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| LI Bolin,CHI Ming,ZHANG Fan.Culture and identification of primary rabbit spinal cord microvascular endothelial cells in vitro[J].Chinese Journal of Spine and Spinal Cord,2025,(5):516-521. |
| Culture and identification of primary rabbit spinal cord microvascular endothelial cells in vitro |
| Received:September 17, 2024 Revised:March 23, 2025 |
| English Keywords:Microvesselsendelial cells of spinal cord Enzymatic digestion method Factor Ⅷ related antigen Immunocytochemistry Japanese large ear white rabbit |
| Fund:国家自然科学基金资助项目（编号：81072714）；福建省自然科学基金资助项目（编号：2017J01545） |
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| 【Abstract】 Objectives: To establish an efficient in vitro culture method for spinal cord microvascular endothelial cells(SCMECs) of rabbits. Methods: 15 Japanese large-ear white rabbits(2-week-old, either sex) were selected. Under sterile conditions, the spinal cord was obtained by incising the back skin, muscle layers and vertebral canal along its longitudinal axis. The spinal cord was minced, and myelin impurities were removed by density gradient centrifugation using bovine serum albumin. The spinal cord microvascular segments were obtained by filtration through a sieve. The enzymatic digestion method was used, with 0.1% type Ⅱ collagenase digesting the microvascular segments for 20min, after which, the segments were seeded into culture flasks, and then placed in a CO2 incubator for static culture for 72h. The above procedures were performed with five rabbits at a time, repeated three times, and the target cells from the third culture were taken as the objects of observation and identification. The growth and morphological characteristics of the cells were observed under an inverted phase-contrast microscope. Immunocytochemical staining was used to detect the expression of factor Ⅷ-related antigen to identify the cultured SCMECs. Results: Microscopically, the segments showed short-rod or branched shapes post-seeding. After 24h of culture, short spindle-shaped cells migrated out from around the microvascular segments and grew in a "budding" manner. After 48h, "island-like" cell colonies formed. After 72h, the cells covered the bottom of the dish, growing in a typical monolayer, "paving stone-like" mosaic pattern. Immunocytochemical staining showed that over 99% of the cells had brownish-red cytoplasm, with positive expression of factor Ⅷ-related antigen, hematoxylin stained the nuclei and was shown in blue. Conclusions: Enzymatic digestion can successfully isolate and culture SCMECs of Japanese large-ear white rabbits with good activity and high purity. |
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