HIGH-EFFICIENCY LOADING, TRANSFECTION, AND FUSION OF CELLS BY ELECTROPORATION IN 2-PHASE POLYMER SYSTEMS

A method to concentrate drugs, DNA, or other materials with target cel ls in two-phase polymer systems for high-efficiency electroloading is described. The two-phase polymer system is utilized for cell and loadi ng material selection, as well as for cell aggregation before electrof usion. The phase mixing of several water-soluble polymers is character ized, and the polyethylene glycol-Dextran (PEG m.w, 8,000 + Dextran m. w. 71,000) mixture is selected to illustrate the advantage of the two- phase systems. Fluorescently labeled Dextran or DNA is loaded into Chi nese hamster ovary (CHO) and JTL cells, using electroporation in eithe r the two-phase polymer system or the conventional single-phase suspen sion, The loading efficiency is 4 to 30 times higher for the two-phase system, with the best advantage at lower applied field range. Transfe ctions of CHO, COS, Melan C, and JTL lymphoid cells using pSV-beta-gal actosidase (for CHO and COS), pBK-RSV-tyrosinase, and pCP4-fucosidase plasmids, respectively, by electroporation in the two-phase polymer sy stem and the conventional single-phase electroporation method, are com pared. The former method is far superior to the latter in terms of eff iciency. The threshold and optimal field strengths for the former are significantly lower than those for the latter method, so the former me thod is more favorable in terms of equipment requirement and safety. E lectrofusion efficiency in the two-phase system is comparable to that in polyethylene glycol suspension alone and is a significant improveme nt from the conventional electrofusion method with dielectrophoresis. The two-phase polymer method is, therefore, a valuable technique for g ene delivery to a limited cell source, as in ex vivo gene therapy.