Khảo sát một số điều kiện nuôi cấy in vitro tế bào đơn nhân được chiết từ máu ngoại vi người

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The purpose of this study is to optimize some conditions for human PBMCs in vitro culture. PBMCs were isolated within 4 hours from the EDTA – coagulated whole blood samples collected from the 10 healthy volunteers. 100 μL of a cell suspension (approximately 5x105 cells/mL)were added to each well in a 96 well microplate and incubated at 37°C, 95±5% humidity and 5% CO2 for 24 hrs. Then the isolation of PBMCs from 6 ml whole blood using 10 mL Leucosep® tube was optimized; cell viability using trypan blue dye was evaluated; the suitable solvents for the dissolution of formazan crystals in the MTT assay and the conditions of PBMCs in vitro culture such as PBMCs density per well, FBS concentration, time for PBMCs culture and PHA mitogen concentration for PBMCs proliferation were examined. The results of this study showed that the centrifuge speed and time for isolating PBMCs from 6 mL whole blood using Leucosep® tube was determined at 1200 xg for 20 minutes. After that, PBMCs viability was found above 95% using trypan blue and hemocytometer. The dissolution of formazan crystals in DMSO/NH3 was better than in either DMSO, isopropanol or isopropanol/HCl. Moreover, the optimal conditions for cell culture media containing 10% FBS, 1.5% PHA mitogen and the range of PBMCs density from 104 – 105 cells/well were determined. These results may be used for the further studies to assess the effects of medicinal plants or new drugs on in vitro PBMCs proliferation.