THE FIBROBLAST-SPECIFIC MAB AS02 - A NOVEL TOOL FOR DETECTION AND ELIMINATION OF HUMAN FIBROBLASTS

The unwelcome presence of fibroblasts in many cell cultures prevents t he long term cultivation of various cell types and work with pure popu lations. Recently, we described a novel fibroblast-specific monoclonal antibody (MAb AS02) that recognises a membrane-bound antigen. We have now developed a method using the fibroblast-specific MAb AS02 immobil ised on goat-anti-mouse-magnetic beads to separate contaminating fibro blasts. An endothelial cell line experimentally contaminated with 5%-5 0% fibroblasts was successfully purified. Additionally, an endothelial cell line with an initial fibroblast contamination of 1.5% was prepar ed. A proportion of each preparation was cultured with no separation s tep being performed, whereas the remainder was cultured after purifica tion with MAb AS02 to exclude the presence of a minor number of fibrob lasts (<0.1%). The proportion of fibroblasts increased up to 38% in th e fifth passage of culture without elimination of the low initial fibr oblast contamination, whereas in the fraction with the separation step , no fibroblasts were detectable by flow cytometry, even after the fif th passage. We also used the antibody to detect the presence of natura lly contaminating fibroblasts in thyrocyte cultures. After cultivation of thyrocyte cultures over five passages, the number of fibroblasts i ncreased dramatically up to 50%-80% of the whole population. Subsequen tly, we successfully applied the method for complete elimination of na turally contaminating fibroblasts from freshly isolated thyrocyte cult ures from enzymatically digested thyroid glands. Thus, MAb AS02 is a f ibroblast-specific marker that is a useful tool for the detection and elimination of contaminating fibroblasts. The specificity of MAb AS02 permits the universal application of this antibody for human cell cult ures of interest.