3R01AR054458-02S2: Bu çalışma, Ulusal Artrit ve Kas-İskelet Sistemi Enstitüsü ve Cilt Hastalıkları bir hibe ile desteklenmiştir.

1. 1. Gün 1) Plaka hücreleri <ol><li> Etiketleme için en az 18-24 saat önce T75 confluency 80% balonuna Plaka hMSC. Alternatif gemiler için talimat için Tablo 1&#39;e bakınız. </li></ol> 2. 2. Gün 2) etiketleme çözümü hazırlayın. Bu preparat, 400 mikrogram Fe / ml &#39;lik bir konsantrasyon ile% 80 confluency az bir (1) T75 şişesi etiketi. Alternatif gemiler için talimat için Tablo 1&#39;e bakınız. <ol><li> 15 ml konik boru: 1 ml serum özgür medya ve 93.1 ul ferumoxytol (30 mg / ml hisse senedi) karıştırarak bir çözüm (solution 1) oluşturun. </li><li> Ikinci bir 15 ml konik tüp: Serum özgür medya ve protamin sülfat 7 ul (10 mg / ml hisse senedi) 1 ml karıştırılarak ikinci bir çözüm (çözüm 2) oluşturun. </li><li> Her çözüm 5 dakika dinlenmek için izin verin. </li><li> Çözüm 1 ve 2 birlikte ekleyin. Yeni etiketleme çözümü yavaşça karıştırın. 5 dakika dinlenmek için çözüm USPIO-pro izin için izin verbronşial histamin sülfat kompleksleri oluşturur. </li><li> SPIO / son bir hacim 7 ml protamin sülfat çözeltisi karışık 2 ml 5 ml serum ücretsiz medya ekleyin. </li></ol> 3) etiketlenmesi için hücreleri hazırlayın <ol><li> Hücrelerden medya aspire. </li><li> Önceden ısıtılmış Mg / Ca-ücretsiz D-PBS veya serum ücretsiz medya 2-3 ml kontrast madde alımı ve etiketleme verimliliği bozabilecek artık serum proteinlerinin ve diğer medya bileşenleri nazikçe yıkayın hücreleri bir kez yıkayın. Durulama sıvısı aspire. </li></ol> 4) Etiket hücreleri <ol><li> Hücreler için çözüm etiketleme tam 7 ml ekleyin. </li><li> Bir inkübatör (37 ° C /% 5 CO 2) ve hücrelerin, 4 saat boyunca etiketleme çözümü inkübe izin icine koyun hücreleri . </li><li> % 10 son bir serum konsantrasyonu elde etmek için FCS 700 ul hücrelerin etiketleme çözümü ekleyin. Serum hücreleri alışkın olan zenginleştirilmiş ortam yeniden kurmak için, ve mil yardımcı olmak için eklenirani bir kayma serum serbest bir ortamda 24 saat maruz kalma hücre ölümüne neden olabilir nimizing. </li><li> Hücreleri etiketleme çözümü ile ek bir 20 saat süreyle inkübe izin ver. </li></ol> 3. 3. Gün 5) işaretli hücrelerin hazırlayın <ol><li> Etiketleme çözümü hücreleri aspire. </li><li> Önceden ısıtılmış Mg / Ca-ücretsiz D-PBS 2-3 ml hücreleri nazikçe yıkayın. PBS aspire. Bu ücretsiz PBS-Mg / Ca, Mg ve Ca sonraki adımda tripsin verimliliğini olumsuz olarak kullanmak önemlidir. </li><li> Hücrelerin% 0.05 tripsin önceden ısıtılmış 2 ml ekleyin ve balon balonun tüm yüzey sağlamak için ileri ve geri eğim tripsin ince bir tabaka ile kaplıdır. Hücrelerin bir inkübatör (37 ° C /% 5 CO 2) yerleştirin ve hücreleri plaka ayırmak alıncaya kadar hücreleri veya 5-7 dakika dinlenmek için izin. Bu mikroskop altında dekolmanı onaylamak için gerekli olabilir. Yavaşça gerekirse fa balonun iki dokununhücre yüzeyi dekolmanı cilitate. </li><li> Tripsin nötralize etmek için balona 4 ml önceden ısıtılmış eksiksiz bir medya ekleyin. Hafifçe birkaç kez tripsin / medya / hücre çözümü yukarı ve aşağı pipetleme şişeyi çalkalayın. 