[ISOLATION AND IDENTIFICATION OF RAT INTERVERTEBRAL DISC NUCLEUS PULPOSUS CELLS AT DIFFERENT SEGMENTS AND COMPARATIVE...

Corresponding author: WANG Yuntao, E-mail: wangyttod@seu.edu.cn

To isolate nucleus pulposus cells (NPCs) from the caudal and lumbar intervertebral disc of rat, and to identify the morphology and to compare the characteristics.

The whole spine was separated from 8-week-old Sprague Dawley rats under the sterile conditions. NPCs of different segments (lumbar group: L1, 2-L6, S1; caudal group: C1, 2-C17, 18) were cultured by adherent cultivation approach. Cellular morphologic change was noted by HE staining and continuous observation under inverted phase contrast microscope. Besides, the aggrecan and collagen type II expression were examined by toluidine blue and immunocytochemistry staining respectively. The total protein contents, senescence level, and the cell viability of passage 1-5 (P1-5) were detected. The growth curves of the P1 cells in lumbar and caudal groups were determined by cell counting kit 8.

The NPCs were isolated and identified successfully. The adherence time of the primary cells (the cell fusion reached 90%) in lumbar group was significantly longer than that in caudal group in primary generation (P < 0.05). HE staining showed that cytoplasm was pink with the blue nucleus. Lumbar disc NPCs were spindle. The larger caudal disc NPCs were polygonal or irregular. Toluidine blue staining showed that the proteoglycan was stained as blue. In the cytoplasm of cells, collagen type II was stained as brown surround the blue-black nucleus. The cell viability had no significant difference between lumbar and caudal groups and between different passages in the same group (P > 0.05). The caudal disc NPCs reached their logarithmic growth phase after 3 days of culture, while the cells in lumbar segments did after 4-5 days of culture. The cell proliferation in caudal segments was more than that in lumbar segments at 3-9 days (P < 0.05). The difference in the total protein contents was not significant between cells at P1-5 in 2 groups (P > 0.05), and the caudal disc NPCs had higher protein contents than lumbar disc NPCs (P < 0.05). There was no significant difference in cell senescence rate between cells at P1, P2, and P3 in 2 groups (P > 0.05), but significant difference was shown in senescence rate between 2 groups in cells at P4 and P5 (P < 0.05).

Caudal disc NPCs have a better status, which is more suitable for experiment as a seed cell than the lumbar disc NPCs in the same generation.