Microfluidic device for stem cell differentiation and localized electroporation of postmitotic neurons

Abstract

New techniques to deliver of nucleic acids and other molecules for gene editing and gene expression profiling, which can be performed with minimal perturbation to cell growth or differentiation, are essential for advancing biological research. Studying cells in their natural state, with temporal control, is particularly important for primary cells that are derived by differentiation from stem cells and are adherent, e.g., neurons. Existing high-throughput transfection methods either require cells to be in suspension or are highly toxic and limited to a single transfection per experiment. Here we present a microfluidic device that couples on-chip culture of adherent cells and transfection by localized electroporation. Integrated microchannels allow long-term cell culture on the device and repeated temporal transfection. The microfluidic device was validated by first performing electroporation of HeLa and HT1080 cells, with transfection efficiencies of ~95% for propidium iodide and up to 50% for plasmids. Application to primary cells was demonstrated by on-chip differentiation of neural stem cells and transfection of postmitotic neurons with a green fluorescent protein plasmid.

Microfluidic systems for stem cell-based neural tissue engineering

“…Furthermore, PDMS can readily undergo molding, has low levels of auto-fluorescence and good transparency which facilitates cell imaging; these properties make it the first choice of bioengineers 106, 107 . In regard to neuroregeneration, numerous studies have taken advantage of these benefits to investigate the use of PDMS-based microdevices in stem cell biology, and manipulation of their fate 108–110 . However, despite these advantages, there is some difficulty in the handling and fabrication of high aspect-ratio channels, the material has low stiffness, a hydrophobic nature, and can undergo unexpected evaporation due to its porous structure, as well as possessing biocompatibility and sterilization issues, that collectively comprise its challenges 9, 92, 107 .…”

“…Collagen type 1 115 , poly-L-lysine (PLL) 116 and fibronectin 111 coatings have all been used to overcome the shortcomings of PDMS, and other polymeric material-based microdevices. For example Pluronic F-127 treatment was used to prevent absorption of fluorescent molecules into the PDMS matrix, and lessening noise and background during fluorescence imaging 110 .…”

Micro- and Nanoscale Technologies for Delivery into Adherent Cells

“…Several technologies for studying adhered cells are currently being developed, and due to the need for individual cell access and non-destructive probing, micro- and nano-technologies are a natural choice because they interact with cells at the appropriate length scale, reduce the working volume of expensive reagents, require less time and space for replicates, allow for automation and integration of sequential analyses, enable portability, and reduce waste [2, 3]. Here we present an overview of recently developed micro- and nano-tools, with a focus on trends in intracellular delivery for in vitro studies of adhered cells, and highlight major advantages/disadvantages of these technologies with respect to features such as individual cell selectivity, spatial resolution, non-destructive cell analysis, and potential for high throughput or automation.…”

“…Electroporation causes transient nanopores to form in the cell membrane when the cell is subjected to a sufficiently large electric field through which molecules can be delivered inside cells [3, 17, 18, 61–63]. As the electric field is typically created by applying an input voltage between two electrodes, the appropriate placement of the electrodes is crucial for reproducibility and consistent yield.…”

“…These challenges can be overcome by miniaturization to reduce the required input voltage, create a more uniform electric field, and rapidly dissipate heat due to the large surface-to-volume ratio [60]. Applying this miniaturization toward adhered cells has resulted in electroporation methods that have recently been developed in configurations suitable for single cells using fluidic nanoprobes (Figure 2, right column) and for multiple cells using a lab-on-a-chip (LOC) approach (Figure 2, left column) [3, 18, 73]. …”