dağılımı (Şekil 2) onaylamak için yapıldı. 21 günlük kültür dönemi sonunda, gen ekspresyon analizi QRT-PCR tarafından yapıldı. Kondrosit 32 qPCR analizi için en güvenilir referans genlerin biri olarak tespit edilmiştir referans gen GAPDH, tüm PCR normalleşmesi için kullanılır oldu. Şekil bir örnek gösterilmiştir. 3, osteoartriti olan altı hastanın sinovyal sıvı toplanmış kültür insan eklem kondrosit sonuçları analiz edildi. Gerçeği ile de uyumlu olduğundan kondrositLonza, kaçınılmaz olarak de-farklılaşma 33 yol açacak 2B kültürleri, genişletilmiş var, biz o kıkırdak matriks genleri Gün 0 kondrosit minimal düzeyde (Şekil 3) olarak ifade edilmiştir. Sinovyal sıvı ile takviye kondrosit farklılaşmasını orta (Lonza) ya da orta kondrosit 3D kültür kıkırdak kollajen, aggrecan ve MMP13, Day 21 (Şekil 3) 34-farklılaşma kondrosit gösterir gen ekspresyonu önemli ölçüde arttı . Medya sinovyal sıvı oranının arttırılması kıkırdak matriks belirteçler kollajen II ve aggrecan mRNA (Fig.3A 3B) karşılaştırılabilir düzeyde sonuçlandı. Ayrıca, orta yalnız (Fig.3C) kültürlü olanlar ile karşılaştırıldığında% 100 sinovyal sıvıda kültür kondrosit bile kıkırdak parçalayıcı enzim MMP13 mRNA bir düşüş sergiledi. İlginç bir şekilde, yavaş yavaş hücre ölümü göstergesi kaspaz 3 ekspresyon seviyesi düşündüren sinovyal sıvı artan oranları azalmıştır olduğunu synovial sıvı kültür azalmış apoptozis düzeyleri (Şekil 3D) yol açmıştır. Bu nedenle, 3 boyutlu bir ortamda sinovyal sıvıda yüksek düzeyde kültür insan eklem kondrosit uygun bir teknoloji olduğunu göstermektedir. Tablolar ve Şekiller <img alt="Şekil 1." src="/files/ftp_upload/3587/3587fig1.jpg" /> Şekil 1. 3D aljinat boncuk sinovyal sıvıda yüksek oranda insan eklem kondrosit kültürü yöntemi şematik. İlk olarak, kondrosit ve aljinat çözüm karıştırılır. CaCl 2 çözüm açılan akıllıca uygulandığında, kondrosit çapraz Ca-aljinat hidrojel boncuklar içinde hareketsiz. Bu 3 boyutlu yapıları, insan sinovyal sıvı oranları değişen kondrosit farklılaşmasını orta (Lonza) kültür. Kültür sallanan bir koşul altında 21 gün sonra, hücre içeren aljinat boncuk hücreleri gen alınacağı sonra yoğun yıkanırekspresyon analizi. <img alt="Şekil 2." src="/files/ftp_upload/3587/3587fig2.jpg" /> Şekil 2: Aydınlık alan (BF) ve insan eklem kondrosit DAPI görüntüler% 0,% 30,% 50,% 70 ve% 100 sinovyal sıvıda kapsüllü kültürler . Kondrosit tüm kültür koşullarında şeklinde küresel. Aydınlık alan (BF) ve DAPI Görüntüler kondrosit yeri ve dağıtım onaylamak için üst üste. Parçalar, büyütülmüş görüntüler. Ok uçları, BF ve DAPI boyama görüntüleri kondrosit ko. <img alt="Şekil 3." src="/files/ftp_upload/3587/3587fig3.jpg" /> Şekil 3 gün kapsüllü insan eklem kondrosit QRT-PCR analizi 0 (D0) sinovyal sıvı (SF) (% 0,% 30,% 50, 70 arasında değişen oranlar ile desteklenmiş orta kültür (D21) 21 gün sonra % ve% 100). Dört bağımsız örnekler sonuçları burada gösterilmiştir. GAPDH tüm PCR için dahili referans olarak kullanılmıştır. <strong&gt; A. Kollajen II mRNA B. Aggrecan mRNA C. MMP13 mRNA D. Kaspaz 3 mRNA. İstatistiksel anlamlılık Günü 0 örnekler ve% 0 Gün 21 örnek ve INSTAT&#39;ın yazılımı kullanarak% 100 sinoviyal sıvı (SF) kültür değerlendirildi. * Gösterir p &lt;0.05.

