3D Cell Culture Method in Channel‐Free Water‐in‐Oil Droplets

A new channel-free water-in-oil (WO) droplet 3D cell culture method is proposed to address the challenges while maintaining the advantages of the conventional 3D cell culture methods. The proposed WO method can fundamentally solve the constraint of spheroids size, a common challenge in conventional 3D culture, by using droplet size controllability. The 3D cell culture performance of the WO method is verified by comparing it with the conventional 3D cell culture methods. A systematic investigation of the culture conditions of the WO method confirms the working range of cell concentration and droplet size, as well as the scalability of spheroid size. Adjusting droplet size and cell concentration enables rapid spheroid formation with large and high cell concentration droplets or fast spheroid growth with small and low cell concentration droplets, providing control over the spheroid size and growth rate according to the purpose. Furthermore, long-term culture is demonstrated for 1 month with the proposed method, showing the largest spheroid culture and demonstrating the possibility that this method can be used not only for spheroid formation but also for organoid studies. Finally, if a WO-based automated 3D cell culture system is developed, it will be a useful tool for organoid research.