Engineering and Maturation of Cardiac Tissues
Cardiac tissues were made using our established protocol.[25] Differentiated iPSC-CMs were mixed with supporting primary human cardiac fibroblasts (NHCF-V; Lonza, CC-2904) in a 3:1 ratio and resuspended in RPMI-B27 (RPMI 1640 basal medium supplemented with l-ascorbic acid 2-phosphate and B27)....etc
Derivation of Bone Marrow Component Cells
iPSCs (WTC-11 line) were derived as previously described using the STEMdiff Mesenchymal Progenitor Kit (Stem Cell Technologies, 05240), expanded, and frozen.[24] Human umbilical vein endothelial cells (HUVECs; Lonza, C2519A) were expanded according to the manufacturer's instructions...etc
Engineering of Liver Tissues
iPSC-derived human hepatocytes were purchased from Cellular Dynamics (iHeps 2.0; CDI, R1027). Using an AggreWell plate with 400 µm microwells (Stem Cell Technologies, 34411), hepatocytes, and primary human dermal fibroblasts (Lonza) were mixed at a 1:1 ratio for the formation of liver spheroids, as previously described.[19, 55] Spheroids were cultured in a hepatocyte culture medium (Lonza, CC-3198) for two days in the AggreWell plates.
Engineering and Maturation of the Endothelial Barrier
Endothelial mesh inserts made of polyester (PET) with 8 µm pore size were incubated in 70% ethanol overnight and washed three times in 1X PBS prior to use. Barriers were flipped upside down and incubated with 10 µg/mL human fibronectin (Corning, 356008) for 1 h prior to cell seeding, and washed twice with PBS. 400,000 HUVECs and 100,000 BM-MSCs (ATCC, PCS-500-012) were seeded onto the inserts and incubated for 1.5–2 h in 50 µL of EGM-2 media (Lonza, CC-3162) at 37 °C in a humidified incubator at 5% CO2