<ol>
	<li>Centers for Disease Control and P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projected+state-specific+increases+in+self-reported+doctor-diagnosed+arthritis+and+arthritis-attributable+activity+limitations--United+States,+2005-2030.">Projected state-specific increases in self-reported doctor-diagnosed arthritis and arthritis-attributable activity limitations--United States, 2005-2030.</a> MMWR. Morb. Mortal. Wkly. Rep. 56, 423-425 (2007).</li><li>Theis, K. A., Murphy, L., Hootman, J. M., Helmick, C. G., Yelin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+and+correlates+of+arthritis-attributable+work+limitation+in+the+US+population+among+persons+ages+18-64:+2002+National+Health+Interview+Survey+Data.">Prevalence and correlates of arthritis-attributable work limitation in the US population among persons ages 18-64: 2002 National Health Interview Survey Data.</a> Arthritis Rheum. 57, 355-363 (2007).</li><li>Chung, C., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+cartilage+tissue.">Engineering cartilage tissue.</a> Adv. Drug. Deliv. Rev. 60, 243-262 (2008).</li><li>Glowacki, J. <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+engineering+of+cartilage.">In vitro engineering of cartilage.</a> J. Rehabil. Res. Dev. 37, 171-177 (2000).</li><li>Chokalingam, K., Hunter, S. A., Gooch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D-In+vitro+Effects+of+Compression+and+Time+in+Culture+on+Aggregate+Modulus+and+on+Gene+Expression+and+Protein+content+of+Collagen+Type+II+in+Murine+Chondrocytes.">3D-In vitro Effects of Compression and Time in Culture on Aggregate Modulus and on Gene Expression and Protein content of Collagen Type II in Murine Chondrocytes.</a> Tissue Eng. Part A. , (2009).</li><li>Butler, D. L., Goldstein, S. A., Guilak, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+tissue+engineering:+the+role+of+biomechanics.">Functional tissue engineering: the role of biomechanics.</a> J. Biomech. Eng. 122, 570-575 (2000).</li><li>Goldring, M. B., Goldring, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoarthritis.">Osteoarthritis.</a> J. Cell. Physiol. 213, 626-634 (2007).</li><li>Zvaifler, N. J., Firestein, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokines+in+chronic+inflammatory+synovitis.">Cytokines in chronic inflammatory synovitis.</a> Scand. J. Rheumatol. Suppl. 76, 203-210 (1988).</li><li>Rhee, D. K., Marcelino, J., Baker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+secreted+glycoprotein+lubricin+protects+cartilage+surfaces+and+inhibits+synovial+cell+overgrowth.">The secreted glycoprotein lubricin protects cartilage surfaces and inhibits synovial cell overgrowth.</a> J. Clin. Invest. 115, 622-631 (2005).</li><li>Campo, G. M., Avenoso, A., Nastasi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronan+reduces+inflammation+in+experimental+arthritis+by+modulating+TLR-2+and+TLR-4+cartilage+expression.">Hyaluronan reduces inflammation in experimental arthritis by modulating TLR-2 and TLR-4 cartilage expression.</a> Biochim. Biophys. Acta. , (2011).</li><li>van de Lest, C. H., van den Hoogen, B. M., van Weeren, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loading-induced+changes+in+synovial+fluid+affect+cartilage+metabolism.">Loading-induced changes in synovial fluid affect cartilage metabolism.</a> Biorheology. 37, 45-55 (2000).</li><li>Saxne, T., Heinegard, D., Wollheim, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+arthritic+synovial+fluid+influences+proteoglycan+biosynthesis+and+degradation+in+organ+culture+of+bovine+nasal+cartilage.">Human arthritic synovial fluid influences proteoglycan biosynthesis and degradation in organ culture of bovine nasal cartilage.</a> Coll. Relat. Res. 8, 233-247 (1988).</li><li>Lee, D. A., Salih, V., Stockton, E. F., Stanton, J. S., Bentley, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+normal+synovial+fluid+on+the+metabolism+of+articular+chondrocytes+in+vitro.">Effect of normal synovial fluid on the metabolism of articular chondrocytes in vitro.