Name: Murine Adipose-Derived Mesenchymal Cell Isolation, Maintenance and Characterization
Uploaded: 2023-04-07T14:30:00
Description: Watch this Scientific Journal Video about Murine Adipose-Derived Mesenchymal Cell Isolation, Maintenance and Characterization at JoVE.com

The video details a protocol for isolating and culturing mouse adipose tissue cells. It covers steps from tissue removal and washing to collagenase digestion, cell centrifugation, and staining for viability assessment. The process emphasizes maintaining sterile conditions, proper temperature, and monitoring cell growth for successful proliferation and colony formation.

murine adipose-derived mesenchymal cell isolation, maintenance and characterization

Yağ Dokusu Kaynaklı Kök Hücre İzolasyonu ve Karakterizasyonu

Mesenchymal stem cells (MSCs) have strongly impacted regenerative medicine due to their high capacity for self-renewal, proliferation, migration, and differentiation into different cell lineages1,2. Currently, a great deal of research is focusing on their potential for the treatment and diagnosis of various diseases.
There are different sources of mesenchymal cells: bone marrow, skeletal muscle, amniotic fluid, hair follicles, placenta, and adipose tissue, among others. They are obtained from different species, including humans, mice, rats, dogs, and horses3. Bone marrow-derived MSCs (BMSCs) have been used for many years as a major source of stem cells in regenerative medicine and as an alternative to the use of embryonic stem cells4. However, adipose-derived MSCs, or adipose-derived stem cells (ASCs), are an important alternative with great advantages due to their ease of collection and isolation, as well as the yield of cells obtained per gram of adipose tissue5,6. It has been reported that the harvest rate of ASCs is generally higher than that of BMSCs7. It was initially proposed that the reparative/regenerative capacity of ASCs was due to their ability to differentiate into other cell lineages8. However, research in recent years has reinforced the primary role of paracrine factors released by ASCs in their reparative potential9,10.
Adipose tissue (AT), in addition to being an energy reserve, interacts with the endocrine, nervous, and cardiovascular systems. It is also involved in postnatal growth and development, the maintenance of tissue homeostasis, tissue repair, and regeneration. The AT is composed of adipocytes, vascular smooth muscle cells, endothelial cells, fibroblasts, monocytes, macrophages, lymphocytes, preadipocytes, and ASCs. The latter possess an important role in regenerative medicine due to their low immunogenicity11,12. ASCs can be obtained by enzymatic digestion and mechanical processing or by adipose tissue explants. Primary cultures of ASCs are easy to maintain, grow, and expand. Phenotypic characterization of ASCs is essential to verify the identity of the cells by assessing the expression of specific membrane markers using methods such as immunofluorescence and flow cytometry13. The International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy (ISCT) have defined that ASCs express CD73, CD90, and CD105, while lacking the expression of CD11b, CD14, CD19, CD45, and HLA-DR14. These markers, both positive and negative, are therefore considered reliable for the characterization of ASCs.
This project was focused on describing a procedure for the isolation and identification of adult mesenchymal cells extracted from rats' AT, as this source of cells does not present ethical challenges, unlike embryonic stem cells. This solidifies the procedure as a viable option because of the ease of access and minimally invasive method compared to bone marrow-derived stem cells. 
Mesenchymal cells from this tissue source have an important role in regenerative medicine because of their immunomodulatory capabilities and low immune rejection. Therefore, the present study is a fundamental part of future research into their secretome and their application as regenerative therapy in different diseases, including metabolic diseases such as diabetes.

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

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

Mesenchymal stem cells have a really broad role in cellular communication due to their paracrine function. This protocol obtains adipose-derived mesenchymal stem cells with morphological, immunophenotypical, and adequate functionality for use in studying therapies for pancreatic regeneration and diabetes. There has been increased use of cell therapies in the treatment of diabetes.These therapies include allogenic transplants of pancreatic cells, autologous stem cell-derived products of insulin-producing cells, and 3D-printed pancreatic tissue using biological inks. Most research primarily studied the secretome components and the role in the regulation of different cellular processes. New techniques like microarrays and next-generation sequencing can investigate the function of miRNAs.A challenge in this area is to achieve greater stimulation of mesenchymal cells, reaching precise molecular composition in the secretome, and directing it to the treatment of a specific conditions. We will be evaluating how an miRNAs contribute to pancreatic tissue degeneration exposed to oxidative stress. The study will involve usage of stem cells from adipose tissue.

