Bu çalışma, AB (STREP Proje 241577 &quot;BÜYÜK KALP,&quot; 7. Avrupa Çerçeve Programı, CP), Menarini Uluslararası Operasyonlar Lüksemburg (AM), Telethon GGP07133 (CP) ve Gilead Sciences (AM) tarafından desteklenmiştir.

Insan dokusu üzerinde deneysel protokoller Careggi Üniversitesi Hastanesinde (; 2009 yenilendi Mayıs 2006/0024713) etik komitesi tarafından onaylandı. Her hasta yazılı bilgilendirilmiş onam verdi. 1.. Çözümleri ve Ekipmanları Hazırlık Çözümler, Tablo 1 &#39;de tarif edilmiştir. Hücre izolasyon prosedürü basitleştirilmiş bir akış şeması Şekil 1&#39; de bulunur. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td colspan="2"> Çözüm </td><td> CP </td><td> DB </td><td> KB </td><td> TB </td><td> PS </td><td> EB1 </td><td> EB2 </td></tr><tr><td rowspan="23"> Reaktif (mM) </td><td> KH 2 PO 4 </td><td> 50 </td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> MgSO 4 </td><td> 8 </td><td> 1.2 </td><td> 5 </td><td></td><td></td><td> 1.2 </td><td> 1.2 </td></tr><tr><td> Hepes </td><td> 10 </td><td></td><td></td><td> 10 </td><td> 10 </td><td></td><td></td></tr><tr><td> adenozin </td><td> 5 </td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> glikoz </td><td> 140 </td><td> 10 </td><td> 20 </td><td> 10 </td><td></td><td> 10 </td><td> 10 </td></tr><tr><td> mannitol </td><td> 100 </td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> boğa </td><td> 10 </td><td> 20 </td><td> 5 </td><td></td><td></td><td> 20 </td><td> 20 </td></tr><tr><td> NaCl </td><td></td><td> 113 </td><td></td><td> 136 </td><td></td><td> 113 </td><td> 113 </td></tr><tr><td> KCl </td><td></td><td> 4.7 </td><td> 85 </td><td> 5.4 </td><td> 25 </td><td> 4.7 </td><td> 4.7 </td></tr><tr><td> MgCI2 </td><td></td><td> &lt;/ Td&gt; <td></td><td> 1.2 </td><td> 5 </td><td></td><td></td></tr><tr><td> KH 2 PO 4 </td><td></td><td> 0.6 </td><td> 30 </td><td></td><td></td><td> 0.6 </td><td> 0.6 </td></tr><tr><td> Na 2 HPO 4 </td><td></td><td> 0.6 </td><td></td><td></td><td></td><td> 0.6 </td><td> 0.6 </td></tr><tr><td> NaHCO 3 </td><td></td><td> 12 </td><td></td><td></td><td></td><td> 12 </td><td> 12 </td></tr><tr><td> KHCO 3 </td><td></td><td> 10 </td><td></td><td></td><td></td><td> 10 </td><td> 10 </td></tr><tr><td> Na-piruvat </td><td></td><td> 4 </td><td></td><td></td><td></td><td> 4 </td><td> 4 </td></tr><tr><td> BDM </td><td></td><td> 10 </td><td></td><td></td><td></td><td> 10 </td><td> 10 </td></tr><tr><td> BHBA </td><td></td><td></td><td> 5 &lt;/ Td&gt; <td></td><td></td><td></td><td></td></tr><tr><td> süksinik asit </td><td></td><td></td><td> 5 </td><td></td><td></td><td></td><td></td></tr><tr><td> EGTA </td><td></td><td></td><td> 0.5 </td><td></td><td></td><td></td><td></td></tr><tr><td> K 2-ATP </td><td></td><td></td><td> 2 </td><td></td><td></td><td></td><td></td></tr><tr><td> piruvik asit </td><td></td><td></td><td> 5 </td><td></td><td></td><td></td><td></td></tr><tr><td> kreatin </td><td></td><td></td><td> 5 </td><td></td><td></td><td></td><td></td></tr><tr><td> KMES </td><td></td><td></td><td></td><td></td><td> 115 </td><td></td><td></td></tr><tr><td rowspan="2"> Enzimler (U / ml) </td><td> Kolajenaz Tip V </td><td></td><td></td><td></td><td></td><td></td><td> 250 &lt;/ Td&gt; <td> 250 </td></tr><tr><td> Proteaz tipi XXIV </td><td></td><td></td><td></td><td></td><td></td><td> 4 </td><td></td></tr><tr><td> pH </td><td></td><td> 7.4 KOH </td><td> 7.3 NaOH </td><td> 7.1 KOH </td><td> 7.35 NaOH </td><td> 7.2 KOH </td><td> 7.3 NaOH </td><td> 7.3 NaOH </td></tr></tbody></table> .. Tablo 1 örnek toplama, hücre izolasyonu ve miyositlerin fonksiyonel karakterizasyonu CP = kardioplejik çözümü için kullanılan solüsyonlar; DB = ayrışma tamponu; KB = Kraft-Brühe çözümü; TB = Tyrode tamponu; PS = pipet çözüm; EB1 = enzim tamponu 1; EB2 = enzim tamponu 2. <ol><li> Kardioplejik (CP) çözeltisi hazırlayın. CP çözelti kadar 1 hafta boyunca 4 ° C&#39;de saklanabilir. </li><li> Ca 2 + ücretsiz ayrışma tamponu (DB) hazırlayın. Bu çözelti bir gün içinde kullanılmalıdır. </li><li> Kraft-Brühe (KB) çözeltisi hazırlayın. KB çözelti kadar 1 çiş boyunca 4 ° C&#39;de saklanabilirk. </li><li> Ca 2 + ücretsiz Tirod tampon (TB) hazırlayın. Bu çözelti bir gün içinde kullanılmalıdır. </li><li> Kullanmadan önce şırınga filtreleri kullanarak tüm çözümleri filtre. </li><li> Sindirim cihazı (Şekil 2), bir elektrik motoru ile döndürüldüğünde biri silikon elastomer birbirine bakan iki fırçalar, yapılmış bir kazıma kap hazırlayın. Sindirim Cihaz özel yapılır. Sindirim aygıtında ayrıntıları Şekil 8 vardır; Cihazın görüntüleri, Şekil 2 ve 2B C içindedir. % 70 etanol ve su ile doku odası yıkayın. </li></ol> Miyokard Örneklerinin 2.. Toplama ve İşleme <ol><li> 50 ml&#39;lik bir tüp içinde cardiplegic (CP) çözeltisi 40 ml dökün ve hücre izolasyon laboratuara ameliyat oda numune taşıma için buz içinde depolar. </li><li> Buz ile yıkayın, hemen çıkarıldıktan sonra ameliyat odasından ventriküler miyokard örnek toplamakCP çözeltisi ve bu tüp içinde saklayın. Açık kalp ameliyatı sırasında üst arası ventriküler septum, ağırlık&gt; 100 mg kesilip endokardiyal örnekleri kullanın. </li><li> Hızla laboratuar alanına numune aktarmak; Numune eksizyondan 10 dakika içinde numune işleme başlar. </li><li> Buz gibi soğuk CP tamponu örnek tutarken, dikkatli bir stereo mikroskop altında ince bir makas kullanarak endokardiyal fibrotik tabakayı kaldırmak; sonra, küçük parçalara (uzun 2-3 mm) miyokard doku kesti. Doku numunesi büyüklüğüne bağlı olarak, her bir izolasyon için 100 mg ila 1 g arasında ventriküler miyokardın toplam miktarı kesti. </li><li> Doku kıyma haline gelme işleminin tamamlanması üzerine, buz gibi soğuk temiz CP çözeltisi ile, sindirim cihazı içine miyokard parçalar aktarın. Artık toplam doku g 1&#39;den kullanarak, miyokard parçaları ile iki silikon fırça arasındaki tüm hacmi (3-4 ml) doldurmaktan kaçının. </li></ol> 3.. Yıkama ve Miyokard topakları Sindirim <ol><li> Kıçtaparçalar sindirim cihazın kazıma odasına transfer edilir, er, soğuk Ca2 + içermeyen ayrışma tamponu (DB) ile oda içindeki CP tampon değiştirin. </li><li> Banyosu içinde ısıtılmış, su (Şekil 1) ile temas halinde olması için bölme için sırayla, termostatik banyoda sindirim yerleştiriniz. 