This work was supported by National Institute of Health (1R01DK103150-01A1, T32 DK007639, R01CA175803, K08CA125209) and the Institute for Translational Sciences at the University of Texas Medical Branch, and a Clinical and Translational Science Award (8UL1TR000071).

Atılan insan cerrahi hastaların doku ve primer kültürlerinde kurulmasını elde etmek için protokol Texas Medical Branch ve New Mexico Kurumsal İnceleme Kurulları Üniversitesi Üniversitesi tarafından onaylanmıştır. İnsan doku örneklerinin alımı için genel şartlar aşağıda açıklanmıştır. MFS donmuş dokunun, biyobankalar ve ticari satıcılardan izole edilebilir çünkü bu da uygun bir seçenek olduğuna dikkat edin. 1. İnsan Dokusu edinin <ol><li> Onay için kurumsal inceleme kuruluna insan kolon dokusu elde etmek için bir protokol gönderin. </li><li> Araştırma için insan dokusu elde etmek amacıyla, genel cerrahlar ve / veya kolorektal cerrah ile işbirliği kurmak. Onlar tanı için gerekli değildir ve araştırma için belirlenmiş olabilir doku belirleyici olacak gibi patoloji bölümü ile işbirliği de zorunludur. </li><li> Grossl sonra neoplazma doku sağlamak ve patolog önerintümörden y, normal mukoza en az 5 cm. Hemen buz soğukluğunda yıkama ortamı örnekleri yerleştirin ve buz üzerine yerleştirin. Patolog güvenli araştırma için verilebilir doku miktarını belirleyecektir. MF izolasyonu için gerekli olan minimum doku 1,5 mm 2 &#39;dir. </li><li> Gelecekteki izolasyonu için -80 ° C &#39;de primer kültürler ya da dondurma ve yeri oluşturmak için elde edilen doku ya hemen kullanın. <ol><li> -80 ° C&#39;de dondurularak ortam (aşağıya bakınız), ve mağaza 1 ml kriyojenik bir tüp içine yerleştirin 2 mm 2 adet - daha sonra izole edilmesi için dondurma ise, yaklaşık 1 doku kesmek. Not: Bu protokol kullanarak, 4 yılı aşkın süredir donmuş dokudan MFS başarılı izolasyon gözlenmiştir. </li></ol></li></ol> 2. Reaktifler hazırlayın <ol><li> Kollajenaz çözümü için: Ca2 + ile Hank&#39;in dengeli tuz çözeltisi (HBSS) içinde 100 U / ml kollajenaz I, II ve IV Hazırlama / Mg2 + (Malzeme bakınız Tablo </strong&gt;). DİKKAT: Kollajenaz aktif ünite konsantrasyon ve saflık olabilir mukozal hücre yüzeyinde epitop doku sindirim ve / veya korunması etkinliğini engelleyebilir satıcıdan satıcıya, değişir. Bu potansiyel tuzaklar önlemek için, saflık (≥95%) en yüksek derecesi ile kollajenaz kullanımı ve etkin birimler konsantrasyonuna dayalı stok solüsyon hazırlanır. </li><li> 10 mg / ml (3.55 UA / mg) Su bazlı DNase stok çözelti hazırlayın. </li><li> Yıkama ortamı hazırlayın: (MEM: 89 ml, antibiyotik antimikotik, 100x: 1 ml ısı ile inaktive edilmiş FCS: 10 mi) </li><li> MEM: Fibroblast büyüme ortamı hazırlayın 500 ml şişe; L-glutamin (200 mM): 5 mi; MEM esansiyel olmayan amino asitler, 100x çözeltisi: 5 mi; siprofloksasin HCI (10 mg / ml): 570 ul; Antibiyotik antimikotik, 100x çözeltisi: 10 mi; sodyum piruvat (100 mM): 5 mi; ısı ile inaktive edilmiş FCS: 50 mi </li><li> Dondurulmuş ortamı hazırlayın: Fibroblast büyüme ortamı,% 10 ilave Dimetil Sülfoksit (DMSO) ile (reaktif 2.4), daha sonra filtre edilir-ster0.22 um filtre ile ilize. </li></ol> 3. enzimatik Digest İnsan Doku <ol><li> Doku ılık (~ 37 ° C) suda donma medya, yer cryovial donmuş ise doku ve medya çözülmüş kadar. </li><li> 4 için - doku 2 mm 2 adet 5, Ca2 + / Mg2 + olmaksızın iki kez HBSS 10 ml doku yıkayın. </li><li> Doku yerçekimi tarafından yerleşmiş sonra, dikkatlice numuneyi sıkma yapmadan süpernatant atın. </li><li> Örnek kolajenaz çözeltisi 10 ml ilave edilir ve steril bir DNase / RNase içermeyen bir tüp içine doku çözelti karışımı transferi yerleşik örnek ayrılması için rotorlar. Epitel hücrelerinin bazal yönünü çevreleyen ve mukozal lamina propria hücresel olmayan kısmını oluşturan hücre dışı matriks ayırmak için kollajenaz kullanın. </li><li> Sıkıca kapatın tüp ve dissociator (örneğin, gentleMACS) koluna üzerine baş aşağı ekleyin. Laboratuar t erişimi yoksaO, ekipman yukarıda değindiğimiz karşılaştırılabilir sonuçlar elde etmek için her sindirim aşamasının süresini artırın. </li><li> Program h_tumor_01 çalıştırın önceden ayarlanmış profil makinesi (- vadede başına 268 mermi ile 4000 rpm, değişen aralıklı darbeleri ile 36 saniyelik toplam süresi, 1000 dan itibaren) ile birlikte. </li><li> Programın sona ermesinden sonra, dissociator tüp ayırın. </li><li> 140 rpm&#39;de çalkalayıcı üzerinde sürekli dönme altında 37 ° C &#39;de 45 dakika boyunca örnek inkübe edin. Not: doku örnekleri değişen miktarlarda için, sindirim zaman orantılı olarak azaltılabilir veya arttırılabilir (yani, doku daha fazla miktarlar ise ek süre gerektirebilir). </li><li> Dissociator koluna üzerine baş aşağı tüp takın. </li><li> (- Vadede başına 235 mermi ile, 4000 rpm 1000 arasında değişen aralıklı darbeleri ile, 37 sn toplam süresini) seçin ve Program h_tumor_02 çalıştırın. </li><li> Programın sona ermesinden sonra, dissociator tüp ayırın. </li><li> DNaz stok solüsyonu 50 ul ekleyin. / Ayırmak ölü hücreleri ve topaklanma hücre açan hücresel enkaz DNA kısmını kaldırmak için DNAz kullanın. </li><li> 60 rpm&#39;de çalkalayıcı üzerinde sürekli dönme altında 37 ° C&#39;de 30 dakika boyunca örnek inkübe edin. </li><li> Yine dissociator koluna üzerine baş aşağı tüp takın. </li><li> (- Vadede başına 168 mermi ile, 4000 rpm 1000 arasında değişen aralıklı darbeleri ile, 37 sn toplam süresini) seçin ve Program h_tumor_03 çalıştırın. Not: doku tamamen sindirilmiş değilse, 20 ° C &#39;de 10 dakika boyunca 250 x g&#39;de santrifüj hücre ayrışma çözeltisi 2 ml süpernatant, tekrar süspansiyon atmak ve 10 dakika boyunca oda sıcaklığında inkübe edilir. </li><li> Steril 70 mikron hücre süzgecinden hücre süspansiyonu geçirin. </li><li> 20 ° C &#39;de 10 dakika boyunca 250 x g&#39;de santrifüje hücre süspansiyonu. Aspire tamamen süpernatanlarına. </li><li> Yıkama hücresi Ca2 + / Mg2 + olmaksızın iki kez HBSS 25 ml pelet ve supernatant atın. </li><li> Son öncesinde olduh laboratuvarda mevcut otomatik hücre sayım sistemi kullanılarak ya da elle hemasitometre kullanarak hücre sayısını. MF primer kültür nesil için analiz için adım 3.19.1 sürmesine veya adım 3.19.2 sürmesine akım sitometri kullanılarak MFS sıralama. <ol><li> Izolasyon ve saf MF kültürünün büyüme (Şekil 1) için, ortam 3 ml 4 x 10 6 hücre kadar tohum için gerekli fibroblast izolasyon ortamının uygun miktarda Son santrifüjleme, tekrar süspansiyon hücre topağından süpernatan atılır well başına 6 de, hücre kültürü ile muamele edilmiş plakalar. 3.20 adıma geçin. </li><li> Analizi için veya flow sitometri 24 fibroblast izolasyon medyanın 2 ml 2 x 10 6 yerleştirebilirsiniz iyi, düşük bağlama plakası ve% 5 CO 2 ile 37 ° C O / N inkübe kullanarak MFS sıralama. Bu enzimatik prosedür ile etkilenebilir hücre yüzey epitoplarını sağlamaktır. Sonra hücre süspansiyonu toplamak ve kurtarılan hücrelerin sayısı ve devamve immün analiz 8 akım sitometri. </li></ol></li><li> % 5 CO2 ile 37 ° C &#39;de hücreler büyütün. % 80 confluent MF tek tabaka ~ oluşumu (Şekil 1D-E) kadar 3 gün - her 2 ortamı değiştirmek. </li><li> Passage 6 plaka dan oranını 1 ile T25 matara hücreleri: 1 ve ~ 10 büyümeye -% 100 confluency - fibroblast büyüme medyada 14 gün 80 elde etmek. </li><li> 2: 1 oranında at T75 hücreleri T25 hücreleri Pasajlanması kültür genişletin. Hücreler konfluense ulaştıktan sonra, daha önce tarif edildiği gibi 5 akış sitometresi ile kullanılarak izole edilmiş MF saflık analizi için bir şişesi kullanın. 3: 1 oranında MF kültürünün başka T75 balon dondurmak veya geçit. Not: EpCAM-, CD31, CD45-, vimentin +, α-SMA +, CD90 +: flow sitometri ile incelendiğinde, mezenkimal kökenli GI mukoza stromal MF kültüründe büyüyen aşağıdaki fenotip olacak o zaman bekleniyor. </li></ol>

