中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (27): 5002-5006.doi: 10.3969/j.issn.1673-8225.2011.27.014

• 干细胞培养与分化 stem cell culture and differentiation • 上一篇    下一篇

高效制备昆明鼠胚胎成纤维细胞饲养层

腊晓琳，田海清，蔡  霞   

  1. 新疆医科大学一附院生殖中心，新疆维吾尔自治区乌鲁木齐市830011
  • 收稿日期:2011-01-06 修回日期:2011-03-21 出版日期:2011-07-02 发布日期:2011-07-02
  • 通讯作者: 蔡霞，博士，主任医师，博士生导师，新疆医科大学一附院生殖中心，新疆维吾尔自治区乌鲁木齐市 830011 caixia5512@126.com
  • 作者简介:腊晓琳☆，女，1971年生，河南省鹤壁市人，汉族，2008年新疆医科大学毕业，博士，副主任医师，主要从事生殖助孕与干细胞研究。 xiaolinla@hotmail.com
  • 基金资助:

    新疆医科大学一附院科研专项基金资助项目(2007-GXB-01)；项目名称：不同来源胚胎干细胞建系培养和鉴定；课题名称：昆明鼠胚胎干细胞培养和鉴定。

Efficient method for establishing mouse embryonic fibroblast feeder layers

La Xiao-lin, Tian Hai-qing, Cai Xia   

  1. Reproductive Center, the First Affiliated Hospital of Xinjiang Medical University, Urumqi  830011, Xinjiang Uygur Autonomous Region, China
  • Received:2011-01-06 Revised:2011-03-21 Online:2011-07-02 Published:2011-07-02
  • Contact: Cai Xia, Doctor, Chief physician, Doctoral supervisor, Reproductive Center, the First Affiliated Hospital of Xinjiang Medical University, Urumqi 830011, Xinjiang Uygur Autonomous Region, China caixia5512@126.com
  • About author:La Xiao-lin☆, Doctor, Associate chief physician, Reproductive Center, the First Affiliated Hospital of Xinjiang Medical University, Urumqi 830011, Xinjiang Uygur Autonomous Region, China xiaolinla@hotmail.com
  • Supported by:

    the Specific Research Fund of the First Affiliated Hospital of Xinjiang Medical University, No. 2007-GXB-01*

PDF

438

被引次数

6

摘要：

背景：小鼠胚胎成纤维细胞作为饲养层是胚胎干细胞培养最常用的方法，能有效抑制胚胎干细胞分化并促进其增殖，但其制备过程繁琐，工作量大，准备周期长。
目的：探索建立小鼠胚胎成纤维细胞饲养层简单、高效的培养体系。
方法：取13.5 d胎龄胎鼠用改良组织块法及简化酶消化法分离培养原代成纤维细胞，倒置显微镜下观察不同方法培养的原代和传代鼠胚胎成纤维细胞的生长形态、结构及细胞数量变化。收集鼠胚胎成纤维细胞进行冻存，复苏后细胞经不同浓度作用时间的丝裂霉素C处理，制备饲养层。
结果与结论：两种简化方法培养的原代鼠胚胎成纤维细胞生长状态良好，得到高效优质足量细胞，操作过程简单，省去了多次消化、离心、细胞计数等繁琐操作，均适宜于鼠胚胎成纤维细胞的原代培养。简化复苏法复苏后的细胞，按其生长汇合情况，以丝裂霉素C 10 mg/L作用1.5~2.0 h或1 mg/L培养过夜，省时并可获得细胞生长状态最佳的饲养层。

关键词: 鼠胚胎成纤维细胞, 饲养层, 胚胎干细胞, 丝裂霉素C, 细胞培养, 小鼠

Abstract:

BACKGROUND: Mouse embryonic fibroblasts (MEFs) feeder is more frequently used to culture embryonic stem cells (ESCs) and can retain ESCs proliferative potential in an undifferentiated state. But the preparation of feeder cells is elaborate and multiplicity.
OBJECTIVE: To develop a simple and efficient method to establish MEFs feeder layers in vitro.
METHODS: Mouse fetuses of 13.5-day gestational age were chosen to isolate MEFs as feeder layer. MEFs were isolated using two simply methods in vitro. The change of cells about appearance and amount was observed by inverted microscope; frozen MEFs were thawed through different methods, and the survival rate of MEFs was contrasted. MEFs were inactivated with different concentration mitomycin C and different time treatment to prepare MEFs feeder layer.
RESULTS AND CONCLUSION: The primary MEFs isolated using the two simple methods were in the best state and adequate. The two methods were better to culture primary mouse embryonic fibroblast cells, and complex manipulate protocol could be avoided. After thawed by the simple method, MEFs were treated with 10 mg/L mitomycin C for 1.5-2.0 hours or 1 mg/L mitomycin C overnight based on different confluence conditions.

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