37.5 ° C banyo ayarlayın ve yavaş yavaş doku odasının sıcaklığını yükseltmek için, açın. 1 devir / saniye dönme hızını ayarlayarak, sindirim cihazının motora çevirin. </li><li> Her 8 dakika temiz DB ile oda içindeki solüsyonu değişen, DB ile 3 yıkama çevrimi gerçekleştirin. DB (37 ° C) ısıtılır ve oksijen miyokard parçalar ile temas etmeden önce doymuş. </li><li> DB çözeltisine kollajenaz Tip V ve 4 U / ml Proteaz tipi XXIV 250 U / ml eklenerek enzim tamponu 1 (EB1) hazırlayın. DB çözüm 250 U / ml kollajenaz Tip V ekleyerek enzim tamponu 2 (EB2) hazırlayın. (37 ° C) ve oxygenat ısınmake EB1 ve EB2. </li><li> (37 ° C)% 100 oksijenli EB1 ile dönen sindirim cihazda sindirim iki 12 dakika döngüleri yapın. Her bir döngüde, bir EB1 ~ 3 ml kullanın. Pipet aspirasyonu ile çözüm çıkarın ve her döngüden sonra atın. </li><li> Hücresi ile toplama ve ~ tamponlar elüsyon için soğuk (4 ° C) KB çözeltisi 80 ml için 6 15 ml tüpler hazırlayın. </li><li> 37 ° C de 3 ml% 100 oksijene EB2 ile bir birinci 15 dakika sindirim çevrimi gerçekleştirme Sindirim devresini sonra, 15 ml bir tüp içinde birinci ayrışmış miyositleri içeren çözelti toplamak ve 12 ml soğuk KB çözeltisi ile hücre süspansiyonu seyreltin. Oda sıcaklığında tüp düz saklayın. </li><li> Aşağıdaki sindirim devir için V kolajenaz konsantrasyonunun yarıya amacıyla DB eşit bir miktarı ile geri kalan EB2 çözeltisi ile seyreltilir. </li><li> 37 ° C de 3 ml EB2 diğer 5 12 dakika sindirim döngüleri yapın; bunların her biri ardından 15 ml konik bir tüp içinde tampon içeren miyosit ve toplamak ve12 ml KB çözeltisi ile seyreltilir. 30 dakika boyunca oda sıcaklığında, 6 hücre ihtiva eden tüpler saklayın. </li></ol> 4. Hücre tabanda ve Ca 2 + READAPTAYON <ol><li> 20 ml Ca 2 + içermeyen Tyrode tamponu (TB) ve 1 mg / ml sığır serum albümini (BSA) ilave edin. Çözüm Filtre. </li><li> Yerleşmek için miyositleri zorlamak için 5 dakika boyunca 100 x g&#39;de altı miyosit içeren konik santrifüj tüpleri. Süpernatantı ve oda sıcaklığında TB içeren BSA (verim bağlı olarak 1-3 mi) değişken bir miktarı ile, her bir tüp içinde tekrar süspansiyon hücreleri. </li><li> Yavaş yavaş 100 mmol / L CaCI2 çözeltisi, az miktar eklenerek hücre ihtiva eden tampon içinde Ca2 + konsantrasyonu artar. Birinci ve ikinci adım olarak Ca2 + konsantrasyonu sırasıyla 50 umol / L ve 100 umol / L &#39;ye kadar yükseltilir. Aşağıdaki Ca 2 + ekleme adımlarını her 5 dakikada gerçekleştirilir ve konsantrasyon her adım t 100 umol / L ile yükseltilir0.9 mmol / L OA nihai konsantrasyonu </li><li> Izolasyon prosedürü verimini değerlendirmek. Mikroskop cam alt bölmesi üzerine miyosit ihtiva eden bir çözeltinin 0.5 ml aktarın. 10x objektif büyütmede 15 mikroskop alanları değerlendirmek ve (net izlerse ve anlamlı içerikleri ile, örneğin çubuk biçimli hücreler, Şekil 2) sağlıklı miyositlerin yüzdesini hesaplamak. Beklenen verim% 20 civarındadır. </li></ol> İzole kardiyomiyositlerinin 5. Fonksiyonel Değerlendirme. Aşağıdaki protokol hareket potansiyellerine ve hücre içi Ca2 + akıları eş zamanlı kayıt dahil olmak üzere insan kardiyomiyosit fonksiyonel değerlendirmesi örneğidir. <ol><li> Delikli yama yapılandırmada yama kelepçe deneyler için pipet çözüm (PS) hazırlayın. Çözelti, 3 aya kadar -20 ° C&#39;de muhafaza edilebilir. </li><li> Ca 2 + ücretsiz Tirod tampon (TB) 1.8 mmol / L CaCl 2 ekleyin. Use patch clamp / floresan deneyleri sırasında kardiyomiyositlerin süperfüzyon için bu çözüm. </li><li> 1.5 ml tüp aktarın hücre süspansiyonu 1 ml ve 10 umol / L ve 10 ul Fluoforte Powerload Konsantre ekleyin. Oda sıcaklığında 30 dakika boyunca inkübe edin. Daha sonra, dikey konumda boru seti ve 5 dakika için yerine getirilmesi için hücreyi terk; Ca hücreleri tekrar süspansiyon 2 + TB içeren. </li><li> (: 37 ± 0.5 ° C sıcaklık), küçük bir (0.5 mi) hücre süspansiyonu, 0.25 ml transfer, sıcaklık kontrollü mikroskop 0.3 ml / dk &#39;lık bir akış hızında ısıtılmış microperfusor sistemi ile yer çekimi ile süperfüze kayıt odası, monte edilebilir. </li><li> Mikropipet çektirmenin kullanarak, 3-5 um&#39;lik bir uç çapı ve PS ile dolu 3 M 4.5 arasında bir direnç ile yama kelepçe pipetler hazırlar. </li><li> PS (250 ug / ml) içinde bir toplu amfoterisin B ekleyin ve elektrotlar doldurmak için kullanın. </li><li> Kapanım yoksun açık desenli bir çubuk şeklinde hücreyi, fo seçinerişim direnci altında 20 MQ düşene kadar rm, 5 ile 10 dakika giga mühür ve bekleyin. </li><li> Stimülasyon farklı frekanslarda (0.2 Hz, 0.5 Hz, 1 Hz, her bir frekansta 1 dakika) kısa darbeleri (&lt;3 msn) kullanılarak geçerli kelepçe modunda aksiyon potansiyelleri toplayın. Kayıt aşamasında, 492 ± 3 nm parlak alan aydınlatma açmak ve 505-520 nm Fluoforte floresan algılar. Floresan Edinme ve Digidata 1440A ve pClamp 10.0 yazılımını kullanarak potansiyel sinyalleri membran. Gerekirse kayıt dizisini birden çok kez tekrarlayın; Ancak, her hücre için 15 dakika aşağıda toplam kayıt süresini tutmak. </li></ol>