</a> Clin. Orthop. Relat. Res. , 228-238 (1997).</li><li>Schalkwijk, J., Joosten, L. A., van den Berg, W. B., van de Putte, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondrocyte+nonresponsiveness+to+insulin-like+growth+factor+1+in+experimental+arthritis.">Chondrocyte nonresponsiveness to insulin-like growth factor 1 in experimental arthritis.</a> Arthritis Rheum. 32, 894-900 (1989).</li><li>Schalkwijk, J., Joosten, L. A., van den Berg, W. B., van Wyk, J. J., van de Putte, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin-like+growth+factor+stimulation+of+chondrocyte+proteoglycan+synthesis+by+human+synovial+fluid.">Insulin-like growth factor stimulation of chondrocyte proteoglycan synthesis by human synovial fluid.</a> Arthritis Rheum. 32, 66-71 (1989).</li><li>Joosten, L. A., Schalkwijk, J., van den Berg, W. B., van de Putte, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondrocyte+unresponsiveness+to+insulin-like+growth+factor-1.+A+novel+pathogenetic+mechanisms+for+cartilage+destruction+in+experimental+arthritis.">Chondrocyte unresponsiveness to insulin-like growth factor-1. A novel pathogenetic mechanisms for cartilage destruction in experimental arthritis.</a> Agents Actions. 26, 193-195 (1989).</li><li>Schuerwegh, A. J., Dombrecht, E. J., Stevens, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synovial+fluid+and+peripheral+blood+immune+complexes+of+patients+with+rheumatoid+arthritis+induce+apoptosis+in+cytokine-activated+chondrocytes.">Synovial fluid and peripheral blood immune complexes of patients with rheumatoid arthritis induce apoptosis in cytokine-activated chondrocytes.</a> Rheumatol. Int. 27, 901-909 (2007).</li><li>Hegewald, A. A., Ringe, J., Bartel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronic+acid+and+autologous+synovial+fluid+induce+chondrogenic+differentiation+of+equine+mesenchymal+stem+cells:+a+preliminary+study.">Hyaluronic acid and autologous synovial fluid induce chondrogenic differentiation of equine mesenchymal stem cells: a preliminary study.</a> Tissue Cell. 36, 431-438 (2004).</li><li>Xu, Q. R., Dong, Y. H., Chen, S. L., Bao, C. D., Du, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degeneration+of+normal+articular+cartilage+induced+by+late+phase+osteoarthritic+synovial+fluid+in+beagle+dogs.">Degeneration of normal articular cartilage induced by late phase osteoarthritic synovial fluid in beagle dogs.</a> Tissue Cell. 41, 13-22 (2009).</li><li>Kruger, J. P., Endres, M., Neumann, K., Haupl, T., Erggelet, C., Kaps, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondrogenic+differentiation+of+human+subchondral+progenitor+cells+is+impaired+by+rheumatoid+arthritis+synovial+fluid.">Chondrogenic differentiation of human subchondral progenitor cells is impaired by rheumatoid arthritis synovial fluid.</a> J. Orthop. Res. 28, 819-827 (2010).</li><li>Steinhagen, J., Bruns, J., Niggemeyer, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion+culture+system:+Synovial+fibroblasts+modulate+articular+chondrocyte+matrix+synthesis+in+vitro.">Perfusion culture system: Synovial fibroblasts modulate articular chondrocyte matrix synthesis in vitro.</a> Tissue Cell. 42, 151-157 (2010).</li><li>Yang, K. G., Saris, D. B., Verbout, A. J., Creemers, L. B., Dhert, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+synovial+fluid+from+injured+knee+joints+on+in+vitro+chondrogenesis.">The effect of synovial fluid from injured knee joints on in vitro chondrogenesis.</a> Tissue Eng. 12, 2957-2964 (2006).</li><li>Skoog, V., Widenfalk, B., Ohlsen, L., Wasteson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+growth+factors+and+synovial+fluid+on+chondrogenesis+in+perichondrium.">The effect of growth factors and synovial fluid on chondrogenesis in perichondrium.</a> Scand. J. Plast. Reconstr. Surg. Hand. Surg.. 24, 89-95 (1990).