A mechanical non-enzymatic method for isolation of mouse embryonic fibroblasts.

Mouse embryonic fibroblasts (MEFs) accessibility coupled with their simple generation make them as a typical embryonic cell model and feeder layer for in vitro expansion of pluripotent stem cells (PSCs). In this study, a mechanical isolation technique was adopted to isolate MEFs and the efficiency of this technique was compared with enzymatic digestion method. The suspended MEFs were prepared either by mechanical method or 0.25% trypsin enzymatic digestion. The effect of tissue processing on cell apoptosis/necrosis, morphology, viable cell yield, population doubling time, surface marker expression, and the capacity to support PSCs were determined. The mechanical method yielded a significantly higher number of viable cells. However, it showed similar morphology and proliferation characteristics as compared to enzymatic digestion. The mechanical method induced slight apoptosis in MEFs; however, it did not exert the necrotic effect of trypsinization. Treatment of tissue slurry with trypsin solution caused cell lysis and subsequently cell clump formation. Mechanically isolated cells exhibited a higher expression of the MEF surface antigens Sca1, CD106, and CD105. The PSCs on mechanically isolated MEFs displayed a higher expression of pluripotency genes, and formed more compact colonies with a stronger tendency to crowding compared with those cultured on cells isolated by enzymatic digestion. The mechanical method based on tissue inter-syringe processing is relatively a rapid and simple method for MEF isolation. Compared to the enzymatic digestion, the cells obtained from this method show higher expression of embryonic fibroblasts markers and a more functional capacity in supporting PSCs culture.