Optimization and Characterization of Calcium Phosphate Transfection in Mesenchymal Stem Cells.
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ABSTRACT:
SUBMITTER: Lo CW
PROVIDER: S-EPMC6761597 | biostudies-literature | 2019 Sep
REPOSITORIES: biostudies-literature
Publications
Optimization and Characterization of Calcium Phosphate Transfection in Mesenchymal Stem Cells.
Lo Chi-Wen CW Lin Tzuhua T Ueno Masaya M Romero-Lopez Monica M Maruyama Masahiro M Kohno Yusuke Y Rhee Claire C Yao Zhenyu Z Pérez-Cruz Magdiel M Meyer Everett E Goodman Stuart B SB
Tissue engineering. Part C, Methods 20190901 9
<h4>Impact statement</h4>Mesenchymal stem cells (MSCs) are a promising tool for cell therapy, and gene-modified MSCs further expand their applications. To take full advantage of MSCs as a therapeutic approach, developing effective gene transfer methods is critical. Calcium phosphate transfection is well-established and safe, but the protocols need to be optimized according to different cell types. Currently, there is no optimized protocol for MSCs. This study optimized the protocol of calcium ph ...[more]
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Project description:BackgroundCalcium phosphate mediated transfection has been used for delivering DNA into mammalian cells in excess of 30 years due to its most low cost for introducing recombinant DNA into culture cells. However, multiple factors affecting the transfect efficiency are commonly recognized meanwhile for years, the low transfection efficiency of this approach on higher differentiated and non-tumor cells such as CHO and C2C12 limits its application on research.ResultsIn this paper, we systematically evaluated the possible factors affecting the transfection rate of this approach. Two categories, calcium phosphate-DNA co-precipitation and on-cell treatments were set for optimization of plasmid DNA transfection into CHO and C2C12 cell-lines. Throughout experimentation of these categories such as buffer system, transfection media and time, glycerol shocking and so on, we optimized the best procedure to obtain the highest efficiency ultimately. During calcium phosphate DNA-precipitation, the transfection buffer is critical condition optimized with HBS at pH 7.10 (P = 0.013 compared to HEPES in CHO). In the transfection step, FBS is a necessary component in transfection DMEM for high efficiency (P = 0.0005 compared to DMEM alone), and high concentration of co-precipitated particles applied to cultured cells in combination with intermittent vortexing is also crucial to preserve the efficiency. For 6-well culture plates, 800 µl of co-precipitated particles (11.25 µg/mL of cDNA) in 1 well is the optimal (P = 0.007 compared to 200 µl). For the highest transfection efficiency, the most important condition is glycerol in shock treatment (P = 0.002 compared to no shock treatment in CHO, and P = 0.008 compared to no shock treatment in C2C12) after a 6 h incubation (P = 0.004 compared to 16 h in CHO, and P = 0.039 compared to 16 h in C2C12) on cultured cells.ConclusionsCalcium phosphate mediated transfection is the most low-cost approach to introduce recombinant DNA into culture cells. However, the utility of this procedure is limited in highly-differentiated cells. Here we describe the specific HBS-buffered saline, PH, glycerol shock, vortex strength, transfection medium, and particle concentrations conditions necessary to optimize this transfection method in highly differentiated cells.
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