mouse epididymal adipose tissue extraction and stem cell isolation for flow cytometric and functional characterization

Fare Epididiminden Elde Edilen Yağ Dokusu Kaynaklı Kök Hücrelerin Primer Kültürü

Mesenchymal stem cells (MSCs) are adult multipotential cells differentiating into cells such as osteoblast, chondroblast, and adipocyte1,2. These cells reside in several organs, and because of that, they can be extracted from adult tissues such as bone marrow, muscle, fat, hair follicle, tooth root, placenta, dermis, perichondrium, umbilical cord, lung, liver, and spleen3,4.
The effects of MSCs on physiology and the immune system have been reported5,6. These cells have been promising for treating several diseases, both in human and veterinary medicine. The MSCs can control inflammation and promote angiogenesis and tissue homeostasis through different mechanisms, such as cell-cell contact, soluble factors, and small extracellular vesicles7,8,9,10. Furthermore, the MSCs are immune-privileged cells capable of surviving in immunologically incompatible allogeneic transplant recipients because these cells show low expression of class I major histocompatibility complex (MHC) molecules and are used in cell-based therapy for allogeneic transplant11,12. The low immunogenicity combined with the regenerative potential makes MSCs ideal candidates for cell therapy, such as graft-versus-host disease (GvHD)13, systemic lupus erythematosus (SLE)14, and multiple sclerosis15, among others16,17.
Despite the fact that MSCs reside in several adult tissues, the adipose tissue offers advantages over other sources, such as accessibility for harvesting, with minimal surgical intervention; large number of available cells with high expansion rate; and easy in vitro expansion using an easy-to-perform protocol without the need for specific equipment and low-cost materials18,19,20. Once extracted, the adipose tissue-derived stem cells (ADSCs) must be characterized as established by the International Society for Cellular Therapy (ISCT)21. Thus, MSCs must show morphology fibroblast-like, adherence to plastic culture, expressing a high percentage (&#8805;95%) of mesenchymal markers such as endoglin (CD105), ecto-5&#39;-nucleotidase (CD73) and Thy-1 (CD90), and low percentage (&#8804;2%) of hematopoietic markers such as leukocyte common antigen (CD45), transmembrane phosphoglycoprotein (CD34), glycolipid-anchored membrane glycoprotein (CD14), integrin alpha M (CD11b), B-cell antigen receptor complex-associated protein alpha chain (CD79&#945;) or B-lymphocyte surface antigen B4 (CD19) and class II human leukocyte antigen (HLA-II). Furthermore, a functional characterization is required, and the cells should be able to differentiate into osteoblast, chondroblast, or adipoblast cells21.
Here, we show how to obtain the MSCs from epididymal adipose tissue using mechanical dissociation and enzymatic digestion for in vitro studies and the morphological characterization preconized by ISCT.

Yazar Gündemi: Yağ Kaynaklı Kök Hücrelerle Tedavi Yaklaşımlarının Geliştirilmesi

Fare Epididimal Yağ Dokusundan Mezenkimal Kök Hücrelerin İzolasyonu, İn Vitro Genişletme ve Karakterizasyonu

My research focus on adipose-derived mesenchymal stem cells as a new approach treatment associated or not associated with other therapies to improve the inflammation from several disease. We demonstrated that using adipose mesenchymal stem cells in conjunction with Praziquantel has significantly controlled the granulomatous reaction in Schistosoma mansoni infection, highlighting the benefits of this combined treatment approach. Our protocol address the research gap in the meticulous collection of epididymal adipose tissue under aseptic condictions, that the physical processing involving vigorous shaking, and identifies the optimal time for utilizing adipose-derived stem cells.Our protocol offers significant advantages, notably the abundant availability and the potential strength for isolation of mesenchymal stem cells. Additionally, mesenchymal stem cells poses potent therapeutic properties including anti-inflammatory and tissue regeneration capabilities.