</li><li>Nuver-Zwart, I., Schalkwijk, J., Joosten, L. A., van den Berg, W. B., van de Putte, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+synovial+fluid+and+synovial+fluid+cells+on+chondrocyte+metabolism+in+short+term+tissue+culture.">Effects of synovial fluid and synovial fluid cells on chondrocyte metabolism in short term tissue culture.</a> J. Rheumatol. 15, 210-216 (1988).</li><li>Beekhuizen, M., Bastiaansen-Jenniskens, Y. M., Koevoet, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoarthritic+synovial+tissue+inhibits+proteoglycan+production+in+human+osteoarthritic+cartilage;+Establishment+and+characterisation+of+a+long-term+coculture.">Osteoarthritic synovial tissue inhibits proteoglycan production in human osteoarthritic cartilage; Establishment and characterisation of a long-term coculture.</a> Arthritis Rheum. , (2011).</li><li>Rodrigo, J. J., Steadman, J. R., Syftestad, G., Benton, H., Silliman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+human+knee+synovial+fluid+on+chondrogenesis+in+vitro.">Effects of human knee synovial fluid on chondrogenesis in vitro.</a> Am. J. Knee. Surg. 8, 124-129 (1995).</li><li>van den Hoogen, B. M., van de Lest, C. H., van Weeren, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loading-induced+changes+in+synovial+fluid+affect+cartilage+metabolism.">Loading-induced changes in synovial fluid affect cartilage metabolism.</a> Br. J. Rheumatol. 37, 671-676 (1998).</li><li>Webb, G. R., Westacott, C. I., Elson, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoarthritic+synovial+fluid+and+synovium+supernatants+up-regulate+tumor+necrosis+factor+receptors+on+human+articular+chondrocytes.">Osteoarthritic synovial fluid and synovium supernatants up-regulate tumor necrosis factor receptors on human articular chondrocytes.</a> Osteoarthritis Cartilage. 6, 167-176 (1998).</li><li>Kook, S. H., Son, Y. O., Lee, K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+affects+positively+the+proliferation+of+bovine+satellite+cells+and+their+myogenic+differentiation+through+up-regulation+of+MyoD.">Hypoxia affects positively the proliferation of bovine satellite cells and their myogenic differentiation through up-regulation of MyoD.</a> Cell. Biol. Int. 32, 871-878 (2008).</li><li>Knobloch, T. J., Madhavan, S., Nam, J., Agarwal, S., Agarwal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+chondrocytic+gene+expression+by+biomechanical+signals.">Regulation of chondrocytic gene expression by biomechanical signals.</a> Crit. Rev. Eukaryot. Gene. Expr. 18, 139-150 (2008).</li><li>Guo, J. F., Jourdian, G. W., MacCallum, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+and+growth+characteristics+of+chondrocytes+encapsulated+in+alginate+beads.">Culture and growth characteristics of chondrocytes encapsulated in alginate beads.</a> Connect. Tissue. Res. 19, 277-297 (1989).</li><li>Toegel, S., Huang, W., Piana, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+reliable+reference+genes+for+qPCR+studies+on+chondroprotective+action.">Selection of reliable reference genes for qPCR studies on chondroprotective action.</a> BMC. Mol. Biol. 8, 13-13 (2007).</li><li>Lin, Z., Fitzgerald, J. B., Xu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiles+of+human+chondrocytes+during+passaged+monolayer+cultivation.">Gene expression profiles of human chondrocytes during passaged monolayer cultivation.</a> J. Orthop. Res. 26, 1230-1237 (2008).</li><li>Goessler, U. R., Bieback, K., Bugert, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+chondrocytes+differentially+express+matrix+modulators+during+in+vitro+expansion+for+tissue+engineering.">Human chondrocytes differentially express matrix modulators during in vitro expansion for tissue engineering.</a> Int. J. Mol. Med. 16, 509-515 (2005).</li><li>Anat, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 121, 107-118 (1976).</li></ol>

