Biz minnetle onun teknik destekler için onların eleştirel discus ve Sayın Mohammad Reza Khadem Şerif için Dr Leila Shakeri &amp; Dr Aref Dournaei kabul ediyorsunuz.

1. Enzim Çözüm ve Çoğalma Orta (PM) hazırlayın <ol><li> Kollajenaz Tip I olun Çözüm: kollajenaz tip I (12 mg / ml) tartılır ve bir 0.2 mikron şırınga filtresi kullanarak 1 ml PBS ve filtre içinde çözülür. Sonra 15 ml tüp yerleştirin ve gerektiği kadar -20 ° C&#39;de saklayın. </li><li> Çözelti dispase edin: dispase tartılır (16 mg / ml) ve 1 ml PBS ve filtre bir 0.2 um şırınga filtresi kullanılarak çözülür. Sonra 15 ml tüp yerleştirmek ve gerektiğinde ° C ye kadar 4 başında tutmak. </li><li> Enzim çözeltisi edin: 2 ml steril PBS 100 mg / ml penisilin, 100 mg / ml streptomisin içeren içine 1 ml kollajenaz tip bir eriyik (12 mg / ml) ve 1 ml dispase çözeltileri (16 mg / ml) ilave edilir. Nihai hacim içinde dispase ve kollajenaz bir toplam konsantrasyon receptively 4 mg / ml ve 3 mg / ml olmalıdır. Sonra, dört 15 ml tüp, her birinde 1 ml enzim çözeltisi için kısım bu hacim. Her bir tüp, bir kağıt hamuru sindirim için kullanılabilecek. </li><li> Yıkama çözeltisi edin: 100 mg / ml penisilin, PBS içinde 100 mg / ml streptomisin ekleyin. </li><li> Temel ortam edin: Eagle ortamı alfa modifikasyonu (α-MEM)% 10 FBS ve 100 birim / ml penisilin, 100 mg / ml streptomisin ile desteklenmiş. </li><li> % 10 FBS, 100 uM L-askorbik asit 2-fosfat, 2 mM L-glutamin, 100 birim / ml penisilin, 100 mg / ml ile desteklenmiş Eagle ortamı (α-MEM) &#39;in alfa modifikasyonu: Proliferasyon Ortam (PM) yapmak streptomisin ve 0.25 mg / ml amfoterisin B </li><li> Odontojenik medya edin: Eagle ortamı alfa modifikasyonu (α-MEM)% 10 FBS, 100 uM L-askorbik asit 2-fosfat, 2 mM L-glutamin, 100 birim / ml penisilin, 100 mg / ml streptomisin, 0.01 uM ile desteklenmiş Deksametazon, 5 mM β-gliserol fosfat, 1.8 mM Monopotasyum fosfat. </li></ol> 2. Diş Pulp Kök Hücre Isolatio İnsan Dental Pulpa Dokusu hazırlayınn <ol><li> Normal insan üçüncü molar diş onaylı Kurumsal İnceleme Kurulu (KİK) kapsamında İmam Ali&#39;nin Diş Kliniği&#39;nde yetişkin (yaş 21-29 yıl) toplanmıştır. </li><li> Dişler temel ortam (MEM α-% 10 FBS ile takviye edilmiş) içine yerleştirildi ve 4 ° C de laboratuvar aktarıldı </li><li> Steril şartlarda, bir biyolojik tehlike laminer akış kaputu içinde çalışan, set-up işlenecek her diş için bir 100 mm Petri kapları yapıldı. Diş yüzeyleri% 70 etanol ile temizlendi. </li><li> Petri-yemekleri birinde çalışırken, diş diş forseps ile tutuldu ve Bistüri enkaz ve kökleri üzerinde periodontal ligament kapalı temizlemek için kullanılmıştır. </li><li> Mine-sement yavaş hamuru oda ortaya çıkarmak için sterilize diş disk kullanılarak kesildi. Bu kesme prosesi diş dokusu aşırı yavaş yavaş azaltmak için yapılması gereken olduğu göz önünde bulundurulmalıdır. </li><li> Pulp