Fig. 1: Conceptual diagram of a conventional method and a proposed method.

a Representative phase-contrast microscopy images of bovine myogenic cells cultured by a conventional serum-free culturing method. On uncoated culture dishes, serum-free culture medium does not induce cell proliferation or even cell adhesion. b Fabrication of the serum-free co-culture medium conditioned by human hepatocellular carcinoma-derived cell line HepG2 and mouse skin-derived cell line NIH/3T3 cells. c Fabrication of serum-free co-culture medium in different ways. In the co-culture group, HepG2 and NIH/3T3 cells were seeded together in same culture dish to perform contact co-culture. In the mix group, HepG2 and NIH/3T3 supernatants were mixed at a 1:1 ratio to create the culture medium after collecting supernatants from each culture dish with HepG2 and NIH/3T3 respectively. In the non-contact co-culture groups, HepG2 and NIH/3T3 cells were seeded on either the bottom surface of a cell culture insert and the bottom of a well plate, respectively. The group with HepG2 cells seeded on the insert bottom was designated as the non-contacted coculture (H-N) group, while the group with the reversed configuration, where NIH/3T3 cells were seeded on the insert bottom, was designated as the non-contacted coculture (N-H) group. In the diluted co-culture group, the culture medium prepared by contact co-culture was mixed with serum-free culture medium (DMEM + 1% PS) at a 1:1 ratio to create a 2-fold diluted culture medium.