dissection and isolation of murine perivascular adipose tissue (pvat)

Murin Perivasküler Yağ Dokusundan İzole Edilen Preadipositlerin Kültürlenmesi

Perivascular adipose tissue (PVAT), a perivascular structure composed of a mixture of mature adipocytes and a stromal vascular fraction (SVF), is believed to interact with the adjacent vessel wall via its secretome paracrineally1. As a critical regulator of vascular homeostasis, PVAT dysfunction is implicated in the pathogenesis of cardiovascular diseases2,3,4. The SVF of adipocyte tissue consists of several expected cell populations, including endothelial cells, immune cells, mesothelium cells, neuronal cells, and adipose stem and progenitor cells (ASPCs)5,6. It is well known that ASPCs residing in the SVF of adipose tissue can give rise to mature adipocytes5. SVF is inferred to be a critical source of mature adipocytes in PVAT. Several studies have shown that PVAT-SVF can differentiate into mature adipocytes under specific induction conditions6,7,8.
Currently, there are two isolation systems for isolating SVF from adipose tissue, one is enzymatic digestion and the other is non-enzymatic9. Enzymatic methods typically result in a higher yield of nucleated progenitor cells10. To date, the benefits of SVF in promoting vascular regeneration and neovascularization in wound healing, urogenital, and cardiovascular diseases have been widely demonstrated11, especially in dermatology and plastic surgery12,13. However, the clinical application prospects of PVAT-derived SVF have not been well explored, which may be attributed to the lack of a standardized method for the isolation of SVF from PVAT. The objective of this protocol is to establish a standardized approach for the isolation, culture, and adipogenic induction of SVF-derived preadipocytes from mouse PVAT surrounding the thoracic aorta, enabling further investigation of PVAT function. This protocol optimizes tissue processing and cell differentiation techniques for culturing periaortic adipocytes obtained from young mice.

Yazar Spotlight: Fare Perivasküler Yağ Dokusundan SVF'den Türetilmiş Preadipositlerin İzolasyonu, Kültürü ve Adipojenik İndüksiyonunun Önemi 

Fare Periaortik Yağ Dokusundan Stromal Vasküler Fraksiyon Kaynaklı Preadipositlerin İzolasyonu, Kültürü ve Adipojenik İndüksiyonu

We aim to establish a standardized approach for the isolation culture and adipogenic induction of stromal vascular fraction-derived preadipocytes from mass PVAT. This protocol enables us to conduct further investigations into PVAT function and into the communication between periaortic adipocytes and vascular cells in vitro. The main challenges we currently face in our experiments include the restricted of availability of PVAT and the requirements for additional human and the material resources when compared to using immortalized preadipocytes.Our protocol allows for the study of perivascular adipose tissue function and it's relationship with vascular cells. We also offers a basis for studying PVAT ring and genes, and heterogeneity, and provides a platform for exploring the important regulator of PVAT differentiation and the adipogenesis in vitro.

Watch this Scientific Journal Video about Dissection and Isolation of Murine Perivascular Adipose Tissue PVAT at JoVE.com

Murin Perivasküler Yağ Dokusunun Diseksiyonu ve İzolasyonu (PVAT)

Cildi dezenfekte etmek için uygun şekilde ötenazi yapılmış fareye %75 alkol püskürterek başlayın. Ardından fareyi sırtüstü pozisyona getirin. Hayvanın karın bölgesinin derisini kaldırın ve cerrahi makas kullanarak küçük bir kesi yapın.Daha sonra makası kullanarak, ventral orta hat boyunca cildi ve karın kaslarını pelvisten boyuna kadar kör bir şekilde ayırın. Orta hattın her iki tarafındaki diyaframı ve kaburgaları keserek kalbi ve akciğerleri ortaya çıkarın. Ardından, bağırsak duvarına zarar vermekten kaçınarak karaciğeri, dalakları, bağırsakları ve böbreği dikkatlice çıkarın.Bunu takiben, akciğerleri ve yemek borusunu çıkarın. Şimdi bir elinizde tutulan forseps kullanarak kalbi nazikçe kaldırın ve diğer elinizde tutulan cerrahi makasla aortu omurgadan ayırın. Daha sonra kalbi ve aortu antibiyotik takviyeli PBS içeren 60 milimetrelik bir petri kabına yerleştirin.Mikrocerrahi forsepsleri ve mikrocerrahi makası alkolden alın ve fazla alkolü gidermek için 25 mililitre PBS'de durulayın. Stereo mikroskop altında çalışarak, timus ve diğer dokuları kalpten ve aorttan çıkarın. Daha sonra kalbi çıkarın, aortu perivasküler yağ dokuları veya PVAT'lerle bırakın.Aortu PVAT'larla birlikte antibiyotik takviyeli PBS içeren 60 milimetrelik temiz bir petri kabına aktarın. Bir çift mikrocerrahi forseps kullanarak aortu tutun ve başka bir çift kullanarak, PVAT'ları aorttan ayırmak için bağlı yağ dokularını çekin. PVAT'lara minimum zarar verirken mümkün olduğunca fazla vasküler doku çıkarın.Son olarak, toplanan PVAT'ları, buz üzerine yerleştirilmiş antibiyotik takviyeli, yüksek glikozlu, DMEM içeren iki mililitrelik bir mikrosantrifüj tüpüne aktarın.

