RecCD47 ile 1. değiştirme Polimer Yüzeyler NOT:. Protokol, Şekil 1 &#39;de şematik olarak özetlenmiştir Şekil 1A, tiol-reaktif polimer yüzeylerinin oluşumunu göstermektedir Şekil 1B, tiol-reaktif recCD47 yaratılışını gösterir.. <ol><li> 1. Gün <ol><li> , Steril su içinde bir PDT-BzPh çözeltisi (1 mg / ml) ve potasyum bikarbonat (KHCO 3) (0.7 mg / ml) hazırlayın. 4 ° C (ışıktan koruma) gece boyunca karıştırın. </li></ol></li><li> Gün 2 <ol><li> (Dönen tekerlekler etrafında sığacak kadar uzun) 40 cm uzunluğunda parçalar halinde polimerik borunun kesin. </li><li> Bir çalkalama ya da Chandler döngü aygıtı üzerinde oda sıcaklığında 90 dakika boyunca hexacylpyridinium bir% 0.1 sulu çözelti içinde tüpler bekletin. 90 dakika bekletin sonra steril su 3x tüpleri durulayın. </li><li> (% 15 sulu potasyum fosfat monobazik eklenerek Gün 1 PDT BzPh çözeltisinin asitleştirilmesi KH 2 </sub&gt; PO 4). Bulanık bir misel çözeltisinin oluşturulması, PDT BzPh ml&#39;si başına 50 ul KH 2 PO 4 ekleyin. </li><li> Bir çalkalama veya aparat üzerinde oda sıcaklığında 40 dakika boyunca asidifiye PDT BzPh çözelti içinde tüpler bekletin. </li><li> Bir defa, seyrek asetik asit (1.000 1) ile boru durulayın. </li><li> 60 dakikalık bir toplam süresi boyunca UV-ışını tüpleri için Açığa. Tüm yüzey alanını aydınlatmak için her 15 dakikada ¼ tüpler döndürmek. </li><li> Bir çalkalama veya aparat üzerinde oda sıcaklığında 20 dakika boyunca, 20 mg / ml potasyum bikarbonat çözeltisine (KHCO 3) tüpler bekletin. </li><li> , Steril su içinde, 4 ° C&#39;de steril su ve 3 x deposuyla tüpleri durulayın. </li></ol></li><li> Gün 3 <ol><li> (: Çeker ocak içinde gerçekleştirme Dikkat) 200 ul dimetilformamid (DMF) içerisinde 5 mg Sülfo-SMCC çözülür. </li><li> RecCD47 poli-lizin çözeltisi 0.1 mg / ml aşama 1 &#39;de hazırlanmış olan Sülfo-SMCC çözeltisi 50 ul, ve RO&#39; de 60 dakika karışmasınaom sıcaklığı. </li><li> 60 dakika süreyle karıştırılmaya sırasında gazını Dulbecco Fosfat Tamponlu Salin (DPBS) ve bir kenara, steril. </li><li> Saflaştınlır Sülfo-SMCC, üretici talimatlarına göre bir 7K molekül ağırlıklı bir kesim sıkma tuz giderme kolonu kullanılarak recCD47 tepki gösterdi. Nihai akış yoluyla (yüksek kaliteli tiol-reaktif recCD47) toplayın ve kat DPBS ile borunun iç için gerekli hacme kadar seyreltin. </li><li> Steril ile 2 gün tüpleri durulayın, DPBS 3x gazı alındı. </li><li> En az 2 dakika süreyle gazdan arındırılmış DPBS içinde 20 mg / ml &#39;si ile tris (2-karboksietil) fosfin hidroklorür (TCEP) bir çözeltisi ile, tüplerin olarak değiştirilmiş bir yüzeyi React. Steril gazı alınmış DPBS 4x&#39;te tüpleri durulayın. </li><li> Tüplerine Sülfo-SMCC-reaksiyona recCD47 ekleyin ve bir çalkalama cihazında ya da 4 ° C&#39;de gece boyunca inkübe edilir. </li></ol></li></ol> Modifiye Yüzeyler üzerinde recCD47 2. İmmünotest Kantitasyonu <ol><li> Modifiye tüpleri durulayın Gün 3 zekâ gelen sayısal