Name: immunomagnetic separation of fat depot-specific sca1high adipose-derived stem cells (ascs)
Uploaded: 2016-08-11T14:00:00.000+00:00
Duration: 8 min 52 s
Description: Watch this Scientific Journal Video about Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs at JoVE.com

Bu eser (THC) NIH DK095137 tarafından desteklenmektedir. Biz anlatılan yöntemlerin geliştirilmesine ve gelişmişliği katkıda mevcut ve eski laboratuar üyelerine teşekkür ederim.

Etik Beyanı: Hayvanların Kullanımı ve Bakımı Michigan Komitesi Üniversitesi (UCUCA) (Laboratuar Hayvan Araştırma Enstitüsü, Ulusal Araştırma Konseyi) Laboratuvar Hayvanları Bakım ve Kullanım Kılavuzu uyarınca tüm yöntemleri ve protokolleri onayladı. Fareler bir Michigan Üniversitesi vivari tutulur ve gıda ve suya ücretsiz erişim verildi ve 12 saat aydınlık / karanlık döngüsü üzerinde tutulur. 1. hazırlıklar <ol><li> DMEM,% 10 FBS, 1x P / S / G, ve 1x antibiyotikler / antifungal ile primer kültür ortamı hazırlayın. Bu daha önce sindirime dokuları tutmak için kullanılır. 37 ºC su banyosunda stok ve kısım yerleştirin. </li><li> 5 mg / ml (+ Ca, Mg), yağ yastığı her bir tipi için beş fare kadar yalıtıldıktan HBSS içinde çözülmüş tip III kolajenaz çözeltisi, beş mililitre hazırlayın. Tek bir genotipten SQ ve VIS izole edilirken, vahşi tip ve mutant kare olduğu ve VIS, 20 ml yapmak, örneğin, 10 ml yapmak. 7.4 Ste pH ayarlamaFiltreyi rile. 50 ml tüpler içine kısım. kenara ışıktan uzakta ayarlayın. </li><li> Cerrahi alan dezenfekte% 70 etanol kullanın. aşağı silin ve mavi ped ile kaplayın. Mavi pad üzerinde bazı etanol püskürtün. </li><li> strafor kurulu bir emici ped aşağı pin ve makasla kesme 22 G iğne kullanın. sonra mavi pad üzerinde kurulu ayarlayabilir ve diseksiyon başlamadan kadar başka bir mavi ped ile kaplayın etanol ile pedi püskürtün. </li><li> cerrahi alan bitişik bir stand etanol ve yer ile iki 50 ml tüpler doldurun. Etanol makas ve forseps yerleştirin. </li><li> cerrahi aletleri etanol kapalı durulama için PBS artı 1x anti-anti başka 50 ml tüp doldurun. </li><li> kaputu, her bir doku tipi etiket 60 mm yemekleri ve 37 ºC ısıtılmış kültür ortamı ile doldurun. Cerrahi alana bitişik tabak yerleştirin. </li></ol> Subkütan (SQ) Yağ Pads 2. izolasyonu <ol><li> izofluran ve pnömotoraks aşırı dozda fare Euthanize. </li><li> Nazikçe% 70 etanol ile fare sprey ve sırtüstü yatıyordu. 