<ol>
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X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+2+early+afterdepolarization+as+a+trigger+of+polymorphic+ventricular+tachycardia+in+acquired+long-QT+syndrome+:+direct+evidence+from+intracellular+recordings+in+the+intact+left+ventricular+wall.">Phase 2 early afterdepolarization as a trigger of polymorphic ventricular tachycardia in acquired long-QT syndrome : direct evidence from intracellular recordings in the intact left ventricular wall.</a> Circulation. 103, 2851-2856 (2001).</li><li>Yue, L., Feng, J., Li, G. R., Nattel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+outward+and+delayed+rectifier+currents+in+canine+atrium:+properties+and+role+of+isolation+methods.">Transient outward and delayed rectifier currents in canine atrium: properties and role of isolation methods.</a> The American Journal of Physiology. 270, 2157-2168 (1996).</li><li>Li, G. R., Feng, J., Yue, L., Carrier, M., Nattel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+two+components+of+delayed+rectifier+K++current+in+human+ventricular+myocytes.">Evidence for two components of delayed rectifier K+ current in human ventricular myocytes.</a> Circulation research. 78, 689-696 (1996).</li><li>Viswanathan, P. C., Shaw, R. M., Rudy, Y. <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+IKr+and+IKs+heterogeneity+on+action+potential+duration+and+its+rate+dependence:+a+simulation+study.">Effects of IKr and IKs heterogeneity on action potential duration and its rate dependence: a simulation study.</a> Circulation. 99, 2466-2474 (1999).</li><li>Volders, P. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+contribution+of+IKs+to+canine+ventricular+repolarization:+key+role+for+beta-adrenergic+receptor+stimulation.">Probing the contribution of IKs to canine ventricular repolarization: key role for beta-adrenergic receptor stimulation.</a> Circulation. 107, 2753-2760 (2003).</li><li>Sanguinetti, M. C., Jurkiewicz, N. K., Scott, A., Siegl, P. 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Mechanism of action.</a> Circulation Research. 68, 77-84 (1991).</li><li>Coppini, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+translational+approach+to+treatment+of+hypertrophic+cardiomyopathy:+pre-clinical+rationale+and+design+of+a+prospective+randomized+pilot+trial+with+ranolazine.">A translational approach to treatment of hypertrophic cardiomyopathy: pre-clinical rationale and design of a prospective randomized pilot trial with ranolazine.</a> Circulation. 125, 1 (2012).</li></ol>

Yazarlar, hiçbir rakip mali çıkarlarını olmadığını beyan ederim.

