Application: Chemyx Fusion 4000 Acoustofluidic Sonoporation For Gene Delivery To Human Hematopoietic Stem And Progenitor Cells

The acoustofluidic devices are comprised of a piezoelectric lead zirconate titanate (PZT) transducer (SMPL26W16T07111, StemInc), a functionalized glass microcapillary, and a glass slide that provides a supporting substrate. The PZT transducers were mounted onto the glass slide with a thin layer of Devcon 5-min epoxy adhesive (300007-392, VWR) after soldering 30-gauge wire to the front and back electrodes of the PZT transducer. A functionalized glass microcapillary was attached onto the transducer with adhesive and cured for 30 min. Polyethylene tubing (PE-50, Instech) was connected to both ends of the microcapillary and sealed with small drops of epoxy. After curing, the tubing was secured to the glass slide with double-sided tape and tested for leaks. The resonant frequency for each device was determined with a vector network analyzer (VNA-120, Array Solutions).

Fabricated acoustofluidic devices were vertically aligned in a custom-built stage that aligned the cross-section of the microfluidic channel within the optical path of a Nikon TE300 optical microscope. Tubing was connected to a syringe by inserting a 23-gauge needle adapter, and the syringe flow rate was controlled with a syringe pump (Fusion 4000, Chemyx). The PZT transducers were excited with a sinusoidal wave at the desired frequency and an amplitude of 40 V_p-p with a signal generator (81150A, Agilent) and a broadband amplifier (25A250B, Amplifier Research).

The APTES-treated glass capillaries were prerinsed with 5 mL of 70% ethanol, followed by 3 mL of 1× phosphate-buffered saline (PBS) solution (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, Gibco) before introducing plasmid DNA. The eGFP expression vector (pCMV-GFP, Plasmid 11153, Addgene) or Cy3-labeled DNA (Integrated DNA Technologies) was diluted to 50 ng/mL in 1× PBS and was zone-loaded with a flow a rate of 3.33 μL/min for 30 min with a three-way valve (CMA 110, Harvard Apparatus) connected to the acoustofluidic device. Cells were then dispersed at a density of 3 million cells/mL (except for CD34⁺ HSPCs) in a delivery medium consisting of 1% (vol/vol) Pluronic F-68 (Gibco), 1× PBS, and 0.1 mg/mL of eGFP-expressing plasmid and collected into a 1-mL syringe. Cells were introduced into the glass capillary at the designated flow rate, and acoustofluidic-treated cells were collected into a sterile tube. The peak-to-peak input voltage was applied to the PZT transducer after the initial drop of solution was collected to ensure that cells were under continuous acoustofluidic treatment while passing through the device. Additional PBS was flowed through the device for 3 min after the cell syringe reached depletion to collect any remaining cells in the dead volume of the device. Upon collection, cells were incubated in the delivery medium for 10 min to facilitate membrane recovery and biomolecule diffusion. Cells were then centrifuged at 500 × g for 5 min and dispersed in their respective culture media. Viability was determined using a Cell Countess II (Invitrogen) and 0.4% trypan blue (Invitrogen).