A multi-chamber single-use bioreactor for cell culture expansion has bag assembly and a rigid support structure defining a bag receiving space. The bag assembly disposed in the bag receiving space of the rigid support structure and supported by the rigid support structure. The bag assembly has at least a first flexible bag and a second flexible bag. The first bag defines a first reaction chamber, and the second bag defines a second reaction chamber. The first reaction chamber has a first volume, a first inlet, and a first outlet, and the second reaction chamber has a second volume different from the first volume, a second inlet, and a second outlet. The second inlet of the second bag is fluidically connected to the first outlet of the first bag so liquid in first reaction chamber can be transferred to the second reaction chamber.