Modified culture method and prolonged action fluorescent labeling of bone marrow mesenchymal stem cells from mice

Meihua WANG; Kaixun HU; Zewu QIU

ABSTRACT

BACKGROUND:

Bone marrow mesenchymal stem cells (BMSCs) of mice are different from human and rats.The difficulties of culture,sustaining impermanency activity of BMSCs after passage and short-term effect of stem cells tracking limited the study of mice.

OBJECTIVE:

To obverse modified isolated culture method of BMSCs of mice and the feasibility of long-term fluorescent labeling stem cells.DESIGN,TIME AND

SETTING:

The experiments were conducted at the Affiliated Hospital of Academy of Military Medicine Science from June to December in 2008.MATERIALSC57BL/6 mice,males and females,4-6 weeks of age,mean weighing 20 g,were used.

METHODS:

Stem cell culture fluid serum and liquid change manner were optimized using adherent screening and Percoll separation method.Rat BMSCs were incubated.In accordance with previous experiences of MSCs between human and macaque,Hyclonehigh-grade fetal bovine serum was selected for mouse MSC incubation.Serum was 10% of the medium.Bone marrow cells were washed out using LG-DMEM to filter bone dregs and small muscle blocks.Subsequently,samples were added on the Percoll separating medium at relative density of 1.082,and then incubated in 75 cm~2 culture flask at the concentration of 1.5×10~6/cm~2.BMSCs at the second passage were labeled with CM-Dil.MAIN OUTCOME

MEASURES:

Morphological changes in primary cultured and subcultured mouse BMSCs were measured.BMSC surface antigen of the second passage of mice (CD105,CD44,CD25,CD34) were determined using flow cytometry.The modified method was assessed to harvest the purity of stem cells.Activity of mouse BMSCs was identified using adipogenic and osteoplastic differentiation.The strength of fluorescent cells following multiple passage was observed.

RESULTS:

①The attached cells were observed 48 hours after primary cells culture and changed shuttles,triangles and flats at 7 days after culture.The figure become bunches and radial pattam at 3 passages.②MSCs highly expressed CD105,CD44 phenotypes and seldom expressed CD34 and CD45.③Spindle shape of cells gradually disappeared,with increased cell body.Some cells grew in cluster.MSCs changed figures to multilayer knots 10 days after inducing osteoplastic differentiation.MSCs became roundness and appeared fat drops in cells 9 days after inducing adipogenic differentiation.Red fat particles were shown following oil O staining.④The labeling cells gave out oranges light,and marked rates were over 80% in MSCs under the fluorescent microscope.The labeling cells were over 47% in 4 passages MSCs using flow cytometry.

CONCLUSION:

The modified method gained high-dosage cells in shorten culture time at passage 2 and made CM-Dil long lime labeling cells,which made more convenient for MSCs experiment on mice and stem cells tracer experiment in vivo.