Introduction: A crucial factor in cell culture technology is the use of appropriate culture medium which can promote cell growth and cellular functions. Development of serum free chemically defined medium enables the researchers to conduct the experiment in a more controlled manner. Myofibroblasts of the tumour microenvironment drive the colorectal carcinogenesis. In vitro study of the tumour-myofibroblast interaction using serum free medium may give a better insight into potential treatment for colorectal cancer (CRC) in the future. This study aims to establish serum free chemically defined medium to study the interplay between myofibroblast and CRC cells. Methods: A myofibroblast-specific serum free culture medium named as M-CIL, was developed to study the interactions between myofibroblasts and CRC cell lines in vitro. The influence of substrate (collagen type I) and subculturing of cells under incubation with M-CIL medium were also analysed. The effect of M-CIL medium on CRC cell growth also was studied. Gene expression analysis using quantitative real time polymerase chain reaction on amine oxidase, copper containing 3 (AOC3) was conducted to investigate the effect of individual components of the medium on myofibroblasts. Results: M-CIL medium supports the proliferation of myofibroblasts and produce minimal effect on CRC cells’ growth. Our data also shows the influence of M-CIL components on gene expression in myofibroblasts. Conclusion: M-CIL culture medium, which was designed with known and defined components, proved to be a suitable alternative to complete medium (DMEM + 10% FBS) for co-culture experiments of myofibroblasts and CRC cell lines.