doku hafifçe taç ve kök ayrıldı. </li&gt; <li> Pulp doku bir neşter bıçak yardımı ile küçük parçalar halinde kesilmiş. </li></ol> 3. İnsan Diş Selüloz İzolasyon <ol><li> Pulp dokuların hDPSCs izolasyonu için pulpa dokusu eksplantlarından pulpa dokusu veya doğrudan hücre akıbet birini enzimatik ayrışma uygulandı. </li><li> Pulpa dokusunun Enzimatik ayrışma: <ol><li> Kağıt hamuru dokuların küçük parçaları 37 ° C sıcaklıkta 1 saat için enzim solüsyonu içine aktarıldı Doku kesiliyor yardım Vorteks her 30 dk. </li><li> Daha sonra, bir 70 uM hücre süzgecinden hücreleri geçirilerek büyük hücre agregatları çıkarıldı ve tek hücre süspansiyonları elde edildi. </li><li> Tek hücre süspansiyonları oda sıcaklığında 5 dakika için 1200 rpm&#39;de santrifüjlenmiştir. </li><li> Süpernatantlar dikkatle kapalı pipetle alındı ​​ve pelet 1 ml çoğalması orta (PM) yeniden askıya alınmıştı. (Not: yayılması orta FBS sonlandırmak dis enzimatiksociation.) </li><li> Diş hamuru Tek hücre süspansiyonları PM ile 25 cm 2 kültürü şişesi içine ekilmiş ve daha sonra% 5 CO2 içinde 37 ° C de inkübe edildi. </li><li> Hücre konfluense elde edilene kadar kültür ortamı her üç günde bir değiştirildi. </li></ol></li><li> Pulpa dokusu eksplantlarından Doğrudan hücre akıbet: <ol><li> Selüloz doku 1-2 mm&#39;lik parçalara kıyılmış edildi ve her bir parça PM ile 25 cm 2 kültür kaplarına yerleştirilir ve daha sonra 37 ° C&#39;de inkübe% 5 CO2 içinde. Bu hücre akıbet için PM toplam hacmi daha fazla hücre akıbet için hamuru parçaları (şişesi başına 2-3 ml) eki desteklemesi gerektiğini dikkate alınmalıdır. </li><li> Filiz gözlenmiştir sonra ortam değiştirilmiştir. </li><li> Izdiham geçmek hücreleri 1:4 oranı (geçit 1) alt-kültür vardı. </li></ol></li></ol> 4. Immünofeotipleme <ol><li> / 250,000 hücrelerpasaj 3&#39;de hücrelerinin her iki türdeki boru 4 ° C karanlık bir yerde, 30 dakika için PE ya da izotipleri ile FITC-konjüge anti-insan antikor ile enkübe edildi. (Not:. Antikorların konsantrasyonu üretimi protokolüne uygun olarak uygulanmıştır) </li><li> İnkübasyon süresinden sonra, 100 ul PBS ya da FACS seyreltme çözeltisi her bir tüpe eklendi ve karanlık bir yerde, 5 dakika için 1200 rpm&#39;de santrifüjlenmiştir. </li><li> Süpernatantlar çıkarıldı ve granül 100 ul PBS veya karanlık bir yerde FACS seyreltme yeniden askıya alındı. </li><li> Hedefli yüzey belirteçleri (CD marker) BD FACSCalibur tarafından değerlendirildi. </li></ol> 5. Mineralize Hücre içine DPSCs Farklılaşma İndüksiyon &amp; Alizarin Red S Assay Sayısallaştırılmış <ol><li> Pulpa dokusu eksplantları (DPSC-OG) gelen pulpa dokusunun enzimatik ayrışma (DPSC-ED olarak da bilinir) veya doğrudan hücre akıbet biri tarafından alındı ​​DPSCs 3 geçişleri için alt-kültür vardı. </li><li> At Passage 3, hücre 5.000 hücre / kuyu başında 6 plaka içine tohum edildi. </li><li> % 60 