edilecekh DPBS 3x. Mağaza tüpler 4 ° C&#39;de DPBS ölçümü için kullanılmaz. </li><li> 96 çukurlu bir levhanın gözleri içine biyopsi zımbaları çift boru örnekleri yapmak ve yerleştirmek için 4 mm biyopsi zımbası kullanın. </li><li> Tespit antikoru ile tedavi edilmeyen modifiye edilmemiş bir boru numunesinin oluşan bir negatif kontrol hazırlayın. Tespit antikoru ile tedavi edilmiş bir modifiye edilmemiş boru numunesi aşağıdakilerden oluşan bir antikor kontrolü hazırlayın. Tespit antikoru ile muamele edilmiş tadil edilmiş boru test numunelerini hazırlayın. </li><li> Bir karıştırıcısı üzerinde oda sıcaklığında 60 dakika boyunca DPBS içinde% 0.4 büyükbaş hayvan serum albümini (BSA) ile blok örnekleri. </li><li> Engelleme aspirat BSA ve tris-tamponlu tuzlu su ve% 1 Tween-20 (TBST) 3x, bir çalkalayıcı üzerinde 10 dakika her biri ile yıkayın. </li><li> Bağışıklık için üreticinin tavsiye edilen seyreltme göre% 0.4 BSA içinde antikorun bir seyreltme çalışma hazırlayın. İnsan CD47 Antikor B6H12-FITC 1 seyreltilir: 100% 0.4 BSA içinde. </li><li> Uygun oyuklara antikor seyreltme 200 ul ilave edin. Tekni% 0.4 BSA, sadece Te negatif kontrol eder. Bir çalkalayıcı üzerinde 60 dakika boyunca (ışıktan koruma) için oda sıcaklığında inkübe edilir. </li><li> TBST&#39;dir 3x, 10 dakika bir çalkalayıcı her ile durulayın. Son TBST&#39;dir durulama aspire ve tüp numune kuyulara 200 ul DPBS ekleyin. </li><li> Nihai akış yoluyla yüksek kalitede tiol-reaktif rekombinant CD47 bir toplayın ve kat DPBS ile tüplerin iç için gerekli hacme kadar seyreltin. </li><li> FITC uyarma (485 nm) ve emisyon (538 nm) ayarları ile bir mikrotablaya okuyucu kullanarak FITC sinyal yoğunluğu okuyun. </li><li> Hesaplayın recCD47 otomatik floresans ve non-spesifik antikor, kontrol örneğinden bağlayıcı oluşturacak şekilde standart değerlere dayalı polimer yüzeyine bağlanır. </li></ol> 3. Chandler Döngü Aparatı Protokolü Kan toplama protokolü Kurumsal Değerlendirme Kurulu (KİK) onayı ve bilgilendirilmiş onam evrak önce insan kan numunelerinin toplanması başlamadan isteyin. Bilgi edininonaylanmamis bir insan kanı donörden onayı. Not: aparatı gösteren bir diyagramı Şekil 2 de gösterilmiştir. <ol><li> Yaklaşık olarak 1/2 tekerleklerinin çapı kadar damıtılmış su ile doldurun su banyosu içine monte edilmiştir. % 10 ağartıcı çözelti yapmak için su banyosu için yeterli çamaşır suyu ilave edin. Ayarlama, su 37 ° C banyo ısısı ve dengelenmeye gelmeye bırakın. </li><li> Metal (Şekil 1 B &#39;de gösterildiği gibi) bağdaştırıcılar, modifiye edilmemiş ve modifiye edilmiş boru boru toplayın, ve Şekil 1C&#39; de gösterildiği gibi, cihaz tekerlekleri etrafında bir araya getirin. Bu boru yerine metal adaptör ile tekerleğin etrafında kuytu uyduğundan emin olun. </li><li> Sitrat ya da diğer antikoagülan, 2 ml ile doldurulmuş bir şişe içinde, bir IRB onaylı protokol kullanılarak 30 ml&#39;lik bir kan örneği elde edilir, ve