Bu tarif edilen ve insan ventriküler miyokardın cerrahi örneklerden uygun miyositleri izole etmek için bir yöntem onaylamıştır. Başarılı bir şekilde hastalıklı ventriküler miyokardın ikinci bir uygun miyosit ayrılması geliştirilmiş ve ince ayarlı edildi izin vermek için atrial cerrahi örnekler, bu teknikle izole edilmiş hücrelere kullanılmıştır, daha önce tarif edilen protokollere başlayarak. Erken raporlar seçici, fizyolojik uyaran 8,24 ile değiştirilmiş elektrofizyolojik ?...

Miyokardın elektrofizyolojik özelliklerinin diseksiyonu tek kardiyak miyosit izolasyon tekniklerinin geliştirilmesi sonra belirgin ilerlemiştir. Kardiyak Uyarma Daralma Kavrama (EC-Kavrama) anlayışı son gelişmeler de sağlam doku tüm fizyolojik özelliklerini muhafaza uygulanabilir tek kardiyomiyositlerin izole yeteneği ile mümkün olmuştur. Yama kelepçe yöntemleri rutin kardiyak sarkolemmal iyon akımlarının fonksiyonu ve farmakolojik modülasyon çalışma istihdam edilmektedir. Ca 2 + duyarlı boyalar ile hücre içi kalsiyum dinamikleri kayıtları da düzenli EC-Kuplajın fizyolojisi yanı sıra hücre içi Ca 2 + patolojik değişiklikler ile ilgili önemli veriler sağlayan, sağlıklı ve hastalıklı modeller çeşitli tek miyositlerindeki yapılmaktadır mekanik düşüklüğü ve kalp hastalıklarında artış aritmojen yüküne yol açan homeostasis. InformaBu çalışmalardan elde edilen tion klinik ortamda ilaçların elektrofizyolojik ve mekanik etkilerini anlamak için çok önemlidir. Ancak, zar-ötesi akımları ve kardiyak ve kardiyak hareket potansiyeli mekaniği belirli özellikler için hesap, AT-Coupling proteinlerde türe özel farklılıklar vardır. Insan olmayan memelilerden izole miyosit çalışmalar biyofiziksel özellikleri ve özel zar iyon kanalları ve EM-Coupling proteinlerinin fizyolojik rolleri durulaştırmamızdan Böylece, bu durum, insan kardiyak miyosit uygun modeller sağlamaz. İnsan miyokardiyumundan canlı miyositlerin izolasyonu tam kalp hastalıklarının patofizyolojisi anlamak ve yeni tedavi yaklaşımları doğrulamak için elzemdir. Atriyal uzantıları genellikle cerrahi işlemler sırasında atılır gibi insan atriyal doku kolayca kullanılabilir. Yetişkin insan kalp aksiyon potansiyelleri ve iyonik cu ilk nicel çalışmalarrrents enzimatik olarak izole edilmiş atrial hücreler 1-4 kullanılabilir. Aksiyon potansiyelleri veya izole yetişkin insan ventriküler hücrelerinden akımların kayıtları sonradan 3,5-10 bildirilmiştir. Bu çalışmaların çoğu nakledilmiş kalplerinden edilen ve izole edilmiş hücreleri elde etmek için kolajenaz için koroner arter bölümü ya da kesilen doku nispeten büyük miktarlarda maruz kolajenaz perfüzyon ya da kullanılan hücreleri kullandık. Bu çalışmalar, sağlıklı kalpleri insan ventriküler kardiyomiyositlerinde gelen ve terminal kalp yetmezliği olan hastaların transmembran iyon akımların bir dizi ayrıntılı bir karakterizasyonu izin verdi. L-tipi Kayıtlar Ca 2 + akımı (I Ca-L), 5-7, geçici dışa potasyum akımı (I için) 8, potasyum rektifayer akım içeri doğru (I κ1) 8, geciktirilmiş düzeltici potasyum akımının farklı bileşenlerinin (I κ ) 9 bildirilmiştir. Avanslar ve rafine birizolasyon prosedürü 10, aksiyon potansiyeli uzamasını 11 içeren terminali, kalp yetmezliği, artan aritmojen potansiyelinin iyonik temeli net bir karakterizasyonu izin depolarizasyonlarından 12 sonra gecikmeli ve diyastolik depolarizasyon ve erken atım giden komik geçerli 13 arttı. Yetişkin kardiyak miyositler, normalde çeşitli enzim karışımları ile bütün kalp, Ca 2 +-hücrelerinin toleranslı 14 arasında yüksek verimler üreten bir tekniğin retrograd perfüzyon göre küçük hayvanlardan izole edilir. Doku fragmanlarından kardiyak miyosit izolasyonu muhtemelen koroner arter perfüzyon ile elde edilene kıyasla tek miyositlerine enzimlerin sınırlı erişim doğal olarak daha az başarılı olmuştur. Çünkü kullanılmayan donör kalplerinin çok sınırlı durumu, düzenli olarak normal insan ventriküler hücreleri elde etmek tek pratik yolu enzimatik digestio tarafındanelektif cerrahi işlemler sırasında kesilip genellikle çok küçük doku parçalarının n. Iyice hücre düzeyinde karakterize edilmiştir sadece insan hastalık modeli nakledilen kalpleri erişilebilirlik nedeniyle terminali kalp yetmezliğidir. Ancak, Terminal kalp yetmezliği hastalarının bir kısmında görülür ve genellikle altta yatan neden 15 nispeten bağımsız olan miyokard hücrelerinin ciddi yeniden, ortak bir yol içerir. Hastalığın erken bir sigara başarısız aşamada hastaların tek kardiyomiyositlerin fonksiyonunu değerlendirmek için yetenek, farklı kalıtsal veya edinsel koşulların belirli patofizyolojisini anlamak için çok önemlidir. Hipertrofik kardiyomiyopati (HKM) söylüyorum örnektir. HKM, ortak bir (1/500 kişi) kardiyak hipertrofisi ile karakterize kalıtsal kardiyak durum nedeniyle çıkış yolu tıkanıklığı ve diyastolik disfonksiyon 16 aritmojen risk ve kasılma değişiklikler artar. HKM kalplerinden Kardiyomiyositler uHücre yapısındaki değişiklikler (hipertrofisi, miyofibriler kargaşa) ve EC-Kuplajını 17 içeren karmaşık bir yeniden süreçleri ndergo. Ancak, HKM&#39;de miyositlerdeki disfonksiyon en çok bilgi transgenik hayvan modellerinde geldi. HKM hastaların sadece bir azınlık terminali kalp yetmezliği doğru geliştikçe ve kalp nakli gerektirdiğinden, HKM kalpleri standart yöntemlerle hücre izolasyonu için çok nadiren mevcuttur. Ancak, HKM hastaların en az% 30 sistolik (HKM) 18 sırasında masif septal hipertrofi değiştiren çıkış yolu kan akışı nedeniyle obstrüktif belirtiler gelişir. HKM&#39;de tıkanıklığın giderilmesi için en etkili tedavi seçeneği mevcut cerrahi septal miyektomi: Bu cerrahi işlem sırasında, üst septum değişken ölçekli bölümü trans aort yaklaşımla kaldırılır. Hipertrofik septum bu bölümü taze dokudan izole edilmesi için, bu nedenle hücre kullanılabilir. İnsan ventricula izolasyonu için bir yöntem,r tek, küçük transvenöz endomiyokardiyal biyopsi ile miyositler önce geliştirilen ve 19 yayımlanmıştır. Biz septal miyektomi ve kapak replasmanı işlemleri uygulanan hastalarda uygulanan HKM hastalar dahil olmak üzere kalp ameliyatı geçiren hastaların ventrikül myokard örneklerinden tek septal miyositleri izole etmek için bir yöntem uyguladı. Izolasyon protokol, temsili elektrofizyolojik ve Ca 2 + floresan ayrıntılı bir açıklama ilaveten ölçümler izole edilmiş beşeri ventriküler miyositlerde yaşayabilirliği ve yama kelepçe ve hücre içi Ca2 + çalışmalar fizibilitesini gösteren sunulmaktadır.

