Improvement of in vitro culture method for umbilical cord blood mesenchymal stem cells and study of biological characteristics of cultured cells
- Author:
Qi WANG
1
Author Information
1. Department of Orthopaedics
- Publication Type:Journal Article
- Keywords:
Cells;
cultured;
Immunophenotyping;
Mesenchymal stem cells;
Umbilical cord blood
- From:
Academic Journal of Second Military Medical University
2010;28(1):27-31
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To improve the traditional in vitro culture method for umbilical cord blood mesenchymal stem cells (UCB-MSCs) in an effort to increase the successful culture rate and to study the biological characteristics of the cultured cells. Methods: Full-term UCB samples were obtained with the mothers' consent and processed within 12 h of collection (n=28). Mononuclear cells (MNCs) were isolated from UCB by centrifugation at 2 500 r/min for 20 min and were suspended in presence of α-minimum essential medium(α-MEM) containing 100% fetal bovine serum (FBS) and 100 U/ ml penicillin. The medium was hall changed after 5-7 days' primary culture and then was totally changed at a 3-4 days' interval. After the cells adhered to the culture wall, the following procedures were carried out according to our improved methods. In group I, the suspension containing detached mesenchymal-like cells were removed onto a new culture disb when osteoclast like cells reached over 80% confluence. In group II, 15% calf serum instead of 10% FBS was used in the culture medium when osteoclast-like cells were growing confluence, and α-MEM medium containing 10% PBS was used again until most of the osteoclast-like cells were detached. The morphology of UCB-MSCs was observed under microscope and the phenotypes of them were examined by flow cytometry. The 5th passage cells were cultured with osteogenic medium and adipogenic medium separately and cytochemical staining was carried out subsequently. Results: Adhered cells were cultured from 20 of the 28 samples. UCB-MSCs were cultured from 13 of the 20 samples, which could grow to confluence and could be stably passaged to P22. The cultured UCB MSCs were all positive for MSC-related antigens, such as CD29 and CD105, but negative for CD34, CD45, and CD106. We also found that the cultured UCB-MSCs could differentiate into osteoblasts and fat cells under specified conditions. Conclusion: MSCs do exist in UCB, and improvement of culture method can increase the yielding of UCB MSCs. The cultured cells, with the similar phenotype and differentiation potential as those derived from other resources, can be amplified easily and passaged stably in vitro, making them promising seeds cells for future bone tissue engineering.