izdiham, odontojenik orta konvansiyonel PM değiştirilmiştir. Üç kuyu büyüme ortamı ilave edilerek bir negatif kontrol olarak kaldı. </li><li> Orta günde 21 kadar 3 gün boyunca her değiştirin. </li><li> 21. günde, hücreler PBS ile yıkandı ve 1 ml / kuyucuk oda sıcaklığında 15 dakika için% 10 paraformaldehit ile fikse edildi. </li><li> Daha sonra paraformaldehid dikkatli bir şekilde, kapalı pipetlendi ve hücreler damıtık H 2 O ile 3 kez (her zaman için 5-10 dk) yıkandı (Not: tek tabaka rahatsız kaçının.) </li><li> Yıkandıktan sonra, 1 ml / Alizarin kırmızı S oda sıcaklığında 20 dakika boyunca ilave edildi. </li><li> Hücreler herhangi bir ek Alizarin kırmızı uzaklaştırmak için deiyonize H2O dört kez (nazik sallama ile, her seferinde 5 dakika) ile yıkandı. </li><li> 1 ml / H2O kurumasını engellemek için hücrelerin ilave edildi. </li><li> Plakaları görsel ins kabul edildipection ve / veya görüntü alma. </li><li> Alizarin Kırmızı S miktar tayini için, H2O sallama ile oda sıcaklığında 30 dakika için 6 plaka ve inkübe her bir kuyucuğa 1 ml% 10 asetik asit ile değiştirilmiştir. </li><li> Sonra, iyi hücre monolayer / kazıma ve asetik asit ve hücreler iki ayrı 1.5 ml mikro-santrifüj tüplerine aktarıldı vardı. (Not:. Her iyi her ED ve RG grupları için ayrı bir tüp yani üç test kuyuları ve üç kontrol kuyulara transferi olmalıdır) </li><li> Tüpler 30 saniye boyunca kuvvetli bir şekilde vortekslenmiş edildi. </li><li> 85 ° C ısı 10 dakika için. (Not: buharlaşma önlemek için, tüpleri parafilm ile imzalandı.) </li><li> Isıtmadan sonra, tüp 5 dakika için buz içine transfer edildi. (Not: soğutmalı hale gelene kadar Tüpler açılmamalıdır.) </li><li> Bulamaçları 15 dakika için 20,000 x g&#39;de santrifüjlenmiştir. Santrifüj iken, Alizarin Kırmızı standartları yapılmıştırkadar. </li></ol> Alizarin Kırmızı S standart ilk seyreltme tamponu seyreltilmesi ve tablosuna göre 2-kat seri seyreltiler yapmak için kullanılan ile hazırlanmıştır. <table border="1"><tbody><tr><td colspan="7"> Boya Yüksek Oranda konsantrasyonu </td><td colspan="7"> Boya Düşük Aralığı konsantrasyonu </td></tr><tr><td> 2 mM </td><td> 1 mM </td><td> 500 uM </td><td> 250 uM </td><td> 125 mcM </td><td> 62.5 uM </td><td> 31.3 uM </td><td> 15.6 uM </td><td> 7.8 uM </td><td> 3.9 uM </td><td> 1.9 uM </td><td> 0.9 uM </td><td> 0.4 uM </td><td> Boş </td></tr></tbody></table><ol start="16"><li> Santrifüj sonra süpernatant / tüp 1 ml yeni ayrı 1.5 ml mikro-santrifüj tüplerine aktarıldı. </li><li> PH ~ 375 ul 10 ile nötralize edildi;Tüp başına% amonyum hidroksit. (Not: öfke pH 4.1-4.5 olmalıdır.) </li><li> Standart ve numune (ayrı testler ve kontroller dahil) 150 ul 96-plaka opak-duvarlı, şeffaf alt içine ilave edildi. </li><li> Absorbansı 405 nm&#39;de okunmuştur ve örnekleri OD değerleri, daha fazla analiz için standart arsa ile karşılaştırılmıştır. </li></ol>

