room temperature after centrifugation 35 ml of supernatant are transferred to a static culture flask t 175 with a filter containing vented cap and 35 ml of basal culture medium supplemented with deoxyribonucleosides ribonucleosides glutamine antibiotics and 10 of foetal bovine serum fbs are added the flask is then incubated at 37 u00b0 c in a humidified atmosphere containing 7 co 2 and the total culture medium changed every 72 h in order to promote cell adhesion and expansion multiplication until maximum surface confluence is achieved cell populations obtained from the first and second phases are cryopreserved after the first adhesion and expansion multiplication round end of p0 this consists on the direct cryopreservation in the vapour phase of liquid nitrogen of a mixture of 0 5 ml of cell suspension containing the desired total cell number and the same volume of a solution of foetal bovine serum fbs containing 10 of dimethyl sulfoxyde dmso to obtain a final concentration of approximately 3 u00d7 10 6 cells ml in a 1 5 ml sterile cryovial containing therefore 1 0 ml cell suspension and 0 5 ml headspace the third and final phase of cell recovery consists on direct cryopreservation of the cell pellet obtained by the previously described centrifugation of the digestion supernatant resuspended in 2 ml of a solution consisting of foetal bovine serum fbs containing 10 of dimethyl sulfoxide dmso using a controlled rate freezer at a temperature decreasing rate of 1 u00b0 c min u2212 1 down to u2212 80 u00b0 c in a 2 5 ml sterile cryovial containing 2 ml cell suspension and 0 5 ml headspace cryopreserved cells can be recovered whenever necessary through a fast thawing process in a water bath at 37 u00b0 c the cells are.