15 ml konik bir tüp içinde tüm çözüm toplayın. 5 dakika boyunca 400 RCF hücre çözümü santrifüjleyin. </li><li> Hücre pelletini bozmadan süpernatantı dikkatlice aspire. Medya 5 ml (serum içeren veya serum) hücrelerin tekrar 5 dakika boyunca 400 RCF hücre çözümü santrifüjleyin. </li><li> Adım 5.5 &#39;de açıklanan hücre yıkama tekrarlayın. Toplamda, hücreler üç kez medya ve hücre yüzeyinde kalan, ücretsiz kontrast madde kaldırmak için durulanır olacaktır. </li><li> Hücreleri önceki yıkama esnasında kaybolacak gibi, en doğru hücre sayısını elde etmek için, bu noktada hücreleri güvenin. Bir hücre canlılığı testi yapın. Hücreler artık bir sonraki analiz ya da deneysel uygulama için hazır. MR görüntüleme ve T1-w ile yapılabilir<html> T2-w parametreleri, ferumoxytol T2-w kontrast madde olmasına rağmen, çünkü ferumoxytol de T1 etkileri sergiler. Görüntü ferumoxytol etiketli hücreler için kullanılabilir Temsilcisi T2-w MR parametreler şunlardır: 60-80 ms 2000-2500 ms ve TE TR FSE veya SE_Multislice dizileri. T1-w bir görüntü elde etmek için, FSE TE değeri veya SE_Multislice dizisi azaltmak veya yüksek TR ve TE düşük GRE dizisi kullanmak. Şekil 4, in vivo uygulama etiketli kök hücre görüntüleme için önemli bir potansiyel göstermektedir . </li></ol> 4. Temsilcisi Sonuçlar: Etiketli hücreleri T2 ağırlıklı MR görüntülerinde önemli koyulaşması veya negatif kontrast etkisi ve T1 ağırlıklı MR görüntülerinde bir parlatma veya pozitif kontrast etkisi (Şekil 2) göstermektedir. Boyuna MR ve canlılık testleri, 5 gün etiketleme anlamlı MR sinyal ve etiketsiz denetimleri (veriler gösterilmemiştir) ile karşılaştırıldığında canlılığı üzerinde anlamlı bir etkiye gösterdi yapıldı. jove_content &quot;&gt; Etiketli kök hücreler daha sonra çeşitli hücre tipleri ayırt edilmesini veya intravenöz in vivo soruşturma enjekte . <img alt="Şekil 1" src="/files/ftp_upload/3482/3482fig1.jpg" /> Şekil 1, kök hücreler, demir oksit nanoparçacık, ferumoxytol etiketlenmesi için şematik zaman çizgisi. <img alt="Şekil 2" src="/files/ftp_upload/3482/3482fig2.jpg" /> Şekil 2. hücre pelletini hücreleri ile sagittal kesitte thrrough Eppendorf tüpleri MR görüntüleri. A) Etiketsiz kontrolleri. B) veya negatif kontrast madde etkisi önemli bir T2 T2 ağırlıklı FSE SE_Multislice görüntüleme ve T1 ağırlıklı GRE sekansında T1 ya da pozitif bir kontrast madde etkisi gösteren Etiketli hücreleri. <img alt="Şekil 3" src="/files/ftp_upload/3482/3482fig3.jpg" /> Şekil 3 Eppendorf tüpleri hücreleri ile hücre pel sagittal kesitlersağlar. Az 10.000 ferumoxytol etiketli hücreleri, T2-w MR ile tespit edilebilir. <img alt="Şekil 4" src="/files/ftp_upload/3482/3482fig4.jpg" /> Şekil 4 ferumoxytol etiketli kök hücrelerin MR görüntüleme fare serebral ventriküller içine enjekte. A) Aksiyel hiçbir USPIO etiketli kök hücre enjeksiyonu ile fare Barin, MR görüntüsü. B) Koronal Aksiyal, C), ve D) sagital MR görüntüleri bir fare beynine enjekte USPIO etiketli kök hücreler (beyaz ok) T2, negatif kontrast etkisi gösteren. <img alt="Tablo 1" src="/files/ftp_upload/3482/3482table1.jpg" /> Tablo 1. alternatif damarlarında hücrelerin etiketlenmesi için Miktar adaptasyonlar.