<table border="0" cellpadding="0" cellspacing="0" width="1059">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Potassium phosphate monobasic (KH2PO4)</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:139px;"><a href="http://www.sigmaaldrich.com/catalog/product/sigma/p9791?lang=it&amp;region=IT">P9791&#160;</a></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium sulfate heptahydrate(MgSO4 * 7H2O)</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/m1880?lang=it&amp;region=IT">M1880&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/h3375?lang=it&amp;region=IT">H3375&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adenosine</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/a9251?lang=it&amp;region=IT">A9251&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-(+)-Glucose</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/g8270?lang=it&amp;region=IT">G8270&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mannitol</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/m4125?lang=it&amp;region=IT">M4125&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Taurine</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td>T0625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium hydroxide (KOH)</td>
			<td>Sigma-Aldrich</td>
			<td>P5958</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/s7653?lang=it&amp;region=IT">S7653</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/p9333?lang=it&amp;region=IT">P9333&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium phosphate dibasic (Na2HPO4)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/s7907?lang=it&amp;region=IT">S7907&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium bicarbonate (NaHCO3)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/s6297?lang=it&amp;region=IT">S6297&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium bicarbonate (KHCO3)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/237205?lang=it&amp;region=IT">237205</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/p2256?lang=it&amp;region=IT">P2256&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2,3-Butanedione monoxime</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/b0753?lang=it&amp;region=IT">B0753&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium hydroxide(NaOH)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/s8045?lang=it&amp;region=IT">S8045&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Glutamic acid monopotassium salt monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/fluka/49601?lang=it&amp;region=IT">49601</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pyruvic acid</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/aldrich/107360?lang=it&amp;region=IT">107360</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3-Hydroxybutyric acid</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/aldrich/166898?lang=it&amp;region=IT">166898</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adenosine 5&#8242;-triphosphate dipotassium salt dihydrate (K2-ATP)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/a8937?lang=it&amp;region=IT">A8937</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Creatine</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/c0780?lang=it&amp;region=IT">C0780&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Succinic Acid</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/s3674?lang=it&amp;region=IT">S3674&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8242;,N&#8242;-tetraacetic acid (EGTA)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/e0396?lang=it&amp;region=IT">E0396&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Albumin from bovine serum</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/a0281?lang=it&amp;region=IT">A0281</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium chloride (MgCl2)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/m8266?lang=it&amp;region=IT">M8266&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase from Clostridium histolyticum, Type V</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/c9263?lang=it&amp;region=IT">C9263&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proteinase, Bacterial, Type XXIV</td>
			<td>Sigma-Aldrich</td>
			<td>P8038</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium chloride solution, ~1 M in H2O</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/21115?lang=it&amp;region=IT">21115</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium chloride 0.1 M solution</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/53704?lang=it&amp;region=IT">53704</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium methanesulfonate</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/aldrich/83000?lang=it&amp;region=IT">83000</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FluoForte Reagent</td>
			<td>Enzo Life Sciences</td>
			<td>ENZ-52015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Powerload concentrate, 100X</td>
			<td>Life Technologies</td>
			<td>P10020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Perfusion Fast-Step System</td>
			<td>Warner Instruments</td>
			<td>VC-77SP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amphotericin B solubilized</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/a9528?lang=it&amp;region=IT">A9528&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multiclamp 700B patch-clamp amplifier</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digidata 1440A</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pClamp10.0&#160;</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="108">
			<td height="108" style="height:108px;">Digestion Device</td>
			<td>CUSTOM</td>
			<td>CUSTOM</td>
			<td style="width:255px;">The device is custome made in our laboratory using plastic tubes, cast Sylgard and a motor; it is described in detail in Fig 1 C-D and in Fig.7. We can provide further details if requested</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silicone elastomer for the digestion device&#39;s brushes</td>
			<td>Dow Corning</td>
			<td>SYLGARD&#174; 184</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Variable speed rotating motor for the digestion device</td>
			<td>Crouzet</td>
			<td>Crouzet 178-4765&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Mold for brushes casting</td>
			<td>N.A.</td>
			<td>N.A.</td>
			<td style="width:255px;">The mold is custom made from standard PTFE 2.5 cm diameter rods</td>
		</tr>
	</tbody>
</table>