Akış Sitometrik ve Fonksiyonel Karakterizasyonu için Fare Epididimal Yağ Dokusu Ekstraksiyonu ve Kök Hücre İzolasyonu

Başlamak için, ötenazi yapılmış bir fareyi ventral tarafı yukarı bakacak şekilde bir mantar panoya yerleştirin. Cımbız kullanarak, traş edilmiş cildi karnın ortasına kaldırın ve bir çift steril makasla bir kesi yapın. Cildi peritondan ayırmak için kesin ve açın.Sonra periton tabakasını kaldırın ve kesin. Yeni bir steril cımbız ve makas seti ile testislerin üzerinde bulunan tüm epididimal yağ dokusunu nazikçe kavrayın, kaldırın ve kesin. Eksize edilen yağı, buz üzerinde bazal ortam içeren 50 mililitrelik steril konik bir tüpe yerleştirin.Daha sonra, epididimal dokuyu PBS ile yıkayın. Steril cımbızla, epididimal yağ dokusunu 15 ila 20 mililitre sindirim çözeltisi içeren bir tüpe aktarın. Dokuyu steril makasla parçalara ayırın.Daha sonra doku parçalarını bir saat boyunca 37 santigrat derecede inkübe edin. Kollajenazı etkisiz hale getirmek için numuneyi eşit hacimde bazal ortamda seyreltin. Süspansiyonu 250 G'de dört santigrat derecede 10 dakika santrifüjleyin.Süpernatanı attıktan sonra, peleti bir mililitre bazal ortamda yeniden askıya alın. Daha sonra, izole edilmiş hücrelere sahip üç mililitre bazal ortam içeren altı oyuklu bir plakanın kuyusuna bakın. Hücreleri gece boyunca 37 santigrat derecede% 5 karbondioksit ile inkübe edin.Ertesi gün, PBS ile yıkanmış hücrelere üç mililitre bazal ortam ekleyin. Yağ dokusundan ekstrakte edilen hücreler mezenkimal kök hücre morfolojisi eşleşmeleri gösterdi.

mouse-epididymal-adipose-tissue-extraction-stem-cell-isolation-for

Research

JoVE Journal

Biology

Mouse Epididymal Adipose Tissue Extraction and Stem Cell Isolation for Flow Cytometric and Functional Characterization

112 Görüntüleme.  State University of Santa Cruz - UESC. Başlamak için, ötenazi yapılmış bir fareyi ventral tarafı yukarı bakacak şekilde bir mantar panoya yerleştirin. Cımbız kullanarak, traş edilmiş cildi karnın ortasına kaldırın ve bir çift steril makasla bir kesi yapın. Cildi peritondan ayırmak için kesin ve açın.Sonra periton tabakasını kaldırın ve kesin. Yeni bir steril cımbız ve makas seti ile testislerin üzerinde bulunan tüm epididimal yağ dokusunu nazikçe kavrayın, kaldırın ve kesin. Eksize edile...

Video: Akış Sitometrik ve Fonksiyonel Karakterizasyonu için Fare Epididimal Yağ Dokusu Ekstraksiyonu ve Kök Hücre İzolasyonu

Isolation, In Vitro Expansion, and Characterization of Mesenchymal Stem Cells from Mouse Epididymal Adipose Tissue

Mesenchymal stem cells (MSCs) have been extensively studied as a new therapeutic approach, mainly to stop exacerbated inflammation due to their potential to modulate the immune response. The MSCs are immune-privileged cells capable of surviving in immunologically incompatible allogeneic transplant recipients based on low expression of class I major histocompatibility complex (MHC) molecules and in the use of cell-based therapy for allogeneic transplant. These cells can be isolated from several tissues, the most commonly used being the bone marrow and adipose tissues. We provide an easy protocol to isolate, culture, and characterize MSCs from epididymal adipose tissue of mice. The epididymal adipose tissue is surgically excised, physically fragmented, and digested with 0.15% collagenase type II solution. Then, primary adipose tissue-derived stem (ADSCs) cells are cultured and expanded in vitro, and the phenotypic characterization is performed by flow cytometry. We also provide the steps to differentiate the ADSCs into osteogenic, adipogenic, and chondrogenic cells, followed by functional characterization of each cell lineage. The protocol provided here can be used for in vivo and ex vivo experiments, and as an alternative, the adipose-derived stem cells can be used to generate MSCs-like immortalized cells.

The adipose tissue is an excellent source of mesenchymal stem cells. Here, we bring the step-by-step extraction, cultivation, and characterization of adipose tissue-derived stem cells (ADSCs) from Swiss mice epididymal adipose tissue.

Mesenchymal stem cells (MSCs) have been extensively studied as a new therapeutic approach, mainly to stop exacerbated inflammation due to their potential ...