<ol>
	<li>Narsinh, K. H., Plews, J., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+human+induced+pluripotent+and+embryonic+stem+cells:+fraternal+or+identical+twins?.">Comparison of human induced pluripotent and embryonic stem cells: fraternal or identical twins?.</a> Mol Ther. 19, 635-638 (2011).</li><li>Bulte, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+MRI+cell+tracking:+clinical+studies.">In vivo MRI cell tracking: clinical studies.</a> AJR. Am. J. Roentgenol. 193, 314-325 (2009).</li><li>Henning, T. D., Boddington, S., Daldrup-Link, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labeling+hESCs+and+hMSCs+with+Iron+Oxide+Nanoparticles+for+Non-Invasive+in+vivo+Tracking+with+MR+Imaging.">Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging.</a> J. Vis. Exp. (13), e685-e685 (2008).</li><li>Tallheden, T., Nannmark, U., Lorentzon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+MR+imaging+of+magnetically+labeled+human+embryonic+stem+cells.">In vivo MR imaging of magnetically labeled human embryonic stem cells.</a> Life. Sci. 79, 999-1006 (2006).</li><li>Jung, C. W., Jacobs, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+and+chemical+properties+of+superparamagnetic+iron+oxide+MR+contrast+agents:+ferumoxides,+ferumoxtran,+ferumoxsil.">Physical and chemical properties of superparamagnetic iron oxide MR contrast agents: ferumoxides, ferumoxtran, ferumoxsil.</a> Magn. Reson. Imaging. 13, 661-674 (1995).</li><li>Coyne, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ferumoxytol+for+treatment+of+iron+deficiency+anemia+in+patients+with+chronic+kidney+disease.">Ferumoxytol for treatment of iron deficiency anemia in patients with chronic kidney disease.</a> Expert. Opin. Pharmacother. 10, 2563-2568 (2009).</li><li>Li, Z., Suzuki, Y., Huang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+reporter+gene+and+iron+particle+labeling+for+tracking+fate+of+human+embryonic+stem+cells+and+differentiated+endothelial+cells+in+living+subjects.">Comparison of reporter gene and iron particle labeling for tracking fate of human embryonic stem cells and differentiated endothelial cells in living subjects.</a> Stem Cells. 26, 864-873 (2008).</li><li>Metz, S., Bonaterra, G., Rudelius, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capacity+of+human+monocytes+to+phagocytose+approved+iron+oxide+MR+contrast+agents+in+vitro.">Capacity of human monocytes to phagocytose approved iron oxide MR contrast agents in vitro.</a> Eur. Radiol. 14, 1851-1858 (2004).</li><li>Nedopil, A., Klenk, C., Kim, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MR+signal+characteristics+of+viable+and+apoptotic+human+mesenchymal+stem+cells+in+matrix-associated+stem+cell+implants+for+treatment+of+osteoarthritis.">MR signal characteristics of viable and apoptotic human mesenchymal stem cells in matrix-associated stem cell implants for treatment of osteoarthritis.</a> Invest. Radiol. 45, 634-640 (2010).</li><li>Kraitchman, D. L., Heldman, A. W., Atalar, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+magnetic+resonance+imaging+of+mesenchymal+stem+cells+in+myocardial+infarction.">In vivo magnetic resonance imaging of mesenchymal stem cells in myocardial infarction.</a> Circulation. 107, 2290-2293 (2003).</li><li>Stuckey, D. J., Carr, C. A., Martin-Rendon, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+particles+for+noninvasive+monitoring+of+bone+marrow+stromal+cell+engraftment+into,+and+isolation+of+viable+engrafted+donor+cells+from,+the+heart.">Iron particles for noninvasive monitoring of bone marrow stromal cell engraftment into, and isolation of viable engrafted donor cells from, the heart.</a> Stem Cells. 24, 1968-1975 (2006).</li><li>Henning, T. D., Sutton, E. J., Kim, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+ferucarbotran+on+the+chondrogenesis+of+human+mesenchymal+stem+cells.">The influence of ferucarbotran on the chondrogenesis of human mesenchymal stem cells.</a> Contrast. Media. Mol. Imaging. 4, 165-173 (2009).</li><li>Arbab, A. S., Yocum, G. T., Kalish, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+magnetic+cell+labeling+with+protamine+sulfate+complexed+to+ferumoxides+for+cellular+MRI.">Efficient magnetic cell labeling with protamine sulfate complexed to ferumoxides for cellular MRI.</a> Blood. 104, 1217-1223 (2004).</li><li>Nedopil, A. J., Mandrussow, L. G., Daldrup-Link, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implantation+of+Ferumoxides+Labeled+Human+Mesenchymal+Stem+Cells+in+Cartilage+Defects.">Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects.</a> J. Vis. Exp. (38), e1793-e1793 (2010).</li><li>Arbab, A. S., Yocum, G. T., Wilson, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+transfection+agents+in+forming+complexes+with+ferumoxides,+cell+labeling+efficiency,+and+cellular+viability.">Comparison of transfection agents in forming complexes with ferumoxides, cell labeling efficiency, and cellular viability.</a> Mol Imaging. 3, 24-32 (2004).</li><li>Babic, M., Horak, D., Trchova, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(L-lysine)-modified+iron+oxide+nanoparticles+for+stem+cell+labeling.">Poly(L-lysine)-modified iron oxide nanoparticles for stem cell labeling.</a> Bioconjug Chem. 19, 740-750 (2008).</li><li>Golovko, D. M., T, H. e. n. n. i. n. g., Bauer, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+stem+cell+labeling+with+ferucarbotran+and+protamine.">Accelerated stem cell labeling with ferucarbotran and protamine.</a> Eur. Radiol. 20, 640-648 (2010).</li><li>Lu, M., Cohen, M. H., Rieves, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FDA+report:+Ferumoxytol+for+intravenous+iron+therapy+in+adult+patients+with+chronic+kidney+disease.">FDA report: Ferumoxytol for intravenous iron therapy in adult patients with chronic kidney disease.</a> Am. J. Hematol. 85, 315-319 (2010).</li></ol>

