Optimizing Transfection of Umbilical Cord Mesenchymal Stem Cells Utilizing Minicircle plasmid/Lipofectamine LTX Complex

Document Type: Original Article

Authors

1 Institute of Bioscience, Universiti Putra Malaysia, 43400 Selangor, Selangor, Malaysia

2 Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

3 Faculty of Medicine, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

Abstract

Lipofectamine LTX is a cationic based transfection reagent that offers high levels of different transgenes expression in a huge number of cell types in vitro utilizing an easy and rapid protocol. The level of transgenes expression depend on some
experimental variables containing cell density, lipofectamine and DNA concentrations, lipofectamine -DNA complexing time, and the presence or absence of medium at the transfection time and antibiotics in medium. The significance of these factors in lipofectamine LTX based transfection will be considered. The main objective of this study is optimizing different situations for transfection of umbilical cord mesenchymal stem cells (UC-MSCs) with lipofectamine LTX. For this purpose, UC-MSCs were transfected with lipofectamine LTX utilizing minicircle plasmid containing green fluorescent protein reporter gene. In this study, the medium existence on cell transfection efficiency as well as effects of time for culture, the ratio of lipofectamine LTX to plasmid DNA, the repetition times of transfection and volume of medium on transfection efficacy were evaluated, and the transfection efficacies were also compared. The growth rate of MSCs was associated with the density of the cells. The medium changing 4 hours after transfection certified the normal cells growth. Nevertheless, the freshness and amount of medium demonstrated no substantial effect on the cell state. The 1:2 ratio of plasmid to ipofectamine LTX (3ng:6µl), the transfection efficacy was pleasing. The transfection cells with different ratios of plasmid to lipofectamine LTX displayed substantial differences18 h post transfection. The different conditions of MSCs transfection with lipofectamine LTX was optimized, which presented a reference for various adherent cells transfection.

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