In vitro improved culture and biological Characteristics of umbilical cord blood mesenchymaI stem cells

Qi WANG; Qiming ZU; Liangbi XIANG; Xianmin LIU; Songbo LIU; Sanhuai GOU

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.MATERIALSA 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.