Mesenchymal stem cell cultivation

Human bone marrow (Cambrex) was mixed 1:2 within 24 hours of collection with Hank's balanced salt solution without Ca2+ or Mg2+ [5-6]. Mononuclear cells were isolated by passage over Histopaque density gradient (1.077) and centrifuging. Washed mononuclear cells were labeled with anti-CD34+ antibody (Miltenyi Biotec) and passed over a midi-Max magnetic bead column (Miltenyi Biotec). CD34- cells were collected, washed 1x with DPBS and centrifuged. Pelleted cells were resuspended in Advanced DMEM with 10% FBS, seeded into a T-75 flask, and incubated at 37°C, 5%CO2 for 24 hours. Non-attached cells were removed and the flask washed 1x with DPBS. Fresh Advanced DMEM (10% FBS) was added and the flask incubated until the cells reached 80-90% confluency with re-feeding every 5-6 days. Passaged MSC were plated into six well plates at a concentration of 4 x 103 cells per mL. Cells were grown for 6 days at 37°C, 5% CO2. Cells were harvested by trypsinization and counted electronically (Coulter).

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