Method for cryopreservation of human spermatozoa free from seminal plasma using a fast and simple aseptic vitrification-devitrification process; portable kit for carrying out the method; and use of the same for treatment of disorders related to reproductive failures

Method for cryopreservation of human spermatozoa free from seminal plasma using a fast and simple aseptic vitrification-devitrification process, wherein said method comprises:

• (a) providing spermatozoa free from seminal liquid;
• (b) resuspending and mixing the spermatozoa obtained in step (a) in a 1:1 volumetric ratio with a vitrification medium comprising:
• i) buffering medium for spermatozoa,
• ii) a cryoprotectant, and
• iii) a dextran supplemented serum;
• (c) mixing and adjusting the final concentration of spermatozoa within a range from 0.2×106 to 1.8×106 spermatozoa per 100 μl of vitrification medium;
• (d) placing 100 μl of the mixture of spermatozoa and vitrification medium immediately after step (c) in a 0.25 ml receptacle or straw, then placing this straw inside other 0.5 ml straw to be sealed;
• (e) immersing the 0.5 ml straw with the sperm content in horizontal position in liquid N2 for no more than 5 seconds;
• (f) keeping the sperm sample inside the straws at a temperature lower than −75° C.;
• (g) devitrifying the sample by introducing the straw containing the sperm sample in a devitrification medium comprising:
• i) buffering medium for spermatozoa,
• ii) HTF supplemented with 1% w/v BSA, and
• iii) a substituting serum supplemented with dextran, incubated at a temperature ranging from 36.5 to 37.5° C.