Supplementary Materials for

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1 advances.sciencemag.org/cgi/content/full/4/8/eaat1659/dc1 Supplementary Materials for Acoustophoretic printing Daniele Foresti*, Katharina T. Kroll, Robert Amissah, Francesco Sillani, Kimberly A. Homan, Dimos Poulikakos, Jennifer A. Lewis* *Corresponding author. (D.F.); (J.A.L.) The PDF file includes: Published 31 August 2018, Sci. Adv. 4, eaat1659 (2018) DOI: /sciadv.aat1659 Materials and Methods Supplementary Analysis Fig. S1. Horizontal acoustophoretic printing of liquid droplets. Fig. S2. Acoustophoretic printing of viscous fluids. Fig. S3. The Fabry-Perot resonator. Fig. S4. Acoustophoretic 3D printing of aqueous polymer solution. Fig. S5. Droplet trajectory accuracy and distribution. Fig. S6. Acoustophoretic printing of honey droplets. Fig. S7. Acoustophoretic bioprinting. Fig. S8. Classical acoustophoretic levitator. Fig. S9. Scaling and nozzle effects on acoustophoretic forces. Fig. S10. Pressure drop as function of nozzle diameter. Table S1. Primary antibodies and markers of interest. Legend for Movies S1 to S5 Other Supplementary Material for this manuscript includes the following: (available at advances.sciencemag.org/cgi/content/full/4/8/eaat1659/dc1) Movie S1 (.mp4 format). Acoustophoretic printing of liquids using different nozzle diameters d and equivalent accelerations g eq. Movie S2 (.mp4 format). Horizontal acoustophoretic printing of a 1:1 water-glycerol mixture. Movie S3 (.mp4 format). Acoustophoretic printing of single droplet of honey compared to simple dripping. Movie S4 (.mp4 format). Confocal z-stack movie and 3D renderings of acoustophoretically printed droplets composed of hmscs suspended in a collagen I matrix. Movie S5 (.mp4 format). Acoustophoretic printing of liquid metal droplets, in which 3D structures are assembled in a contact-free manner (real time).

2 Materials and Methods Supplementary Analysis Role of capillary forces and pressure drops. To decrease droplet size at detachment, one can either alter the capillary force F c or the gravity force g, i.e., an external force that acts on the pendant drop. Independently of the host medium, the nozzle diameter d can be varied from less than one micron to several millimeters allowing one to tailor the drop volume, V. Unfortunately, for small d, the pressure drop is quite large. The total pressure drop p tot arising from two contributions: the capillary pressure drop p σ and the viscous pressure drop p µ. p σ is determined by the Young-LaPlace equation, p σ = σ (1/R + 1/R ), where R and R are the two radii describing the drop curvature at the nozzle tip. In our typical axisymmetric configuration, R = R. When the meniscus at the liquid-gas interface approaches its minimum diameter corresponding to 2R= 2R =2R =d, the capillary pressure reaches its maximum value of, p σ = 4σ/d. The viscous pressure losses can be modeled by the Hagen-Poiseuille equation, dp µ /dz = µq/2µd i (z) 4, where the inner diameter of the nozzle d i varies as a function of height (z). By integrating this equation over the nozzle length L n along the z-axis, the total p µ is obtained. Figure S10 shows pressure drops for a typical tapered nozzle configuration. Even for a low viscosity liquid (e.g., water) and a relatively low flow rate (q= 10 µl/min), the total pressure losses quickly increases when d < 10 µm. Importantly, when printing complex fluids, such as those that contain colloidal particles or human cells, the nozzle diameter should be at least one order of magnitude higher than then characteristic size of these building blocks to avoid clogging (1). Acoustophoresis. For in-air acoustophoresis (15), in which the acoustic host medium is a gas, force enhancement can be achieved by generating an acoustic standing wave, which is

3 established between an emitter and a reflector (29), Fig. S8B. The resonant condition requires the distance H between the oscillating source and the reflective surface to be a multiple of half of the acoustic wavelength. When H /2, a pressure node is generated in the middle of the levitator: small samples (R< /2) are pushed towards its center at about H /4. Injection and ejection of liquid samples in and out an acoustic levitator are typically performed by manually inserting a needle at the location of the levitated sample (15). However, this simplistic approach to manually introducing droplet in the acoustic field through a needle circumvents a crucial limitation of standing waves levitators used for drop handling, i.e., outcoupling of the sample from the levitating device (Fig. S8C). Indeed, after overcoming the capillary forces, the acoustic force traps the ejected drop at the location of the acoustic node, precluding any form of droplet ejection (15). New approaches utilizing acoustic tweezers, both in a liquid (13,14), and a gaseous (12) medium, may represent an interesting alternative to classical levitators. Since they do not require a reflector, they possess the advantage of generating a trapping force with a single-sided array of ultrasonic emitters or pre-programmed hologram. However, the magnitude of the applied acoustic force is currently limited (i.e., 1-10 g, levitation of water), and while it can be localized, one must use an open field, since any interaction with a substrate would be strongly coupled to the ultrasonic field (i.e., reflected waves) hindering both drop ejection and accurate deposition on a given substrate.

4 Supplementary Figures Fig. S1. Horizontal acoustophoretic printing of liquid droplets. (A) Acoustophoretic forces act along the nozzle axis. When the acoustophoretic forces are significantly higher than the gravitational force, different ejection angles δ can be achieved. (B) The ballistic trajectory of the ejected droplets (composed of a 1:1 water-glycerol mixture) enables accurate printing even in the direction normal to gravity. [Note: The orange dashed lines aid the visualization of the droplet trajectories.]

