Xiao Yuzhou, Yao Yunfeng, Pan Gongping
Department of Orthopedics, Affiliated Hospital of Bengbu Medical College, Bengbu Anhui, 233004, PR China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2006 Aug;20(8):801-4.
To investigate an effect of different temperature cryopreservation of the two-step freezing method on the Schwann cell biological activity in the peripheral nerve of the rat.
Eighty female SD rats were randomly divided into 8 groups of 10 rats each. One was the control group and 7 were the experimental groups. Two 2-cm-long sciatic nerve segments were respectively taken from both legs of each rat. In the control group, the sciatic nerve segments did not undergo the treatment of cryopreservation; however, in the 7 experimental groups, the sciatic nerve segments respectively underwent the different temperature cryopreservation of the two-step freezing method at -20 degrees C, -30 degrees C, -40 degrees C, -50 degrees C, -60 degrees C, -70 degrees C and -80 degrees C. The sciatic nerve segments were cryopreserved for 2 hours,and then placed into the liquid nigrtrogen at -196 degrees C. After 48 hours of storage, the nerve segments were thawed quickly in the 37 degrees C water bath box for 1 minute. Then, the sciatic nerve segments each group were harvested. The cells of the sciatic nerve were incubated with Calcein-AM for 15 minutes. The average fluorescence intensity of the cells was measured by the flow cytometry. The nerve fibers were also incubated with Calcein-AM for 15 minutes. The fluorescence intensity of the cells was analyzed by the confocal fluorescence microscope. The Schwann cell biological activity intensity was measured.
The fluorescence intensity in the -40 degrees C group was the strongest and the Schwann cell biological activity in this group was the best among all the groups (P < 0.01). The fluorescence intensity in the 8 groups measured by the flow cytometry was as follows: 242.5220 +/- 9.5684 in the control group, 168.6770 +/- 10.2070 in the -20 degrees C group, 214.9920 +/- 8.3291 in the -30 degrees C group, 235.5260 +/- 9.2805 in the -40 degrees C group, 222.4340 +/- 8.5155 in the -50 degrees C group, 217.4090 +/- 9.5157 in the -60 degrees C group, 132.3760 +/- 13.4597 in the -70 degrees C group, and 108.1320 +/- 16.0331 in the -80 degrees C group. The fluorescence intensity detected by the confocal fluorescence microscope was as follows: 143.7000 +/- 5.5678 in the control group, 119.7000 +/- 5.161 5 in the -20 degrees C group, 121.3000 +/- 4.3474 in the -30 degrees C group, 139.7000 +/- 5.0122 in the -40 degrees C group, 121.0000 +/- 4.5461 in the -50 degrees C group, 118.4000 +/- 4.9261 in the -60 degrees C group, 81.2000 +/- 5.1164 in the -70 degrees C group, and 79.0000 +/- 5.7164 in the -80 degrees C group.
The Schwann cell biological activity treated by the two-step freezing method can be preserved and the activity is cryopreserved best at -40 degrees C.
探讨两步冷冻法不同温度冻存对大鼠周围神经中雪旺细胞生物学活性的影响。
80只雌性SD大鼠随机分为8组，每组10只。1组为对照组，7组为实验组。分别从每只大鼠的双腿取下两段2 cm长的坐骨神经。对照组坐骨神经节段不进行冻存处理；然而，在7个实验组中，坐骨神经节段分别在-20℃、-30℃、-40℃、-50℃、-60℃、-70℃和-80℃下采用两步冷冻法进行不同温度的冻存。坐骨神经节段冻存2小时，然后置于-196℃的液氮中。储存48小时后，将神经节段在37℃水浴箱中快速解冻1分钟。然后，收集每组的坐骨神经节段。将坐骨神经细胞与钙黄绿素-AM孵育15分钟。通过流式细胞术测量细胞的平均荧光强度。神经纤维也与钙黄绿素-AM孵育15分钟。通过共聚焦荧光显微镜分析细胞的荧光强度。测量雪旺细胞的生物学活性强度。
-40℃组的荧光强度最强，该组雪旺细胞生物学活性在所有组中最佳（P<0.01）。流式细胞术测量的8组荧光强度如下：对照组为242.5220±9.5684，-20℃组为168.6770±10.2070，-30℃组为214.9920±8.3291，-40℃组为235.5260±9.2805，-50℃组为222.4340±8.5155，-60℃组为217.4090±9.5157，-70℃组为132.3760±13.4597，-80℃组为108.1320±16.0331。共聚焦荧光显微镜检测的荧光强度如下：对照组为143.7000±5.5678，-20℃组为119.7000±5.1615，-30℃组为121.3000±4.3474，-40℃组为139.7000±5.0122，-50℃组为121.0000±4.5461，-60℃组为118.4000±4.9261，-70℃组为81.2000±5.1164，-80℃组为79.0000±5.7164。
两步冷冻法处理可保存雪旺细胞生物学活性，在-40℃时活性冻存效果最佳。
