PMC

Electroporation of functional siRNAs bound within microwell arrays and evaluation of cellular phenotypes. A) Left-Schematic showing that the full area microwell arrays were coated with either siRPS27a or siControl. Right-Death phenotypes were observed only in the siRPS27a-coated microwells and only under electroporation conditions. No death was observed in siRPS27a-coated microwells without electroporation. B) Left-Schematic showing half areas of the microwell array coated with siRPS27a or siControl. Right-Death phenotypes were observed only on the siRPS27a-coated half. C) Left-Schematic showing quadrant areas of the microwell array coated with siRPS27a or siControl. Right-Death phenotypes were observed only in quadrants containing siRPS27a. Experiments were performed with HeLa cells and phenotypes assessed 48 h post-electroporation.

In situ electroporation of functional siRNAs printed onto microwells arrays. A) Top left-Electroporation and assessment of phenotypes using Design pattern 1 with HeLa cells within microwell arrays loaded with siControl (red areas) and siRPS27a (green areas). Quadrant areas are depicted by Q1 & Q4 (for siControl) and Q2 & Q3 (for siRPS27a). Areas Q12 and Q34 consist of bordering siControl and siRPS27a microwells. Bottom-Phase contrast images of all areas taken 48 h post-electroporation. Top right-Magnified image of area Q12 where death phenotypes are observed only in microwells printed with siRPS27a. B) Top left-Electroporation and assessment of phenotypes using Design pattern 2 with HeLa cells within microwell arrays loaded with siControl (red areas) and siRPS27a (green areas). Areas A1, A2 and A3 were chosen to assess phenotypes. Bottom-Phase images of areas A1, A2, and A3 at 48 h post-electroporation. Top right-Magnified image of area A2 with checkered death phenotypes obtained in siRPS27a-printed microwells.

Microwell array design and biocompatibility. A) Top-Image of a complete, fabricated microwell array on ITO-coated glass slides. Sixteen devices consisting of a 9 × 9 microwell array each are spaced for dicing and individual experimentation. Lower left-Image of a single device. Lower right-Magnified image of a microwell array where the opaque (black) areas are the patterned titanium/gold electrode grid and the insulating microwell material surrounding the grid made of SU-8. B) Image of HEK 293 cells, C) HeLa cells, and D) HeLa-GFP cells cultured in microwell arrays 48 h post-seeding. E) Primary mouse macrophages derived from a GFP transgenic mouse line cultured and imaged in phase (left) and fluorescence (right) in a microwell array.

Registered loading of siRNAs into microwell arrays using a piezo-based inkjet printer. A) Schematic showing alignment of piezo-dispense head and a microwell array prior to dispensing of siRNA. Nanolitre volumes (8 nL) are dispensed into each microwell, coating the entire bottom surface. B) Left-Alternating design pattern programmed into the inkjet microarrayer. Right-Scan of a single microwell array loaded with Alexa-fluor-488 (green) and Cy5 (red) conjugated siRNAs using the alternating pattern. C) Top left-Design pattern 1 comprising of a quadrant to be printed into the microwell array. Top right-Scanned image of a single microwell array loaded with the quadrant pattern. Bottom-Scanned image of an entire slide (16 devices) printed in the quadrant pattern. D) Top left-Design 2 consisting of a checkered pattern to be printed into the microwell array. Top right-Scanned image of a single microwell array loaded with the checkered pattern. Bottom-Scanned image of an entire slide (16 devices) printed with the checkered pattern. Concentrations of fluorescently labeled siRNAs loaded were 5 μM in (B) and 1 μM in (C) and (D).

Schematic of the electroporation protocol in miniaturized electroporation-ready microwell arrays. Left column – top view of microwell array; Center column – cross-sectional view of a single microwell; Right column – magnified view of surface interactions. First row – microwell array consisting of an ITO-coated glass surface patterned with an electrode grid, which is further insulated by a pattern of thick photoresist (SU-8). The ITO layer is amine-functionalized to generate a positively charged surface. Second row – registered and multiplexed loading of nucleic acids (siRNA) into the microwell array. Negatively charged siRNAs electrostatically bind the positive surface. Third row – cell seeding onto the microwell array in electroporation buffer by sandwiching the plated volume at a precise distance from the bottom ITO surface. Cells attach to the bottom of the microwells and are in proximity to the bound siRNA. Fourth row – using the top sandwich electrode as an anode and the bottom ITO layer as the cathode, an electroporation pulse is applied to all microwells simultaneously. Current flows from the patterned electrode grid into the ITO layer and through the electroporation buffer. The microwell array material (SU-8) insulates the grid and prevents direct current flow into the buffer. Fifth row – cells are allowed to recover and strengthen attachment post-electroporation. Sixth row – the microwell array is incubated for the period of the assay and imaged for cellular phenotypes.