the single cell suspension are plated in culture dishes containing cell culture medium the cultures are maintained in an incubator wherein the concentration of co 2 and o 2 may be kept constant the cell debris present in the cultures should be gently removed as necessary and fresh cell culture medium should be added regularly small colonies will start to form and these colonies subsequently will increase in size to merge with each other when confluence of the colonies is attained the cells may be subcultured the cells are subcultured by incubating them with trypsin e g 0 25 trypsin in the cell medium in the incubator for a period of time ranging from approximately 5 minutes to approximately 15 minutes preferably approximately 10 minutes the effect of trypsin is arrested by adding fresh fetal calf serum e g 10 then the cells are scraped off the culture surface and transferred to a plate with multiple wells approximately 1000 to 100 000 cells are added to each well after a periodof time ranging from approximately 5 days to approximately 10 days preferably 7 days the cultured cells are transferred from the plate to a medium sized flask and then to a large sized flask the expanded culture which is maintained in the large size flask constitutes the 34 first passage 34 approximately every 10 days the cells may be passaged again the true identity of the passaged cells may be routinely monitored by indirect immunofluorescence using as a probe a tumor specific monoclonal antibody the ability of the primary tumor cell culture to form colonies in soft agaris a characteristic feature of transformed cells and therefore the presence of tumor cells may be checked by growing the cells in soft agar in the conventional way the.