Name: isolation of cd 90+ fibroblast/myofibroblasts from human frozen gastrointestinal specimens
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Description: Watch this Scientific Journal Video about Isolation of CD 90+ Fibroblast/Myofibroblasts from Human Frozen Gastrointestinal Specimens at JoVE.com

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><tbody><tr><td>MEM, 1x</td><td>Corning</td><td>MT-10-010-CV</td><td></td></tr><tr><td>Hanks&#039; Balanced Salt Solution</td><td>Sigma-Aldrich</td><td>H6648</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma-Aldrich</td><td>S8636</td><td></td></tr><tr><td>Antibiotic-Antimycotic, 100x</td><td>Gibco</td><td>15240-062</td><td></td></tr><tr><td>Ciprofloxacin HCl</td><td>Corning</td><td>61-277-RF</td><td></td></tr><tr><td>L-Glutamine, 100x</td><td>Corning</td><td>25-005-CI</td><td></td></tr><tr><td>MEM Nonessential Amino Acids</td><td>Corning</td><td>25-025-CI</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO) Hybri-Max</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>DL-Dithiothreitol solution (DTT)</td><td>Sigma-Aldrich</td><td>646563</td><td></td></tr><tr><td>0.5 M EDTA, pH 8.0</td><td>Cellgro</td><td>46-034-CI</td><td></td></tr><tr><td>DNAse</td><td>Worthington</td><td>LS002139</td><td></td></tr><tr><td>Collagenase from&amp;nbsp;Clostridium histolyticum, Type I</td><td>Sigma-Aldrich</td><td>C1639</td><td></td></tr><tr><td>Collagenase from&amp;nbsp;Clostridium histolyticum, Type II</td><td>Sigma-Aldrich</td><td>C1764</td><td></td></tr><tr><td>Collagenase from Clostridium histolyticum, Type IV</td><td>Sigma-Aldrich</td><td>C5138</td><td></td></tr><tr><td>Accumax cell dissociation solution</td><td>Sigma-Aldrich</td><td>A7089</td><td></td></tr><tr><td>gentleMACS C Tubes</td><td>Miltenyi Biotec</td><td>130-093-237</td><td></td></tr><tr><td>gentleMACS Dissociator</td><td>Miltenyi Biotec</td><td>130-093-235</td><td></td></tr><tr><td>Countess Automated Cell Counter</td><td>Invitrogen</td><td>C10227</td><td></td></tr><tr><td>Cell Strainer (70 &amp;micro;m)</td><td>Corning</td><td>352350</td><td></td></tr><tr><td>PE Mouse Anti-Human Vimentin</td><td>BD Biosciences</td><td>562337</td><td>Clone RV202</td></tr><tr><td>FITC Monoclonal Anti-Actin, &amp;alpha;-Smooth Muscle</td><td>Sigma-Aldrich</td><td>F3777</td><td>Clone 1A4</td></tr><tr><td>APC Mouse Anti-Human CD90</td><td>BD Biosciences</td><td>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

9.6K 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

Murine Dermal Fibroblast Isolation by FACS

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast physiology and pathology underlie a spectrum of clinical entities, including fibroses in multiple organs, hypertrophic scarring following burns, loss of cardiac function following ischemia, and the formation of cancer stroma. However, fibroblasts remain a poorly characterized type of cell, largely due to their inherent heterogeneity. Existing methods for the isolation of fibroblasts require time in cell culture that profoundly influences cell phenotype and behavior. Consequently, many studies investigating fibroblast biology rely upon in vitro manipulation and do not accurately capture fibroblast behavior in vivo. To overcome this problem, we developed a FACS-based protocol for the isolation of fibroblasts from the dorsal skin of adult mice that does not require cell culture, thereby preserving the physiologic transcriptional and proteomic profile of each cell. Our strategy allows for exclusion of non-mesenchymal lineages via a lineage negative gate (Lin-) rather than a positive selection strategy to avoid pre-selection or enrichment of a subpopulation of fibroblasts expressing specific surface markers and be as inclusive as possible across this heterogeneous cell type.

Fibroblast behavior underlies a spectrum of clinical entities, but they remain poorly characterized, largely due to their inherent heterogeneity. Traditional fibroblast research relies upon in vitro manipulation, masking in vivo fibroblast behavior. We describe a FACS-based protocol for the isolation of mouse skin fibroblasts that does not require cell culture.

FACS ile Murin Dermal fibroblast İzolasyonu

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast ...

Isolation of Papillary and Reticular Fibroblasts from Human Skin by Fluorescence-activated Cell Sorting

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have traditionally been attributed to the superficial papillary or lower reticular dermis according to their histological localization. In mouse dermis, papillary and reticular fibroblasts originate from two different lineages with diverging functions regarding physiological and pathological processes and a distinct cell surface marker expression profile by which they can be distinguished.
Importantly, evidence from explant cultures from superficial and lower dermal layers suggest that at least two functionally distinct dermal fibroblasts lineages exist in human skin dermis as well. However, unlike for mouse skin, cell surface markers enabling the discrimination of different fibroblast subsets have not yet been established for human skin. We developed a novel protocol for the isolation of human papillary and reticular fibroblast populations via fluorescence-activated cell sorting (FACS) using the two cell surface markers Fibroblast Activation Protein (FAP) and Thymocyte antigen 1 (Thy1)/CD90. This method enables the isolation of pure fibroblast subsets without in vitro manipulation, which was shown to affect gene expression, thus permitting accurate functional analysis of human dermal fibroblast subsets in regard to tissue homeostasis or disease pathology.

