Protocol for culture and maintenance of canine induced pluripotent stem cells

Here, we present a protocol for a feeder-free culture system for canine induced pluripotent stem cells (ciPSCs) using a recently developed medium, termed AR medium, on extracellular matrix (ECM)-coated dishes. We describe steps for thawing frozen ciPSC stocks, medium exchange, passaging, and cryopreservation. This technique enables stable colony formation, high viability, and consistent proliferation of ciPSCs during long-term maintenance. It provides a reproducible platform for downstream applications of ciPSCs, such as cell differentiation, gene editing, and disease modeling. For complete details on the use and execution of this protocol, please refer to Nishimura et al. 1 • Steps for thawing and cryopreserving canine induced pluripotent stem cells (ciPSCs) • Instructions for maintaining ciPSCs using AR medium under feeder-free conditions • Guidance on passaging ciPSCs while preserving pluripotency and colony morphology Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Here, we present a protocol for a feeder-free culture system for canine induced pluripotent stem cells (ciPSCs) using a recently developed medium, termed AR medium, on extracellular matrix (ECM)-coated dishes. We describe steps for thawing frozen ciPSC stocks, medium exchange, passaging, and cryopreservation. This technique enables stable colony formation, high viability, and consistent proliferation of ciPSCs during long-term maintenance. It provides a reproducible platform for downstream applications of ciPSCs, such as cell differentiation, gene editing, and disease modeling.