Increased delivery efficiency with high cell viability in serpentine device
Nucleofection and viral transduction are effective methods for intracellular delivery of gene into different cells (Bilal, Vacaflores, & Houtman, 2015)´(Kebriaei et al., 2016) but because of substantial reduction in cell viability and recovery of cells using this conventional transfection methods ,we sought to investigate if comparable intracellular delivery efficiencies and higher viability could be achieved via our pattern.
For our proof-of-concept study, we selected an approximately 11.0 kB GFP construct including anti-BCMA gene to transfect into myeloma cell via serpentine microfluidic device. We observed an increased level of GFP expression compared to cells processed through nucleofection. (Fig. 2)
To confirm that lower DNA volumes in our pattern is required compared to nucleofection, various plasmid concentration was applied to the device. Also, the effects of different speeds were evaluated on the percentage of GFP positive cells as shown in the diagram (fig 3). Our data showed that delivery efficiency increased with increasing flow rate across designed pattern but viability in flow rate of 13 ml/h decreased compared to lower speeds. However, viability impacts on expression of critical genes associated with function, we observed that cell viability and delivery efficiency in 9 ml/h had better results.
GFP expression levels ranging from 19 % to 55.7 % in live cells were observed approximately 24hours post-transfection, with significant increase in expression detected in cells processed with different flow rates. (fig3 A). Interestingly, the GFP expression observed in the live cell population was linearly correlated with increasing flow rate until 11 ml/h, indicating that the overall expression level of GFP by augmenting chaotic advection in channel has been enhanced.
While delivery efficiency and viability are important success metrics for cell engineering, unintended changes to cell phenotype may adversely impact functional potential. Accompanying the significant decrease in viability, cell death is remarkable in nucleofected cells and progresses days after transfection (Zhang et al., 2014).In comparison, intracellular delivery of gene via our design showed only slight decreases in cell viability two days after transfection compared to nucleofection. Quantifying the GFP expression levels in transfected cells revealed a peak transfection efficiency of 55.7 % when applying 0.5 μg/ml plasmid. (fig3 B) We found that the levels of GFP expression were significantly higher across all concentrations and speeds compared to nucleofection. Cumulatively, these results suggest that delivery of plasmid DNA at various concentration and different flow rates via our microfluidic device varies the transfection efficiency and cell viability with optimal levels achieved when delivering 0.5 μg mL−1 DNA in 9 ml/h speed.