numune, bir kez tıkama bilmek çevirerek karıştırın. </li><li> Tüp içinde bir hava bırakarak metal valfi ve bir şırınga kullanarak her bir tüp için kan yaklaşık 10 ml ilave edilir. Valf kapağını ve r Secure3 saat boyunca otate. </li><li> % 10 çamaşır suyu çözeltisi ile muamele edilmiş bir atık behere kan akıtmak (ya da kurumsal politika tarafından gerekli). </li><li> Yavaşça bütün kan izlerini kaldırmak için DPBS boru içini durulayın. Akış yoluyla atık beher toplayın. Kurumsal politikası uyarınca kan imha edin. </li><li> Aparatı ve yüzeyleri% 10 çamaşır suyu çözeltisi kullanılarak ya da kurumsal politikaya göre temas herhangi bir kan dezenfekte edin. </li><li> Fluoresan mikroskobu ya da tarama elektron mikroskobu için işlem örnekleri aşağıda tarif edildiği gibi. </li></ol> 4. Floresan Mikroskopi ve Hücre Sayım <ol><li> % 4 paraformaldehid (PFA) solüsyonu (Dikkat: davlumbaz gerçekleştirin) hazırlayın. </li><li> 4 ° C de bir gece% 4 PFA çözeltisi içinde boru inkübe edin. </li><li> Gece boyunca inkübasyondan sonra,% 4 PFA çıkarın ve DPBS filmler çok yavaşça çalkalayın. </li><li> Boyama için lümen yüzey ortaya bölüme hortumu kesilir. </li><li> LekeOda sıcaklığında 30 dakika boyunca 4 &#39;, 6-diamidino-2-fenilindol (DAPI) ile ortam monte edilmesi için bir kaç damla ise, borunun bir bölümü (ışıktan koruma). Boyama sonra, arka plan DAPI sinyali azaltmak için DPBS ile çok yavaşça tüp durulayın. </li><li> DAPI filtre ve dijital kamera ile donatılmış bir floresan mikroskop kullanılarak görüntü 200X büyütmede görüntüsü altında 9 körü körüne seçilen alanlarda hücrelerin sayısını saymak için. </li><li> Tutanak hücre görüş alanı başına sayar ve sonuçların istatistiksel önemini belirlemek için uygun istatistiksel analizler gerçekleştirmek. </li></ol> 5. Taramalı Elektron Mikroskobu <ol><li> % 2 glutaraldehit çözeltisi (Dikkat: davlumbaz gerçekleştirin) hazırlayın. </li><li> Gece boyunca 4 ° C&#39;de% 2 glutaraldehid solüsyonunda boru inkübe edin. </li><li> Gece boyunca inkübasyondan sonra, yavaşça DPBS ile boru 3 kez yıkayın. </li><li> Ozmiyum tetroksit bir% 1 çözeltisi hazırlayın (Dikkat: Şiddetli inhalasyon tehlike! ) Davlumbaz kullanın. </li><li> Oda sıcaklığında 15 dakika boyunca ozmiyum tetroksit bir% 1 çözeltisi içinde boru inkübe edin. </li><li> Yavaşça DPBS boru 3x durulayın. </li><li> Etanol konsantrasyonları bir dizi (% 25,% 50,% 75,% 95 ve% 100 etanol) hazırlayın. </li><li> Etanol konsantrasyonu dizi içinde inkübe edilerek, boru kurutmak. % 25,% 50,% 75, ve 20 dakika her biri için% 95 etanol içinde inkübe edin. 30 dakika boyunca% 100 etanol içinde inkübe edin. </li><li> Süper kritik nokta nemi çıkarmak için 45 dakika boyunca CO2 ile boru kurutun. </li><li> Gümüş macun veya grafit ile bir örnek saplama Mount tüp bölümleri. </li><li> Altın paladyum 12.5 nm ile kaplayın tüp bölümleri. </li><li> Bir taramalı elektron mikroskobunda inceleyin. </li></ol>