Biz ifşa etmek başka bir şey var.

Bu raporda, insan sinovyal sıvıda yüksek konsantrasyonlarda içeren ortamda 3 boyutlu bir ortamda insan eklem kondrosit kültür sağlayan bir yöntem geliştirdi. Sinovyal sıvı eklem boşluğu, eklem kondrosit bulunduğu doğal ortamı oluşturan önemli bileşenlerinden biridir. Ancak, sinovyal sıvıda viskozite kondrosit üç boyutlu, uzun vadeli kültür için önemli bir sorun olmuştur. 3D viskoz bir ortamda inşa etmek ve önlemek için agregasyonu bile besin dağılımı sürdürmenin zorluğu aşmak için...

Özel reaktifler ve ekipman Tablo: <table border="1"><tr><th> Reaktif Adı </th><th> Şirket </th><th> Katalog Numarası </th><th> Yorumlar </th></tr><tr><td> Aljinat için (Aljinik asit sodyum tuzu) </td><td> Sigma </td><td> A2158-250G </td><td> 40 ° C&#39;de depolanan% 2.4 çözümü </td></tr><tr><td> Kalsiyum Klorür Dihidrat, Granüler </td><td> JT Baker </td><td> A19339 </td><td></td></tr><tr><td width="239" rowspan="2" > Chondrogenic Büyüme ortamı </td><td width="254" rowspan="2" > Lonza </td><td width="182" > CC-3156 (baz medya) </td><td width="141" rowspan="2" ></td></tr><tr><td width="182" > CC-4409 (ek) </td></tr><tr><td width="239" rowspan="2" > Chondrogenic Farklılaşma Media </td><td width="254" rowspan="2" > Lonza </td><td width="182" > CC-3226 (baz medya) </td><td width="141" rowspan="2" ></td></tr><tr><td width="182" > CC-4408 (ek) </td></tr><tr><td> İnsan eklem kondrosit </td><td> Lonza </td><td > CC-2550 </td><td></td></tr><tr><td> DAPI (4 &#39;,6-Diamidino-2-phenylindole dihidroklorür) </td><td> Sigma-Aldrich </td><td > D9542 </td><td></td></tr><tr><td> RNeasy mini kiti (RNA ekstraksiyonu için) </td><td> Qiagen </td><td > 74104 </td><td></td></tr><tr><td> PCR: SYBR-yeşil </td><td> Kuantum </td><td > 95053-500 </td><td></td></tr><tr><td> 12 ml şırınga </td><td> Tyco-Kendall-Monoject </td><td > 512852 </td><td></td></tr><tr><td> 22-Gague Hipodermik İğne </td><td> Tyco-Kendall-Monoject </td><td > 8881 </td><td></td></tr><tr><td> Mikroskop </td><td> Olympus </td><td > IX71 </td><td></td></tr><tr><td> Platform rocker </td><td> Thermoscientific thermolyne </td><td > Vari-mix </td><td></td></tr><tr><td></td><td></td><td ></td><td></td></tr><tr><td> Astarlar dizileri </td><td></td><td ></td><td></td></tr><tr><td> Kollajen IIa ileri </td><td> 5&#39;-TTC ATC CCA CCC TCT CAC AGT-3 &#39; </td><td ></td><td></td></tr><tr><td> Kollajen IIa-ters </td><td> 5&#39;-CCTCTGCCTTGACCCGAA-3 &#39; </td><td ></td><td></td></tr><tr><td> MMP13 ileri </td><td> 5&#39;-TGT GCC CTT CTT CAC ACA GAC ACT-3 &#39; </td><td ></td><td></td></tr><tr><td> MMP13 geri </td><td> 5&#39;-GAG AGC AGA CTT TGA GTC ATT GCC-3 &#39; </td><td ></td><td></td></tr><tr><td> Kaspaz 3-ileri </td><td> 5&#39;-TCA TTA TTC AGG SKK GCC GTG GTA-3 &#39; </td><td ></td><td></td></tr><tr><td> Kaspaz 3 geri </td><td> 5&#39;-TGG ATG AAC CAG GAG CCA TTK TTT -3 &#39; </td><td ></td><td></td></tr></table>