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Palacky Olomouc Czech Repub. 153, 31-35 (2009).</li><li>d'Aquino, 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=Human+postnatal+dental+pulp+cells+co-differentiate+into+osteoblasts+and+endotheliocytes:+a+pivotal+synergy+leading+to+adult+bone+tissue+formation.">Human postnatal dental pulp cells co-differentiate into osteoblasts and endotheliocytes: a pivotal synergy leading to adult bone tissue formation.</a> Cell Death Differ. 14, 1162-1171 (2007).</li><li>Atari, M., 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=Isolation+of+pluripotent+stem+cells+from+human+third+molar+dental+pulp.">Isolation of pluripotent stem cells from human third molar dental pulp.</a> Histol. Histopathol. 26, 1057-1070 (2011).</li></ol>

Çıkar çatışması ilan etti.

Bu protokol iki yöntem, enzimatik ayrışma ve pulpa dokusu eksplantlar alınan kök hücrelerin direkt sonucudur kullanarak diş hamurundan hDPSCs izolasyonu ve karakterizasyonu süreci anlatılmaktadır. Buna ek olarak, odontoblastlar bu hücrelerin in vitro olarak farklılaşma, kantitatif Alizarin Kırmızı S tahlil ve QPCR ile değerlendirildi. İnsan dişinden pulpa dokusu izole etmek için mevcut protokoller pense (kemik forseps) 9, imha iğne 29, Gracey küret <...

Kök hücreler, çok kudret ve kendini yenileme 15 olarak bilinen iki önemli özellik, sahip clonogenic hücrelerdir. Farklı replikatif potenslerine Tüm kök hücreler arasında, postnatal kök hücreler olarak diş kök hücreleri nedeniyle erişilebilirlik, esneklik, ve diğer yetişkin kök hücreleri 16 ile karşılaştırıldığında yüksek proliferatif yeteneği son yıllarda dikkat çekmektedir. Karakteristik olarak, mezenkimal kök hücreler ile benzer şekilde, diş özü kök hücrelerinin farklılaşmasını çoklu mezenşim içine kapasitesi ve / veya in vitro ve in vivo hem de non-mezenşim soy sahip yapışık clonogenic hücrelerdir. 1-8,17,18 DPSCs ile tanımlanır olumsuz hematopoetik antijenlerin ekspresyonu (örneğin, CD45, CD34, CD14) ve CD90 pozitif ifadesi, CD29, CD73, CD105, CD44 ve STRO-1. 19,20 Minimum ağrı ve morbidite ile kolay elde potansiyeli valu olarak insan DPSCs yapmakMKH yapabiliyor kaynağı kemik iliği mezenkimal kök hücre 21 olarak sıradan kaynaklarına kıyasla. Genel olarak, DPSCs ya akıbet yöntemi, yani, pulpa dokusu eksplantlar (DPSC-OG) 22-24, ve / veya enzimatik sindirim (DPSC-ED) 4,6,25 kök hücrelerin göç ile izole edilmiştir. Önceki çalışmalar izolasyon yöntemi ve kültür koşullarında in vitro pasajlar 26,27 bölgesindeki altında farklı popülasyonlarda veya soy neden olduğunu göstermiştir. Kalıcı dişlerin (pDPSCs) söz konusu olduğunda, Huang ve ark enzimatik sindirimi pDPSCs geçmek DPSCs kıyasla daha yüksek çoğalma potansiyeli olduğunu ortaya koymuştur. Ayrıca 26, dişlerin (dDPSCs) söz konusu olduğunda, bu gösterilmiştir ki STRO-1 ve CD34 belirteçleri dDPSC-OG ile karşılaştırıldığında dDPSC-ED daha dile getirdi. Buna ek olarak, dDPSC-ED tanımlanan osteo / odonto ortam içinde daha yüksek bir mineralizasyon hızı görüntülenir. Bu nedenle 27 sebebiyle r DPSCs olağanüstü potansiyeliegenerative tıp, daha fazla çalışmaları farklı izolasyon yöntemleri türetilmiştir olası çeşitli popülasyonlarda daha iyi anlaşılması için gerekli olacaktır. İşte, bu hamuru çıkarma sürecini kolaylaştırmak için bir adım diş elmas disketini kullanarak, hamuru ekstraksiyon kolay bir şekilde tanıtmak için bir girişimdi. Ayrıca ED veya OG yöntemleri uygulayarak insan pulpa kaynaklı kök hücrelerin izolasyonu sonra, iki grup arasında genel özellikleri ve farklılaşma kapasitesi de incelenmiştir.