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Bu yayında rapor Araştırma National, ödül sayısı T32 HL007915 (JBS ve RJL) kapsamında, ödül sayısı R21 EB015612 (SJS) ve Ulusal Kalp, Akciğer ve Kan Enstitüsü bünyesinde Biyomedikal Görüntüleme ve Biyomühendislik, Ulusal Enstitüsü tarafından desteklenen Sağlığı Enstitüsü.

(Şekil 1 &#39;de özetlenen veriler) foto-aktifleştirme kimyası PDT-BzPh eki ve daha sonra UV ışınlaması kolaylaştırmak için yeterli hidrokarbonlara hemen hemen herhangi bir polimer yüzeyinin değişimine olanak sağlar PDT BzPh fotoğrafı dışı bırakacaktır. Reaktif tiyol grupları ile polimerik yüzey fonksiyonalize ilgi test edilebilir bir molekül, bir dizi daha sonra eklenmesine izin verir. Bizim özel çalışmalarda biz rekombinant CD47 11-13 seçti. Bu iki fonksiyonlu ...

Örneğin kardiyopulmoner baypas ve böbrek diyalizi gibi pek çok klinik uygulamalar ise, polimerik kan kanalların kullanılmasını gerektirir ve genellikle işlem sonrası komplikasyonlara 1 ile ilişkilidir. Kan ile perfüze edilmiş olduğunda, bu polimerler, kan proteinleri ve trombositler, monosit / makrofaj yapışma emdirme ile sonuçlanır, yabancı cisim reaksiyonu (FBR) kural dışı, ve işlem sonrası komplikasyonlara ve katkı yapan tüm bu pro-enflamatuar sitokinlerin salınması / veya aygıt hatası 2,3. Böylece, bu sorunu çözmek için stratejiler biomateryaller önemli bir araştırma ve devam eden alan kalır. Müfettişler biyoaktif veya biyoinert molekülleri 4-6 kan temas yüzeyleri değiştirerek bu sorunu çözmek için çalıştılar. Laboratuvarımızda Araştırma FbR hafifletmek ve bu malzemelerin etkinliğini artırmak için bir strateji olarak polimerik biyomalzeme rekombinant CD47 (recCD47) ekleyerek odaklanmıştır. CD47 bir yerde ifade transmembr olduğuhücreleri 7-10 ve gösterileri ifade üzerine bağışıklık kaçırma bilinen bir rol kazandıran &quot;öz&quot; durumu ile ane protein polimerik yüzeyler 11-13 eklendiği takdirde biyouyumluluk veren de söz veriyorum. Sinyal-düzenleyici proteini alfa (SIRPα), CD47 için ortak kökenli alıcı, ve zar-ötesi proteinlerin ailesi-ihtiva-eden immüno reseptör tirosin bazlı inhibisyon motifi (İTİM) üyesi, miyeloid kökenli 14 hücreleri üzerinde ifade edilir. Daha önce CD47, SIRPα aracılı hücre sinyal iletimi yoluyla, in vitro, ex vivo olarak, poliüretan (PU) ve polivinil klorür (PVC) için bağışıklık tepkilerini aşağı regüle eder ve in vivo modellerde 11-13 olduğunu göstermiştir. Yapılan incelemelerde Orta kimyasal olarak reaktif tiyol gruplar kovalent olarak bir çok fonksiyonlu bir polimer (PDT Bz ile reaksiyona sokulması suretiyle borunun polimerik boru eklenmiş olduğu, burada tarif edilen bir nispeten yeni bir foto-aktifleştirme kimyası, bir2-piridilditiyo (PDT) oluşan) Ph, fotoreaktif benzofenon (BzPh) ve bir karboksi-değiştirilmiş polialilamin 11-13. Tris kovalent ekteki PDT gruplarının indirgenmesi (2-karboksietil) fosfin hidroklorür (TCEP); 11 sonradan olarak bir yüzey tiollenmiş terapotık kısımlar ile reaksiyona verir. Bu tarifnamede daha önce ayrıntılı 12,13, ayrıca bir C-terminali poli-lisin 12,13 kuyruk ilavesiyle modifiye recCD47, Sülfosukinimidil-4-[N -maleimidomethyl] sikloheksan-1-karboksilat ile reaksiyona sokulur (sülfo-SMCC) 1 saat boyunca ve boru 11 arasında bir recCD47 monosulfide bağ oluşumuna müsaade eden, tiol-reaktif gruplar oluşturmak için. CD47 fonksiyonalize yüzeylerinin anti-enflamatuar kapasitesi orjinal trombotik pıhtılaşma 15 bir in vitro modeli olarak, 1958 de tarif edilmiştir bütün insan kanı ile Chandler döngü aygıtı kullanılarak ex viv o test edilmiştir. Aparat bir dayanırkapalı bir boru sisteminin kısmen hava ve boru 15 yoluyla kan dolaşımı için bir döner motor ile doldurulur. Bu deneysel modelde, modifiye edilmiş ve modifiye edilmemiş yüzeyleri üzerine kan maruz kalma etkisi ve aynı zamanda kan hücrelerinin fizyolojisi üzerine, bu yüzey modifikasyon etkisini incelemek için bir fırsat sağlar. recCD47 Bu aktifleştirme kimyası kullanılarak polimer çeşitli yüzeyler eklenmiş olabilir, ve anti-enflamatuar kapasitesi polimer yüzeyleri 11,12 üzerinden kan perfüzyonunun taklit eden klinik olarak anlamlı bir eks vivo modeli kullanılarak değerlendirilebilir. Aparat insan kanı maruz kaldığında, değiştirilmemiş polimerlere kıyasla recCD47 modifiye klinik sınıf kan kanalları önemli ölçüde daha az trombosit ve inflamatuar hücre eki göstermektedir. Bu modifikasyon işleminin bir adım-adım bir açıklaması aşağıda ayrıntılı olarak açıklanmıştır.

