Study on the preparation of mouse embryonic stem cell feeder layer

WANG Lu; et al

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Study on the preparation of mouse embryonic stem cell feeder layer

更新时间：2024-01-17

- Study on the preparation of mouse embryonic stem cell feeder layer
- Journal of Chongqing Medical University Issue 1, Pages: 77-81(2008)
- 作者机构：
  
  1. 重庆医科大学基础医学院组织胚胎学教研室
  2. 重庆医科大学基础医学院组织胚胎学教研室,重庆,400016
- 作者简介：
- 基金信息：
- DOI：
  CLC： Q813
- Published：2008，
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[1]王璐,王亚平.小鼠胚胎干细胞饲养层制备条件的研究[J].重庆医科大学学报,2008(01):77-81.

WANG Lu, et al. Study on the preparation of mouse embryonic stem cell feeder layer. [J]. Journal of Chongqing Medical University (1):77-81(2008)
[1]王璐,王亚平.小鼠胚胎干细胞饲养层制备条件的研究[J].重庆医科大学学报,2008(01):77-81. DOI：

WANG Lu, et al. Study on the preparation of mouse embryonic stem cell feeder layer. [J]. Journal of Chongqing Medical University (1):77-81(2008) DOI：

摘要

目的:建立稳定高效的小鼠胚胎成纤维细胞饲养层培养体系

用于分离克隆胚胎干细胞（Embryonic stem cell

ES细胞）。方法:取妊娠13.5～14.5d的胎鼠

采用组织消化法结合组织块培养法分离、培养小鼠成纤维细胞;MTT法测定细胞生长曲线

结合倒置显微镜观察筛选小鼠成纤维细胞饲养层的最佳种植密度;MTT法筛选丝裂霉素C作用的最佳浓度和时间。取妊娠3.5d的小鼠囊胚在经丝裂霉素C处理的饲养层上培养

观察胚胎干细胞生长情况。结果:组织消化法结合组织块培养法是分离培养小鼠成纤维细胞的较好的方法;1×105～2×105个/ml种植密度适于饲养层铺板;丝裂霉素C 10μg/ml作用3.5h或20μg/ml作用2.5h能抑制成纤维细胞增殖

细胞至少在7d内维持稳定的水平;妊娠3.5d小鼠囊胚在饲养层上培养能形成ES集落。结论:制备的胎鼠成纤维细胞饲养层能有效地支持小鼠胚胎干细胞的生长。

Abstract

Objective:To establish a stable and effective system of mouse embryonic fibroblasts(MEFs) feeder layer in order to separate and culture mouse embryonic stem cells(ESC) in vitro.Methods:the MEFs were isolated from the mouse embryos at 13.5~14.5dpc(days post-coitus) through primary tissue digestion and tissue piece culture.The optional planting density of cell was determined according to the cellular growth curve detected by MTT and the morphology in the culture dish under inverted microscope.The optional concentration of mitomycin C and the time treating with mitomycin on MEFs were determined by MTT assay.Murine blastcysts(3.5dpc) were cultured on the feeder layer treated by mitomycin C and ESC growth was observed.Result:The method of tissue digestion and tissue piece culture is an efficient way to obtain primary MEFs;1×105~2×105/ml cells is an optional planting density;MEFs proliferation could be effeciently repressed after being treated with mitomycin C 10μg/ml for 3.5 hours or 20μg/ml for 2.5 hours

and the quantity of MEFs could maintain at least for 7 days.ES clone could be obtained from the mouse blastcysts(3.5dpc) after being cultured on the feeder layer.Conclusion:MEFs feeder layer could effectively support mouse embryonic stem cells to grow.

关键词

胚胎干细胞饲养层胚胎成纤维细胞小鼠

Keywords

Embryonic stem cellfeeder layerEmbryonic fibroblastmouse

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