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

摘要

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 gamma-radiation 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.