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The authors have nothing to disclose.

Bu protokol, potansiyel bir kanserli prognostik biyolojik ve terapötik bir hedef olarak stromal hücrelerin kritik önemi giderek artan ışığında, sadece araştırma uygulamaları için geliştirilen birlikte, daha sonra kullanılmak üzere &quot;işlevsel&quot; biospecimens dondurma kabiliyeti önemli bir avantaj sunmaktadır. Bu kişiselleştirilmiş tıp 10, 11 gelişimini ilerletmek için hizmet olarak ek araçlar hizmet edebilir, hangi klinik biorepository yaratılması için benzersiz bir avantaj o...

Miyofibroblastlar / fibroblastlar (MFS), gastrointestinal (Gİ) mukoza hücrelerinin bol nüfusu temsil etmektedir. Bu stromal hücreler sadece epiteli altında bulunan ve bağırsaktaki mukozal lamina propria içinde birbirine bir ağ oluştururlar. MFS olmayan hücre dışı matrisin birikmesine sadece sorumludur, ancak parakrin düzenleme içinden geçen bitişik epitel 1, 2 elektrolit taşıma, iadesini ve bariyer işlevini etkileyebilir. Dahası, MFS enflamasyon ve doku önemli bir rol oynadığı gösterilmiştir 3, 4 biçimlenme. miyofibroblastlar onlar da kanserle ilişkili fibroblastlar olarak bilinir tümör mikro, kritik bir parçası olan ve tümör hücre büyümesine katkı ve kanser kök hücreleri 3 için niş olarak hizmet vermektedir. Ortaya çıkan veriler, MFS ayrıca yerel antijen sunan hücreler hizmet olabileceğini düşündürmektedir. Ayrıca, MFS işlevi doğal ve kazanılmış bağışıklık tepkilerinin önemli düzenleyiciler, süredensitokinler ve büyüme faktörleri 5 çeşitli dans ediyorum. Sağlıklı bireylerde, MFS hücreleri Bu hücreler aynı zamanda vimentin için pozitif. Mezenkimal kök hücrelerinden ayırt ve hücre yüzeyinde, mezenkimal marker, CD90 3, 5 tarihinde ifade inanılmaktadır, ancak epitelyal ve hematopoetik hücre belirteci, EpCAM ve CD45 negatif sırasıyla. Miyofibroblastların fibroblastlar aktive edilmiş bir şekli olması önerilmektedir ve α-SMA 5 ifadesi ile aktif olmayan fibroblastlardan ayırt edilebilir. Geçtiğimiz on yıl içinde, insan kolon mukozasının gelen miyofibroblastlar izolasyonu için birden yaklaşımlar yayınlanan-çoğunlukla başlangıçta Mahida ve ark., 5-8 açıklanan yönteme dayalı olarak oylandı. Bireysel çalışmalar, çeşitli bağırsak mukozasında, çoklu gastrointestinal alanlar (yani, mide, küçük ve l&#39;den MFS izolasyonu için hiçbir evrensel protokolü izolasyon prosedürleri rapor ederkenARGE intestinal mukoza) yayınlanmıştır. Bu tarifnamede sunulan protokol test edilmiş ve başarılı bir şekilde, yukarıda belirtilen dokuların her üç tip kullanılmaktadır. . Ayrıca, dondurulmuş GI mukozasından MF oranı pratisyen izole edilmesi için prosedürlerin rapor edilmemiştir. Burada, enzimatik sindirimi dayalı optimize edilmiş bir yöntem sunmak ve aynı zamanda kültür için insan bağırsak mukozal MFS izolasyonu sağlar ve taze sindirilmiş, tek hücre, mukozal hazırlıkları analiz akım sitometri. Bu teknik, güvenilir bir şekilde bir MF fenotipi olan primer kültürleri elde edilir. Bundan başka, aynı yöntemler yanı sıra, mide ve ince bağırsak dokularının dondurulmuş örneklerden MF oranı pratisyen izole etmek için kullanılabilir. Taze GI dokusundan rolü in izole edilmesi, daha önce tarif edilmiştir; Bununla birlikte, dondurulmuş örneklerin kullanımı birçok avantaj sunar. Yani, araştırmacılar capabi sahip dünya çapında işbirlikçileri herhangi bir sayıda toplamak ve depolamak dokuları mümkün olacaktırlity dondurulmuş doku örnekleri gemi. Ayrıca, araştırmacılar, mevcut deney programı ile çatışmaya izolasyon doku işleme ameliyathane ve / veya endoskopi paketi ve acil atılır doku toplama bulabilirsiniz. Ayrıca, cerrahi öngörülemeyen doğası nedeniyle, doku tedarik işleme doku için kalan süre sınırı, hangi çok geç gün oluşabilir. Daha sonra işlenmek için doku Donma bu zorlukları hafifleten. Son olarak, bu yöntemler, başarılı şekilde kolorektal karsinom ve iltihaplı bağırsak hastalıkları gibi hastalık durumlarında, kolon, mide ve ince bağırsak rolü in izolasyon kullanılmıştır.