5 Fig. S2. Acoustophoretic printing of viscous fluids. (A) Maximum ejection frequency for liquids of varying viscosity calculated for Ca < 1, nozzle diameter d = 140 µm and surface tension = 35 mn/m. (B) Semilog plot of droplet volume as a function of surface tension, σ, for water-sds solutions using a nozzle diameter, d = 60 µm. (C) Time evolution of ejection of a single droplet of honey using a glass nozzle, d = 70 µm. Simple dripping (g eq = 1) and acoustophoretic ejection (g eq = 36) show a similar behavior, with a thin neck forming upon droplet detachment.

6 Fig. S3. The Fabry-Perot resonator. Validation of the numerical model reported by Chistensen et al, which is based on a square cavity. We obtain an equivalent resonator with a circular cross section by equating their cross-sectional areas. [Note: Chistensen et al calculated the transmittance (normalized to the cavity cross sectional area) of the cavity, while we report the peak of the P rms within the cavity]. To optimize the subwave for a fixed frequency, it is more convenient to consider the H h -d h parametric space (right). The parametric space H h -d h identifies optimal Fabry-Perot resonances. The maximum P rms within the cavity is plotted for each combination of parameters. The circle indicates the parameters used for the subwave.

7 Fig. S4. Acoustophoretic 3D printing of aqueous polymer solution. (A) The print path is obtained directly from the CAD file through the software Slic3r. The distance between adjacently printed features takes into account droplet spreading on the glass substrate, which leads to a final diameter of roughly 500 µm. Each layer has an approximate thickness of 200 µm. (B) Image sequence highlighting the evolution of a 3D polymer architecture in the form of an H-shape, as sequential layers of an aqueous polyethylene glycol (PEG) solution are printed.

8 Fig. S5. Droplet trajectory accuracy and distribution. A Gaussian distribution is obtained along the print path, in which the distributions of droplet positions broadens with an increase nozzle-substrate distance L s. Ink: water-glycerol 50%, nozzle d=55 µm, and g a = 38 g. Fig. S6. Acoustophoretic printing of honey droplets. Optical images of honey droplets printed on the surface of white filling within an Oreo cookie.

9 Fig. S7. Acoustophoretic bioprinting. (A) A acoustophoretic printing apparatus for patterning pure and stem-cell laden collagen-based inks, equipped with a computer-controlled motion-stages, syringe pump, acoustophoretic force generator, ink temperature and humidity. UV air purification ensures a sterile environment during the printing process. (B) (left) hmscs in printed droplets (g eq = 43 g) showing immunofluorescence staining panels in which appropriate multipotency markers are maintained, CD105 + /CD90 + /CD45 -, akin to the same cells grown on a 2D control (plastic substrate, right).

10 Fig. S8. Classical acoustophoretic levitator. (A) In this case, liquid droplets are trapped within the acoustic field, i.e. outcoupling is not possible. (B) A classical standing wave levitator prevents the outcoupling of the sample from the system (C) Acoustic levitator and subwave geometries of the respective numerical models. (D) A highly localized field is required for droplet outcoupling and accurate deposition. (E) The acoustic chamber is custom designed and manufactured in the form of a rectangular chamber (15x65x7.5 mm 3 ) using CNC milled acrylic parts. The Fabry-Perot resonator is drilled within the bottom of the acoustic chamber (Fig 2B). The emitter (15x15mm plate) is designed to resonate at a driving frequency of around 25 khz. The magnetostrictive transducer excites the emitter.

11 Fig. S9. Scaling and nozzle effects on acoustophoretic forces. (A) Quadratic relationship between voltage u and g eq, which gives rise to a F a P 2 dependence within the subwave (nozzle d = 13 µm; water). (B) Calculated acoustophoretic force, F a, acting on a pendant sphere as a function of the ratio of unconstrained drop of radius, R, to the nozzle diameter, d. (C) Schematic view of subwave device showing the drop position relative to the nozzle tip, where the drop center (z-height) at detachment is given by the drop radius R plus the nozzle tip height. Fig. S10. Pressure drop as function of nozzle diameter. Meniscus ( p σ ), viscous ( p µ ), and total ( p tot ) pressure losses as function of d. Tapered nozzle with a tapering length of 3 mm and d i = 0.58 mm. Fluid: water (µ = 1mPa s); flow rate: 10 µl/min. In our typical configurations, the viscous losses in the cylindrical part of the glass nozzle and tubing are negligible.

12 Supplementary Tables Table S1. Primary antibodies and markers of interest. Antibody Source Catalog # Host Species & Reactivity Dilutio n CD45 ThermoFisher MA5- Rat anti-human 1: CD90 Abcam ab Rabbit antihuman 1:200 CD105 Abcam ab11414 Mouse antihuman 1:250 Donkey anti-rat Life Technologies A Donkey anti-rat 1:500 AlexaFluor488 Donkey antirabbit AlexaFluor555 Life Technologies A Donkey antirabbit 1:500 Donkey antimouse AlexaFluor647 Life Technologies A Donkey antimouse 1:500

13 Supplementary Movies Movie S1. Acoustophoretic printing of liquids using different nozzle diameters d and equivalent accelerations g eq. Movie S2. Horizontal acoustophoretic printing of a 1:1 water-glycerol mixture. Movie S3. Acoustophoretic printing of single droplet of honey compared to simple dripping. Movie S4. Confocal z-stack movie and 3D renderings of acoustophoretically printed droplets composed of hmscs suspended in a collagen I matrix. Movie S5. Acoustophoretic printing of liquid metal droplets, in which 3D structures are assembled in a contact-free manner (real time).