寿光黑鸡成纤维细胞的体外培养与冷冻保存

Culture in vitro and Cryopreservation of Shouguang Black Chicken Fibroblasts

[目的]建立鸡成纤维细胞的体外培养体系。[方法]用组织块法和消化法分别分离培养鸡皮肤成纤维细胞;比较细胞生长速率及冻存复苏率。[结果]酶消化培养的成纤维细胞原代生长较快,约5 d便可形成单层;2种方法传代细胞的生长速度相似,仅需2-3 d就可形成单层;使用酶消化法和反复贴壁法,经3-4代后,可获得纯化的成纤维细胞;成纤维细胞经冷冻复苏后有75%-80%存活;分离纯化的胚胎和皮肤成纤维细胞的生长曲线均正常,可传至12代,传代后染色体数目不变。[结论]采用组织块法及酶消化法培养鸡成纤维细胞均可获得ES细胞建系所需的饲养层细胞。该研究为ES细胞系的成功建立奠定了基础。

[ Objective] The aim of this study was to establish the in vitro culture system of chicken fibroblasts. [ Method ] Tissue explant method and enzymatic digestion method were used to separate and culture chicken fibroblasts respectively, and the cell growth rates and the cryopreservation and recovery rates were compared. [ Result ] The primary chicken fibroblasts derived from enzymatic digestion grew faster than those derived from tissue explant culture and formed confluent monolayer after culture for 5 days. However, the passage fibroblasts derived from the two methods had similar growth rates, which could be cultured in confluence within 2 to 3 days. Homogeneous fibroblasts could be obtained after the mixture of fibroblasts and epithelial cells were treated by the combined use of typsin digestion and repeated attachment method for 3 or 4 subcultures. There were 75% -80% of cells survived after frozen and thawed. The seperated and purified embryonic and skin fibroblasts showed normal and similar growth curves, both of which could be subcultured to the twelfth generation and still retain the normal chromosome number. [Conclusion] The feeder layer cells needed in ES cell line establishment could be obtained by cuhuring chicken fibroblasts using the tissue explant method and enzymatic digestion method. This study laid a foundation for the successful establishment of ES cell line.