Cryopreservation of the single cells from seven-day-old mouse seminiferous epithelia

The single cells from 7 day old mouse seminiferous epithelia were cryopreserved,and the cell recovery rates were examined by trypan blue exclusion staining after thawing.The freezing solutions were DMEM/10% NBS supplemented with dimethyl sulphoxide(DMSO),propanediol(PG),ethylene glycol(EG) and glycerol(G) at concentrations of 5%,10%,15%,20% and 25% respectively.The cell recovery rates of DMSO at concentrations ranging from 5% to 25% were 77 1%,88 2%,86 5%,73 5% and 65 5% respectively.There were significant differences between the cell recovery rates of DMSO at 10% concentration and at other concentrations.The cell recovery rates of PG at concentrations ranging from 5% to 25% were 66 2%,84 3%,72 1%,69 9% and 47 5% respectively.There were significant differences between the cell recovery rate of PG at 10% concentration and at other concentrations.The cell recovery rates of EG at concentrations ranging from 5% to 20% were 64 9%,81 6%,60 9% and 44 7% respectively.There were significant differences between the cell recovery rate of EG at 10% concentration and at other concentrations.The cell recovery rates of G at concentrations ranging from 5% to 25% were all below 10%.The highest cell recovery rates of the four cryoprotectants showed difference or significant difference between them.The minimum cell loss of DMSO,PG,EG and G were 8 6%,12 5%,15 2% and 80% respectively.The results suggest that 10% DMSO,10% PG and 10% EG are suitable for cryopreservation of mouse spermatogonia,and 10% DMSO is the best,while G is unsuitable.The two step freezing in 10% DMSO cryopreservative solution,liquid nitrogen storing and 37℃ water bath thawing would be considered a cryopreservative protocol with high cell recovery rate for mouse sprmatogonia.