Bu çalışmada, tüm katkıda açıklamalar sunuyoruz.

Kök hücre engraftments etkinliğini artırma rejeneratif tıbbın ilerlemesi için kritik öneme sahiptir. In vivo kök hücreleri için bir non-invaziv görüntüleme tekniği önemli ölçüde başarılı engraftman sonuçlara yol açan mekanizmaları anlamak için yeteneğimizi artırır. Manyetik etiketleme için prosedür olarak ortaya koyduk MR görselleştirme, MR görüntüleme ile kök hücrelerin in vivo takibi sağlar. Manyetik etiketli kök hücreleri hedef dokulara daha önce nakledilen ...

<table border="1"><tbody><tr><td> Reaktifi Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td> D-MEM Yüksek Glikoz </td><td> Sigma </td><td> D5648 </td><td> Kullanılmak istenen kök hücre hattı veya diğer taban orta </td></tr><tr><td> D-PBS (Ca + +, Mg + + ücretsiz) </td><td> Gibco </td><td> 14190-144 </td><td></td></tr><tr><td> Tripsin-EDTA% 0.05 </td><td> Invitrogen </td><td> 25300-120 </td><td></td></tr><tr><td> Fetal sığır serumu (FBS) </td><td> Hyclone </td><td> SH30071.03 </td><td></td></tr><tr><td> Ferumoxytol (Feraheme) </td><td> AMAG </td><td> 59338-0775-01 </td><td></td></tr><tr><td> Protamin Sülfat </td><td> APP Ecz. </td><td> 22930 </td><td></td></tr>
</tbody>
</table>