isolation and functional characterization of human ventricular cardiomyocytes from fresh surgical samples

Hastalıklı kalplerinden Kardiyomiyositler hücre yapısındaki değişiklikler, eksitasyon kasılma kaplin ve membran iyon akımları içeren karmaşık yeniden işlemlere tabi tutulur. Bu değişiklikler, artmış aritmojenik riski ve kalp hastalarında sistolik ve diyastolik fonksiyon bozukluğu yol açan büzülme değişikliklerden sorumlu olması muhtemeldir. Bununla birlikte, kalp hastalıklarında miyosit fonksiyonu değişiklikler üzerinde en çok bilgi hayvan modellerinde geldi. Burada tarif ve kardiyak cerrahi operasyon geçiren hastaların ventriküler miyokardın küçük cerrahi örneklerinden canlı miyositleri izole etmek için bir protokol doğrulamak. Protokol detaylı olarak tarif edilmektedir. Elektrofizyolojik ve hücre içi kalsiyum ölçümleri, bu yöntem ile elde edilen insan ventriküler kardiyomiyositlerde tek hücre ölçümler bir dizi uygulanabilirliğini göstermek için rapor edilmiştir. Protokol he bildirdiYeniden farklı kalp hastalıklarının mevcudiyetinde insan kalbinin fonksiyonel değişiklikler hücresel ve moleküler temelinin gelecek çalışmalar için yararlı olabilir. Bundan başka, bu yöntem, hücresel seviyede yeni terapötik hedeflerini tanımlamak için ve doğrudan translasyonel değeri, insan kardiyomiyositler yeni bileşiklerin etkinliğini test etmek için de kullanılabilir.

Olmayan başarısız olmayan hipertrofik cerrahi hastalarında 21 ile karşılaştırıldığında, yukarıda açıklanan yöntem, miyektomi operasyon yapıldı hipertrofik kardiyomiyopati (HKM) hastalarda interventriküler septum izole kardiyomiyositlerin fonksiyonel anormalliklerini karakterize etmek için kullanılmıştır. Bu bölümde yer alan sonuçlar, çalışma 21 elde edilir ve bu teknik kardiyak hastalık koşullarında miyokard hücresi işlevinin değişiklikler karakterize etmek içi...

Watch this Scientific Journal Video about Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples at JoVE.com

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Kalp hastalıklarının hücre temelinde Mevcut bilgi çok hayvan modelleri üzerinde yapılan çalışmalara dayanmaktadır. Burada açıklamak ve insan ventrikülün küçük cerrahi örneklerinden tek bir canlı kardiyomiyositlerin elde etmek için yeni bir yöntem doğrulamak. İnsan ventriküler miyositler elektrofizyolojik çalışmalar ve uyuşturucu testi için kullanılabilir.

Taze Cerrahi Örneklerden İzolasyon ve İnsan Ventriküler kardiyomiyositlerin Fonksiyonel Karakterizasyonu

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

isolation-functional-characterization-human-ventricular

Research

JoVE Journal

Medicine

17.1K Görüntüleme.  University of Florence.  Kalp hastalıklarının hücre temelinde Mevcut bilgi çok hayvan modelleri üzerinde yapılan çalışmalara dayanmaktadır. Burada açıklamak ve insan ventrikülün küçük cerrahi örneklerinden tek bir canlı kardiyomiyositlerin elde etmek için yeni bir yöntem doğrulamak. İnsan ventriküler miyositler elektrofizyolojik çalışmalar ve uyuşturucu testi için kullanılabilir. 

Video: Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Hasta Türetilen Hücre Kültürü ve CD133 İzolasyonu<sup&gt; +</supMelanom dan&gt; sağlayan olası Kanser Kök Hücreleri

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Tıp

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

İnsan Prostat Kanseri Örneklerinden Kanser Kök Hücreleri İzolasyon

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.

The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays.

Toplama, İzolasyon, ve İnsan endoservikal Akışı Sitometrik Analizi

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female ...