a 3d system for culturing human articular chondrocytes in synovial fluid

Kıkırdak imha osteoartrit merkezi bir patolojik özellik, ABD&#39;nin önde gelen sakatlık nedenidir. Kıkırdak yetişkin in vivo olarak çok verimli bir şekilde yeniden değildir; ve bunun sonucu olarak, osteoartrit, kıkırdak kaybı geri döndürülemez açar ve kronik ağrı ve hareketsizlik 1,2 eşlik ediyor . Kıkırdak doku mühendisliği, doku işlevini yeniden ve geri yüklemek için umut verici bir potansiyel sunmaktadır. Bu teknoloji, genellikle doğal veya sentetik iskeleleri ve kültür içine çıkan 3D 3-6 in vivo bir defekt içine nakledilen bir biyokimyasal ve biyomekanik olgun doku mühendisliği amacı ile bir süre içinde dengeli bir orta inşa ekim kondrosit içerir . Kondrosit büyüme ve matriks birikimi için optimal bir durum ulaşmak kıkırdak doku mühendisliği başarısı için esastır. Arktik doğal eklem boşluğu, kıkırdakkemik bilirsiniz yüzey sinovyal sıvıdaki yıkanıyor. Bu berrak ve yapışkan sıvı besin sağlar avasküler eklem kıkırdağı ve büyüme faktörleri, sitokinler ve kondrosit metabolizması 7,8 için önemli olan enzimleri içerir. Ayrıca, eklem sıvısı ile kıkırdak yüzeyler arasında esas olarak iki temel bileşen, hyaluronan ve lubricin 9 10 salgılayan düşük sürtünme hareketi kolaylaştırır. Buna karşın, doku mühendislik kıkırdak en sık yapay medyada kültüre. Bu medya muhtemelen kondrosit metabolizması eğitim için daha fazla tanımlanmış koşullar sağlamak mümkün iken, eklem sıvısı, eklem kondrosit içinde bulunduğu doğal çevreye en doğru yansıtan Gerçekten de, sinoviyal sıvı almak ve saklamak için kolay olma avantajı vardır ve genellikle düzenli olarak vücut tarafından doldurulan olabilir. Çeşitli gruplar büyüyen insan, sığır, tavşan ve köpek c sinovyal sıvı ile kültür ortamı takviyehondrocytes, ancak çoğunlukla sadece sinoviyal sıvı (% 20 altında) 11-25 düşük düzeylerde kullanılır. Tavuk, at ve insan kondrosit sinovyal sıvıda yüksek yüzdesi ile orta kültüre alınmış olsa da, bu kültür sistemleri, iki boyutlu 26-28 edildi. Burada 21 günlük bir süre içinde sinoviyal sıvının yüksek bir yüzdesi (% 100&#39;e kadar) insan eklem kondrosit kültür yöntemi ile 3D sistemi mevcut. Bunu yaparken, biz üstesinden sinovyal sıvıda yüksek viskozite tarafından sunulan önemli bir engel. Bu sistem, 3 boyutlu bir ortamda, daha doğal ortam taklit kıkırdak için doğal ortamı oluşturan diğer iki önemli faktörler (oksijen basıncı ve mekanik yükleme) 29,30 ile kombine edilebilir sinoviyal sıvı insan kondrosit eğitim imkanı sağlar kıkırdak büyüme. Ayrıca, Bu sistem kondrosit sinoviyal sıvı aktivitesi assaying için kullanılır ve geliştirmek için bir platform sağlamak olabilirkıkırdak rejenerasyon teknolojileri ve artrit için tedavi seçenekleri.

Watch this Scientific Journal Video about A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid at JoVE.com

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

Sinovyal sıvıda yüksek düzeyde insan eklem kondrosit kültür 3D sistemi açıklanmıştır. Sinovyal sıvı eklem kıkırdağı için en doğal mikroçevresinin yansıtır ve kolayca elde ve saklanabilir. Bu sistem, böylece kıkırdak rejenerasyon eğitim ve artrit tedavisi için tarama terapötikler için kullanılabilir.

Sinovya Sıvısı Kültürleme İnsan Eklem kondrosit 3D Sistemi

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not ...

a-3d-system-for-culturing-human-articular-chondrocytes-synovial

Research

JoVE Journal

Biology

20.7K Görüntüleme.  Tufts University School of Medicine. Sinovyal sıvıda yüksek düzeyde insan eklem kondrosit kültür 3D sistemi açıklanmıştır. Sinovyal sıvı eklem kıkırdağı için en doğal mikroçevresinin yansıtır ve kolayca elde ve saklanabilir. Bu sistem, böylece kıkırdak rejenerasyon eğitim ve artrit tedavisi için tarama terapötikler için kullanılabilir.