labeling stem cells with ferumoxytol, an fda-approved iron oxide nanoparticle

Kök hücre temelli tedaviler rejeneratif tıp alanında önemli bir potansiyel sunmaktadır. Ancak, çok nakledilen hücrelerin in vivo kinetiği ile ilgili olarak anlaşılmalıdır kalır. In vivo olarak nakledilen kök hücrelerin arka arkaya izlemek için bir non-invaziv yöntem, araştırmacılar kök hücre nakli doğrudan izlemek için izin ve başarılı veya başarısız engraftman sonuçların belirlenmesi . Kök hücrelerin geniş bir yelpazede sayısız uygulamalar için soruşturma devam ediyor. Insan embriyonik böbrek, 293 (HEK293) hücreler, insan mezenkimal kök hücreler (hMSC) ve uyarılmış pluripotent kök (iPS) hücreleri: Bu protokol, 3 farklı kök hücre popülasyonlarının üzerinde duruluyor. HEK 293 hücreler makaslanmış adenovirüs 5 DNA kültür yetiştirilen insan embriyonik böbrek hücreleri elde edilir. Bu hücreler kolayca kültüre, çünkü araştırma yaygın olarak kullanılan, hızlı büyümek ve kolayca transfekte. hMSCs yetişkin kemik iliği bulunur. Bu hücreler ca,multipotency veya sınırlı sayıda hücre kaderi içine ayırt etmek için potansiyel korurken n farklılaşmamış hücreler olarak çoğaltılan. hMSCs osteoblastlar, adipositler, kondrosit, tendon, kas ve kemik iliği stroma içeren mezenkimal doku, soy ayırt edebilirsiniz. iPS hücreleri, genetik, genler ve benzeri faktörler, embriyonik kök hücreleri tanımlayan özellikleri ifade etmek için değiştirilmiş yetişkin hücreler reprogrammed. Bu hücreler, tüm hücre soyları 1 içine ayırt etmek için kapasitesine sahip pluripotent anlamı vardır . Her iki hMSCs ve iPS hücreleri, doku yenileyici kapasitesi in vivo göstermiştir . Süperparamanyetik demir oksit (SPIO) nanoparçacık hücre etiketleri kullanımı ile birlikte manyetik rezonans (MR) görüntüleme yakın mikroskobik anatomik çözünürlük nedeniyle kök hücrelerin in vivo takibi için etkili olduğu kanıtlanmıştır, bu yarı-ömrü daha uzun bir kan uzunlamasına görüntüleme ve izin yüksek sensitivity SPIO MR tarafından sağlanan hücre tespiti için 2-4 nanopartiküller. Buna ek olarak, SPIOs kullanımı ile MR görüntüleme klinik olarak çevrilebilir. SPIOs bioreactive demir çekirdek plazma bileşenleri içeren hizmet veren bir dekstran, carboxydextran veya nişasta yüzey tabakası ile demir oksit çekirdek oluşur. Bu ajanlar, T2-ağırlıklı MR görüntüleri 5 bir azalma sinyal yol yerel manyetik alan homojen olmayan düzensizlikleri oluşturun . Ne yazık ki, SPIOs artık üretilmektedir. İkinci nesil ultrasmall SPIOs (USPIO), ancak, bir alternatif sunuyoruz. Ferumoxytol (FerahemeTM) polyglucose sorbitol carboxymethylether kat ile çevrili olmayan bir stokiyometrik manyetit çekirdek oluşan bir USPIO. Ferumoxytol kolloidal, parçacık boyutu, ışık saçılması tarafından belirlenen 17-30 nm. Molekül ağırlığı 750 kDa ve 2T MR alanında sabit relaksivite 58,609 mM-1 sn-1 gücü 4. Ferumoxytol yakın zamanda FDA onaylı bir, böbrek yetmezliği 6 olan hastalarda demir eksikliği tedavisi için bir demir takviyesidir . Grubumuz, bu ajan uygulanan hücre etiketleme uygulamaları için &quot;off label&quot; kullanım vardır. Bizim teknik, MR görüntüleri etiketli hücrelerinin önemli MR sinyali etkilere yol açar ferumoxytol ile kök hücreleri etkin bir etiketleme göstermektedir. Bu teknik, kök hücre tedavileri, klinik öncesi ve klinik ortamlarda non-invaziv izlenmesi için uygulanabilir.

Watch this Scientific Journal Video about Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle at JoVE.com

Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle

Biz, FDA onaylı, süperparamanyetik demir oksit (SPIO), ferumoxytol (Feraheme) ile kök hücre etiketleme ve izleme için kullanılan bir tekniktir açıklar. Hücresel görüntüleme tekniği, manyetik rezonans görüntüleme (MR) görüntüleme için kullanır Bu, başarılı ya da başarısız hastalarda kök hücre engraftments uzun vadeli izleme ve teşhis için kolay erişilebilir.