<table border="0" cellpadding="0" cellspacing="0" width="802">
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			<td height="21" style="height:21px;width:269px;">Name</td>
			<td style="width:175px;">Company</td>
			<td style="width:107px;">Catalog Number</td>
			<td style="width:251px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">16% Paraformaldehyde (PFA)</td>
			<td style="width:175px;">Thermo Scientific</td>
			<td style="width:107px;">58906</td>
			<td>Caution! Use in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">25% Glutaraldehyde</td>
			<td style="width:175px;">VWR</td>
			<td style="width:107px;">AAA17876-AP&#160;</td>
			<td>Caution! Use in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">2-pyridyldithio,benzophenone (PDT-BzPH)</td>
			<td style="width:175px;">Synthesized in lab</td>
			<td style="width:107px;">N/A</td>
			<td></td>
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			<td height="21" style="height:21px;width:269px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">A3059-100G</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Citrate</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">S5770-50ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Digital Camera</td>
			<td style="width:175px;">Leica</td>
			<td style="width:107px;">DC500</td>
			<td>Out of production</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Dimethylformamide (DMF)</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">270547-100ML</td>
			<td>Caution! Use in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Dulbecco&#8217;s Phosphate Buffered Saline (DPBS)</td>
			<td style="width:175px;">Gibco/Life Technologies</td>
			<td style="width:107px;">14190-136</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Fluorescent Microscope</td>
			<td style="width:175px;">Nikon</td>
			<td style="width:107px;">TE300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Glacial Acetic Acid</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:107px;">A38-212</td>
			<td>Caution! Use in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Human CD47 (B6H12) &#8211; FITC Antibody</td>
			<td style="width:175px;">Santa Cruz Biotechnology</td>
			<td style="width:107px;">SC-12730</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Osmium Tetroxide</td>
			<td style="width:175px;">Acros Organics</td>
			<td style="width:107px;">197450050</td>
			<td>Caution! Use in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Potassium Bicarbonate (KHCO3)</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">237205-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Potassium Phosphate Monobasic (KH2PO4)</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">P5655-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">PVC Tubing (Cardiovascular Procedure Kit)</td>
			<td style="width:175px;">Terumo Cardiovascular Systems</td>
			<td style="width:107px;">60050</td>
			<td>Most clinical-grade tubing will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Scanning Electron Microscope</td>
			<td>JEOL</td>
			<td>JSM-T330A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:269px;">Sodium Chloride (NaCl)</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td>BP358-212</td>
			<td></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:269px;">Microplate Reader</td>
			<td style="width:175px;">Molecular Devices</td>
			<td style="width:107px;">Spectramax Gemini EM&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Sulfo-SMCC</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">M6035-10MG</td>
			<td>Moisture Sensitive!</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">tris (2-carboxyethyl) phosphine (TCEP-HCl)</td>
			<td style="width:175px;">Thermo Scientific</td>
			<td style="width:107px;">20491</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:269px;">Tris Base</td>
			<td style="width:175px;">Sigma</td>
			<td>T1503-100G</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:269px;">Tween-20</td>
			<td>Bio-Rad</td>
			<td>170-6531</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Vectashield with DAPI</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:107px;">H-1200</td>
			<td>Light sensitive!</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Zeba Spin Desalt Columns &#8211; 7K MWCO</td>
			<td style="width:175px;">Thermo Scientific</td>
			<td style="width:107px;">89891</td>
			<td></td>
		</tr>
	</tbody>
</table>

the use of the ex vivo chandler loop apparatus to assess the biocompatibility of modified polymeric blood conduits

