*2.3. Bioprinting Process*

This sca ffold-free bioprinter was controlled using an Arduino (Arduino Uno Rev3, Arduino, Somerville, MA, USA) and a custom-written script. It controlled the stepper motor (NEMA 14, Lin Engineering, Morgan Hill, CA, USA) for moving the print head, the servomotor (5V servomotor, Arduino, Somerville, MA, USA) for moving the spheroid stage, the vacuum pump for aspirating the spheroids, and the end stop (Mechanical End Stop, SparkFun, Niwot, CO, USA) mechanism for calibrating the stepper motor prior to beginning the printing process. The bioprinter was designed to first automatically calibrate to ensure that the print head is in the proper position before beginning. Once it has calibrated, the Arduino program prompts the user to load the spheroids into the spheroid stage in the round bottom plate (Figure 2B). Next, the user is asked to input the exact number of layers to print, between one and five. Once this value is entered, the printing process begins (Video S1). First, the print head is lowered down into the spheroid container, and the vacuum pump is activated to aspirate one layer of spheroids (Figure 2C,D). While the vacuum is still on, the print head is raised out of the container, and the spheroid stage is moved out of the way. The print head with spheroids is lowered straight down onto the needles in the array bath (Figure 2E). Once the print head is placed in the correct position, the vacuum pump is turned o ff, and the spheroids are left on the needles. This process is repeated until all layers have been placed on the needles, with each subsequent layer being placed ~800 μm higher than the previous layer. After all layers are completed, the array bath is removed from the system and placed in the incubator for at least 24 h for fusion to take place. Once the spheroids have adequately fused, the stainless steel plate is used to assist in the removal of the fused tissue for further processing (Figure 2F).

**Figure 2.** Bioprinting process. Spheroid culture can take place in ( **A**) a bioreactor or a standard round-bottom culture plate, and once the spheroids are approximately 800 μm in diameter, they can be placed in (**B**) the round-bottom dish on the spheroid stage. Once the desired number of spheroids are placed in the dish, ( **C**) the print head is lowered down to the center of the dish, and the vacuum is turned on, capturing ( **D**) a single layer of spheroids. With the vacuum still on, the print head is lowered down to the needles (**E**), placing the spheroids on them. After the required number of layers is achieved, (**F**) the spheroids are allowed to fuse and then later removed for further processing.

### *2.4. Vacuum and Print Head Testing*

Glass beads were used to determine the e fficiency of the vacuum and print head system. Glass beads approximately 700 μm in diameter (±10% diameter, Zirconia Beads, BioSpec Products, Bartlesville, OK, USA) were used in place of cultured spheroids because of the number of tests that needed to be completed and the non-sterile conditions of the testing area. This diameter was chosen because it is

similar to the 800 μm diameter of the spheroids that our lab has previously produced [27]. These beads were held on the spheroid stage in the PTFE container. To help counteract aggregation during the testing process, the beads were placed in a solution of phosphate-buffered saline with 0.1% Tween 20 (PBST), which also allowed to simulate spheroids in media. To determine the different efficiencies of these experiments, the print head was lowered onto the spheroid stage and the vacuum was turned on, which allowed the print head to pick up the beads. After a given time, the number of aspirated beads were counted and calculated as a percentage of the total number of holes available to collect the beads (e.g., 15 beads aspirated into an array of 16 possible holes equates to an efficiency of 93.8%).

### *2.5. Preparation of Alginate Beads*

Alginate beads were chosen as a reasonable spheroid surrogate to test the ability of the bioprinter to place spheroids on the needle array effectively. As mentioned previously, spheroids that would be used with this system are approximately 800 μm in diameter, so the alginate beads needed to have a similar diameter. These beads were generated by adding a solution of 1.5% sodium alginate (Cat. No. 218295, MP Biomedicals, Irvine, CA, USA) in deionized water (DI) water to a 100 mM solution of calcium chloride (Cat. No. 4901, Sigma Aldrich, St. Louis, MO, USA) in DI water [28]. The alginate solution was added using a 22-gauge serological needle dropwise into the calcium chloride solution which was being stirred using a magnetic stir bar. The beads were separated according to their approximate diameter and measured using image analysis.

### *2.6. Preparation of Human Induced Pluripotent Stem Cell (hiPSC) Spheroids*

Human induced pluripotent stem cells (hiPSCs) were cultured on Matrigel-coated dishes at 37 ◦C under 5% CO2 with mTeSR (Stem Cell Technologies, Vancouver, BC, Canada). Fresh medium was added daily until the cells reached 90% confluency. The cells were seeded and grown in suspension according to the manufacturer's protocol [29]. Briefly, the cells were passaged by treating with Gentle Cell Dissociation Reagent (Stem Cell Technologies, Vancouver, BC, Canada) for 7 min. The cells were carefully scraped off the dish and seeded at a density of 5 × 10<sup>5</sup> cells per mL in 30 mL of mTeSR 3D Seed Media (Stem Cell Technologies, Vancouver, BC, Canada). This solution was then cultured in a 125 mL shaker flask at 70 rpm with daily media additions of 3.4 mL. Prior to their use with the bioprinter, the spheroids were filtered through a 500 μm reversible strainer (pluriSelect, El Cajon, CA, USA) and collected in 5 mL of mTeSR.

### *2.7. Statistical and Image Analysis*

Data are shown in the form mean ± standard error of the mean (SEM). Significance was chosen as p < 0.05. This was determined using both one- and two-way analysis of variance (ANOVA) along with the Student's t-test. These analyses were performed utilizing Microsoft Excel's data analysis software package.

For image analysis, ImageJ (Version 1.52o with the addition of plug-ins for colocalization (Colocalization Finder, Version 1.2, Institut de Biologie Moleculaire des Plantes, Strasbourg, France) was used to estimate the overlap coefficient between each layer of beads and to generate representative pictures. Also, it was used in the diameter measurements of the alginate beads that were synthesized. Images and videos in this experiment were taken with a stereomicroscope (Olympus SZ61, Olympus, Center Valley, PA, USA), a phase-contrast microscope (AMG EVOS FL Imaging System, Thermo Fisher Scientific, Waltham, MA, USA), and a Canon camera (Canon EOS Rebel T7i, Canon, Tokyo, Japan).