Kök Hücreler Ferumoxytol, FDA Onaylı Demir Oksit Nanopartikül Etiketleme

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo ...

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20.7K Görüntüleme.  Molecular Imaging Program at Stanford (MIPS). Biz, FDA onaylı, süperparamanyetik demir oksit (SPIO), ferumoxytol (Feraheme) ile kök hücre etiketleme ve izleme için kullanılan bir tekniktir açıklar. Hücresel görüntüleme tekniği, manyetik rezonans görüntüleme (MR) görüntüleme için kullanır Bu, başarılı ya da başarısız hastalarda kök hücre engraftments uzun vadeli izleme ve teşhis için kolay erişilebilir.

Video: Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

İnsan Diş Pulp Kök Hücre İzolasyonu, Karakterizasyonu ve Karşılaştırmalı Farklılaşma İki Farklı Yöntemler Kullanılarak Daimi Dişler türetilen

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

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Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pankreas Kanseri Yeni Tedavi Stratejileri Geliştirme Hücre Özellikleri Kök incelenmesi

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

Forskolin kaynaklı bağırsak Organoids şişme: Bir<em&gt; İn Vitro</em&gt; Deney Kistik Fibrozis Hastalarında İlaç Tepki değerlendirilmesi için

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Continuous Manual Exchange Transfusion for Patients with Sickle Cell Disease: An Efficient Method to Avoid Iron Overload

Children with sickle cell anemia (SCA) may be at risk of cerebral vasculopathy and strokes, which can be prevented by chronic transfusion programs. Repeated transfusions of packed red blood cells (PRBCs) is currently the simplest and most used technique for chronic transfusion programs. However, iron overload is one of the major side effects of this therapy. More developed methods exist, notably the apheresis of RBC (erythrapheresis), which is currently the safest and most efficient method. However, it is costly, complicated, and cannot be implemented everywhere, nor is it suitable for all patients. Manual exchange transfusions combine one or more manual phlebotomies with a PRBC transfusion.
At the Reference Center of Sickle Cell Disease, we set up a continuous method of manual exchange transfusion that is feasible for all hospital settings, demands no specific equipment, and is widely applicable. In terms of HbS decrease, stroke prevention, and iron overload prevention, this method showed comparable efficiency to erythrapheresis. In cases where erythrapheresis is not available, this method can be a good alternative for patients and care centers.

We have outlined a method of continuous manual exchange transfusion for the treatment of sickle cell disease in patients. This safe protocol was designed to effectively limit iron overload in patients in need of chronic transfusions and can be used extensively without any special equipment.

Orak Hücre Hastalığında için sürekli Manuel Değişim Transfüzyon: Demir birikimini önlemek için Etkin Bir Yöntem

Children with sickle cell anemia (SCA) may be at risk of cerebral vasculopathy and strokes, which can be prevented by chronic transfusion programs. ...

Synthesis, Characterization, and Application of Superparamagnetic Iron Oxide Nanoprobes for Extrapulmonary Tuberculosis Detection

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was synthesized to enhance imaging sensitivity for extrapulmonary tuberculosis (ETB). An SPIO nanoprobe was synthesized and conjugated with MtbsAb. The purified SPIO-MtbsAb nanoprobe was characterized using TEM and NMR. To determine the targeting ability of the probe, SPIO-MtbsAb nanoprobes were incubated with Mtb for in vitro imaging assays and injected into Mtb-inoculated mice for in vivo investigation with magnetic resonance (MR). The contrast enhancement reduction on magnetic resonance imaging (MRI) of Mtb and THP1 cells showed proportional to the SPIO-MtbsAb nanoprobe concentration. After 30 min of intravenous SPIO-MtbsAb nanoprobe injection into Mtb-infected mice, the signal intensity of the granulomatous site was enhanced by 14-fold in the T2-weighted MR images compared with that in mice receiving PBS injection. The MtbsAb nanoprobes can be used as a novel modality for ETB detection.

To improve serological diagnostic tests for Mycobacterium tuberculosis antigens, we developed superparamagnetic iron oxide nanoprobes to detect extrapulmonary tuberculosis.

