In vitro improved culture and biological Characteristics of umbilical cord blood mesenchymaI stem cells
10.3969/jissn.1673-8228.2009.32.038
- VernacularTitle:改良法体外培养脐带血间充质干细胞及其生物学特性分析析☆
- Author:
Qi WANG
;
Qiming ZU
;
Liangbi XIANG
;
Xianmin LIU
;
Songbo LIU
;
Sanhuai GOU
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2009;13(32):6383-6387
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND:Studies have demonstrated that the success rate of isolation of umbilical cord blood mesenchymal stem cells (UCB-MSCs)is low,which also lacks of unified identification method.OBJECTIVE:To modify the traditional in vitro isolation and culture method of UC8-MSCs to obtain a higher success rate and to observe its biological characteristics.DESIGN,TIME AND SETTING:In vitro observation of cytology.The study was performed at the Ninth People's Hospital of Shanghai Jiao Tong University Medical College between April 2006 and January 2007.MATERIALS:A totaI of 28 UCB samples were obtained from full-term normal delivery.Department of Matemity,Shanghai Red House Hospital.The written informed consent was obtained from the puerpera and their families.METHODS:NeonataI umbilical cord blood was collected under sterile condition.Mononuclear cells (MNCs) were separated from using lymphocyte isolation medium by centrifugation and suspended in α-minimum essential medium(α-MEM)containing 10% fetal bovine serum.The medium was half exchanged after 5-7 days of primary culture and then was totally exchanged every 3-4 days.The confluent cells were divided into 2 groups.In group 1.when the round megakaryocytes were confluent and fusiform fibroblast-like cells were fell off.the cell suspension was transferred to new culture dish;in group 2.when the round megakaryocytes dominated the majodty,the culture medium was replaced by α-MEM containing 15% calf serum,followed by culture in α-MEM containing 10% fetal bovine serum when the round megakaryocytes fell off.The fifth passage of UCB-MSCs was harvested for subsequent osteoinduction in vitro.MAIN OUTCOME MEASURES:The cell morphology was observed by microscopy;Flow cytometry was used to examine the surface antigen phenotype;alkaline phosphatase and oil red staining was performed to detect cell differentiation capacity.RESULTS:Of 28 samples of UCB,attaching cells were obtained from 20 samples(6/10 in group 1,14/18 in group 2),fibroblast-like cells that could passage steadily(4 samples in group 1,9 in group 2)were cultured from 13 0f 20 samples with success rate of 46.4%,among which MSCs were passaged steadily up to P22.UCB-MSCs were all positive for MSC-related antigens such as CD29 and CD105,but negative for CD34,CD45 and CD106.Incubation of UCB-MSCs under special condition resulted in a differentiation of osteoblast and adipocyte.CONCLUSIONMSCs exist in UCB,which have multi-differentiation capacity,and passage steadily.The modified in vitro culture method improves culture success rale of UCB-MSCs.