<table border="0" cellpadding="0" cellspacing="0" width="719">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">MEM, 1X</td>
			<td style="width:111px;">Corning</td>
			<td style="width:119px;">MT-10-010-CV</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Hanks&#39; Balanced Salt Solution</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:119px;">H6648</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Sodium pyruvate</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:119px;">S8636</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Antibiotic-Antimycotic, 100X</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:119px;">15240-062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Ciprofloxacin HCl</td>
			<td style="width:111px;">Corning</td>
			<td style="width:119px;">61-277-RF</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">L-Glutamine, 100x</td>
			<td style="width:111px;">Corning</td>
			<td style="width:119px;">25-005-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">MEM Nonessential Amino Acids</td>
			<td style="width:111px;">Corning</td>
			<td style="width:119px;">25-025-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Dimethyl Sulfoxide (DMSO) Hybri-Max</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:119px;">D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">DL-Dithiothreitol solution (DTT)</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:119px;">646563</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">0.5M EDTA, pH 8.0</td>
			<td style="width:111px;">Cellgro</td>
			<td style="width:119px;">46-034-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">DNAse</td>
			<td style="width:111px;">Worthington</td>
			<td style="width:119px;">LS002139</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">Collagenase from&#160;Clostridium histolyticum, Type I</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:119px;">C1639</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">Collagenase from&#160;Clostridium histolyticum, Type II</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:119px;">C1764</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">Collagenase from Clostridium histolyticum, Type IV</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:119px;">C5138</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Accumax cell dissociation solution</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:119px;">A7089</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">gentleMACS C Tubes</td>
			<td style="width:111px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-093-237</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">gentleMACS Dissociator</td>
			<td style="width:111px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-093-235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Countess Automated Cell Counter</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:119px;">C10227</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Cell Strainer (70&#181;m)</td>
			<td style="width:111px;">Corning</td>
			<td style="width:119px;">352350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">PE Mouse Anti-Human Vimentin</td>
			<td style="width:111px;">BD Biosciences</td>
			<td style="width:119px;">562337</td>
			<td>Clone RV202</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">FITC Monoclonal Anti-Actin, &#945;-Smooth Muscle</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:119px;">F3777</td>
			<td>Clone 1A4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">APC Mouse Anti-Human CD90</td>
			<td style="width:111px;">BD Biosciences</td>
			<td style="width:119px;">559869</td>
			<td>Clone 5E10</td>
		</tr>
	</tbody>
</table>

isolation of cd 90+ fibroblast/myofibroblasts from human frozen gastrointestinal specimens

