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。冰冻对生命系统也是致命的，这也是事实。在冷冻过程中，细胞内外结冰、渗透水渗入细胞膜、细胞变形等行为在微观上发生，对活体细胞的生死存亡造成严重伤害，主动控制结晶是一种很有前途的冷冻保存技术。本项目的目的是研究超声辐照对生物材料冻结过程中结冰的影响。在实验中，琼脂凝胶在不同的超声辐照方式下冷冻，频率为28 kHz。利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态，减小冰晶的尺寸。然而，在较高的超声波功率下，由于吸收了超声波而降低了冰晶的冷却速度，因此可以观察到冰晶的肥化现象。组织中的冰晶大小与超声功率和组织大小有关。其次，对不同的辐照方法进行了试验。结果表明，脉冲辐照可以减小由于超声波吸收引起的冰晶肥化，有利于冰晶尺寸的减小。从超声功率、开关频率和辐照时间三个方面讨论了生物组织中超声结晶的机理。

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.