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog veya Lot. numara </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td> α-MEM </td><td> GIBCO </td><td> 11900-073 </td><td></td></tr><tr><td> Kollajenaz tip I </td><td> Sigma-Aldrich </td><td> C0130-100mg </td><td></td></tr><tr><td> Dispase </td><td> GIBCO </td><td> 17105-041 </td><td></td></tr><tr><td> Penisilin / streptomisin </td><td> GIBCO </td><td> 15140-122 </td><td></td></tr><tr><td> Amfoterisin B </td><td> GIBCO </td><td> 15290-018 </td><td></td></tr><tr><td> Fetal Bovine Serum tanımlı (FBS) </td><td> HyClone </td><td> SH30070.03 </td><td></td></tr><tr><td> 2-fosfat L-askorbik asit </td><td> Sigma </td><td> A8960-5G </td><td></td></tr><tr><td> L-glutamin </td><td> GIBCO </td><td> 25030-024 </td><td></td></tr><tr><td> Deksametazon </td><td> Sigma </td><td> D4902 </td><td></td></tr><tr><td> β-gliserol fosfat disodyum tuzu hidrat, BioUltra </td><td> Sigma </td><td> G9422-100G </td><td></td></tr><tr><td> Potasyum fosfat tekbazlı </td><td> Sigma-Aldrich </td><td> P5655 </td><td></td></tr><tr><td> Osteogenezis Assay Kit </td><td> Millipore </td><td> PS01802031 </td><td></td></tr><tr><td> Fare IgG2b-PE izotip kontrol </td><td> BD Pharmingen </td><td> 50808088029 </td><td></td></tr><tr><td> FITC fare IgG2b izotip kontrol </td><td> BD Pharmingen </td><td> 559532 </td><td></td></tr><tr><td> FITC fare IgG1 κ izotip </td><td> BD Pharmingen </td><td> 11471471 </td><td></td></tr><tr><td> FITC / PE fare anti-insan CD34/CD45 </td><td> BD Pharmingen </td><td>341071 </td><td></td></tr><tr><td> PE anti-insan CD146 </td><td> BD Pharmingen </td><td> 550315 </td><td></td></tr><tr><td> Monoklonal fare anti-insan CD90/FITC </td><td> Daka </td><td> 00034418 </td><td></td></tr><tr><td> PE fare anti-insan CD73 </td><td> BD Pharmingen </td><td> 550257 </td><td></td></tr><tr><td> Anti-h CD105/Endoglin PE </td><td> BD Pharmingen </td><td> FAB10971P </td><td></td></tr><tr><td> PE fare anti-insan CD11b/Mac1 </td><td> BD Pharmingen </td><td> 5553888 </td><td></td></tr><tr><td> CD44 PE fare anti insan </td><td> BD Pharmingen </td><td> 555479 </td><td></td></tr><tr><td> Fosfat tampon çözeltisi (PBS) </td><td> GIBCO </td><td> 003000 </td><td></td></tr><tr><td> 70 mikron hücre süzgecinden </td><td> Şahin </td><td> 352360 </td><td></td></tr><tr><td&gt; 0.2 um şırınga filtresinden </td><td> Millex-GV </td><td> SLGV033RB </td><td></td></tr><tr><td> 25 cm 2 kültürü şişesi </td><td> Sigma-Aldrich </td><td> Z707481 </td><td></td></tr><tr><td></td><td></td><td></td><td> EKİPMAN </td></tr><tr><td> BD FACSCalibur </td><td> BD </td><td> 342975 </td><td></td></tr><tr><td> Multiskan mikroplak spektrofotometre </td><td> Termo bilimsel </td><td> 51119200 </td><td></td></tr><tr><td> Fleurcense Mikroskop </td><td> Olimpos </td><td></td><td></td></tr><tr><td> Yazılım sürümü 2.3.1 Flowing </td><td></td><td></td><td></td></tr></tbody></table>

isolation, characterization and comparative differentiation of human dental pulp stem cells derived from permanent teeth by using two different methods

