Transfection of Cultured Primary Neurons

Improved CaP Nanoparticles for Nucleic Acid and Protein Delivery to Neural Primary Cultures and Stem Cells

“…Moreover, the CaP particle size and number were positively and negatively correlated with calcium concentrations (Figure A–C). Importantly, these ∼100 nm CaP nanoparticles are small enough to perform extensive Brownian motions (Videos S1–S3), which is significantly different from the lipofectamine-produced liposome and conventional CaP precipitates (precipitating on the bottom with slight shaking). ,,− …”

“…Despite many suggestions from previous protocols, ,,− we discovered that calcium concentration and culture medium are critical factors that influence CaP nanoparticle formation and efficiency of transfection (Figure ). In our system, CaP nanoparticles were created by mixing 50–70 mM calcium solution with 2× HEPES-buffered saline (HeBS) using either vortexing or pipetting, without the need for any further incubation, in just two simple steps (Supporting Information Figure S1 and Table S4).…”

“…Importantly, these ∼100 nm CaP nanoparticles are small enough to perform extensive Brownian motions ( Videos S1–S3 ), which is significantly different from the lipofectamine-produced liposome and conventional CaP precipitates (precipitating on the bottom with slight shaking). 4 , 16 , 19 − 25 …”

“…However, cultures of primary neurons are notoriously difficult to transfect, limiting their usability. Although numerous commercialized kits and experimental approaches have been developed to improve transfection efficiency, cytotoxicity, stress response induction, poor reproducibility, and excessive price remain limiting factors. − CaP is arguably the simplest and cheapest reagent for cell transfection but suffers from about 1% poor efficiency and low cell viability (reagent consistency is critical for reproducibility) after transfection in primary neuron cultures. , Many modified CaP-transfection protocols have been developed, but they can only be applied to very specific conditions because of the microscale size of CaP precipitates (uncontrolled growth of CaP nanoparticles), i.e., microisland and low-density cultures, ,,− which constrains their applicability (Table S3).…”

“… 12 − 18 CaP is arguably the simplest and cheapest reagent for cell transfection but suffers from about 1% poor efficiency and low cell viability (reagent consistency is critical for reproducibility) after transfection in primary neuron cultures. 12 , 13 Many modified CaP-transfection protocols have been developed, but they can only be applied to very specific conditions because of the microscale size of CaP precipitates (uncontrolled growth of CaP nanoparticles), i.e., microisland and low-density cultures, 4 , 16 , 19 − 25 which constrains their applicability ( Table S3 ).…”