Most high throughput/high content cell-based screening assays rely on exogenous gene expression such as reporter genes, expression of fusion proteins, artificially engineered proteins or overexpression of a target of interest. Gene expression for these assays has historically been approached by either creating stable cell lines or using transient transfection. Stable cell lines have the advantage of long term protein expression; however, their creation is a costly, time consuming and labor intensive process that requires multiple rounds of selection and clonal isolation. Whether constructed internally or outsourced, the average time to create a stable cell line is approximately 10 to 15 weeks. This prolonged time between assay conceptualization and conducting a functional screen is eliminated using transiently transfected cells. Additional considerations of stable cell line usage include the complexity of expressing multiple targets or multi-subunit protein complexes, as well as potentially toxic targets. Due to time and budgetary constraints, many pharmaceutical companies have streamlined their compound screening and profiling initiatives by incorporating transient transfection systems within their drug discovery processes.
Transient transfection technologies have evolved from simple chemical carriers such as DEAE-dextran and calcium phosphate into sophisticated, highly engineered methodologies such as lipid-based reagents, viral-mediated delivery and high throughput electroporation (2). Newer technologies provide superior performance and broader applicability allowing introduction of DNA, RNA, siRNA and proteins into mammalian cells. A variety of transient transfection options are available, each having specific strengths and limitations. Researchers are tasked with matching their needs to the strengths of a specific technology. Factors to consider range from scientific performance such as cell viability, transfection efficiency, reproducibility and induction of off-target effects to more practical considerations such as ease of use, system flexibility and cost.
Additional factors must be taken into consideration for any assay technology to be successfully integrated within a screening environment. Most importantly, they must have high throughput capacities and reproducible results, while remaining easy to use and affordable. Chemical carriers, while affordable, are prone to high levels of variability and are thus not generally used in higher throughput applications. In contrast, lipid-mediated transfection, virus delivery and electroporation are more amenable to higher throughput settings. Second generation lipid-based technologies have relatively high transfection efficiencies and the ability to transfect a range of cells. Lipid transfections, however, require re-optimization of transfection conditions for higher throughputs and can become cost prohibitive.
While higher throughput viral delivery methods can achieve very high levels of transfection efficiency, vector type can limit cell compatibility. Additionally, creation of viral vectors and production of viral stocks can invoke biosafety considerations, often require a high level of user knowledge and are labor and time intensive.
MaxCyte’s flow electroporation technology enables transfection of a large number of cells in a very short period of time. This technology combines broad applicability, ease of use and superior performance with the capacity to transfect 2 x 1010 cells in 20-30 minutes. This platform provides a cost effective solution that uniquely fulfills the demands of early phase drug development activities.