Chronic Thromboembolic Pulmonary Hypertension and Assessment of Right Ventricular Function in the Piglet

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. Pulmonary Hypertension (PH) was induced in 3-week-old piglets by a progressive obstruction of the pulmonary vascular bed. A ligation of the left Pulmonary Artery (PA) was performed first through a mini-thoracotomy. Second, weekly embolizations of the right lower pulmonary lobe were done under fluoroscopic guidance with n-butyl-2-cyanoacrylate during 5 weeks. Mean Pulmonary Arterial Pressure (mPAP) measured by ritght heart catheterism, increased progressively, as well as Right Atrial pressure and Pulmonary Vascular Resistances (PVR) after 5 weeks compared to sham animals. Right Ventricular (RV) structural and functional remodeling were assessed by transthoracic echocardiography (RV diameters, RV wall thickness, RV systolic function). RV elastance and RV-pulmonary coupling were assessed by Pressure-Volume Loops (PVL) analysis with conductance method. Histologic study of the lung and the right ventricle were also performed. Molecular analyses on RV fresh tissues could be performed through repeated transcutaneous endomyocardial biopsies. Pulmonary microvascular disease in obstructed and unobstructed territories was studied from lung biopsies using molecular analyses and pathology. Furthermore, the reliability and the reproducibility was associated with a range of PH severity in animals. Most aspects of the human CTEPH disease were reproduced in this model, which allows new perspectives for the understanding of the underlying mechanisms (mitochondria, inflammation) and new therapeutic approaches (targeted, cellular or gene therapies) of the overloaded right ventricle but also pulmonary microvascular disease.

Chronic Thromboembolic Pulmonary Hypertension (CTEPH) and Right Ventricular (RV) dysfunction were induced in piglets by progressive obstruction of the pulmonary arteries. Consequences were remarkably similar to those observed in CTEPH patients. This animal model would be a very useful tool for pathophysiology and therapeutic experiments on CTEPH and RV failure.

Kronik tromboembolik pulmoner hipertansiyon ve Piglet Sağ Ventrikül Fonksiyonu Değerlendirilmesi

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. ...

Assessment of Sarcoplasmic Reticulum Calcium Reserve and Intracellular Diastolic Calcium Removal in Isolated Ventricular Cardiomyocytes

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum (SR) serves as a Ca2+ reservoir for contraction, which reuptakes intracellular Ca2+ during relaxation. The SR Ca2+ reserve available for beats is determinate for cardiac contractibility, and the removal of intracellular Ca2+ is critical for cardiac diastolic function. Under some pathophysiological conditions, such as diabetes and heart failure, impaired calcium clearance and SR Ca2+ store in cardiomyocytes may be involved in the progress of cardiac dysfunction. Here, we describe a protocol to evaluate SRCa2+ reserve and diastolic Ca2+ removal. Briefly, a single cardiomyocyte was enzymatically isolated, and the intracellular Ca2+ fluorescence indicated by Fura-2 was recorded by a calcium imaging system. To employ caffeine for inducing total SR Ca2+ release, we preset an automatic perfusion switch program by interlinking the stimulation system and the perfusion system. Then, the mono-exponential curve fitting was used for analyzing decay time constants of calcium transients and caffeine-induced calcium pulses. Accordingly, the contribution of the SR Ca2+-ATPase (SERCA) and Na+-Ca2+ exchanger (NCX) to diastolic calcium removal was evaluated.

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Here, we describe a protocol to evaluate sarcoplasmic reticulum Ca2+ reserve and diastolic calcium removal function in cardiomyocytes by a calcium imaging system.

Sarcoplasmic retikulum kalsiyum koruma ve hücre içi diyastolik kalsiyum kaldırma izole Ventriküler Cardiomyocytes olarak değerlendirilmesi

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum ...

Isolation and Cultivation of Adult Rat Cardiomyocytes

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise investigation of the behavior of these cells under specific treatments and environments is possible. This manuscript presents a protocol for successful isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC).
The rat is sacrificed by cervical dislocation under deep anesthesia. Then, the heart is extracted and the aorta is uncovered. Subsequently, perfusion on the Langendorff perfusion system with calcium depletion and collagenase treatment is performed. Afterwards, ventricular tissue gets minced, re-circulated, and filtered, followed by three centrifugation steps with gradual addition of CaCl2 until physiological calcium concentration is reached. ARVC are plated on cell culture dishes. After refreshing the cell culture medium, ARVC can be cultivated for up to six days without changing the serum-containing culture medium. Isolation of ARVC is a calcium sensitive process. Small changes in the intracellular calcium concentration cause a decrease in the quality and viability of the isolated cells.
Freshly isolated ARVC are rod shaped. Within the first days of cultivation they lose the rod-shaped morphology and form pseudopodia-like structures (spreading). During this morphological formation ARVC initially degrade their contractile elements followed by a reformation through actin stress fibers and de novo sarcomerogenesis. After one week of cultivation, most ARVC show a widespread appearance with a clearly detectable cross striation. This process is sensitive to intracellular calcium concentration, as treatment with ionomycin attenuates spreading. Key markers in this process of de- and re-differentiation are &#946;-myosin heavy chain (&#946;-MHC), oncostatin M (OSM), and swiprosin-1 (EFHD2). Recent studies have suggested that cardiac re- and de-differentiation occurring under culture conditions mimics features seen in vivo during cardiac remodeling. Therefore, isolation and cultivation of ARVC play a key role in understanding the biology of cardiomyocytes.

Here, we present a protocol for the isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC). Isolated ARVC can be used for short and long-term cultivation. The isolation and cultivation of ARVC can play a key role in developing new treatment regimens for cardiac diseases.

Yalıtım ve yetişkin fare Cardiomyocytes tarımı

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise ...