22 G iğne ile köpük kuruluna pençeleri sabitleyin. </li><li> Alt karın derisinde küçük bir kesim yapın. forseps ile kesilen üst Holding, makas almak ve periton cilt ayırmak. </li><li> Cilt karından göğüse periton ayrılır sonra, vücudun yan tarafında deride yan keser yaparak uzak kafasına doğru periton cilt yansıtır. </li><li> vücuttan uzak kasık yağ tutma kalan cilt yansıtır ve 22 G iğne ile kuruluna aşağı pin. </li><li> ince makas ve forseps geçin. periton yakın kökeni forseps ile kasık yağ yastığı tut ve deri ile SQ kirletici kaçınarak kasık doğru ilerleyen deri ve kasık yağ arasında uzak snip. </li><li> etiketli 60 mm çanak güneşe maruz kasık yağ yastığı yerleştirin. </li></ol> Visseral (VIS) Yağ Pads 3. İzolasyon <ol><li> Benzer bir şekilde, 2.5 adım yağ yastıkları ve bağırsak visseral maruz açık periton kesmek için. </li><li> uzakta toraks doğru gut hareket ettirin. </li><li> uzak ucunda VIS yağ yastığı tut ve yavaşça yukarı doğru çekin. doku (dişi fareler kullanıyorsanız, ya da rahim) epididimal dışlamak için dikkat ederek VIS yağ yastığı parçalara ayır. </li><li> etiketli çanak ve kalan VIS pad adımı yineleyin 3.3 yerleştirin. </li></ol> Yağ Pads 4. kollajenaz sindirimi <ol><li> doku kültürü kaputu 60 mm yemekleri taşımak ve medya aspire. </li><li> kavisli makas sonra kollajenaz çözüm eklemek ile yağ yastıkları kıyma. etanol ve PBS ile doku tipleri arasındaki makas ve forseps durulama emin olun. </li><li> dokular dijeste kadar 300 rpm&#39;de 10-20 dakika boyunca 37 ° C&#39;de çalkalanır. SQ bazı parçaları hala görünür eğer kabul edilebilir. Bu bir sonraki adımda ele alınacaktır. </li><li> collag durdurmak için kollajenaz çözeltisine kültür ortamı 25 ml ilave edilir10 ml&#39;lik serolojik pipet ile enase tedavi ve pipet yukarı ve aşağı 10 kez sindirilmiş dokuları dağıtmak için. </li><li> 100 mikron hücre süzgeç kullanarak Gerilme hücreleri. 300 x g, 10 dakika boyunca santrifüj. </li><li> Dikkatle pelet rahatsız ve yavaşça hücrelerin askıya pipet pelet steril su 5 ml ekleyin ve için medyayı kapalı süzün. eritrositleri lizise uğratmak üzere 2 dakika bekleyin. </li><li> yeni bir 100 mikron hücre süzgecinden% 10 FBS ve şekil hücreleri ile ortam 25 ml ilave edilir. </li><li> 300 x g, 10 dakika boyunca santrifüj. Durusu medya ve kültür ortamı 1 ml tekrar süspansiyon pelet. </li><li> 1 tripan mavi: 1 kullanarak bir hemasitometre ile hücre sayımı. </li><li> 6 oyuklu bir plaka üzerinde, bir oyuk Levha 1 x 10 6 hücre. </li></ol> 5. Manyetik Hücre Ayırma <ol><li> Hücre yapışması, yaklaşık 4 ila 6 saat sonra, HBSS (-Ca, -mg) ile iki kez hücreleri yıkayın. % 0.05 tripsin kullanılarak yapışkan hücreleri ayırmak. 1 ml ayırma tamponu ve spin tripsin hücre süspansiyonu seyreltilir300 x g 5 dak. </li><li> Süpernatant aspire. 500 ul tampon içinde süspanse edin hücreleri. 10 ul kısım çıkarın ve hemasitometre kullanarak hücreleri saymak. </li><li> 300 x g, 5 dakika boyunca Spin hücreleri. </li><li> Aspire tamamen süpernatanlarına. 90 ul tampon içinde süspanse edin hücreleri. 