Robotic handling preserves iPSC-derived vascular smooth muscle cell differentiation.

To tackle the global cardiovascular disease epidemic, vascular cell culture requires scalable, efficient, and reproducible methods for differentiating cells. However, no studies have validated whether automation, essential for scale-up, preserves differentiation quality of cells. Here, we developed a differentiation protocol for induced pluripotent stem cell (iPSC)-derived vascular smooth muscle cells (iVSMCs) that is applicable to both manual, and automated robotic culture. iPSC lines generated from male and female donors across various ages were used to optimize the generation of iVSMCs. iVSMCs had consistent morphology and protein expression, which were largely similar to those of primary VSMCs, including expression of myosin heavy chain-11 (MYH11), α-smooth muscle actin (α-SMA), transgelin (TAGLN) and calponin (CNN1). Functionally, these iVSMCs responded to the vasoconstrictor carbachol. Coupling this protocol with an automated Hamilton liquid-handling robotics system allowed the generation of iVSMCs in large quantities, that were morphologically similar to manually differentiated iVSMCs. Comparative proteomic analysis confirmed that protein expression was substantially the same between automated and manual differentiation methods. Automation markedly reduced manual labor and facilitated increased production without sacrificing cell quality. This study demonstrates the feasibility of automating iVSMC differentiation, marking a significant step towards scalable VSMC manufacture for three-dimensional applications, and for the ever-growing demands of organoid and tissue engineering applications.