| 破骨细胞的传代、冻存与复苏 |
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投稿时间：2017-02-07
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| 作者 | Author | 单位 | Address | E-Mail |
| 俞索静 |
YU Suo-jing |
浙江中医药大学 浙江省骨伤研究所, 浙江 杭州 310053 |
Institute of Orthopaedics and Traumatology, Zhejiang Chinese Medical University, Hangzhou 310053, Zhejiang, China |
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| 吴承亮 |
WU Cheng-liang |
浙江中医药大学 浙江省骨伤研究所, 浙江 杭州 310053 |
Institute of Orthopaedics and Traumatology, Zhejiang Chinese Medical University, Hangzhou 310053, Zhejiang, China |
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| 金红婷 |
JIN Hong-ting |
浙江中医药大学 浙江省骨伤研究所, 浙江 杭州 310053 |
Institute of Orthopaedics and Traumatology, Zhejiang Chinese Medical University, Hangzhou 310053, Zhejiang, China |
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| 胡雪琴 |
HU Xue-qin |
浙江中医药大学 浙江省骨伤研究所, 浙江 杭州 310053 |
Institute of Orthopaedics and Traumatology, Zhejiang Chinese Medical University, Hangzhou 310053, Zhejiang, China |
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| 肖鲁伟 |
XIAO Lu-wei |
浙江中医药大学 浙江省骨伤研究所, 浙江 杭州 310053 |
Institute of Orthopaedics and Traumatology, Zhejiang Chinese Medical University, Hangzhou 310053, Zhejiang, China |
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| 童培建 |
TONG Pei-jian |
浙江中医药大学 浙江省骨伤研究所, 浙江 杭州 310053 |
Institute of Orthopaedics and Traumatology, Zhejiang Chinese Medical University, Hangzhou 310053, Zhejiang, China |
tongpeijian@163.com |
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| 期刊信息：《中国骨伤》2017年，第30卷，第5期，第463-469页 |
| DOI：10.3969/j.issn.1003-0034.2017.05.014 |
| 基金项目:国家自然科学基金面上项目（编号：81373669）；浙江省科技厅重大专项（编号：2014C03035） |
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中文摘要:
目的：探讨破骨细胞传代、冻存与复苏的可行性，为开展破骨细胞的相关研究提供新的技术手段与方法。
方法：破骨细胞的传代：取成年SPF 级雄性SD 大鼠1只，体重250 g，腹主动脉采血，分离周围血单个核细胞，经RANKL、M-CSF诱导培养2周，形成多核破骨细胞后，采用胰蛋白酶消化、震荡、吹打法，制成细胞悬液，离心后用含RANKL、M-CSF的α-MEM完全培养基重悬，接种至6孔培养板与直径35 mm培养皿中。破骨细胞的冻存：取上述细胞悬液，离心后用DMSO、FBS、α-MEM（1：2：7）冻存液，按常规程序将细胞冻存于-196 ℃液氮中。破骨细胞的复苏：从液氮中取出冻存的破骨细胞，37 ℃水浴中快速解冻，加PBS离心清洗后，用含RANKL、M-CSF的α-MEM完全培养基重悬，接种至6孔细胞培养板与直径35 mm培养皿中。同时对传代与复苏培养中破骨细胞的消化与贴壁过程，采用倒置相差显微镜与活细胞工作站进行观察记录与动态成像，3 d后作TRAP染色。
结果：破骨细胞经胰蛋白酶消化、震荡、吹打后，细胞基本上脱壁、悬浮，可以制成细胞悬液，用于传代与冻存。破骨细胞传代培养后，大多在2 h内开始贴壁，贴壁后胞内细胞核清晰可见。经液氮冻存的破骨细胞，复苏培养后2～3 h开始贴壁，胞质展开，可见多个细胞核。传代与冻存复苏培养后的破骨细胞，TRAP染色呈阳性反应。
结论：破骨细胞虽然贴壁比较牢固，但经适当的消化、震荡及吹打，多数可脱壁悬浮，并能实现传代培养，同时还可采用常规方法进行细胞冻存与复苏培养，经传代与冻存的破骨细胞仍可保持破骨细胞的活性。破骨细胞的传代与冻存，可以按实验计划与用量需求，随时进行分量传代与复苏培养，为开展破骨细胞的相关研究提供新的技术手段与方法。 |
| 【关键词】破骨细胞 培养 传代 冻存 复苏 |
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| Subculture,cryopreservation and recovery of osteoclasts |
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ABSTRACT
Objective: To explore the feasibility of passage,cryopreservation,and recovery of osteoclasts in order to develop new techniques facilitating osteoclast research.
Methods: Passage of osteoclasts:adult male SD rat(SPF grade,weight of 250 g) was sacrificed and the abdominal aorta was exposed for blood draw. Monocytes isolated from peripheral circulation was treated with RANKL and M-CSF for 2 weeks. After formation of osteoclasts,they were trypsinized with pipetting,centrifuged,re-suspended with α-MEM containing RANKL and M-CSF,and cultured in 6 well-plates and 35 mm culture dishes. Freezing of osteoclasts:trypsinized osteoclasts were centrifuged and resuspended with DMSO,FBS,α-MEM (1:2:7),and were stored in liquid nitrogen(-196 ℃). Recovery of osteoclasts:frozen osteoclasts were taken out of liquid nitrogen tank and thawed quickly at 37 ℃ in water bath. After wash with PBS,the cells were resuspended with α-MEM containing RANKL and M-CSF,and were cultured in 6 well dishes and 35 mm culture dishes. Meanwhile,cells were checked with inverted phase contrast microscope and observed in the live cell station for real time imaging. TRAP staining was performed 3 days after plating.
Results: Trypsinization together with pipetting and shaking can detach the adherent osteoclasts,and the resuspended cells can be used for passage and storage in liquid nitrogen. The passaged cells became fully attached to the culture dishes in 2 hours,and the multinucleated feature could be clearly seen. The osteoclasts recovered from liquid nitrogen could completely spread out for 2 to 3 hours so that the multinucleated cells were clearly seen. These cells were still TRAP positive.
Conclusion: Although osteoclasts strongly adhere to the bottom of culture dishes,a large majority of the osteoclasts can be detached after appropriate digestion with trypsin,pipetting and shaking. These cells can be used for passage and cryopreservation. After recovering from liquid nitrogen,these cells still preserve the viability and the feature of osteoclasts. The results provide a new and powerful tool for future study of osteoclast biology. |
| KEY WORDS Osteoclast Culture techniques Subculture Cryopreservation Recovery |
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| 引用本文，请按以下格式著录参考文献： |
| 中文格式: | 俞索静,吴承亮,金红婷,胡雪琴,肖鲁伟,童培建.破骨细胞的传代、冻存与复苏[J].中国骨伤,2017,30(5):463~469 |
| 英文格式: | YU Suo-jing,WU Cheng-liang,JIN Hong-ting,HU Xue-qin,XIAO Lu-wei,TONG Pei-jian.Subculture,cryopreservation and recovery of osteoclasts[J].zhongguo gu shang / China J Orthop Trauma ,2017,30(5):463~469 |
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