10 ul anti-Sca1-FITC ile primer antikor ekleyin. İyice karıştırın ve karanlıkta 4 ° C&#39;de 10 dakika inkübe edilir. </li><li> mi tampon ve santrifüj hücreler, 300 x g, 5 dakika için 1 hücreleri yıkayın. tamamen süpernatantı. </li><li> 80 ul tampon içinde süspanse edin hücreleri. 20 ul anti-FITC mikroboncukları ekleyin. İyice karıştırın ve karanlıkta 4 ° C&#39;de 15 dakika inkübe edilir. </li><li> mi tampon ve santrifüj hücreler, 300 x g, 5 dakika için 1 hücreleri yıkayın. </li><li> 500 ul tampon süpernatant ve tekrar süspansiyon pelet çıkarın. </li><li> Manyetik tutucu sütun yerleştirin ve 500 ul tamponu ile sütun yıkayın. </li><li> kolonuna hücre süspansiyonu ekleyin. pipetlenmesinden sırasında kabarcıkları kaçının. </li><li> Etiketsiz SCA1 toplayın - &lt;/ Sup&gt; hücreler ve 500 ul tampon ile kolon 3 kere yıkayın. rezervuar boşken sadece yeni tampon ekleyin. pipetlenmesinden sırasında kabarcıkları kaçının. </li><li> manyetik tutucu sütunu çıkarın ve 15 ml konik tüp içine yerleştirin. </li><li> Kolon rezervuar üzerinden sağlanan pistonu iterek Sca1 + kolona tampon ve Zehir ml 1 ekleyin. </li><li> 300 x g&#39;de 5 dakika boyunca hücre kesirler aşağı doğru döndürün. 1 ml kültür ortamı içinde süspanse hücreleri. </li><li> etiketli ve etiketsiz kesirler 10 ul kısım çıkarın ve hemasitometre kullanarak hücreleri saymak. </li><li> 6 plaka 1 kuyuda her bir hücre fraksiyonu Plate. </li></ol> Akım Sitometrisi ile Sca1 yüksek ACSS ve İmmunomanyetik Ayrılık 6. Doğrulama <ol><li> HBSS (-Ca, -MG) ile iki kez hücreleri durulayın ve% 0.05 tripsin kullanarak hücreleri ayırmak. </li><li> 5 dakika boyunca 300 x g&#39;de santrifüje hücreleri. 1 ml kültür ortamı içinde süspanse edin hücreleri ve hemasitometre kullanarak saymak. </li><li> Elde&gt; 10 6 </sup&gt; 1 ml kültür ortamı içinde hücre ve 5 dakika boyunca 300 x g&#39;de santrifüjleyin. </li><li> süpernatant ve tekrar adım 6.3 iki kez daha çıkarın. </li><li> % 2 keçi serumu, 1 ml +% 2 BSA ile oda sıcaklığında 30 dakika boyunca bir blok ile yeniden süspanse hücreleri. </li><li> 5 dakika boyunca 300 x g&#39;de santrifüje hücreleri. </li><li> çözümü engelleme çıkarın. </li><li> 4 ° C&#39;de% 2 keçi serumu ve% 2 BSA + PBS ile 100 ul PBS içinde: ya da anti-SCA1 Alexa Fluor 647 (400, 0.25 ug, 1): sıçan IgG2a Alexa Fluor 647 (400, 0.25 ug, 1) ekleme karanlıkta 30 dakika karıştırıldı. </li><li> hücreler soğuk PBS ile 1 ml ilave edilir. 4 ° C&#39;de 5 dakika boyunca santrifüje 300 x g. </li><li> süpernatant ve tekrar adım 6.9 iki kez daha çıkarın. </li><li> 1 ml PBS içinde yeniden süspanse hücreleri. akım sitometri analizi için hazırlık 100 mikron hücre süzgecinden hücre süspansiyonları iletin. </li></ol>

