Novel Technology for Cryopreservation of Biological Tissue by Utilizing Ultrasonic lrradiation

冷冻可以减缓或停止某些生物反应，以保存生物细胞。冰冻对生命系统是致命的，这也是事实。在冷冻过程中，细胞外和细胞内的结冰、水渗透细胞膜、细胞变形等行为在微观尺度上发生，对活细胞造成严重的伤害，关系到活细胞的生死。主动控制结晶的方法是一种很有前途的低温保存技术。本课题的目的是研究超声波辐照对生物材料冷冻过程中结冰的影响。在实验中，琼脂凝胶在不同的超声波照射方式下冷冻，频率为28 kHz。用荧光指示剂对冰晶进行了温度测量和显微观察。首先，发现超声可以释放组织中的过冷状态，减小组织中的冰晶尺寸。然而，在较高的超声功率照射下，由于超声波的吸收，冷却速度减慢，观察到冰晶的增肥。澄清了组织中冰晶的大小与超声功率和组织大小的关系。其次，对各种辐照方法进行了试验。结果表明，脉冲辐照减少了冰晶因吸收超声波而产生的增肥现象，有利于减小冰晶的尺寸。探讨了超声功率、开关频率和照射时间与生物组织超声结晶机理的关系。

Freezing can slow down or stop some biological reactions for preserving biological cell. It is also true that the freezing is lethal to the living system. During freezing, the extra- and intra-cellular ice formation, osmotic water permeation through cell membrane, deformation of the cell and other behavior occur at microscale and they bring serious injuries connecting to life-and-death of living cell.A method to actively controlling crystallization is one of promising technique for cryopreservation. The object of this project is to study the effects of ultrasonic irradiation on ice formation during freezing of biological materials. In the experiments, agar gel was frozen under various irradiation methods of ultrasound at frequency of 28 kHz. The measurements of temperature and the microscopic observation of ice crystals by using fluorescent indicator were carried out.Firstly, it was found that ultrasound released the supercooling states, and reduced ice crystal size in the tissue. However, the fattning of ice crystal was observed by irradiation of higher ultrasonic power, since the cooling rate was reduced due to absorption of the ultrasonic wave. The ice crystal size in the tissue was clarified in relation to ultrasonic power and tissue size. Secondly, various irradiation methods were tested. It was found that the pulsed irradiation is useful for reducing ice crystal size, since the fattening of ice crystal due to absorption of ultrasonic wave is reduced. The mechanism of sonocrystallization in biological tissue was discussed in relation to ultrasonic power, on-off frequency and irradiation duration.