Ekstrapulmoner Tüberküloz Tespiti için Süperparamanyetik Demir Oksit Nanoprobukların Sentezi, Karakterizasyonu ve Uygulanması

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was ...

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a promising therapeutic target in many disorders, such as autoimmune diseases, atherosclerosis, and cancer. RNAi-mediated gene silencing is a valuable approach of choice to probe and manipulate macrophage function; however, the transfection of macrophages with siRNA is often considered to be technically challenging, and, at present, few methodologies dedicated to the siRNA transfer to macrophages are available. Here, we present a protocol of using polyethyleneimine-coated superparamagnetic iron oxide nanoparticles (PEI-SPIONs) as a vehicle for the targeted delivery of siRNA to macrophages. PEI-SPIONs are capable of binding and completely condensing siRNA when the Fe:siRNA weight ratio reaches 4 and above. In vitro, these nanoparticles can efficiently deliver siRNA into primary macrophages, as well as into the macrophage-like RAW 264.7 cell line, without compromising cell viability at the optimal dose for transfection, and, ultimately, they induce siRNA-mediated target gene silencing. Apart from being used for in vitro siRNA transfection, PEI-SPIONs are also a promising tool for delivering siRNA to macrophages in vivo. In view of its combined features of magnetic property and gene-silencing ability, systemically administered PEI-SPION/siRNA particles are expected not only to modulate macrophage function but also to enable macrophages to be imaged and tracked. In essence, PEI-SPIONs represent a simple, safe, and effective nonviral platform for siRNA delivery to macrophages both in vitro and in vivo.

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

We describe a method of using polyethyleneimine (PEI)-coated superparamagnetic iron oxide nanoparticles for transfecting macrophages with siRNA. These nanoparticles can efficiently deliver siRNA to macrophages in vitro and in vivo and silence target gene expression.

Polyethyleneimine kaplı demir oksit nano tanecikleri küçük müdahale RNA makrofajlar için teslim için bir araç olarak Vitro ve In Vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a ...

An Enzyme-free Method for Isolation and Expansion of Human Adipose-derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. As a clinically relevant cell type, optimal methods are needed to isolate and expand these cells in vitro. Most methods to isolate adipose-derived MSCs (ADSCs) rely on harsh enzymes, such as collagenase, to digest the adipose tissue. However, while effective at breaking down the adipose tissue and yielding a high ADSC recovery, these enzymes are expensive and can have detrimental effect on the ADSCs &#8212; including the risks of using xenogeneic components in clinical applications. This protocol details a method to isolate ADSCs from fresh lipoaspirate and abdominoplasty samples without enzymes. Briefly, this method relies on mechanical disassociation of any bulk tissue followed by an explant-type culture system. The ADSCs are permitted to migrate out of tissue and onto the tissue culture plate, after which the ADSCs can be cultured and expanded in vitro for any number of research and/or clinical applications.

This protocol provides an enzyme-free method for isolating mesenchymal stem cells from abdominoplasty and lipoaspirate samples using an explant method. The absence of harsh enzymes or centrifugation steps provides for clinically relevant stem cells that can be used for studies in vitro or transferred back to the clinic.

İnsan Yağtüretilmiş Mezenkimal Kök HücrelerinIn İzolasyon ve Genişlemesi için Enzimsiz Bir Yöntem

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. ...

Tracking Superparamagnetic Iron Oxide-labeled Mesenchymal Stem Cells using MRI after Intranasal Delivery in a Traumatic Brain Injury Murine Model

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical hurdles such as invasive cell delivery and tracking with low transplantation efficiency remain challenges in translational stem-based therapy. This article describes an emerging technique for stem cell labeling and tracking based on the labeling of the mesenchymal stem cells (MSCs) with superparamagnetic iron oxide (SPIO)&#160;nanoparticles, as well as intranasal delivery of the labeled MSCs. These nanoparticles are fluorescein isothiocyanate (FITC)-embedded and safe to label the MSCs, which are subsequently delivered to the brains of TBI-induced mice by the intranasal route. They are then tracked non-invasively in vivo by real-time magnetic resonance imaging (MRI). Important advantages of this technique that combines SPIO for cell labeling and intranasal delivery include (1) non-invasive, in vivo MSC tracking after delivery for long tracking periods, (2) the possibility of multiple dosing regimens due to the non-invasive route of MSC delivery, and (3) possible applications to humans, owing to the safety of SPIO, non-invasive nature of the cell-tracking method by MRI, and route of administration.