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Yazarlar ifşa hiçbir şey yok.

Bu yazıda izolasyon ve farklı yağ pedleri gelen fare TSK immunomagnetic hücre ayırma ve in vitro deneyler için onların kullanımını göstermektedir. Sunulan yöntem TSK 9,14 arasında, teknik olarak karmaşık ve pahalı FACS aracılı izolasyon avantajlıdır Sca1 pozitif TSK sayıda hızlı izolasyonu için etkilidir. FACS aksine immünomanyetik hücre ayırma, bir hedef hücre popülasyonunun belirlenmesine ilişkin birden fazla antijenin kullanılmasına izin vermez. Yüzey antijeni iyi ...

Hücre antijeni 1 (SCA1 veya LY6A / e) kök ilk hematopoietik ve mezenkimal kök hücreleri 5,6 ile ifade edilen bir hücre yüzey markerı olarak tespit edilmiştir. Fare yağ depolarından elde edilen yağ dokusu stromal vasküler fraksiyonu (SVF) fibroblastlar, makrofajlar, vasküler endotelyal hücreler, nöronal hücrelerin ve adiposit progenitör hücrelerin 7 hücrelerinden oluşan heterojen bir popülasyonu. Adiposit progenitör hücreleri veya adipoz kaynaklı kök hücreler (TSK) kollajen zengin perivasküler hücre dışı matriks (ECM) 8 ikamet dışı lipid yüklü hücrelerdir. CD34 +: SCA1 + 9 ve CD29 + - yaklaşık SVF% 50 soy-negatif (Lin) olarak karakterize edilmiştir TSK oluşmaktadır. In vitro adiposit farklılaşması edebilen adiposit ataları, - CD24: bu hücrelerin çoğu Sca1 + &#39;dır Bununla birlikte, hücrelerin sadece küçük bir kısmı (SVF 0.08%) SCA1 teşkil <syukarı&gt; +: çoğalan ve in vivo koşullarda 9 adipositlere farklılaşma tamamen yetenekli CD24 + hücreler. CD24 gelen CD24 + hücreler demeden Sca1 + SVF kullanarak potansiyel ihtar rağmen - immunomagnetic hücre ayırma kullanarak yağ depolarından SCA1 + TSK, hücreler izole birincil adiposit progenitör hücrelerin hücre özerk fenotip belirlemek için etkili ve pratik bir yaklaşımdır. Obezite ve diyabet, doku fibrozu ve enflamasyon alanına tip-2 diyabet 3 gelişimi ve korunmasında önemli bir rol oynamaktadır. Son zamanlarda, Tokunaga ve ark. (VIS veya visseral) inguinal (ya da deri altı, SQ) ve perigonadal izole Sca1 yüksek hücreler C57BL6 / J yağ depoları vitro 10 farklı gen imzalar ve ECM biçimlenme sergilerler gösterdi. MMP14 (MT1-MMP), membran-t prototip üyesiYPE matriks metalloproteinaz (MMP) ailesi kolajenolitik aktiviteye 1 ile beyaz adipoz dokuda (WAT) gelişimini aracılık eder. Aşağıdaki protokol ile izole edilmiş ve zenginleştirilmiş hücreleri ile gerçekleştirilebilir deney örnekleri arasında, üç boyutlu bir kültür, farklılaşma çalışmaları, kolajen alçalmasının tahlilleri ve RNA sıralaması 10,11 bulunmaktadır. Parçalanma deneyleri telopeptit 11,12 korunmasını sağlamak için asit ekstre kollajen ile yapılmalıdır. Aşağıdaki protokol farklı yağ depolarından birincil vasküler stroma hücreleri izole ve immunomagnetic hücre ayırma yöntemi adiposit progenitör hücreler zenginleştirmek için yöntemler gösterecektir. Hücre sıralama geçerliliği akım sitometri ile ve bir Sca1 promotör 13 tarafından tahrik SCA1 + hücrelerde GFP ifade Sca1-GFP fareler kullanılarak yoluyla değerlendirilecektir.

