Primary cell culture of a peripheral mononuclear cells of a mouse strain C57BL/6

Uvod: Primarna kultura stanica dobiva se izolacijom stanica izravno iz tkiva. Uzgojem u odgovarajućim uvjetima, primarne stanice rastu i proliferiraju, ali imaju ograničen životni vijek. Osnovni koraci u uspostavi primarne kulture stanica su dobivanje uzorka, izolacija tkiva, disekcija ili razgradnja tkiva te kultivacija u posudicama za kulturu. Cilj: Uspostava primarne kulture mononuklearnih stanica miša te određivanje uvjeta potrebnih za njihov uzgoj. Materijali i metode: U istraživanju je korištena krv iz srca triju ženki soja CB7BL/6N koje smo pulirali. Miševi su uzgojeni u vivariju Medicinskog fakulteta Osijek. Limfociti su iz krvi izolirani metodom gradijenta gustoće pomoću Lymphoprep otopine. Kultivirane stanice čuvaju se u CO_2 inkubatoru (Shel lab) uz atmosferu od 5% CO_2 i temperaturu od 37°C. Za održavanje primarne kulture mononuklearnih stanica koristi se RPMI 1640 definirani medij. Broj živih stanica u kulturi određen je u testu vijabilnosti s bojom tripansko plavilo u Bürker-Türk-ovoj komorici. Rezultati: Uspostavljena je primarna kultura perifernih mononuklearnih stanica. Broj živih stanica u kulturi 0.dan bio je 213,75 x 〖10〗^4 stanica/ml. Zaključak: Uspostava primarne kulture perifernih mononuklearnih stanica miša moguća je pridržavanjem pravila aseptične metode i uzgojem u strogo kontroliranim uvjetima u kojima stanice mogu preživjeti i do osam dana.Introduction: Primary cell culture is obtained by isolation of cells directly from the tissue. By cultivation under appropriate conditions, primary cells grow and proliferate, but have a limited life span. The basic steps in establishing a primary culture cell include obtaining a sample, tissue isolation, dissection or tissue degradation and cultivation in culture vessels. Objective: The aim of the study is to establish the primary culture of mononuclear mouse cells and to determine the conditions necessary for their growth. Materials and methods: The blood from three female CB7BL/6N strains was used in the study. The mice were raised at the vivarium of Faculty of Medicine in Osijek. Lymphocytes were isolated from the blood by the density gradient method using a Lymphoprep solution. Cultivated cells are stored in a CO_2 incubator (Shel lab) under a 5% CO_2 atmosphere and a temperature of 37 ° C. An RPMI 1640 defined medium was used to maintain the primary culture of mononuclear cells. The number of viable cells was determined in a viability test with Trypan blue color in the Bürker-Türk chamber. Results: Primary culture of peripheral mononuclear cells was established. The number of viable cells in culture on the first day was 213,75 x 〖10〗^4 cells/ ml. Conclusion: The establishment of primary culture of peripheral mononuclear cells of a mouse is possible by adhering to the rules of aseptic technique and cultivation under strictly controlled conditions in which cells can survive up to eight days