293细胞悬浮培养的问题

lwdfjw

goast(55001775@QQ.COM) 2009-02-25 09:50:45
请教大家个问题，驯化293悬浮培养，前2代，培养一切正常，大约3天就可以从0.5m到2m每ml.但从第三代开始悬浮的细胞就不再生长
goast(55001775) 2009-02-25 09:50:57
大家觉得是什么原因？

xiaobin

老兄，293cell贴壁的在10%小牛血清中都不好长，你把它驯化成悬浮的是用来做蛋白表达吗？能问一下你的参考文献么？
我看有文献是用微载体悬浮培养293cell的，用无血清培养基
文献如下：
HEK 293 cell suspension culture using fibronectin-adsorbed polymer nanospheres in serum-free medium
Ju Hee Ryu 1 2, Sang-Soo Kim 1 3, Seung-Woo Cho 1 2, Cha Yong Choi 2 3, Byung-Soo Kim 1 *
1Department of Chemical Engineering, Hanyang University, Seoul 133-791, Korea
2School of Chemical Engineering, Seoul National University, Seoul 151-742, Korea
3Interdisciplinary Program for Biochemical Engineering and Biotechnology, Seoul National University, Seoul 151-742, Korea
email: Byung-Soo Kim (bskim@hanyang.ac.kr)

*Correspondence to Byung-Soo Kim, Department of Chemical Engineering, Hanyang University, Seoul 133-791, Korea

Funded by:
Korea Research Foundation; Grant Number: KRF-2003-041-D002200

摘要：Previously, we reported on suspension culture of anchorage-dependent animal cells using plain polymer nanospheres in serum-containing medium. For commercial cell culture, it is more advantageous to use serum-free medium than serum-containing medium. To culture anchorage-dependent animal cells using polymer nanospheres in serum-free medium, the nanospheres need to be coated with cell adhesion proteins. In this study, we utilized fibronectin-adsorbed polymer nanospheres for suspension culture of anchorage-dependent animal cells in serum-free medium. Fibronectin was adsorbed onto poly(lactic-co-glycolic acid) nanospheres (433 nm in average diameter) by immersing the nanospheres in fetal bovine serum. The nanospheres were used to culture human embryonic kidney (HEK) 293 cells in serum-free medium in stirred suspension bioreactors. Nanospheres attached between HEK 293 cells and promoted cell aggregate formation compared with culture without nanospheres. Most cells in the aggregates were viable over a 10-day culture period. Importantly, the use of poly(lactic-co-glycolic acid) nanospheres promoted the cell growth significantly, compared with culture without nanospheres (3.8- vs 1.8-fold growth). The nanosphere culture method developed in this study removes the time-consuming and costly process of adaptation of anchorage-dependent animal cells to suspension culture in serum-free medium. This culture method may be useful for the large-scale suspension culture of various types of anchorage-dependent animal cells in serum-free medium. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res 71A: 128-133, 2004

dingyihe

2# xiaobin


HEK293 本来用小牛血清就养不好的，要用胎牛血清的。
不过悬浮培养是可以做的很好的。

dingyihe

1# lwdfjw

在无血清驯化过程中出现细胞不生长和细胞密度降低都是正常的。