<table><tbody><tr><td>Type 3 Collagenase</td><td>Worthington Biochemical</td><td>LS004182</td><td>Tissue digestion</td></tr><tr><td>DMEM</td><td>Gibco</td><td>11965-092</td><td>High-glucose culture medium</td></tr><tr><td>Pen/Strep/Glutamine (100x)</td><td>Gibco</td><td>10378-016</td><td>Media antibiotic</td></tr><tr><td>Anti-anti (100x)</td><td>Gibco</td><td>15240-062</td><td>Media antifungal</td></tr><tr><td>FBS</td><td>Gibco</td><td>16000-044</td><td></td></tr><tr><td>PBS (1x, pH 7.4)</td><td>Gibco</td><td>10010-023</td><td></td></tr><tr><td>Trypsin (0.05%)</td><td>Gibco</td><td>25300-054</td><td></td></tr><tr><td>Cell strainer</td><td>BD Bioscience</td><td>352360</td><td>100-&amp;mu;m cell strainer</td></tr><tr><td>60 mm plates</td><td>BD Falcon</td><td>353004</td><td></td></tr><tr><td>Scissors</td><td>FST</td><td>14001-12</td><td>Large</td></tr><tr><td>Scissors</td><td>FST</td><td>14091-11</td><td>Fine, curved tip</td></tr><tr><td>Large Forceps</td><td>FST</td><td>11000-12</td><td></td></tr><tr><td>Fine Forceps</td><td>Any vendor</td><td></td><td></td></tr><tr><td>25G 5/8&amp;rdquo; needles</td><td>BD</td><td>305122</td><td></td></tr><tr><td>22G 1.5&amp;rdquo; needles</td><td>BD</td><td>305159</td><td></td></tr><tr><td>15 ml conical tubes</td><td>BD Falcon</td><td>352097</td><td></td></tr><tr><td>50 ml conical tubes</td><td>BD Falcon</td><td>352098</td><td></td></tr><tr><td>MACS separation columns</td><td>Miltenyi Biotec</td><td>130-042-201</td><td></td></tr><tr><td>Anti-Sca1 microbead kit (FITC)</td><td>Miltenyi Biotec</td><td>130-092-529</td><td>FITC-anti-Sca1 1&amp;ordm;Ab and anti-FITC microbeads 2&amp;ordm;Ab</td></tr><tr><td>AutoMACS running buffer</td><td>Miltenyi Biotec</td><td>130-091-221</td><td></td></tr><tr><td>MiniMACS separator</td><td>Miltenyi Biotec</td><td>130-042-102</td><td></td></tr><tr><td>MACS MultiStand</td><td>Miltenyi Biotec</td><td>130-042-303</td><td></td></tr><tr><td>Blue chux pads</td><td>Fisher</td><td>276-12424</td><td></td></tr><tr><td>Absorbent pads</td><td>Fisher</td><td>19-165-621</td><td></td></tr><tr><td>Styrofoam board</td><td></td><td></td><td>Use from 50 ml tubes</td></tr><tr><td>70% ethanol</td><td></td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Any vendor</td><td></td><td></td></tr><tr><td>Rat IgG2a Alexa Fluor 647</td><td>Invitrogen</td><td>R2a21</td><td></td></tr><tr><td>Rat IgG2a anti-mouse Sca1 Alexa Fluor 647</td><td>Invitrogen</td><td>MSCA21</td><td></td></tr><tr><td>Rat IgG2a R-PE</td><td>Invitrogen</td><td>R2a04</td><td></td></tr><tr><td>Rat IgG2a anti-mouse F4/80 R-PE</td><td>Invitrogen</td><td>MF48004</td><td></td></tr><tr><td>Round-bottom tube</td><td>BD Falcon</td><td>352058</td><td></td></tr><tr><td>HBSS (&amp;ndash;Ca, &amp;ndash;Mg)</td><td>Gibco</td><td>14175-095</td><td></td></tr><tr><td>HBSS (+Ca, +Mg)</td><td>Gibco</td><td>14025-092</td><td>For collagenase solution</td></tr><tr><td>Type I collagen (2.7 mg/ml in 37mm acetic acid</td><td>Prepare in house&lt;sup&gt;12&lt;/sup&gt;</td><td></td><td></td></tr><tr><td>10x MEM</td><td>Gibco</td><td>11430-030</td><td></td></tr><tr><td>1 M HEPES</td><td>Gibco</td><td>15630-080</td><td></td></tr><tr><td>0.34 N NaOH</td><td>Prepare in house</td><td></td><td></td></tr><tr><td>Cover slips</td><td>Corning</td><td>2870-22</td><td></td></tr><tr><td>Alexa Fluor 594 carboxylic acid, succinimidyl ester, mixed isomers</td><td>Invitrogen</td><td>A-20004</td><td></td></tr><tr><td>0.89 M NaHCO&lt;sub&gt;3&amp;nbsp;&lt;/sub&gt;</td><td>Gibco</td><td>25080-094</td><td></td></tr></tbody></table>

immunomagnetic separation of fat depot-specific sca1high adipose-derived stem cells (ascs)

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix (ECM) remodeling. In vivo, ECM-rich environment consisting of fibrillar collagens provides a structural support to adipose tissues during the progression and regression of obesity. Physiological ECM remodeling mediated by matrix metalloproteinases (MMPs) plays a major role in regulating adipose tissue size and function1,2. The loss of physiological collagenolytic ECM remodeling may lead to excessive collagen accumulation (tissue fibrosis), macrophage infiltration, and ultimately, a loss of metabolic homeostasis including insulin resistance3,4. When a phenotypic change of the adipose tissue is observed in gene-targeted mouse models, isolating primary ASCs from fat depots for in vitro studies is an effective approach to define the role of the specific gene in regulating the function of ASCs. In the following, we define an immunomagnetic separation of Sca1high ASCs.

