Design of Experiments implementation in a transfection protocol fine-tuning: Design of ransfections (DoT).

Design of Experiments (DoE) is applied for planning, conducting, analysing and interpreting controlled tests in order to identify the factors that influence one or more outputs of interest. Until now DoE has been mostly used in industrial field in order to maximize processes, but recently different studies have demonstrated the advantages of using the DoE approach with respect to the classical ones (in which a single parameter is tested in each assay) also in biomedical research field [1,2]. In our work, we implemented the DoE methodology to optimize a transfection protocol for immortalised neural progenitor cells (NPCs) derived from mesencephalon of mouse embryos [3]. Polyethyleneimine (PEI), a cationic non-lipidic molecule, was chosen as a transfection reagent, because of the NPCs refractory behaviour to traditional lipidic transfection methods [4]. Among the factors relevant for transfection efficiency, we selected three quantitative factors: (i) PEI concentration, (ii) DNA amount, (iii) cell density; and a qualitative one, the PEI type, linear 'L' (22 kDa) vs. branched 'B' (25 kDa) [5]. We used cell profiler software to compute transfection efficiency and a new pipeline was generated ad hoc in order to obtain reliable results and significantly reduce user-variance, avoiding manual counting. We performed first a two-level full factorial design (screening experiment) to identify the most influencing factors and their interactions, second a Box-Behnken design to investigate deeper the interval identified by the screening experiment and select the conditions that maximize the transfection efficiency output. This DoE approach allowed us to develop a very flexible tool named "Design of Transfections" (DoT), extremely powerful for identifying the optimal transfection conditions, suitable for different cell types and transfection reagents.