Fibroblastlar / miyofibroblastlar (MFS) patogenezinde rolleri ve tümör mikro hem yara iyileşmesi ve promosyon katkıları için artan ilgi kazanmaktadır. Şu anda mide-bağırsak (GI) dokulardan MFS izolasyonu için bir çok teknik vardır, ancak bu protokol, dondurulmuş dokudan bu stromal hücrelerin izolasyonu için yeni bir elemanı getirmektedir. GI doku örneklerinin Donma sadece dünya işbirlikçileri, biyobankalar ve ticari satıcılardan örnekleri elde etmek için araştırmacı, aynı zamanda taze örneklerin gecikmeli işleme izin verir. Açıklanan protokol sürekli belirteçleri CD90, α-SMA ve vimentin ifade MF fenotip ile karakteristik iğ şekilli hücreler verecektir. Bu hücreler, hasta örneklerinden türetilmiş olarak, pil kullanımı aynı zamanda hastalık durumlarında-, yani kanser ve enflamatuar barsak hastalıkları arasından yakın taklit MFS&#39;de yararı kazandırmaktadır. Bu teknik arı vardırn mide, ince bağırsak, kolon ve MF primer kültür nesil valide. Birincil MF kültürleri pasaj bir dizi üzerinden deney geniş bir dizi de kullanılabilir ve bunların saflığı hem immünositokimya değerlendirilmiş ve akış sitometri analizine.

Bu enzimatik sindirim protokolü kullanarak, sürekli izole ve GI cerrahi örneklerin ve biyopsi (Şekil 1) MF nüfus gut CD90 + mukozal stromal hücreler büyümeye mümkün olmuştur. 6 yuvalı plakalar (Şekil 1A-B) tek çekirdekli hücre süspansiyonları tohumlama sonra 5 - Görünür MF koloni proliferasyon günde 2 gözlenebilir. MF primer kültürlerinde ulaşmak ~ 50-11 (Şekil 1C-D) - 7. günde% 70 izdiham. <p cl...

Watch this Scientific Journal Video about Isolation of CD 90+ Fibroblast/Myofibroblasts from Human Frozen Gastrointestinal Specimens at JoVE.com

Isolation of CD 90+ Fibroblast/Myofibroblasts from Human Frozen Gastrointestinal Specimens

Here, a protocol to isolate and establish primary fibroblast/myofibroblast (MF) cultures from frozen gastric, small intestinal, and colonic tissue-yielding cells with a MF phenotype-is presented. These cells express CD90, &#945;-SMA and vimentin. MFs can be used for a variety of functional assays including enzymatic activity and cytokine production.

İnsan Frozen Gastrointestinal örneklerden CD 90+ fibroblast / miyofibroblastlar İzolasyonu


Fibroblasts/myofibroblasts (MFs) have been gaining increasing attention for their role in pathogenesis and their contributions to both wound healing and ...

isolation-cd-90-fibroblastmyofibroblasts-from-human-frozen

Research

JoVE Journal

Developmental Biology

9.5K Görüntüleme.  University of Texas Medical Branch. Here, a protocol to isolate and establish primary fibroblast/myofibroblast (MF) cultures from frozen gastric, small intestinal, and colonic tissue-yielding cells with a MF phenotype-is presented. These cells express CD90, α-SMA and vimentin. MFs can be used for a variety of functional assays including enzymatic activity and cytokine production. 