Sentetik yüzey gövdesine içeri getirildiği zaman, yabancı cisim reaksiyon meydana gelir. Bu işlem sonrası komplikasyonlara yol açan, kan proteinleri adsorpsiyon ve trombositlerin arkaya takılmasının ve aktivasyonu, monosit / makrofaj yapışma, ve inflamatuar hücre sinyal olayları ile karakterize edilir. Chandler Döngü Aparatı araştırmacılar kan geniş hacimli polimerik kanallar üzerinde perfüze zaman meydana gelen moleküler ve hücresel etkileşimleri çalışmak için izin veren deneysel bir sistemdir. Bu amaçla, bu cihaz, çeşitli polimer yüzey modifikasyonları, anti-enflamatuar özellikleri tahmin edilmesine izin veren bir ex vivo bir modeli olarak kullanılmıştır. Laboratuvarımız kovalent rekombinant CD47 ile aktifleştirme kimyası ile modifiye kan kanallar, polimerik yüzeylere biyouyumluluk kazandıran göstermiştir. Polimerik yüzeylere CD47 ekleme polimerik kan kanalların etkinliğini teşvik etmek etkili bir araç olabilir. HerEin CD47 değiştirilebilir ve kontrol kanallarına kan etkileşimleri incelemek için klinik olarak anlamlı bir polimer, kan ve boru, bir ex vivo deney modeli olarak Chandler özenin kullanımının, rekombinant CD47 eklemek için kullanılan foto-aktifleştirme kimyası ayrıntılı bir yöntemdir.

SMCC ile tiyol-reaktif recCD47 poli-lisin ile birlikte PDT BzPh ve TCEP kullanımı yoluyla tiol-reaktif polimer yüzeyleri oluşturma polimerik yüzeylere recCD47 eklenmesine izin verir. Modifikasyon işlemi Şekil 1 şematik olarak özetlenmiştir. Bu modifikasyon işleminin rahatlık, bu protein varsayarak, örneğin amin içeren bir lisin ve yeterli kimyasal yönden reaktif gruplar ile modifiye edilebilir, bir çok farklı protein ve bir çok farklı polimerik yüzeylere uygulanabilir olmasıdır , ...

Watch this Scientific Journal Video about The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits at JoVE.com

The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

Blood exposure to polymeric blood conduits initiates the foreign body reaction that has been implicated in clinical complications. Here, the Chandler Loop Apparatus, an experimental tool mimicking blood perfusion through these conduits, is described. Appendage of recombinant CD47 results in decreased evidence of the foreign body reaction on these conduits.

Kullanımı<em&gt; Ex vivo</em&gt; Chandler Döngü Aparatı Modifiye Polimer Kan Kondüit biyouyumuna De¤erlendirilir

The foreign body reaction occurs when a synthetic surface is introduced to the body. It is characterized by adsorption of blood proteins and the ...

the-use-ex-vivo-chandler-loop-apparatus-to-assess-biocompatibility

Research

JoVE Journal

Bioengineering

The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

11.5K Görüntüleme.  The Children's Hospital of Philadelphia. Blood exposure to polymeric blood conduits initiates the foreign body reaction that has been implicated in clinical complications. Here, the Chandler Loop Apparatus, an experimental tool mimicking blood perfusion through these conduits, is described. Appendage of recombinant CD47 results in decreased evidence of the foreign body reaction on these conduits. 

Video: The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

Devirli Basınç Biyoreaktör Tasarım Ex vivo</emAort Kalp Vanalar> Eğitim

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Biyomühendislik

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

Plasenta karşısında Ksenobiyotik ve Nanomalzemelerin Ulaştırma Hızı belirlenmesi kullanarak<em&gt; Ex vivo</em&gt; İnsan Plasental Perfüzyon Modeli

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

Yöntem kan-beyin bariyerini bir insan hücre tabanlı bir İletişim Modelinin Hazırlanması için Geliştirilmiş

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

Genetik olarak tadil edilmiş hücre transplantasyonu için Micropatterned kültür plakalarında Sferoid hücrelere karşı gene

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential to establish an ex vivo model that can evaluate SIM performance accurately, for developing high-performance SIMs. In our previous study, we developed a new ex vivo model that can be used to evaluate the performance of various SIMs in detail by applying constant tension to the specimen&#39;s ends. We also confirmed that the proposed new ex vivo model allows accurate submucosal elevation height (SEH) measurement under uniform conditions and detailed comparisons of the performances of various types of SIMs. Here, we describe the new ex vivo model and explain the detailed setup methodology of this model. Since all parts of the new model were easy to obtain, the setup of the new model could be completed quickly. SEH of various SIMs could be measured more accurately by using the new model. The critical factor that determines SIM performance can be identified using the new model. SIM development speed will drastically increase after the factor has been identified.

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

We developed a new ex vivo model that applies constant tension to the porcine gastric specimen. This development made it possible to evaluate the performance (the height and duration of the submucosal elevation) of various SIMs accurately.&#160; The detailed setup methodology of this new model is explained.

