ObjectiveTo investigate a most suitable method for harvesting mouse embryonic fibroblasts (MEF)). Methods Three different protocols including tissue only, cell-trypsin digestion, and tissue-trypsin digestion were applied for harvesting and culturing MEF. The MEF growth speed and cell morphology were also compared and analyzed respectively among the 3 methods. ResultsThe cell proliferation was slow, cell size was small, but cells of other types were very few in the tissue only method, but the duration of purification was short. In the cell-trypsin digestion method, the cell proliferation was fast, cell size was somewhat large, but there were a little bit more cells of other types and the duration of purification was somehow longer. Finally, in the tissue-trypsin digestion method, the cell proliferation was very slow, cell size was very small, but there were also very few cells of other types. The duration of purification was very short. ConclusionsOur findings indicate that the most efficient method for collecting and culturing mouse embryonic fibroblasts is the protocol of tissue-trypsin digestion.