To create the cardiac spheroids used for Fig. 1, D and E and fig. S2, we adapted a previous protocol by Lian et al. (28). Briefly, PGP1 iPSCs were grown to 80 to 95% confluency on T225 plates in mTeSR1 medium. Cardiac differentiation media was prepared by combining 500 ml of RPMI 1640 (Thermo Fisher Scientific) with either 10 ml of B27 without insulin (to form cardiomyocyte differentiation medium, CDM) or 10 ml of B27 (to form cardiomyocyte maturation medium, CMM). On day 0, differentiation was initiated by switching the mTeSR1 media to CDM and 6 μM CHIR99021 for 24 hours. The following media changes were then made: day 1, CDM; day 3, CDM and 2 μM iWR1 (BioGems; CDMI); days 5 and 7, CDM; day 9 (or by day of beating), CMM; days 11 and 13, RPMI without glucose supplemented with 2% B27 (to purify cardiomyocytes); and day 15, CMM. In a separate T225 flask, primary ventricular normal human cardiac fibroblasts (Lonza) were cultured in stromal cell growth medium (Lonza). Next, cardiomyocytes, at day 17, and fibroblasts were passaged by rinsing with PBS and then incubated with TrypLE for 9 min (for cardiomyocytes) or 5 min (for fibroblasts) at 37°C. Next, the TrypLE was rinsed using 37°C DMEM, and the cells were pelleted into separate 15-ml tubes via centrifugation at 220g for 5 min. Both cell types were resuspended in cardiac spheroid medium (CSM), comprising DMEM with high glucose (American Type Culture Collection) containing 10% fetal bovine serum (FBS) and penicillin-streptomycin (100 U/ml). The cells were counted and combined at 70% cardiomyocytes and 30% fibroblasts and then seeded into molded 800-μm-diameter PDMS microwells at a cell density of 2000 cells per microwell. The microwell arrays were centrifuged at 100g for 3 min to form OBBs from the cells. CSM was half-changed daily, and the cardiac spheroids were observed to be beating by 2 to 4 days after aggregation, at which point they were harvested for SWIFT vascularization.

To demonstrate the versatility of this process, we also generated cardiac OBBs according to a protocol adapted from (27) and (28). BJFF iPSCs were formed into EBs on day −4, as described above. Two T225s (at ~80% confluency) were used to seed four six-well plates of microwells. On day −3, to remove ROCKi, the media was replaced with 4 ml of EBCM. On day −2, EBs from two wells were harvested, combined, and allowed to settle under gravity. The supernatant was aspirated, and the EBs from two wells were resuspended in 12 ml of EBCM and transferred to a single untreated T25 flask. Thus, EBs from four six-well plates (i.e., 24 wells in total) were harvested and placed into 12 T25 flasks. These flasks were incubated on an orbital shaker with a rotational speed of 60 rpm. To maintain and differentiate the cells in the T25 flasks, the following media changes were performed: day −1, 12 ml of EBCM; days 0 and 1, 10 ml of CDM and 5 μM CHIR99021 (BioGems) (CDMC); day 2, 10 ml of CDM; days 3 and 4, 10 ml of CDMI; and day >5, 10 ml of CDM daily. On days 0, 2, and 5, a rinse was performed after aspiration, using 4 ml of the medium that the cells were being changed into, to increase the removal of growth factors or small molecules from the previous medium. After the cells resettled, the rinsing medium was removed before the new culture medium was added. Beating was typically initially observed by day 7, and almost all cardiac OBBs were visibly beating by day 9. SWIFT printing was performed using cardiac OBBs between days 9 and 11, after which the cells were maintained in CMM.

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