Optimization of isolation and culture conditions of rabbit bone marrow mesenchymal stem cells

Junren HE; Ziquan YANG; Bing LI; Xiaochun WEI

ABSTRACT

BACKGROUND:

Different isolation and culture methods for bone marrow mesenchymal stem cell (BMSCs) will result in varying cell activity and purity, which will influence repair effect of tissue engineering.

OBJECTIVE:

To investigate the optimized methods of isolation and culture of BMSCs in vitro and validate the efficacy of cell acquisition. DESIGN, TIME AND

SETTING:

In vitro cytology trial was performed at the laboratory of Department of Orthopedics, Second Hospital of Shanxi Medical University from October to December 2007. MATERIALS Two 3-month-old New Zealand rabbits were provided by Shanxi Institute of Livestock.

METHODS:

Lymphocyte isolation solution was added to DMEM culture solution containing fresh bone marrow. BMSCs were obtained and purified by gradient centrifuge and adhesion culture in vitro. Non-attached cells were moved to new culture flask every 8 hours. The solution was changed firstly after 5 days of culture. Trypsinization was conducted at cell confluence of 90%. The first, third and fifth passages of cells were harvested. MAIN OUTCOME

MEASURES:

The morphology of BMSCs was observed with phase contrast microscope; the growth curve was drawn to determine doubling time. The percentage of the wel1 growth P2 cells were identified by CD44 staining by flow cytometry.

RESULTS:

Primary cultured BMSCs were oval, spindle-shaped or polygonal, and adhered to plastic surface within 48 hours, exhibiting spindle-shaped or polygonal with clear nucleus, and reached 90% confluence within 14 days. The first, third and fifth passages of BMSCs showed S-type, and were in latent phase at 1-3 days, in log phase 3 days later, and cells came into platform phase at 7-8 days. The mean doubling time was 24.22 hours. CD44-positive cells were found in the second passage, with positive rate of 93.0%.

CONCLUSION:

BMSCs are easily isolated and cultured in vitro with good growth and high purity by gradient centrifuge and adhesion culture with lymphocyte isolation solution.