Presented here is a protocol for non-invasive mesenchymal stem cell (MSC) delivery and tracking in a mouse model of traumatic brain injury. Superparamagnetic iron oxide&#160;nanoparticles are employed as a magnetic resonance imaging (MRI) probe for MSC labeling and non-invasive in vivo tracking following intranasal delivery using real-time MRI.

Travmatik Beyin Hasarı Murine Modelinde İntranazal Doğum sonrası MRG kullanarak Süperparamanyetik Demir Oksit etiketli Mezenkimal Kök Hücrelerin Takibi

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical ...

Estimation of Urinary Nanocrystals in Humans using Calcium Fluorophore Labeling and Nanoparticle Tracking Analysis

Kidney stones are becoming more prevalent worldwide in adults and children. The most common type of kidney stone is comprised of calcium oxalate (CaOx) crystals. Crystalluria occurs when urine becomes supersaturated with minerals (e.g., calcium, oxalate, phosphate) and precedes kidney stone formation. Standard methods to assess crystalluria in stone formers include microscopy, filtration, and centrifugation. However, these methods primarily detect microcrystals and not nanocrystals. Nanocrystals have been suggested to be more harmful to kidney epithelial cells than microcrystals in vitro. Here, we describe the ability of Nanoparticle Tracking analysis (NTA) to detect human urinary nanocrystals. Healthy adults were fed a controlled oxalate diet prior to drinking an oxalate load to stimulate urinary nanocrystals. Urine was collected for 24 hours before and after the oxalate load. Samples were processed and washed with ethanol to purify samples. Urinary nanocrystals were stained with the calcium binding fluorophore, Fluo-4 AM. After staining, the size and count of nanocrystals were determined using NTA. The findings from this study show NTA can efficiently detect nanocrystalluria in healthy adults. These findings suggest NTA could be a valuable early detection method of nanocrystalluria in patients with kidney stone disease.

The objective of this study was to determine whether nanoparticle tracking analysis (NTA) could detect and quantify urinary calcium containing nanocrystals from healthy adults. The findings from the current study suggest NTA could be a potential tool to estimate urinary nanocrystals during kidney stone disease.

Kalsiyum Florofor Etiketleme ve Nanopartikül Takip Analizi Kullanılarak İnsanlarda Üriner Nanokristallerin Tahmini

Kidney stones are becoming more prevalent worldwide in adults and children. The most common type of kidney stone is comprised of calcium oxalate (CaOx) ...

Measurement of Tissue Non-Heme Iron Content using a Bathophenanthroline-Based Colorimetric Assay

Iron is an essential micronutrient. Both iron overload and deficiency are highly detrimental to humans, and tissue iron levels are finely regulated. The use of experimental animal models of iron overload or deficiency has been instrumental to advance knowledge of the mechanisms involved in the systemic and cellular regulation of iron homeostasis. The measurement of total iron levels in animal tissues is commonly performed with atomic absorption spectroscopy or with a colorimetric assay based on the reaction of non-heme iron with a bathophenanthroline reagent. For many years, the colorimetric assay has been used for the measurement of the non-heme iron content in a wide range of animal tissues. Unlike atomic absorption spectroscopy, it excludes the contribution of heme iron derived from hemoglobin contained in red blood cells. Moreover, it does not require sophisticated analytical skills or highly expensive equipment, and can thus be easily implemented in most laboratories. Finally, the colorimetric assay can be either cuvette-based or adapted to a microplate format, allowing higher sample throughput. The present work provides a well-established protocol that is suited for the detection of alterations in tissue iron levels in a variety of experimental animal models of iron overload or iron deficiency.

Here, a protocol for the measurement of the non-heme iron content in animal tissues is provided, using a simple, well-established colorimetric assay that can be easily implemented in most laboratories.

Batofenantropin Bazlı Kolorimetrik Tahlil Kullanılarak Doku Non-Heme Demir İçeriğinin Ölçülmesi

Iron is an essential micronutrient. Both iron overload and deficiency are highly detrimental to humans, and tissue iron levels are finely regulated. The ...