<p class="jove_content"&gt; Gelişmekte yirmilik dişler rutin ortodontik tedaviler ile elde edilebilir yetişkinlik döneminde kök hücrelerin kolay erişilebilir bir kaynak vardır. İnsan hamuru kökenli kök hücreleri (hDPSCs) erişkin kök hücrelerin olağan kaynağı karşılaştırmak çok soy farklılaşma kapasitesine sahip yüksek proliferasyon potansiyeline sahip<sup&gt; 1-8</sup&gt;, Bu nedenle hDPSCs doku mühendisliği ve rejeneratif tıp otolog transplantasyon için iyi bir aday olabilir. Bu avantajları ile birlikte, bu tür immunolodulatory etki olarak mezenkimal kök hücreleri (MSC) özellikleri haiz, hatta allograft nakli durumunda, hDPSCs daha değerli yapmak<sup&gt; 6,9,10</sup&gt;. Bu nedenle, kök hücre kaynağı kullanmak için birincil adım pulpa dokusu hDPSCs yalıtmak için en iyi protokolü seçmektir. Bu amaca ulaşmak için, bu tür ortak yüzey belirteçleri ve ayrıca farklılaşma kapasitesi gibi farklı hücresel davranışlar, çeşitli izolasyon koşullarının etkisini incelemek için çok önemlidir.</p&gt;<p class="jove_content"&gt; Böylece, burada gömülü üçüncü molar dişler insan pulpa dokusu ayırmak ve sonra tecrit hDPSCs için literatüre dayalı mevcut protokoller, hem de kullanılan<sup&gt; 11-13</sup&gt;<em&gt; Yani</emDoku eksplantlarından&gt; enzimatik pulpa dokusunun dissosiasyon (DPSC-ED) veya akıbet (DPSC-OG). Bu bağlamda, biz diş elmas disketi kullanarak izolasyon yöntemleri kolaylaştırmak için çalıştı. Sonra, bu hücreler CD146 ve ayrıca STRO-1 gibi stromal ilişkili Markers (CD73, CD90, CD105 ve CD44), hematopoetik / endotel Markers (CD34, CD45 ve CD11b), perivasküler marker açısından karakterize. Daha sonra, bu iki protokol kantitatif polimeraz zincir reaksiyonu (QPCR) ve alizarin kırmızısı boyama her iki tarafından odontoblastlar içine farklılaşma etki göre karşılaştırılmıştır. QPCR mineralizasyon ilişkili genlerin ekspresyonu (; ALP, matris ekstraselüler phosphoglycoprotein; MEPE ve dentin sialophosphoprotein; DSPP alkalen fosfataz) değerlendirilmesi için kullanılmıştır.<sup&gt; 14</sup</p

<ol><li> Enzimatik ayrışma (DPSC-ED) ile elde edildi DPSCs günde 10, 15,18 (Şekil 1) burada gösterilir. Dışlanmasının ardından yaklaşık 3 ila 5 gün oluşmaya başlayan koloniler. </li><li> Geçmek DPSCs (DPSC-OG) günlük 5, 10, 13 ve 18, Şekil 2&#39;de gösterilmiştir. Hücreleri paralel tohumlama sonra yaklaşık 5 gün tarafından sipariş ederek hamuru doku balonun içine göç etmeye başladılar Fibroblast benzeri. </li><li> Her iki yöntem kullanılarak pasaj 3&#39;de DPSCs Şekil ...

Watch this Scientific Journal Video about Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods at JoV

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

Yöntemi kullanarak insan dental pulpa kök hücrelerinin (hDPSCs) izolasyonu ve karakterizasyonu tarif<strongKağıt hamuru&gt; enzimatik ayrışma (DPSC-ED)</strong&gt; Veya<strongPulpa dokusu eksplantları alınan kök hücrelerin&gt; direkt sonucudur (DPSC-OG). Sonra takip</strong&gt;<em&gt; In vitro</emOdontoblastlar içine hDPSCs Her iki tip&gt; karşılaştırmalı farklılaşma.

