Facile Production of Large‐Area Cell Arrays Using Surface‐Assembled Microdroplets

Abstract Techniques that enable the spatial arrangement of living cells into defined patterns are broadly applicable to tissue engineering, drug screening, and cell–cell investigations. Achieving large‐scale patterning with single‐cell resolution while minimizing cell stress/damage is, however, technically challenging using existing methods. Here, a facile and highly scalable technique for the rational design of reconfigurable arrays of cells is reported. Specifically, microdroplets of cell suspensions are assembled using stretchable surface‐chemical patterns which, following incubation, yield ordered arrays of cells. The microdroplets are generated using a microfluidic‐based aerosol spray nozzle that enables control of the volume/size of the droplets delivered to the surface. Assembly of the cell‐loaded microdroplets is achieved via mechanically induced coalescence using substrates with engineered surface‐wettability patterns based on extracellular matrices. Robust cell proliferation inside the patterned areas is demonstrated using standard culture techniques. By combining the scalability of aerosol‐based delivery and microdroplet surface assembly with user‐defined chemical patterns of controlled functionality, the technique reported here provides an innovative methodology for the scalable generation of large‐area cell arrays with flexible geometries and tunable resolution.


S2
We prepared thin PDMS films using a 10:1 base to curing agent ratio, by pouring the degassed prepolymer mixture onto 4-inch silicon wafers (University Wafer Inc.). We used a universal applicator (Zehntner GmbH Testing Instruments, Switzerland) to obtain elastomeric films with a thickness of 500µm. PDMS was cured for 48 hours at 60°C in a convection oven. We cut the prepared films into 30 mm × 6 cm strips and stirred them in ethanol for additional 48 hours at room temperature to remove un-crosslinked monomers, as demonstrated by Regehr et al. [S1] After a rinsing step with DI water, we dried the PDMS strips first under pressurized nitrogen and then inside a vacuum pump for 1 hour to remove ethanol.

Surface functionalization/Micropatterning of elastomeric films
Elastomeric substrates were oxidized in an oxygen plasma chamber (Plasma Etch Inc., Carson City, NV, Model# PE-25 Series) for 10 seconds at a power of 15W, and at ~201.1 mTorr of O2.
Prior to cell deposition, native or oxidized PDMS samples were incubated in PBS for 1 hour at 37°C, rinsed with filtered Millipore water and dried.
For the synthesis of wettability micropatterns, we placed MIMIC masks onto the PDMS surface before the oxidation process. [S2] After removing the masks, we immersed the samples in a PLL solution (50 µg/mL in PBS) for 1 hours at 37°C and rinsed them with Millipore water and dried.

Cell culture
The human epidermoid carcinoma cell line A-431 was obtained as a kind gift from Dr. Kathleen Green's laboratory from Northwestern University. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, and 1% Penicillin Streptomycin (10,000 units mL −1 ), in 75 cm 2 flask sat 37 °C in a humidified atmosphere with 5% carbon dioxide.

S3
Cell lines at 90% confluency were detached from the cell culture flask using trypsin treatment (0.25% Trypsin-EDTA) and resuspended in 5mL DMEM. After a centrifugation (100g, 5 minutes) and a rinsing step with PBS, we discarded the supernatant and re-suspended the cell lines in DMEM supplemented with HEPES (20mM) and 5wt. % Glycerol, pre-warmed at 37°C to a final concentration of 2×10 6 cells/mL.

Spraying, aerosol droplet formation and delivery
We loaded the spraying device with 500 µL of cell suspension at secured it on the syringe pump.
Cell spraying was performed at 0.01-0.1 mL/min and 0-70 kPa. We set a distance of 15 cm between the spray nozzle and the samples. The samples and spraying device were placed in a humidity chamber, equipped with a humidity sensor (RH> 90%) to prevent the evaporation of microdroplets.

Stretching and droplet assembly
We fabricated a stretcher to apply mechanical strain to PDMS strips using solid object printing. [S3] The components were designed in a CAD program (Inventor Professional by Autodesk Inc.) and printed in ABS using a 3D-printer (Dimension Elite, Stratasys Ltd.). ( Figure   S4). We applied a uniaxial strain by stretching the substrate along its length (L). An increase in the initial length (L) to a final length (L') would provide a strain ε = (L'−L)/L.

Microscopic characterization
Bright field and fluorescence microscopy of the functionalized wettability patterns was performed using a Zeiss microscope (Axio Scope.A1) equipped with a AxioCam MRc5 camera, and the ZEN-Pro software (version 2.3).

Fluorescence cell labeling and imaging
To assess cell viability, we labeled living cells with Calcein-AM (0.5 µM, GFP) and dead cells with Propidium Iodide (3µM, TRITC). Cells were incubated in the labeling solution for 30 minutes at 37°C. We then incubated the cells in DMEM at 37°C prior to imaging. We counted the number of live green cells (NLive) and the number of dead red cells (NDead) for each sample S4 through fluorescence imaging and obtained: Viability (%) = NLive /(NLive+NDead)*100. A total of 6 samples were performed for each set of spraying conditions during two independent experiments and an average of 2,000 cells were counted for each sample. Cell viability results obtained through this method are reported in Figure 1D and Figure 2H in the main text and  and concentric circles (C) with A-431 cells stained with CellTracker™ green or CellTracker™ orange, assembled on elastomeric membranes containing 300µm FITC-labeled PLL patterns.
Cross section fluorescence intensity levels for green and red channels extracted from (C).