To generate a single cell suspension of chick embryo wing bud mesenchyme in sucrose electroporation buffer (SEB; 272 mM sucrose and 1 mM MgCl2 in 7 mM K2HPO4, pH 7.4; all reagents from Sigma-Aldrich, St. Louis, MO), we followed a previously detailed method for preparing micromass cultures (12), with minor adjustments. Wing buds were excised from Hamburger-Hamilton stage 23/24 chick embryos, incubated for 1 h in 0.8 units/mL dispase diluted in Hank's enzyme-free cell dissociation buffer, and adherent epithelia were manually removed. Isolated limb mesenchymal tissue was pooled, incubated for 25 min at 37°C in 0.25% trypsin-EDTA, and dissociated into a single cell suspension of 2.0 × 107 cells/mL in room temperature micromass culture medium (MCM) composed of F12 Nutrient Mixture containing L-glutamine, 10% Mesenchymal Stem Cell (MSC) Qualified fetal bovine serum (all reagents from Life Technologies, Grand Island, NY), 100 units/mL penicillin G, and 100 μg/mL streptomycin (both from Sigma-Aldrich). 1.0 × 106 cells were transferred to a 1.5 mL microtube and pelleted by centrifugation at 150 × g for 5 min. MCM supernatant was removed and the pellet was resuspended in 250 μL of 4°C sterile SEB.
Plasmid DNA (1–2 μg) was added to the cells and triturated prior to transferring the entire volume of suspension to a 2.0 mm gap BTX Electroporation Cuvette Plus (Harvard Apparatus, Inc., Holliston, MA) that had been cooled overnight at 4°C. Cuvettes containing cell suspension/DNA mixture were incubated at 4°C for 5 min prior to square wave pulse electroporation. We used the BTX ECM 830 Square Wave Electroporation System (Harvard Apparatus) to pulse the mesenchymal cells suspended in SEB, and we varied the voltage, pulse number, pulse length, and duration between pulses (interval) in order to optimize transfection efficiency and cell viability without affecting chondrogenic differentiation potential (see Figure 1). Immediately following pulse delivery, cuvettes were placed at 4°C for 10 min. This 4°C incubation was followed by a gradual warming of the cells wherein the cuvettes were maintained first at room temperature for 5 min and subsequently at 37°C for an additional 5 min. This 20 min post-electroporation incubation is hypothesized to allow viable cells to sink to the bottom of the cuvette while any non-viable cellular debris floats to the surface (3). The lower four-fifths volume of cell suspension (~200 μL) was subsequently drawn up with a BTX plastic pipette and transferred to a 1.5 mL microtube. Caution was exercised to avoid including any of the top one-fifth volume (~50 μL). A 2 μl aliquot of cell suspension was removed for a hemacytometer count, and the remaining cells were pelleted by centrifugation at 150 x g for 5 min. SEB supernatant was removed, and the cells were resuspended at a density of 2.0 × 107 cells/ mL in 37°C MCM. Micromasses were established by spotting 10 μL drops of cell suspension onto Nunc Permanox chamber slides (ThermoFisher Scientific, Waltham, MA) and incubated at 37°C in a 5% CO2 atmosphere for 60 min to permit cell attachment prior to flooding of the wells with MCM supplemented with 20 μg/mL ascorbate (Sigma-Aldrich). All micromasses were cultured for three days.
We first wished to determine the effect of varying pulse number and voltage on transfection efficiency and cell viability. Mesenchymal cells were pulsed 2 or 3 times (150 μs length, 100 ms interval) with voltages increasing incrementally by 100 V from 200 to 600 V. We transfected the cells with 1 μg of the Monster Green Fluorescent Protein (GFP) Vector (Promega, Madison, WI) in order to monitor transfection efficiency by FACS. FACS analysis carried out with a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) and FlowJo software (Tree Star Inc., Ashland, OR) revealed that 3 – 400 V pulses resulted in a transfection efficiency of ~65%, the highest observed (Figure 1A). As importantly, PicoGreen (Promega) DNA quantitation of 3-day micromasses showed that 3 – 400 V pulses did not affect the number of cells present in culture relative to unelectroporated controls (Figure 1B). In order to evaluate the viability of transfected limb mesenchyme, we carried out an MTS tetrazolium salt assay using the CellTiter 96 AQueous Non-Radioactive Cell Proliferation kit (Promega) and found that 3 – 400 V pulses only slightly reduced the number of viable cells present in 3-day micromass cultures (Figure 1C). After determining that 400 V was an effective voltage with which to pulse embryonic limb mesenchyme, we varied other parameters, such as pulse number, pulse length, interval, and DNA concentration, while maintaining constant voltage. However, we could not significantly improve upon the ~65% efficiency achieved with 3 – 400 V pulses of 150 μs length and 100 ms intervals. Also, changes in methodology often resulted in decreased micromass culture cell density (Figure 1D).