Method for cryopreservation of human sperm-free seminal plasma using a simple and rapid vitrification process-devitrification Aseptic

摘要

Method for cryopreservation of human sperm-free seminal plasma using a simple and rapid vitrification-devitrification aseptic process, said method comprising: (a) providing free spermatozoa seminal fluid; (B) resuspending and mixing the spermatozoa obtained in step (a) in a volume ratio of 1: 1 with an average vitrification comprising: i) buffering means for sperm, ii) a non-permeable cryoprotectant with a final concentration of 0.15 to 0.30 M, and iii) a serum supplemented with dextran (c) mixing and adjust the final sperm concentration to a range of 0.2 x 106 to 1.8 x 106 sperm per 100 .mu.l of said means vitrification; (D) placing 100 ul of the mixture of spermatozoa and through vitrification immediately after step (c) in a 0.25 ml straws, leaving 1 cm free liquid at both ends, positioning the first straw in a second 0.5 ml straw to seal at both ends, positioning the first and second straw horizontally in a thermally conductive receptacle; (E) immersing the straw 0.5ml resulting from step d) with the spermatic content horizontally in liquid N2; (F) maintaining the sperm sample within the straws at a temperature between -75 ° C and -85 ° C; (G) devitrify the sample by introducing the straw containing the semen sample in a medium comprising devitrification; i) buffering means for sperm, ii) human tubal fluid supplemented with bovine serum albumin to 1% w / v, and iii) a serum supplemented with dextran; where devitrification is performed by first placing the samples to devitrify in a container with liquid N2 and then introducing three 0.25 ml straws with sample in a tube with 5 ml of devitrification incubated at a temperature ranging from 36 5 to 37.5 ° C in a heating block.