Highly parallel introduction of nucleic acids into mammalian cells grown in microwell arrays

Abstract

High-throughput cell-based screens of genome-size collections of cDNAs and siRNAs have become a powerful tool to annotate the mammalian genome, enabling the discovery of novel genes associated with normal cellular processes and pathogenic states, and the unravelling of genetic networks and signaling pathways in a systems biology approach. However, the capital expenses and the cost of reagents necessary to perform such large screens have limited application of this technology. Efforts to miniaturize the screening process have centered on the development of cellular microarrays created on microscope slides that use chemical means to introduce exogenous genetic material into mammalian cells. While this work has demonstrated the feasibility of screening in very small formats, the use of chemical transfection reagents (effective only in a subset of cell lines and not on primary cells) and the lack of defined borders between cells grown in adjacent microspots containing different genetic material (to prevent cell migration and to aid spot location recognition during imaging and phenotype deconvolution) have hampered the spread of this screening technology. Here, we describe proof-of-principles experiments to circumvent these drawbacks. We have created microwell arrays on an electroporation-ready transparent substrate and established procedures to achieve highly efficient parallel introduction of exogenous molecules into human cell lines and primary mouse macrophages. The microwells confine cells and offer multiple advantages during imaging and phenotype analysis. We have also developed a simple method to load this 484-microwell array with libraries of nucleic acids using a standard microarrayer. These advances can be elaborated upon to form the basis of a miniaturized high-throughput functional genomics screening platform to carry out genome-size screens in a variety of mammalian cells that may eventually become a mainstream tool for life science research.

Figure 1

Electroporation of exogenous molecules into HEK 293T cells growing on ITO without microwells. A) Phase contrast, transfection assay (Propidium Iodide fluorescence 2 hrs post-EP) and viability assay (Calcein AM 1 day post-EP) images for three different electroporation parameter sets (electric field intensities 100 V cm⁻¹, 500 V cm⁻¹ and 800 V cm⁻¹ respectively, at constant pulse width of 1 ms and 1 square pulse). A scratch (not shown) was made on each of the ITO pieces to locate the culture area for the two assays at different time points. B) Further confirmation of viability post-EP. Cells electroporated with propidium iodide (top row: red) using the optimal electroporation parameter (500 V cm⁻¹, pulse width 1 ms and 1 square pulse) were virally transduced with viral-GFP particles and assayed for viability 24 hr post-EP (bottom row: green). GFP expression is a confirmation of cell viability. C) Electroporation of Alexa Fluor 488- conjugated siRNA molecules using two electroporation parameter sets (100 V cm⁻¹ and 500 V cm⁻¹; constant pulse width of 1 ms and 1 square pulse) and assaying for transfection using Alexa Fluor 488 compatible excitation/emission filters 2 hr post-EP. EP: Electroporation.

Figure 2

Schematic showing the step-by-step process from creation of microwell arrays to image analysis after electroporation. A) Bonding of a pre-cleaned ITO slide with a laser cut microwell array stencil/coverlay. B) Sterilization and coating with fibronectin to enhance cellular adhesion. C) Seeding of mammalian cells into the microwell array by placement of the substrate and cells in a tissue culture dish. 1 hr after seeding, the dish was gently washed to remove unbound cells. Cells inside the microwells experience minimal flow stress and remain attached during this step. D) Electroporate substrate using either a single or double cathode scheme. Post-electroporation incubation. E) Image acquisition of either single microwells under a microscope or whole slide scanning.

Figure 3

Culture and electroporation of HEK 293T cells in microwell arrays on ITO-coated glass slides. A) Culture of HEK 293T cells within individual microwells 1 day after seeding. Top row: Phase contrast image of cells. Bottom row: live assay of cells using Calcein AM within microwells. B) Electroporation of HEK 293T cells growing within microwell arrays. Electroporation parameter set used: 500 V cm⁻¹, 1 ms pulse-width and 1 square pulse. Top row: brightfield image of cells post-electroporation. Bottom row: fluorescence image of cells at appropriate molecule compatible excitation/emission spectra. Left column: electroporation of propidium iodide. Middle column: electroporation of Alexa Fluor 488-labeled siRNA. Right column: electroporation of plasmid encoding GFP. C) Estimation of transfected cell count in an individual microwell using software identification of the microwell edges as physical markers for identification of microscale culture spatial locations.

Figure 4

Electroporation of primary macrophage cells within microwells and automated image analysis of microcultures. A) Electroporation of primary macrophages within microwell arrays. All images taken at 2 hr post-electroporation. Left column: Phase contrast image post-EP. Middle column: Transfection assay using Propidium Iodide post-electroporation. Right column: Live assay with Calcein AM post-EP. Top row: Control pulse (CP) 100 V cm⁻¹, 1 ms pulse-width and 1 square pulse. Bottom row: Electroporation pulse (EP) 600 V cm⁻¹, 1 ms pulse-width and 5 square pulses at 1 Hz. B) Automated microwell edge detection and image analysis estimating total cell nuclei (Hoechst), transfected cells (Propidium Iodide) and viable cells (Calcein AM) in individual microwells of the images shown in Fig. 4A.

Figure 5

Effect of electrode configuration on electroporation efficiency variation across microwell array substrate. A.) Finite Element Analysis FEMLAB (Comsol, CA) simulations of electric field across a 484 microwell array on a conductive microscope slide using either a single or double cathode configuration. B) Analysis of mean and standard deviation, as a result of the simulations, by binning electric fields at the center of each microwell of the array using the single or double cathode configuration. Individual columns are binned at 10 V cm⁻¹. E_t shows the estimated threshold of electric field required for ~50% electroporation efficiency relative to maximum as determined by matching experimental data to simulated electric field values. C) Parallel electroporation of mammalian cells in 400 microwells of the array (the outermost microwells of the 484 microwell array were excluded to avoid potential edge effects) with propidium iodide using previously optimized parameters with the single cathode configuration or D) double cathode configuration. Insets on the left and right show a zoomed out 4×4 array from the left and right sides of the larger 484 microwell array. Bar graphs indicate electroporation efficiency (measured as relative fluorescent units, RFU, of internalized propidium iodide) using a single or double cathode configuration. Individual bars represent mean values across the 20 rows of a single column plotted left to right of the microwell array. E) Cell viability and transfection efficiency of a 400 microwell array 1 hr post-electroporation, with a double cathode set up. The adjacent plots on the top and side of each image indicate fluorescence intensity in the central row and column in each case.

Figure 6

Parallel electroporation of Alexa 488 fluor-siRNA into HEK 293T cells contained within the microwell array. Electroporation was conducted using the double cathode method with an electric field intensity of 500 Vcm⁻¹, 1 ms pulse-width and 1 square pulse. Two hours post-electroporation, the cells were washed in PBS, fixed and scanned for green fluorescence on a ProScanArray HT confocal laser slide scanner (Perkin Elmer, MA). Prior to scanning, the microwell stencil was removed with forceps to eliminate auto-fluorescence from the stencil material. The artifact in the lower-left corner is due to handling with forceps.

Figure 7

Microarraying within microwells using an iterative process of imaging and calibration. A) A blank slide is spotted with printing buffer to determine the X-Y offset error from a microwell array slide to be printed on. Both slides are independently scanned and their images overlaid in software to determine the offset. B) After re-calibrating the microarrayer with the offset error, Alexa Fluor 488-labeled siRNA was spotted directly into alternating wells of the microwell array.