BACKGROUND: Setting of cellular cultures extracted from colorectal cancer tissue represents a valid model for in vitro study of biological and molecular characteristics of each single tumor finalized to obtain a tailored chemiotherapy. The end point of this study is to create primary cellular cultures from “fresh” cancer tissue in different stages of evolution. METHODS: Cancer tissue samples are obtained by means of surgical excisional biopsy or by means of semi-automatic biopsy instrument (Sprig-Cut®). After having compared different approaches, two experimental protocols have been selected to have the highest number or intact cells: enzimatic digestion with trypsin and explantation. RESULTS AND CONCLUSIONS: Primary cell culture free of microbic contamination, obtained mainly by means of SpringCut® methods, underwent immunohistochemical analysis to evaluate what kind of cell have been grown in vitro by measuring the expression of CK20 and GFAP, both resulted positive. The possibility of setting a primary cell culture which represents the cancer of each patient allows a pharmacologic and biomolecular study which can contribute to the development of a tailored adjuvant therapy with many advantages for the patient in terms of positive answer to the treatment and reduced toxicity.