Endoskopik Submucosal enjeksiyon malzemesi performans değerlendirilmesi için yeni bir Ex Vivo Model

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential ...

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome absorbance respectively. In addition, numerous aspects of the mitochondrial metabolic status such as membrane potential and substrate entry can also be estimated. To perform cardiac wall transmission optical spectroscopy, a commercially available side-firing optical fiber catheter is placed in the left ventricle of the isolated perfused heart as a light source. Light passing through the heart wall is collected with an external optical fiber to perform optical spectroscopy of the heart in near real- time. The transmission approach avoids numerous surface scattering interference occurring in widely used reflection approaches. Changes in transmural absorbance spectra were deconvolved using a library of chromophore reference spectra, providing quantitative measures of all the known cardiac chromophores simultaneously. This spectral deconvolution approach eliminated intrinsic errors that may result from using common dual wavelength methods applied to overlapping absorbance spectra, as well as provided a quantitative evaluation of the goodness of fit. A custom program was designed for data acquisition and analysis, which permitted the investigator to monitor the metabolic state of the preparation during the experiment. These relatively simple additions to the standard heart perfusion system provide a unique insight into the metabolic state of the heart wall in addition to conventional measures of contraction, perfusion, and substrate/oxygen extraction.

Here we introduce a method for using an intra-ventricle optical catheter in perfused hearts to perform absorbance spectroscopy across the heart wall. The data obtained provides robust information on tissue oxygen tension as well as substrate utilization and membrane potential simultaneously with cardiac performance measures in this ubiquitous preparation.

Perfkullanılmış Mammalin kalpleri transmural absorbance spektroskopisi kullanarak hücresel metabolizmayı Izleme için kalp içi yan ateş ışık kateter

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome ...

Blood Flow Imaging with Ultrafast Doppler

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler modes, it has several limits. Firstly, a finely tuned signal filtering operation is needed to distinguish blood flows from surrounding moving tissues. Secondly, the operator must choose between localizing the blood flows or quantifying them. In the last two decades, ultrasound imaging has undergone a paradigm shift with the emergence of ultrafast ultrasound using unfocused waves. In addition to a hundredfold increase in framerate (up to 10000 Hz), this new technique also breaks the conventional quantification/localization trade-off, offering a complete blood flow mapping of the field of view and a simultaneous access to fine velocities measurements at the single-pixel level (down to 50 &#181;m). This data continuity in both spatial and temporal dimensions strongly improves the tissue/blood filtering process, which results in an increase sensitivity to small blood flow velocities (down to 1 mm/s). In this method paper, we aim to introduce the concept of ultrafast Doppler as well as its main parameters. Firstly, we summarize the physical principles of unfocused wave imaging. Then, we present the Doppler signal processing main steps. Particularly, we explain the practical implementation of the critical tissue/blood flow separation algorithms and on the extraction of velocities from these filtered data. This theoretical description is supplemented by in vitro experiences. A tissue phantom embedding a canal with flowing blood-mimicking fluid is imaged with a research programmable ultrasound system. A blood flow image is obtained and the flow characteristics are displayed for several pixels in the canal. Finally, a review of in vivo applications is proposed, showing examples in several organs such as carotids, kidney, thyroid, brain and heart.

This protocol shows how to apply ultrafast ultrasound Doppler imaging to quantify blood flows. After a 1 s long acquisition, the experimenter has access to a movie of the full field of view with axial velocity values for each pixel every &#8776;0.3 ms (depending on the ultrasound time of flight).

Ultra Hızlı Doppler ile Kan Akışı Görüntüleme

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler ...

&quot;Avatar&quot;- Optimized Ex Vivo Work Loop Experiment to Emulate In Vivo Muscle Force

"Avatar", a Modified Ex vivo Work Loop Experiments Using In vivo Strain and Activation

