Chinese Journal of Tissue Engineering Research ›› 2011, Vol. 15 ›› Issue (10): 1750-1754.doi: 10.3969/j.issn.1673-8225.2011.10.009

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Biological activity and restriction of human umbilical cord mesenchymal stem cells during long-term culture in vitro

Xu Chao1, Liao Ji-dong1, Liu Jing2, Mao Wen-zhe1, Li Yang-qiu3   

  1. 1Tiankaping Center for Medical Experiments, 2Department of Biochemistry, 3Institute of Hematology, School of Medicine, Jinan University, Guangzhou  510632, Guangdong Province, China
  • Received:2010-12-10 Revised:2011-02-03 Online:2011-03-05 Published:2011-03-05
  • Contact: Liao Ji-dong, Associate researcher, Tiankaping Center for Medical Experiments, School of Medicine, Jinan University, Guangzhou 510632, Guangdong Province, China tliaojd@jnu.edu.cn
  • About author:Xu Chao★, Studying for master’s degree, Tiankaping Center for Medical Experiments, School of Medicine, Jinan University, Guangzhou 510632, Guangdong Province, China xuchao717@126.com

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Abstract:

BACKGROUND: Mesenchymal stem cells (MSCs) possess multi-lineage differentiation potential and immunomodulatory effects, and can be made long-term culture in vitro. However, it is still not very clear about the restriction and biological activity feature of MSCs during long-term culture in vitro.
OBJECTIVE: To investigate changes of biological activity feature and restriction of human umbilical cord MSCs (hUC-MSCs) for long-term culture in vitro.
METHODS: The umbilical cord from cesarean section at term newborns was collected in sterile condition. MSCs were isolated from human umbilical cord by collagenase Ⅱ digestion. The hUC-MSCs were obtained by plastic surface adhesion selection, continuous passage culture by trypsin digestion. The cells surface antigens were identified by flow cytometry, adipogenic and osteogenic ability were determined by specially inducing culture from third generation. A series of different generation cells were collected for following analysis. The cell growth curve was analyzed by MTT assay. The cell cycle and apoptosis rate were measured by flow cytometry. The cell adhesive ability was detected by piece of climbing.
RESULTS AND CONCLUSION: The cultured hUC-MSCs phenotype was CD105+/CD29+/CD44+/CD31-/CD34-/CD45-/HLADR-, and could were induced toward osteoblast and adipocyte differentiation under appropriate experimental conditions. There was no significant change in the cellular morphology and the cell growth curve of subculture 3-23 generation. After subculture 28 generation, cell growth became slowly. When subculture 33 generation was compared with subculture 3 generation, the cellular number of G0/G1 phase was decrease 17.9%, and S+G2/M phase and the cell apoptotic rate were increase 103.4% and 316.7%, respectively. Their diversity was statistical significant (P < 0.01). Within subculture 3-18 generation, their diversity of the cellular number of G0/G1 and S+G2/M phase and the cell apoptotic rate were not statistical significant (P > 0.05). Our data show that following increase of subculture frequency, hUC-MSCs proliferate and adherent capabilities of were decrease, while its apoptosis rate increased, in condition of long-term culture in vitro. It’s all biological activity has gradually decreasing trend. The best biological activity period appeared in 3 to 18 generations.

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