Farklı Yağ Pads gelen Sca1 yüksek TSK zenginleştirilmesi. SQ yağ görüntüleme fibroblast benzeri izole vasküler stromal hücreler SCA1 sentezleme seviyesi (Şekil 1A) bağımsız hücre şekli uzanıyordu. VIS Öte yandan, (eWAT türevi), yüksek Sca1 ve Sca1 düşük hücreleri, hücre şekli belirgin farkı göstermektedir. VIS SQ (IWAT türevi) Sca1 yüksek</sup...

Watch this Scientific Journal Video about Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs at JoVE.com

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs

We present the techniques required to isolate the stromal vascular fraction (SVF) from mouse inguinal (subcutaneous) and perigonadal (visceral) adipose tissue depots to assess their gene expression and collagenolytic activity. This method includes the enrichment of Sca1high adipose-derived stem cells (ASCs) using immunomagnetic cell separation.

Yağ Deposu özgü SCA1 ve İmmunomanyetik Ayırma<sup&gt; yüksek</sup&gt; Yağ türetilmiş Kök Hücreler (TSK)

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix ...

immunomagnetic-separation-fat-depot-specific-sca1high-adipose-derived

Research

JoVE Journal

Developmental Biology

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells (ASCs)

6.8K Görüntüleme.  Biointerfaces Institute, University of Michigan. We present the techniques required to isolate the stromal vascular fraction (SVF) from mouse inguinal (subcutaneous) and perigonadal (visceral) adipose tissue depots to assess their gene expression and collagenolytic activity. This method includes the enrichment of Sca1high adipose-derived stem cells (ASCs) using immunomagnetic cell separation.

Video: Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

İzolasyon ve İnsan zenginleştirilmesi Gelişmiş Osteogenezi için Stromal Hücreleri adipoz-türevi

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

Gelişim Biyolojisi

Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of cardiovascular disease. ADSCs derived from different adipose tissues show distinct features, and those from the PVAT have not been well characterized. In a recent study, we reported that some ADSCs in the periaortic arch adipose tissue (PAAT) descend from the neural crest cells (NCCs), a transient population of migratory cells originating from the ectoderm.
In this paper, we describe a protocol for isolating red fluorescent protein (RFP)-labeled NCCs from the PAAT of Wnt-1 Cre+/-;Rosa26RFP/+ mice and inducing their adipogenic differentiation in vitro. Briefly, the stromal vascular fraction (SVF) is enzymatically dissociated from the PAAT, and the RFP+ neural crest derived ADSCs (NCADSCs) are isolated by fluorescence activated cell sorting (FACS). The NCADSCs differentiate into both brown and white adipocytes, can be cryopreserved, and retain their adipogenic potential for ~3&#8211;5 passages. Our protocol can generate abundant ADSCs from the PVAT for modeling PVAT adipogenesis or lipogenesis in vitro. Thus, these NCADSCs can provide a valuable system for studying the molecular switches involved in PVAT differentiation.

We present a protocol for the isolation, culture, and adipogenic induction of neural crest derived adipose-derived stem cells (NCADSCs) from the periaortic adipose tissue of Wnt-1 Cre+/-;Rosa26RFP/+ mice. The NCADSCs can be an easily accessible source of ADSCs for modeling adipogenesis or lipogenesis in vitro.

Periaortik Yağ Dokusundan Nöral Adipoz-Türetilmiş Kök Hücrelerin İzolasyon, Kültür ve Adipojenik İndüksiyonu

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of ...

Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute functionally to the expansion and remodeling of WAT in adulthood. A tremendous barrier to studying the implications of this cellular heterogeneity is the inability to readily isolate functionally distinct cell subpopulations from WAT SVF for in vitro and in vivo analyses. Single-cell sequencing technology has recently identified functionally distinct fibro-inflammatory and adipogenic PDGFR&#946;+ perivascular cell subpopulations in intra-abdominal WAT depots of adult mice. Fibro-inflammatory progenitors (termed, &#8220;FIPs&#8221;) are non-adipogenic collagen producing cells that can exert a pro-inflammatory phenotype. PDGFR&#946;+ adipocyte precursor cells (APCs) are highly adipogenic both in vitro and in vivo upon cell transplantation. Here, we describe multiple methods for the isolation of these stromal cell subpopulations from murine intra-abdominal WAT depots. FIPs and APCs can be isolated by fluorescence-activated cell sorting (FACS) or by taking advantage of biotinylated antibody-based immunomagnetic bead technology. Isolated cells can be used for molecular and functional analysis. Studying the functional properties of stromal cell subpopulation in isolation will expand our current knowledge of adipose tissue remodeling under physiological or pathological conditions on the cellular level.

This protocol describes the technical approach to isolate adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal white adipose tissue (WAT) depots by fluorescence-activated cell sorting or immunomagnetic bead separation.

Murin İntraabdominal Yağ Depolarından Adipojenik ve Fibroinflamatuar Stromal Hücre Alt Popülasyonlarının İzolasyonu

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute ...

İmmünoloji ve Enfeksiyon

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

İnsan Lipoaspirates den Yağ-türetilen Kök Hücre Manuel İzolasyon

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

Biyoloji

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. ...

Tıp

Technique for Obtaining Mesenchymal Stem Cell from Adipose Tissue and Stromal Vascular Fraction Characterization in Long-Term Cryopreservation

Human mesenchymal stem cells derived from adipose tissue have become increasingly attractive as they show appropriate features and are an accessible source for regenerative clinical applications. Different protocols have been used to obtain adipose-derived stem cells. This article describes different steps of an improved time-saving protocol to obtain a more significant amount of ADSC, showing how to cryopreserve and thaw ADSC to obtain viable cells for culture expansion. One hundred milliliters of lipoaspirate were collected, using a 26 cm three-hole and 3 mm caliber syringe liposuction, from the abdominal area of nine patients who subsequently underwent elective abdominoplasty. The stem cells isolation was carried out with a series of washes with Dulbecco&#39;s Phosphate Buffered Saline (DPBS) solution supplemented with calcium and the use of collagenase. Stromal Vascular Fraction (SVF) cells were cryopreserved, and their viability was checked by immunophenotyping. The SVF cellular yield was 15.7 x 105 cells/mL, ranging between 6.1-26.2 cells/mL. Adherent SVF cells reached confluence after an average of 7.5 (&#177;4.5) days, with an average cellular yield of 12.3 (&#177; 5.7) x 105 cells/mL. The viability of thawed SVF after 8 months, 1 year, and 2 years ranged between 23.06%-72.34% with an average of 47.7% (&#177;24.64) with the lowest viability correlating with cases of two-year freezing. The use of DPBS solution supplemented with calcium and bag resting times for fat precipitation with a shorter time of collagenase digestion resulted in an increased stem cell final cellular yield. The detailed procedure for obtaining high yields of viable stem cells was more efficient regarding time and cellular yield than the techniques from previous studies. Even after a long period of cryopreservation, viable ADSC cells were found in the SVF.

The present protocol describes an improved methodology for ADSC isolation resulting in a tremendous cellular yield with time gain compared to the literature. This study also provides a straightforward method for obtaining a relatively large number of viable cells after long-term cryopreservation.

Uzun Süreli Kriyoprezervasyonda Yağ Dokusundan Mezenkimal Kök Hücre Elde Etme Tekniği ve Stromal Vasküler Fraksiyon Karakterizasyonu

Human mesenchymal stem cells derived from adipose tissue have become increasingly attractive as they show appropriate features and are an accessible ...

Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident adipose progenitor cells (APCs) play a key role in adipose tissue homeostasis and can contribute to the tissue pathology. The growing use of single cell analysis technologies &#8211; including single-cell RNA-sequencing and single-cell proteomics &#8211; is transforming the stem/progenitor cell field by permitting unprecedented resolution of individual cell expression changes within the context of population- or tissue-wide changes. In this article, we provide detailed protocols to dissect mouse epididymal adipose tissue, isolate single adipose tissue-derived cells, and perform fluorescence activated cell sorting (FACS) to enrich for viable Sca1+/CD31-/CD45-/Ter119- APCs. These protocols will allow investigators to prepare high quality APCs suitable for downstream analyses such as single cell RNA sequencing.

We present a simple method to isolate highly viable adipose progenitor cells from mouse epididymal fat pads using fluorescence activated cell sorting.

Fare Epidididimal Yağ Dokularından Yağ Progenitör Hücrelerinin Hazırlanması

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident ...

Yağ Dokusu Kaynaklı Kök Hücre İzolasyonu ve Karakterizasyonu

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in addition to their great capacity for self-renewal, migration, and proliferation. Adipose tissue is one of the least invasive and most accessible sources of mesenchymal cells. It has also been reported to have higher yields compared to other sources, as well as superior immunomodulatory properties. Recently, different procedures for obtaining adult mesenchymal cells from different tissue sources and animal species have been published. After evaluating the criteria of some authors, we standardized a methodology that is applicable to different purposes and easily reproducible. A pool of stromal vascular fraction (SVF) from perirenal and epididymal adipose tissue allowed us to develop primary cultures with optimal morphology and functionality. The cells were observed adhered to the plastic surface for 24 h, and exhibited a fibroblast-like morphology, with prolongations and a tendency to form colonies. Flow cytometry (FC) and immunofluorescence (IF) techniques were used to assess the expression of the membrane markers CD105, CD9, CD63, CD31, and CD34. The ability of adipose-derived stem cells (ASCs) to differentiate into the adipogenic lineage was also assessed using a cocktail of factors (4 &#181;M insulin, 0.5 mM 3-methyl-iso-butyl-xanthine, and 1 &#181;M dexamethasone). After 48 h, a gradual loss of fibroblastoid morphology was observed, and at 12 days, the presence of lipid droplets positive to oil red staining was confirmed. In summary, a procedure is proposed to obtain optimal and functional ASC cultures for application in regenerative medicine.

Yazar Spotlight: Sprague Dawley Sıçanlarının Yağ Dokusundan Türetilen Mezenkimal Kök Hücrelerin İzolasyonu ve Tanımlanması  

This protocol describes a methodology for isolating and identifying adipose tissue-derived mesenchymal stem cells (MSCs) from Sprague Dawley rats.

Sprague Dawley Sıçanlarının Yağ Dokusundan Elde Edilen Mezenkimal Kök Hücrelerin İzolasyonu ve Tanımlanması

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in ...

Yağ Deposu özgü SCA1 ve İmmunomanyetik Ayırma

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

İzolasyon ve İnsan zenginleştirilmesi Gelişmiş Osteogenezi için Stromal Hücreleri adipoz-türevi

Periaortik Yağ Dokusundan Nöral Adipoz-Türetilmiş Kök Hücrelerin İzolasyon, Kültür ve Adipojenik İndüksiyonu

Murin İntraabdominal Yağ Depolarından Adipojenik ve Fibroinflamatuar Stromal Hücre Alt Popülasyonlarının İzolasyonu

İnsan Lipoaspirates den Yağ-türetilen Kök Hücre Manuel İzolasyon

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

Uzun Süreli Kriyoprezervasyonda Yağ Dokusundan Mezenkimal Kök Hücre Elde Etme Tekniği ve Stromal Vasküler Fraksiyon Karakterizasyonu

Fare Epidididimal Yağ Dokularından Yağ Progenitör Hücrelerinin Hazırlanması

Sprague Dawley Sıçanlarının Yağ Dokusundan Elde Edilen Mezenkimal Kök Hücrelerin İzolasyonu ve Tanımlanması

Sprague Dawley Sıçanlarının Yağ Dokusundan Elde Edilen Mezenkimal Kök Hücrelerin İzolasyonu ve Tanımlanması

Sprague Dawley Sıçanlarının Yağ Dokusundan Elde Edilen Mezenkimal Kök Hücrelerin İzolasyonu ve Tanımlanması