Movement behaviors are emergent features of dynamic systems that result from muscle force production and work output. The interplay between neural and mechanical systems occurs at all levels of biological organization concurrently, from the tuning of leg muscle properties while running to the dynamics of the limbs interacting with the ground. Understanding the conditions under which animals shift their neural control strategies toward intrinsic muscle mechanics (&#39;preflexes&#39;) in the control hierarchy would allow muscle models to predict in vivo muscle force and work more accurately. To understand in vivo muscle mechanics, ex vivo investigation of muscle force and work under dynamically varying strain and loading conditions similar to in vivo locomotion is required. In vivo strain trajectories typically exhibit abrupt changes (i.e., strain and velocity transients) that arise from interactions among neural activation, musculoskeletal kinematics, and loads applied by the environment. The principal goal of our &#34;avatar&#34; technique is to investigate how muscles function during abrupt changes in strain rate and loading when the contribution of intrinsic mechanical properties to muscle force production may be highest. In the &#34;avatar&#34; technique, the traditional work-loop approach is modified using measured in vivo strain trajectories and electromyographic (EMG) signals from animals during dynamic movements to drive ex vivo muscles through multiple stretch-shortening cycles. This approach is similar to the work-loop technique, except that in vivo strain trajectories are scaled appropriately and imposed on ex vivo mouse muscles attached to a servo motor. This technique allows one to: (1) emulate in vivo strain, activation, stride frequency, and work-loop patterns; (2) vary these patterns to match in vivo force responses most accurately; and (3) vary specific features of strain and/or activation in controlled combinations to test mechanistic hypotheses.

Author Spotlight: Bridging the Gap Between In Vivo and Ex Vivo Studies with the &quot;Avatar&quot; Technique to Advance Muscle Mechanics Research 

This article details the methodology for emulating in vivo muscle force production during ex vivo work loop experiments using an "avatar" muscle from a laboratory rodent to assess the contributions of strain transients and activation to the muscle force response.

Movement behaviors are emergent features of dynamic systems that result from muscle force production and work output. The interplay between neural and ...

Preparation of Cardiomyocytes and Setting-Up the Apparatus for the Slow Injection Method

Prepare the 10%gelatin stock solution by weighing and dissolving the gelatin in the ADS buffer, and autoclaving the solution. Rewarm the 10%gelatin stock at 37 degrees Celsius, until it becomes liquid. Then dilute the gelatin stock, using a prewarmed ADS buffer, to obtain the final injectable 5%gelatin solution.Suspend 96 cardiomyocyte balls in 100 microliters of prewarmed injection solution. Load the cardiomyocyte ball suspension into the cell injection syringe without aspirating the excess air. Next, hold the syringe vertically, with the needle facing upward, and tap the syringe to collect the bubbles at the upper ridge of the syringe.Also, observe the cardiomyocyte balls gradually settling onto the rubber seal of the plunger. Maintain the vertical position of the syringe, and slowly push the plunger to discard the bubbles and excess cell suspension solution, until 20 microliters of the cell suspension remain in the syringe, and the cardiomyocyte ball remains resting on the rubber seal of the plunger. Once done, immerse the capped syringe in an ice bath for five minutes, and fix the injection syringe apparatus on the animal stage before activating the main syringe pump during percutaneous cell transplantation in an anesthetized rat.

Kardiyomiyositlerin hazırlanması ve yavaş enjeksiyon yöntemi için aparatın ayarlanması

İmmünofloresan Boyama ile Kan-Beyin Bariyer Modellerinin Analizi

Use of the MicroSiM (&#181;SiM) Barrier Tissue Platform for Modeling the Blood-Brain Barrier

The microSiM (&#181;SiM) is a membrane-based culture platform for modeling the blood-brain barrier (BBB). Unlike conventional membrane-based platforms, the&#160;&#181;SiM provides experimentalists with new capabilities, including live cell imaging, unhindered paracrine signaling between &#39;blood&#39; and &#39;brain&#39; chambers, and the ability to directly image immunofluorescence without the need for the extraction/remounting of membranes. Here we demonstrate the basic use of the platform to establish monoculture (endothelial cells) and co-culture (endothelial cells and pericytes) models of the BBB using ultrathin nanoporous silicon-nitride membranes. We demonstrate compatibility with both primary cell cultures and human induced pluripotent stem cell (hiPSC) cultures. We provide methods for qualitative analysis of BBB models via immunofluorescence staining and demonstrate the use of the &#181;SiM for the quantitative assessment of barrier function in a small molecule permeability assay. The methods provided should enable users to establish their barrier models on the platform, advancing the use of tissue chip technology for studying human tissues.

Yazar Spotlight: İnsan Dokularını İncelemek için Doku Çipi Teknolojisinin Kullanımını Geliştirme 

This report provides protocols for assembly, cell culture, and assays on the &#181;SiM platform for the construction of blood-brain barrier models.

Kan-Beyin Bariyerini Modellemek için MicroSiM (μSiM) Bariyer Doku Platformunun Kullanımı

The microSiM (&#181;SiM) is a membrane-based culture platform for modeling the blood-brain barrier (BBB). Unlike conventional membrane-based platforms, ...