Electric Pulses to Prepare Feeder Cells for Sustaining and Culturing of Undifferentiated Embryonic Stem Cells

Current challenges in embryonic-stem-cell (ESC) research include inability of sustaining and culturing of undifferentiated ESCs over time. Growth-arrested feeder cells are essential to the culture and sustaining of undifferentiated ESCs, and they are currently prepared using gammaradiation and chemical inactivation. Both techniques have severe limitations. In this study, we developed a new, simple and effective technique (pulsed-electric-fields, PEFs) to produce viable growth-arrested cells (RTS34st) and used them as high-quality feeder cells to culture and sustain undifferentiated zebrafish ESCs over time. The cells were exposed to 25 sequential 10- nanosecond-electric-pulses (10nsEPs) of 25, 40 and 150 kV/cm with 1s pulse interval, or 2 sequential 50-microsecond-electric-pulses (50μsEPs) of 2.83, 1.78 and 0.7 kV/cm with 5s pulse interval, respectively. We found that cellular effects of PEFs depended directly upon the duration, number and electric-field-strength (E) of the pulses, showing the feasibility of tuning them to produce various types of growth-arrested cells for culturing undifferentiated ESCs. Either 10nsEPs of 40 kV/cm or 50μsEPs of 1.78 kV/cm provided by inexpensive and widely available conventional electroporators, generated high-quality growth-arrested feeder cells for proliferation of undifferentiated ESCs over time. One can now use PEFs to replace radiation methods for preparation of growth-arrested feeder cells for advancing ESC research.