海藻酸钠/壳聚糖微囊冻存的低温显微实验

摘要

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 cryopreservation protocol 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 the cryoprotector. 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.%背景:目前国内外对微囊的低温保存效果均不理想,已成为制约微胶囊广泛应用的一个重要环节.目的:观察不同溶液、不同降温速率冻存海藻酸钠-壳聚糖-海藻酸钠(alginate-chitosan-alitosan-alginate,ACA)微囊时冰晶的特点及其对微囊形态的影响,探索一种适合ACA微囊低温保存的降温方法.设计:观察实验,于2008-02/04在上海理工大学低温显微镜实验室完成.材料:应用自制的高压脉冲微胶囊成型装置制备ACA微囊.方法:在低温显微系统下以1,10,30和100℃/min的降温速率对微囊进行低温保存,最后以50℃/min复温;然后在ACA微囊悬液中加入10%二甲基亚砜,重复实验.主要观察指标:不同降温速率及添加了二甲基亚砜后冰晶生长情况及其对ACA微囊形态的影响;复温后微囊形态的变化及微囊的破损率.结果:在以小于10℃/min降温速率且不添加低温保护剂的情况下,冻结过程中生成的冰晶粗大.冰晶的生长会导致微囊发生形变,当提高降温速率、添加二甲基亚砜后,冰晶变得细小,对微囊的损伤也更小.从复温后微囊的形态来看,以超过30℃/min降温、添加10%二甲基亚砜为低温保护剂时微囊的破损率较小(P＜0.05).结论:微囊在低温保存过程中主要受到由冰晶生长引起的机械性损伤,提高降温速率及添加低温保护剂可以有效抑制冰晶的生长.