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 ...

isolation-characterization-comparative-differentiation-human-dental

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Medicine

26.2K Görüntüleme.  Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran. Yöntemi kullanarak insan dental pulpa kök hücrelerinin (hDPSCs) izolasyonu ve karakterizasyonu tarif enzimatik ayrışma (DPSC-ED) Veya direkt sonucudur (DPSC-OG). Sonra takip In vitro karşılaştırmalı farklılaşma.

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

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 and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

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 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 ...

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell culture systems arising from human endometrial samples are no exception. Applications range from normal cyclic physiological processes to endometrial pathologies such as gynecological cancers, infectious diseases, and reproductive deficiencies. Here, we provide two methods for establishing primary endometrial stromal cells from surgically resected endometrial hysterectomy specimens. The first method is referred to as &#8220;the scraping method&#8221; and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed &#8220;the trypsin method.&#8221; This latter method uses the enzymatic activity of trypsin to promote the separation of cells and primary cell outgrowth. We illustrate step-by-step methodology through digital images and microscopy. We also provide examples for validating endometrial stromal cell lines via quantitative real time polymerase chain reactions (qPCR) and immunofluorescence (IF).

Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human patients.&#160;

Histerektomi Örneklerden İlköğretim İnsan endometrial stroma hücrelerinde Kurulması İçin İki Yöntem

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, synovial-fluid-derived MSCs were reported to have multi-lineage differentiation potential and immunomodulatory features, which indicates that these cells can be used for tissue engineering and systemic treatments. This study presents a protocol for simple and non-invasive isolation of MSCs derived from the bone marrow and synovial fluid of minipigs to analyze cell surface markers for cell phenotyping and in vitro culturing. Using sexually mature six-month-old minipigs, bone marrow was extracted from the iliac crest bone using a bone marrow extractor, and the synovial fluid was aspirated from the femorotibial joint. Procedures for the collection of samples from both sources were non-invasive. The protocols for effective isolation of MSCs from harvested cell sources and for creating in vitro culture conditions to expand stable MSCs from minipigs and the application of systemic autologous treatments are provided. For cell phenotyping, the cell surface markers of both cells were analyzed using flow cytometry. In the results, the MSCs were isolated from the synovial fluid of the minipigs and showed that synovial-fluid-derived MSCs have a similar morphology and cell phenotype to bone-marrow-derived MSCs. Therefore, non-invasively obtained synovial fluid is a valuable source of MSCs.

A protocol establishing mesenchymal stem cells (MSCs) isolated from the synovial fluid and bone marrow of minipigs and non-invasively collected using syringe aspiration is presented. Cellular phenotyping was performed using flow cytometry after cell isolation and in vitro cultivation of bone marrow and synovial fluids derived from MSCs.

Mini-domuzlarda sinovyal sıvı ve Kemik İliği Elde Edilen Mezenkimal Kök Hücrelerin İzolasyonu ve Hücresel Fenotiplendirme

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, ...

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the development of obesity-associated complications such as type 2 diabetes. To quantitatively and qualitatively investigate the immune cell subsets in human AT deposits, we have developed a flow cytometry approach. The stromal vascular fraction (SVF), containing the immune cells, is isolated from subcutaneous and visceral AT biopsies by collagenase digestion. Adipocytes are removed after centrifugation. The SVF cells are stained for multiple membrane-bound markers selected to differentiate between immune cell subsets and analyzed using flow cytometry. As a result of this approach, pro- and anti-inflammatory macrophage subsets, dendritic cells (DCs), B-cells, CD4+ and CD8+ T-cells, and NK cells can be detected and quantified. This method gives detailed information about immune cells in AT and the amount of each specific subset. Since there are numerous fluorescent antibodies available, our flow cytometry approach can be adjusted to measure various other cellular and intracellular markers of interest.

This article describes a method to analyze immune cell content of adipose tissue by isolation of immune cells from adipose tissue and subsequent analysis using flow cytometry.

Akış Sitometresi kullanarak insan yağ dokusu içinde bağışıklık hücreleri karakterizasyonu

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the ...