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Research

JoVE Journal

Biology

Dissection and Isolation of Murine Perivascular Adipose Tissue (PVAT)

405 Görüntüleme.  Shanghai Jiao Tong University School of Medicine. Cildi dezenfekte etmek için uygun şekilde ötenazi yapılmış fareye %75 alkol püskürterek başlayın. Ardından fareyi sırtüstü pozisyona getirin. Hayvanın karın bölgesinin derisini kaldırın ve cerrahi makas kullanarak küçük bir kesi yapın.Daha sonra makası kullanarak, ventral orta hat boyunca cildi ve karın kaslarını pelvisten boyuna kadar kör bir şekilde ayırın. Orta hattın her iki tarafındaki diyaframı ve kaburgaları keserek kalbi ve akciğerleri ortaya...

Video: Murin Perivasküler Yağ Dokusunun Diseksiyonu ve İzolasyonu (PVAT)

Isolation, Culture, and Adipogenic Induction of Stromal Vascular Fraction-derived Preadipocytes from Mouse Periaortic Adipose Tissue

Perivascular adipose tissue (PVAT) is an adipose tissue depot that surrounds blood vessels and exhibits the phenotypes of white, beige, and brown adipocytes. Recent discoveries have shed light on the central role of PVAT in regulating vascular homeostasis and participating in the pathogenesis of cardiovascular diseases. A comprehensive understanding of PVAT properties and regulation is of great importance for the development of future therapies. Primary cultures of periaortic adipocytes are valuable for studying PVAT function and the crosstalk between periaortic adipocytes and vascular cells. This paper presents an economical and feasible protocol for the isolation, culture, and adipogenic induction of stromal vascular fraction-derived preadipocytes from mouse periaortic adipose tissue, which can be useful for modeling adipogenesis or lipogenesis in vitro. The protocol outlines tissue processing and cell differentiation for culturing periaortic adipocytes from young mice. This protocol will provide the technological cornerstone at the bench side for the investigation of PVAT function.

Here, we describe the isolation, culture, and adipogenic induction of stromal vascular fraction-derived preadipocytes from mouse periaortic adipose tissue, allowing for the study of perivascular adipose tissue function and its relationship with vascular cells.

Perivascular adipose tissue (PVAT) is an adipose tissue depot that surrounds blood vessels and exhibits the phenotypes of white, beige, and brown ...

Biyoloji

Begin by spraying the properly euthanized mouse with 75%alcohol to disinfect the skin. Then place the mouse in a supine position. Lift the skin of the animal's abdominal region, and using surgical scissors, make a small incision in it.Then using the scissors, bluntly separate the skin and abdominal muscles from the pelvis to the neck, along the ventral midline. Expose the heart and lungs by cutting the diaphragm and ribs along both sides of the midline. Next, carefully remove the liver, spleen, bowels, and kidney, avoiding damage to the intestinal wall.Following that, remove the lungs and esophagus. Now gently lift the heart using forceps held in one hand and separate the aorta from the spine with surgical scissors held in the other hand. Then place the heart and aorta in a 60 millimeter petri dish, containing antibiotic supplemented PBS.Retrieve the microsurgical forceps and microsurgical scissors from the alcohol, and rinse them in 25 milliliters of PBS to remove excess alcohol. Working under a stereo microscope, remove the thymus and other tissues from the heart and aorta. Then remove the heart, leaving the aorta with the perivascular adipose tissues or PVATs.Transfer the aorta along with the PVATs to a clean 60 millimeter petri dish containing antibiotic supplemented PBS. Using one pair of microsurgical forceps, hold the aorta, and using another pair, pull off the attached adipose tissues to separate the PVATs from the aorta. Remove as much vascular tissue as possible while causing minimal harm to the PVATs.Finally, transfer the collected PVATs into a two milliliter microcentrifuge tube, containing antibiotic supplemented, high glucose, DMEM, placed on ice.

Isolation of Stromal Vascular Fraction (SVF) from Perivascular Adipose Tissue (PVAT)

While working under a class two laminar flow hood, begin by sequentially washing the perivascular adipose tissues or PVATs collected from mice two times. First, using PBS containing 10%penicillin-streptomycin, and, next, using PBS supplemented with 1%penicillin-streptomycin. Transfer the rinsed tissues into a sterile 60-millimeter Petri dish and add 200 microliters of digestion solution to it.Using sterile scissors, mince the tissues into pieces having a dimension of one cubic millimeter. With the help of sterile scissors, cut a plastic pipette tip to broaden its end. Then transfer the minced tissue mix to a 15-milliliter centrifuge tube using the broad-ended pipette tip.Add six milliliters of the digestion solution to the tissues to initiate digestion. After tying the tube horizontally on a rack, incubate it at 37 degrees Celsius in an orbital shaker for 30 to 45 minutes. Every 5 to 10 minutes, remove the tube from the shaker in the incubator and manually shake it up and down vigorously.Pass the digested tissue mix through a 70-micron cell strainer into a 50-milliliter centrifuge tube. Rinse the strainer with an equal volume of culture medium to maximize cell yield and quench digestion. Transfer the filtrate to a new 15-milliliter centrifuge tube.Centrifuge the tube to isolate the stromal vascular fraction or SVF. Invert the tube to discard the supernatant and re-suspend the pellet in five milliliters of PBS. Centrifuge the tube again at 1, 800 G for five minutes before discarding the supernatant.Re-suspend the pellet in an appropriate volume of culture medium. Seed the cells into a collagen-coated 12-well plate and incubate overnight at 37 degrees Celsius in a humid atmosphere with 5%carbon dioxide. On the next day, remove cell debris and red blood cells by aspirating the culture medium and washing the cells with antibiotic-supplemented PBS pre-warmed to 37 degrees Celsius.Finally, add one milliliter of fresh culture medium to each well, and continue culturing the cells until they reach the desired stage of confluency.