Watch this Scientific Journal Video about Murine Adipose-Derived Mesenchymal Cell Isolation, Maintenance and Characterization at JoVE.com

Murine Adipose-Derived Mesenchymal Cell Isolation, Maintenance and Characterization  

Murin Adipoz Kaynaklı Mezenkimal Hücre İzolasyonu, Bakımı ve Karakterizasyonu

Bir biyogüvenlik kabininin içinde, murin yağ dokusunu steril forsepslerle çıkarın. Fazla PBS çıkarıldıktan sonra, dokuyu başka bir petri kabına aktarın ve her biri 10 mililitre PBS AA çözeltisi ile beş dakika boyunca iki kez yıkayın. Bir santimetre kare büyüklüğünde parçalanmış doku ve manyetik bir çubuk içeren bir kristal beher içine 20 mililitre hazırlanmış kollajenaz çözeltisi ekleyin.Kapalı beheri sürekli ajitasyonla 37 santigrat derecede inkübe edin. Homojenatı 40 örgülü 0,38 milimetre paslanmaz çelik açıklıktan filtreleyin Bir petri kabı üzerinde filtreleyin ve hücre süspansiyonunu steril bir 50 mililitrelik konik tüpte toplayın. Stromal vasküler fraksiyonu 20 mililitre sıcak takviyeli DMEM'de dikkatlice askıya alın ve süspansiyonu santrifüj edin.Süpernatanı attıktan sonra, hücreleri 40 mililitre takviye edilmemiş DMEM ortamı ile iki kez hafifçe yıkayın. Damakları beş mililitre takviyeli DMEM'de askıya alın. Süspansiyonu 25 santimetrekarelik bir T şişeye aktarın ve inkübe edin.Hücreleri beş mililitre ılık PBS AA ile üç kez ve takviye edilmemiş DMEM ile iki kez yıkayın. Herhangi bir hücre kalıntılarını ve murin olmayan hücreleri çıkarmak için beş mililitre taze, sıcak takviyeli DMEM ekleyin ve ortamı her üç ila dört günde bir değiştirin. Hücreler% 90 ila% 100 akıcılığa ulaştığında, DMEM ile yıkanmış hücrelere bir mililitre sıcak tripsin EDTA çözeltisi ekleyin.37 santigrat derecede beş ila yedi dakika inkübe edin. Ayrılmış hücre tek katmanına dört mililitre takviyeli DMEM ekleyin ve süspansiyonu yavaşça ayırın. Hücreleri beş mililitre sıcak takviyeli DMEM'de askıya alın.Supernatanı santrifüj edip attıktan sonra, peleti bir mililitre takviyeli DMEM'de yavaşça karıştırın. Hücreleri tripan mavisi ile lekeleyin ve bir Neubauer odasında sayın. T-75 şişelerindeki tohum hücreleri.Koloni oluşumunu ve yüksek proliferasyonu sağlamak için, hematoksilin ve eosin boyalı hücrelerin morfolojisini optik mikroskop altında gözlemleyin. hematoksilin ve eozin boyaması, uzamış uzamalar ve bol miktarda hücre içi mikrofilament ile geniş sitoplazmayı ortaya koymaktadır.

Research

JoVE Journal

Biology

Murine Adipose-Derived Mesenchymal Cell Isolation, Maintenance and Characterization

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 ...

Biyoloji

Murine Adipose Tissue Excision

Murin Yağ Dokusu Eksizyonu

Immunofluorescent Detection of Adipose-Derived Stem Cell Expression Markers

Adipoz Kaynaklı Kök Hücre Ekspresyon Belirteçlerinin İmmünofloresan Tespiti

Flow Cytometric Detection of Adipose-Derived Stem Cell Expression Markers

Yağ Kaynaklı Kök Hücre Ekspresyon Belirteçlerinin Akış Sitometrik Tespiti

Adipose-Derived Stem Cell Differentiation to the Adipogenic Lineage

Adipojenik Soya Adipoz Kaynaklı Kök Hücre Farklılaşması