Video: Isolation of CD 90+ Fibroblast/Myofibroblasts from Human Frozen Gastrointestinal Specimens

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

İnsan İskelet Kası İlköğretim andMyogenic Hücreleri ve Fibroblast İzolasyon ve Kantitatif İmmünositokimyasal Karakterizasyonu

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Gelişim Biyolojisi

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

Periventriküler Yetişkin Rat Bölgesinde ve İnsan Omurilik gelen Nöral Kök / Progenitör Hücreler izolasyonu

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

Bütünleşmeyen episomal plasmidler kullanılarak dondurulmuş Buffy tüylerdeki uyarılmış pluripotent kök hücrelerin üretilmesi

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

İnsan uyarılmış pluripotent kök hücrelerin türetilmiş Piramidal Nöronlar gelen Dendritik Spine Üç boyutlu Niceleme

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human development and investigate the pathophysiology of disease. By employing site-specific nucleases (SSNs) for genome editing, the rapid derivation of new hPSC lines harboring specific genetic alterations in an otherwise isogenic setting becomes possible. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 are the most commonly used SSNs. All of these nucleases function by introducing a double stranded DNA break at a specified site, thereby promoting precise gene editing at a genomic locus. SSN-meditated genome editing exploits two of the cell&#39;s endogenous DNA repair mechanisms, non-homologous end joining (NHEJ) and homology directed repair (HDR), to either introduce insertion/deletion mutations or alter the genome using a homologous repair template at the site of the double stranded break. Electroporation of hPSCs is an efficient means of transfecting SSNs and repair templates that incorporate transgenes such as fluorescent reporters and antibiotic resistance cassettes. After electroporation, it is possible to isolate only those hPSCs that incorporated the repair construct by selecting for antibiotic resistance. Mechanically separating hPSC colonies and confirming proper integration at the target site through genotyping allows for the isolation of correctly targeted and genetically homogeneous cell lines. The validity of this protocol is demonstrated here by using all three SSN platforms to incorporate EGFP and a puromycin resistance construct into the AAVS1 safe harbor locus in human pluripotent&#160;stem cells.

Genome editing of human pluripotent stem cells (hPSCs) can be done quickly and efficiently. Presented here is a robust experimental procedure to genetically engineer hPSCs as exemplified by editing the AAVS1 safe harbor locus to express EGFP and introduce antibiotic resistance.

Genom düzenlenmiş İnsan Pluripotent Kök Hücre Hatları oluşturulması: İzolasyon hedefleyen Gönderen

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

İnsan Plasenta Doku türetilen İzolasyon ve mezenkimal kök Genişleme / Stromal hücreler

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

İnsan Kardiyak Doku perivasküler Multipotent Öncü hücre popülasyonlarının izolasyonu

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

İnsan göbek bağı ve fetal plasentadan Mezenkimal Stromal Hücre İzolasyonu ve Vasıflandırılması

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues1. Later, others documented the existence of similar types of EPCs originating from bone marrow2,3. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs7,8. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques9. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.

The goal of this protocol is to isolate endothelial progenitor cells from umbilical cord blood. Some of the applications include using these cells as a biomarker for identifying patients with cardiovascular risk, treating ischemic diseases, and creating tissue-engineered vascular and heart valve constructs.

Yalıtım endotelyal Progenitor Hücre insan göbek kordon kanı

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of the heart after ischemic injury. When activated, epicardial cells undergo a process known as epithelial to mesenchymal transition (EMT) to provide cells to the regenerating myocardium. Furthermore, the epicardium contributes via secretion of essential paracrine factors. To fully appreciate the regenerative potential of the epicardium, a human cell model is required. Here we outline a novel cell culture model to derive primary epicardial derived cells (EPDCs) from human adult and fetal cardiac tissue. To isolate EPDCs, the epicardium is dissected from the outside of the heart specimen and processed into a single cell suspension. Next, EPDCs are plated and cultured in EPDC medium containing the ALK 5-kinase inhibitor SB431542 to maintain their epithelial phenotype. EMT is induced by stimulation with TGF&#946;. This method enables, for the first time, the study of the process of human epicardial EMT in a controlled setting, and facilitates gaining more insight in the secretome of EPDCs that may aid heart regeneration. Furthermore, this uniform approach allows for direct comparison of human adult and fetal epicardial behavior.

The epicardium plays a crucial role in the development and repair of the heart by providing cells and growth factors to the myocardial wall. Here, we describe a method to culture human primary epicardial cells that enables the study and comparison of their developmental and adult characteristics.

Yalıtım ve kültür insan yetişkin ve Fetal kalp örnekler elde edilen birincil Epicardial hücre

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...