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Objective To develop an efficient cryopreservation method using a single sperm. Design Experimental study. Setting Laboratory of a private institute. Patient(s) A fertile donor. Intervention(s) We produced hollow-core capsules with agarose walls. A single human sperm was injected into each capsule as per the conventional intracytoplasmic sperm injection (ICSI) method. The capsules that contained the spermatozoa were cryopreserved on polycarbonate or nylon mesh sheets using nitrogen vapor. Before their use, the capsules were thawed and recovered. The motile spermatozoa in the capsules were counted. Main Outcome Measure(s) The recovery rates of the agarose capsules and the spermatozoa in these capsules after thawing and the mortality and survival rates of the spermatozoa. Result(s) The recovery rates of the capsules were 91.5% (75/82) using polycarbonate sheets (PS) and 98.3% (59/60) using mesh sheets (MS) after thawing. The recovered capsules were not at all damaged. The recovery rates of the spermatozoa were 91.5% (75/82) using PS and 96.7% (58/60) using MS. Sperm motility rates were 85.3% (64/75) and 82.8% (48/58), whereas the survival rates of the immotile spermatozoa by the hypoosmotic swelling test were 81.8% (9/11) and 50.0% (5/10); furthermore, the total survival rates of the spermatozoa were 97.3% (73/75) and 91.4% (53/58) using PS and MS, respectively. There was no significant difference between the results obtained using PS and MS. Conclusion(s) A cryopreservation method for a single sperm using an agarose capsule has been developed. The method is expected to be useful in ICSI treatment in patients with few spermatozoa.