P 2000

Zygotes were prepared for microinjections with a membrane softening pretreatment. Embryos were exposed to a freshly made 1:1 solution of 1 M sucrose and 0.25 M sodium citrate for 20 s followed by three quick rinses with FSW (Meyer et al., 2010 (link)). Individual zygotes were pressure injected with quartz glass needles (QF100-70-10; Sutter Instruments) that were pulled using a micropipette puller (P-2000; Sutter Instruments). Needles were filled with a cocktail of 800 μM MO antisense oligonucleotides, nuclease-free water and a 1:10 dilution of 20 mg/ml red dextran reconstituted in FSW (Texas Red, Molecular Probes). The volume of microinjected MO cocktail was typically between 0.5 and 2% of the total embryo volume, as estimated from injection into an oil drop, and resulted in a final MO concentration of 4-12 µM. For rescue experiments, needles were filled with 100 ng/μl of Ct-Smad2/3 mRNA plus dextran, 100 ng/μl Ct-Smad2/3 mRNA 3′ 6×His tag plus dextran, or a combination of 100 ng/μl Ct-Smad2/3 mRNA with 800 μM MO plus dextran. Injected and uninjected animals from the same brood were raised in FSW containing 60 μg/ml penicillin and 50 μg/ml streptomycin in separate dishes, and compared to assess overall brood health. A brood was considered healthy if more than 90% of the uninjected animals developed normally.

PB micro‐patterns were printed using a meniscus‐guided printing approach. Glass micropipettes with IDs of 2, 10, 20, and 30 µm were obtained using a pipette puller (P‐2000, Sutter Instruments). The ink was filled through the back of the micropipette using capillary forces with no applied pressure. Indium tin oxide‐coated glass with a resistance of 4–6 Ω cm⁻² (AMG Tech) was used as the printing substrate. During the printing process, the pipette motion corresponding to the printed paths was determined using parameterized G‐code scripts and controlled by three‐axis stepping motors with 250 nm positioning accuracy. The nozzle motion corresponds to the printed paths. The printing process was observed in situ using a high‐resolution monitoring system consisting of an optical objective lens (50 ×, Mitutoyo) and complementary metal oxide semiconductor camera (BFS‐U3_200S6C, Flir). The printed patterns were treated thermally on a hot plate for 9 s at 120°C for reduction.

The Institutional Animal Care and Use Committee of Xuzhou Medical University provided full approval for this research (IACUC-2202049). BALB/c nude mice were acquired from GemPharmatech, Inc. (Nanjing, China). BALB/c nude mice were obtained from GemPharmatech, Inc. (Nanjing, China). The participants were randomly divided into two groups (n = 5, with no blinding performed in either group). Cells were injected subcutaneously into the axilla of the nude mice, with each mouse receiving a dosage of 1 × 10⁷ cells. The maximum diameter of the tumor was measured every 2 days after implantation. After 12 days, the mice were euthanized, and the tumor was removed and weighed. AB line zebrafish were acquired from China Zebrafish Resource Center (Wuhan, China). They were randomly divided into two groups (n = 16, no blinding was performed, respectively). After A549 cells incubated with CFSE (Thermo Fisher, Waltham, MA, USA), xenograft perivitelline injection were conducted in 2 day-post-fertilization zebrafish via a Picoliter Microinjector (PLI-100A; Warner Instruments, Hollister, MA, USA) with a glass capillary needle (Sutter, Q100-50-10) made on a laser-based needle puller (P-2000; Sutter, Sacramento, CA, USA). Observed cell extravasation in the caudal vasculature at 3 days post-transplant and imaging on a Zeiss AxioZoom V16 fluorescence microscope.

Volumetric changes in the droplets were used to characterize the injection volume of the micropipette. The micropipettes were routinely fabricated from glass capillary tubes (BF100-50-15, Sutter Instruments) using a programmable laser-base pipette puller (P-2000, Sutter Instruments). The tip diameter of the fabricated micropipette was measured by scanning electron microscopy, and micropipettes with a tip inner diameter of 0.5 μm were selected. After back filling with 1 μL of deionized water, the micropipette was fixed to the microrobot and connected to the microinjector, which controls the injection pressure and time. The pipette tip was then immersed into the mineral oil and water droplets were dispensed by adjusting the injection pressure or time. Since the droplets formed were very small, multiple injections were required to produce visible size changes in the droplets. Droplets were assumed to be spherical, and their volumes were determined by measuring the area of each droplet from captured images (See Supplementary Fig. 4a). The amount of injected substance could be determined by quantitatively injecting a certain volume of a substance with known concentration. Once the calibration results of injection volume versus injection pressure and time were obtained, the calibrated micropipette could be used for cell injection.