Oral Biofilm Formation on Different Materials for Dental Implants

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low microbial adhesion may reduce the prevalence and progression of peri-implant diseases. In view of the oral environment complexity and oral biofilm heterogeneity, microscopy techniques are needed that can enable a biofilm analysis of the surfaces of teeth and dental materials. This article describes a series of protocols implemented for comparing oral biofilm formation on titanium and ceramic materials for prosthetic abutments, as well as the methods involved in oral biofilms analyses at the morphological and cellular levels. The in situ model to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments as described in this study provides a satisfactory preservation of the 48 h biofilm, thereby demonstrating methodological adequacy. Multiphoton microscopy allows the analysis of an area representative of the biofilm formed on the test materials. In addition, the use of fluorophores and the processing of the images using multiphoton microscopy allows the analysis of the bacterial viability in a very heterogeneous population of microorganisms. The preparation of biological specimens for electron microscopy promotes the structural preservation of biofilm, images with good resolution, and no artifacts.

Here, we present a protocol to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments, including the analysis of bacterial cells viability and morphological characteristics. An in situ model associated with powerful microscopy techniques is used for the oral biofilm analysis.

Diş implantları için farklı malzemeler üzerinde sözlü biyofilm oluşumu

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low ...

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. ...

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow transplantation. While UCB has several unique advantages, the limited numbers of HSCs within each UCB unit limits their use in regenerative medicine and HSC transplantation in adults. Efficient expansion of functional human HSCs can be achieved by ex vivo culturing of CD34+ cells isolated from UCBs and treated with a deacetylase inhibitor, valproic acid (VPA). The protocol detailed here describes the culture conditions and methodology to rapidly isolate CD34+ cells and expand to a high degree a pool of primitive HSCs. The expanded HSCs are capable of establishing both short-term and long-term engraftment and are able to give rise to all types of differentiated hematopoietic cells. This method also holds potential for clinical application in autologous HSC gene therapy and provides an attractive approach to overcome the loss of functional HSCs associated with gene editing.

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Here, we describe the ex vivo expansion of hematopoietic stem cells from CD34+ cells derived from umbilical cord blood treated with a combination of a cytokine cocktail and VPA. This method leads to a significant degree of ex vivo expansion of primitive HSCs for either clinical or laboratory applications.

EX Vivo insan göbek kordon kanı elde edilen CD34 hematopoetik kök hücrelerden genişlemesi+ hücreleri kullanarak Valproik asit

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow ...

Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central nervous system (CNS). However, further optimization of these technologies would benefit from methods allowing rapid and streamline determination of the extent of CNS and cell-specific targeting upon administration of viral vectors or nanoparticles in the body. Here, we present a protocol that takes advantage of the high throughput and multiplexing capabilities of flow cytometry to allow a straightforward quantification of different cell subtypes isolated from mouse brain or spinal cord, namely microglia/macrophages, lymphocytes, astrocytes, oligodendrocytes, neurons and endothelial cells. We apply this approach to highlight critical differences between two tissue homogenization methods in terms of cell yield, viability and composition. This could instruct the user to choose the best method depending on the cell type(s) of interest and the specific application. This method is not suited for analysis of anatomical distribution, since the tissue is homogenized to generate a single-cell suspension. However, it allows to work with viable cells and it can be combined with cell-sorting, opening the way for several applications that could expand the repertoire of tools in the hands of the neuroscientist, ranging from establishment of primary cultures derived from pure cell populations, to gene-expression analyses and biochemical or functional assays on well-defined cell subtypes in the context of neurodegenerative diseases, upon pharmacological treatment or gene therapy.

We present a flow cytometry method to identify simultaneously different cell types retrieved from mouse brain or spinal cord. This method could be exploited to isolate or characterize pure cell populations in neurodegenerative diseases or to quantify the extent of cell targeting upon in vivo administration of viral vectors or nanoparticles.

Farklı Homojenizasyon Yöntemleri ile Fare Beyin/Omurilikten Alınan Çoklu Hücre Tiplerinin Eşzamanlı Akış Sitometrik Karakterizasyonu

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central ...