[An effect of the two-step freezing method on the Schwann cell biological activity in the peripheral nerve of the rat].
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Xiao
Yuzhou
Y
Department of Orthopedics, Affiliated Hospital of Bengbu Medical College, Bengbu Anhui, 233004, PR China. XiaoYuzhou1@yahoo.com.cn
Yao
Yunfeng
Y
Pan
Gongping
G
D000818:Animals; D015925:Cryopreservation / Q000379:methods*; D005260:Female; D009416:Nerve Regeneration; D051381:Rats; D017207:Rats, Sprague-Dawley; D012583:Schwann Cells; D012584:Sciatic Nerve / Q000166:cytology