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state against self-antigens as well as antigens derived from commensal organisms. The disruption of this immune homeostasis can lead to chronic inflammation and to the development of autoimmunity. Dendritic cells (DCs) are the professional antigen-presenting cells of the innate immune system involved in activating na&#239;ve T cells to initiate immune responses against foreign antigens. However, DCs can also be differentiated into TolDCs that act to maintain and promote T cell tolerance and to suppress effector cells contributing to the development of either autoimmune or chronic inflammation conditions. The recent advancement in our understanding of TolDCs suggests that DC tolerance can be achieved by modulating their differentiation conditions. This phenomenon has led to tremendous growth in developing TolDC therapies for numerous immune disorders caused due to break in immune tolerance. Successful studies in preclinical autoimmunity murine models have further validated the immunotherapeutic utility of TolDCs in the treatment of autoimmune disorders. Today, TolDCs have become a promising immunotherapeutic tool in the clinic for reinstating immune tolerance in various immune disorders by targeting pathogenic autoimmune responses while leaving protective immunity intact. Although an array of strategies has been proposed by multiple labs to induce TolDCs, there is no consistency in characterizing the cellular and functional phenotype of these cells. This protocol provides a step-by-step guide for the development of bone marrow-derived DCs in large numbers, a unique method used to differentiate them into TolDCs with a synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-difluoro-propyl-amide (CDDO-DFPA), and the techniques used to confirm their phenotype, including analyses of essential molecular signatures of TolDCs. Finally, we show a method to assess TolDC function by testing their immunosuppressive response in vitro and in vivo in a preclinical model of multiple sclerosis.

Here, we present a protocol to develop and characterize tolerogenic dendritic cells (TolDCs) and evaluate their immunotherapeutic utility.

Geliştirme ve işlevsel fare Tolerogenic dendritik hücrelerin karakterizasyonu

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state ...

Generation of Ventricular-Like HiPSC-Derived Cardiomyocytes and High-Quality Cell Preparations for Calcium Handling Characterization

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a valuable human source for studying the basic science of calcium (Ca2+) handling and signaling pathways as well as high-throughput drug screening and toxicity assays. Herein, we provide a detailed description of the methodologies used to generate high-quality iPSC-CMs that can consistently reproduce molecular and functional characteristics across different cell lines. Additionally, a method is described to reliably assess their functional characterization through the evaluation of Ca2+ handling properties. Low oxygen (O2) conditions, lactate selection, and prolonged time in culture produce high-purity and high-quality ventricular-like cardiomyocytes. Similar to isolated adult rat cardiomyocytes (ARCMs), 3-month-old iPSC-CMs exhibit higher Ca2+ amplitude, faster rate of Ca2+ reuptake (decay-tau), and a positive lusitropic response to &#946;-adrenergic stimulation compared to day 30 iPSC-CMs. The strategy is technically simple, cost-effective, and reproducible. It provides a robust platform to model cardiac disease and for the large-scale drug screening to target Ca2+ handling proteins.

Here we describe and validate a method to consistently generate robust human induced pluripotent stem cell-derived cardiomyocytes and characterize their function. These techniques may help in developing mechanistic insight into signaling pathways, provide a platform for large-scale drug screening, and reliably model cardiac diseases.

Ventriküler Benzeri HiPSC Türevi Kardiyomiyositlerin Ve Kalsiyum Kullanımı Karakterizasyonu için Yüksek Kaliteli Hücre Preparatlarının Üretimi

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a valuable human source for studying the basic science of calcium (Ca2+) ...

Functional Characterization of Endogenously Expressed Human RYR1 Variants

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia susceptibility, core myopathies and centronuclear myopathy. Because of the diverse phenotypes linked to RYR1 mutations it is fundamental to characterize their functional effects to classify variants carried by patients for future therapeutic interventions and identify non-pathogenic variants. Many laboratories have been interested in developing methods to functionally characterize RYR1 mutations expressed in patients&#39; cells. This approach has numerous advantages, including: mutations are endogenously expressed, RyR1 is not over-expressed, use of heterologous RyR1 expressing cells is avoided. However, since patients may present mutations in different genes aside RYR1, it is important to compare results from biological material from individuals harboring the same mutation, with different genetic backgrounds. The present manuscript describes methods developed to study the functional effects of endogenously expressed RYR1 variants in: (a) Epstein Barr virus immortalized human B-lymphocytes and (b) satellite cells derived from muscle biopsies and differentiated into myotubes. Changes in the intracellular calcium concentration triggered by the addition of a pharmacological RyR1 activators are then monitored. The selected cell type is loaded with a ratiometric fluorescent calcium indicator and intracellular [Ca2+] changes are monitored either at the single cell level by fluorescence microscopy or in cell populations using a spectrofluorometer. The resting [Ca2+], agonist dose response curves are then compared between cells from healthy controls and patients harboring RYR1 variants leading to insight into the functional effect of a given variant.

Here methods used to study the functional effect of RYR1 mutations endogenously expressed in Epstein Barr Virus immortalized human B-lymphocytes and muscle biopsy derived satellite cells differentiated into myotubes are described.

Endojen Olarak İfade Edilen İnsan RYR1 Varyantlarının İşlevsel Karakterizasyonu

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia ...

Modifications of the Langendorff Method for Simultaneous Isolation of Atrial and Ventricular Myocytes from Adult Mice

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction and electrical activity. Hence, isolating viable, high-quality cardiomyocytes from the heart is the initial and most crucial experimental step. Comparing the various protocols for isolating the cardiomyocytes of adult mice, the Langendorff retrograde perfusion is the most successful and reproducible method reported in the literature, especially for isolating ventricular myocytes. However, isolating quality atrial myocytes from the perfused heart remains challenging, and few successful isolation reports are available. Solving this complicated problem is extremely important because apart from ventricular disease, atrial disease accounts for a large part of heart diseases. Therefore, further investigations on the cellular level to reveal the mechanisms are warranted. In this paper, a protocol based on the Langendorff retrograde perfusion method is introduced and some modifications in the depth of aorta cannulation and the steps that may affect the digestion process to isolate atrial and ventricular myocytes were simultaneously made. Moreover, the isolated cardiomyocytes are confirmed to be amenable to patch clamp investigation.

This protocol describes modifications of the Langendorff method including the depth of aorta cannulation for simultaneous Isolation of atrial and ventricular myocytes from adult mice.

Yetişkin Farelerden Atriyal ve Ventrikül Miyositlerinin Eşzamanlı İzolasyonu için Langendorff Yönteminin Modifikasyonları

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction ...