Electroporation Optimization

@mastersthesis{posadas2022,
  title = {Electroporation Optimization},
  author = {Posadas, Olivia},
  year = {2022},
  month = aug,
  publisher = {Lehigh University},
  keywords = {Electroporation, fusion, Heat shock, lysis, Osmotic shock, shape factor},
  abstract = {Electroporation is a physical method of transfection of molecules of interest through an electrical impulse. Settings that would increase transfection by enhancing polarization or permeabilization of the membrane has to be balanced against the risk of irreversible electroporation. Irreversibly electroporated cells die for a number of reasons including hyperthermia, osmotic shock, pore expansion, and electrolysis. Altering one variable does not just increase or decrease cell death by that particular mode it has systemic effects. Understanding the specific cause of cell death for the particular cell line utilized allows electroporation protocols to be re-designed to counterbalance the cytotoxic effects of that setting while still enhancing the ability to transform the cell. The changes made to electroporation settings can be quantified into single measures to analyze the outcomes of different shocks when multiple variables differ. Electroporation methods were evaluated using NIH 3T3 cells and the plasmid pCAG-GFP plasmid \# 11150 from Addgene. Initially, electroporations were performed using the pre-set protocol of Bio-Rad GenePulser Xcell electroporation system with PBS (1X) and Opti-MEM and then Bio-Rad Electroporation Buffer for mammalian cells in 4 mm cuvettes. To improve transfection rates alterations were made to the pre-set protocol first with manufacturer suggested changes to machine settings. Other changes were made including electric field via cuvette size or voltage setting which had different outcomes because the shocked cells are affected by the suspension resistance which is dimensional dependent. Electroporation buffers were prepared based off of additional solutions suggested by Bio-Rad in the GenePulser Xcell electroporation system manual. Resistance of both the buffer used and the resistor setting were modified. Each buffer has its own characteristics including resistance and thus conductivity, and osmolarity. Cells performed best in hypo-osmotic solutions in terms of rates of transfection to population. Bio-Rad electroporation buffer was amongst the most successful buffers to use for exponential decay; one setting had a day one transfection rate of ~27.5\%. The highest final transfection rate, which is the percentage of GFP positive cells at the end of the experiment compared to the initial survival, was 60\%. However, under different circumstances square wave electroporations with HEPES sorbitol 43 mM was able to reach over 30\% GFP expression after one day incubation post shock, and an 80\% final transfection rate. These were not transfections performed with higher DNA concentrations, so DNA content is not the limiting factor for transformation, other modifications play a greater role. To further improve transfection efficiency more modifications to electroporation protocols can be evaluated.},
}