BACKGROUND: Presently, there is not an optimal cryopreservation protocol of the microcapsules, which has restrained the application of the microcapsules. OBJECTIVE: To investigate the characteristics of ice crystal and the morphology of alginate-chitosan-alginate (ACA) microcapsules cryopreserved at different solutions and different cooling rates, and to explore the optimal cryopreservationprotocol for ACA microcapsules. DESIGN, TIME AND SETTING: An observational study was performed at the Laboratory of Cryomicroecope in Shanghai University of Science and Technology (China) from February to April in 2008. MATERIALS: The high-voltage pulsing microcapsule shaping device was used to prepare ACA microcapsules.METHODS: The ACA microcapsules were preserved at different cooling rates (1 ℃/minute, 10 ℃/minute, 30 ℃/minute and 100 ℃/minute) by the cryomicroscopy system and then rawarmed at 50 ℃/minute. The protocols were repeated after the supplement of 10% dimethyl sulphoxide. MAIN OUTCOME MEASURES: The growth of ice crystals and the morphology of ACA microcapsules were checked at different cooling rates and in different solutions. The changes of forms and the rates of damage were checked after the microcapsules were rewarmed.RESULTS: The ice crystals grew into big crystals at the freezing process when the cooling rate was low than 10 ℃/minute and cryoprotector was not used. The growth of ice crystals would result in the distortion of microcapsules. It also could reduce the cryodamage of the microcapsules. The size of the ice crystals would grow down when raising the cooling rate and using thecryoprotector. The post-thaw ACA microcapsules were intact when dimethyl sulphoxide was used at a concentration of 10% and the cooling rate was higher than 30 ℃/minute (P ＜ 0.05). CONCLUSION: Mechanical damage occurs mainly during the growing of ice crystals at the time of microcapsules cryopreservation process. The growth of the ice could be restrained effectively by raising the cooling rate and using the cryoprotector.