This manuscript describes a FACS-based protocol for isolation of papillary and reticular fibroblasts from human skin. It circumvents in vitro culture which was inevitable with the commonly used isolation protocol via explant cultures. The emanating fibroblast subsets are functionally distinct and display differential gene expression and localization within the dermis.

Floresan-aktif hücre sıralama ile ınsan derisinden papiller ve Reiküler fibroblastların izolasyonu

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have ...

Genetik

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

Hastalık Modelleme için kas biyopsisi Hasta türetilmiş Hücre Hatları İzolasyonu ve Ölümsüzleştirme

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

Tıp

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

Erişkin İnsan Dermal Fibroblastlarının Abdominal Deriden İzolasyon ve İndüklenen Pluripotent Kök Hücrelerin Indüklenen Alacalı Kök Hücrelerin Indükleyici Olmayan Yöntemle Üretilmesi

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Biyomühendislik

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker &alpha;-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFR&alpha; as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFR&alpha; is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to &alpha;-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFR&#x03B1; as a surface marker.

Floresans Aktif Hücre Ayırma (FACS) Taze Dokular Normal ve Kanser ilişkili Fibroblastlar İzolasyonu

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

Biyoloji

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

Fare ve İnsan Kolon Doku Primer miyofibroblastlar İzolasyonu

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Ultrasonic-augmented Primary Adult Fibroblast Isolation

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary fibroblasts cannot divide indefinitely due to myofibroblast differentiation or senescence induction, new cultures must be established regularly. However, there are several obstacles to overcome during the processes of developing a reliable isolation protocol and primary fibroblast isolation itself: the method&#8217;s degree of difficulty (especially for beginners), the risk of bacterial contamination, the required time until primary fibroblasts can be used for experiments, and subsequent cell quality and viability. In this study, a fast, reliable and easy-to-learn protocol to isolate and culture primary adult fibroblasts from mouse heart, lung, liver and kidney combining enzymatic digestion and ultrasonic agitation is provided.

We present a protocol to isolate primary adult fibroblasts in an easy, fast and reliable way, performable by beginners (e.g., students). The procedure combines enzymatic tissue digestion and mechanical agitation with ultrasonic waves to obtain primary fibroblasts. The protocol can easily be adapted to specific experimental requirements (e.g., human tissue).

Ultrasonik-Augmented primer yetişkin fibroblast yalıtım

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary ...

Isolation of Myofibroblasts from Mouse and Human Esophagus

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, and subjected to enzymatic digestion with collagenase and dispase for 25 min. Human esophageal resection specimens are stripped of muscularis propria and adventitia and the remaining mucosa is minced, and subjected to enzymatic digestion with collagenase and dispase for up to 6 hr. Cultured cells express &#945;-SMA and vimentin and express desmin weakly or not at all. Culture conditions are not conducive to growth of epithelial, hematopoietic, or endothelial cells. Culture purity is further confirmed by flow cytometric evaluation of cell surface marker expression of potential contaminating hematopoietic and endothelial cells. The described technique is straightforward and results in consistent generation of non-hematopoieitc, non-endothelial stromal cells. Limitations of this technique are inherent to the use of primary cultures in molecular biology studies, i.e., the unavoidable variability encountered among cultures established across different mice or humans. Primary cultures however are a more representative reflection of the in vivo state compared to cell lines. These methods also provide investigators the ability to isolate and culture stromal cells from different clinical and experimental conditions, allowing comparisons between groups. Characterized esophageal stromal cells can also be used in functional studies investigating epithelial-stromal interactions in esophageal disorders.

We present a protocol to generate primary cultures of murine and human esophageal stromal cells with a myofibroblast phenotype. Cultured cells have spindle shaped morphology, express &#945;-SMA and vimentin, and lack epithelial, hematopoietic and endothelial cell surface markers. Characterized stromal cells can be used in functional studies of epithelial-stromal interactions.

Fare ve İnsan Özofagus gelen miyofibroblastların izolasyonu

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, ...

Isolation, Culture, and Characterization of Primary Dermal Fibroblasts from Human Keloid Tissue

Fibroblasts, the major cell type in keloid tissue, play an essential role in the formation and development&#160;of keloids. The isolation and culture of primary fibroblasts derived from keloid tissue are the basis for further studies of the biological function and molecular mechanisms of keloids, as well as new therapeutic strategies for treating them. The traditional method of obtaining primary fibroblasts has limitations, such as poor cellular state, mixing with other types of cells, and susceptibility to contamination. This paper describes an optimized and easily reproducible protocol that could reduce the occurrence of possible issues when obtaining fibroblasts. In this protocol, fibroblasts can be observed 5 days after isolation and reach nearly 80% confluency after 10 days of culture. Then, the fibroblasts are passaged and verified using PDGFR&#945; and vimentin antibodies for immunofluorescence assays and CD90 antibodies for flow cytometry. In conclusion, fibroblasts from keloid tissue can be easily acquired through this protocol, which can provide an abundant and stable source of cells in the laboratory for keloid research.

This study describes an optimized protocol for establishing primary fibroblasts from keloid tissues that can effectively and steadily provide pure and viable fibroblasts.

İnsan Keloid Dokusundan Primer Dermal Fibroblastların İzolasyonu, Kültürü ve Karakterizasyonu

Fibroblasts, the major cell type in keloid tissue, play an essential role in the formation and development&#160;of keloids. The isolation and culture of ...