Video: A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

Newt Lens Rejenerasyon Eğitim Kültürleme İris Pigment Epitel Hücreler için Sistem

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

Biyoloji

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Küçük Molekül İndüksiyon Pluripotent İnsan Embriyonik Kök Hücreleri İnsan Kardiyak Öncüleri ve kardiyomiyositlerde Verimli türetilmesi

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Için bir sistem<em&gt; Ex vivo</emEmbriyonik Pankreas&gt; ekimi

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer's Vesicle

Internal organs such as the heart, brain, and gut develop left-right (LR) asymmetries that are critical for their normal functions1. Motile cilia are involved in establishing LR asymmetry in vertebrate embryos, including mouse, frog, and zebrafish2-6. These 'LR cilia' generate asymmetric fluid flow that is necessary to trigger a conserved asymmetric Nodal (TGF-&beta; superfamily) signaling cascade in the left lateral plate mesoderm, which is thought to provide LR patterning information for developing organs7. Thus, to understand mechanisms underlying LR patterning, it is essential to identify genes that regulate the organization of LR ciliated cells, the motility and length of LR cilia and their ability to generate robust asymmetric flow.
In the zebrafish embryo, LR cilia are located in Kupffer's vesicle (KV)2,4,5. KV is comprised of a single layer of monociliated epithelial cells that enclose a fluid-filled lumen. Fate mapping has shown that KV is derived from a group of ~20-30 cells known as dorsal forerunner cells (DFCs) that migrate at the dorsal blastoderm margin during epiboly stages8,9. During early somite stages, DFCs cluster and differentiate into ciliated epithelial cells to form KV in the tailbud of the embryo10,11. The ability to identify and track DFCs&mdash;in combination with optical transparency and rapid development of the zebrafish embryo&mdash;make zebrafish KV an excellent model system to study LR ciliated cells.
Interestingly, progenitors of the DFC/KV cell lineage retain cytoplasmic bridges between the yolk cell up to 4 hr post-fertilization (hpf), whereas cytoplasmic bridges between the yolk cell and other embryonic cells close after 2 hpf8. Taking advantage of these cytoplasmic bridges, we developed a stage-specific injection strategy to deliver morpholino oligonucleotides (MO) exclusively to DFCs and knockdown the function of a targeted gene in these cells12. This technique creates chimeric embryos in which gene function is knocked down in the DFC/KV lineage developing in the context of a wild-type embryo. To analyze asymmetric fluid flow in KV, we inject fluorescent microbeads into the KV lumen and record bead movement using videomicroscopy2. Fluid flow is easily visualized and can be quantified by tracking bead displacement over time.
Here, using the stage-specific DFC-targeted gene knockdown technique and injection of fluorescent microbeads into KV to visualize flow, we present a protocol that provides an effective approach to characterize the role of a particular gene during KV development and function.

Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer&#039;s Vesicle

Cilia-generated fluid flow in Kupffer&#x2019;s Vesicle (KV) controls left-right patterning of the zebrafish embryo. Here, we describe a technique to modulate gene function specifically in KV cells. In addition, we show how to deliver fluorescent beads into KV to visualize fluid flow.

Kupffer en veziküllü Gen Fonksiyonu ve Cilia Oluşturulan Akışkanın Görselleştirme Analizi

Internal organs such as the  heart, brain, and gut develop left-right (LR) asymmetries that are critical for  their normal functions1. Motile cilia are ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

Hava Sıvı Arayüz İnsan Burun Epitel Hücrelerinin ekimi

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

Kültürleme Embriyonik için bir yöntem<em&gt; C. elegans</em&gt; Hücreler

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

Gap Junctional Hücreler arası iletişim: Doğal Ürünler Toksikantların ve Toksinler Yan Etkileri değerlendirmek için Fonksiyonel Biyomarker ve Sağlık Faydaları

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

Montaj ve &#39;Kayma Rings&#39; Uygulama: Orbital tek yönlü ve Periyodik Akışkan Akışının ve Kayma Stres için Yeni Bir Endotel Modeli

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

Manyetik aktif hücre stratejileri yalıtmak ve Sinovyal sıvı elde edilen mezenkimal kök hücre bir tavşan modelinden arındırmak için sıralama

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...