Objective    To develop new methods to cultivate

retrieve and purify mouse mesenchymal stem cells (mMSCs).     Methods    Bone marrow was collected from 2-month-old Kunming mice by flushing femurs and tibias with complete medium of DMEM-LG. Cells were plated in a Petri dish. After 24 hours

non-adherent cells were removed by two to three washes with PBS

adherent cells were further cultured in complete medium and retrieved by trypsinisation with 0.25% trypsin for 5 min at 37 ℃. The treated adherent cells were cultivated with 3×dilution for further generations. CD11b-negative cells were retrieved from the collected adherent cells of 3

rd

generation by using immunomagnetic microbeads

and continued t

o be cultured in complete medium. After the cultured cells were retrieved

their morphology and their ability of osteoblastic differentiation and adipocytic differentiation were examined.      Results    Most of mMSCs from 1

st

generation were of shuttle shape

some of irregular shape. After treatment with magnetic microbeads and several generations

mMSCs were of spindle

star and irregular shape. These cells were of rich cytoplasma

clear nucleolus

and grew in parallel or vortex. The cultured adherent cells from the first and subsequent generations had plenty of CD11b-positive blooding-making cells. After 20-day osteoblastic induction

mMSCs differentiated into bone cells

which showed orange phosphate in extracellular matrix by Alizarin red S staining. mMSCs could differentiate into lipocytes. The size of cells increased along with fat-developing induction period. These cells showed many orange fatty follicles with O Red Oil dyeing.      Conclusion    Pure mMSCs can not be retrieved by either adhering method or generation cultivation method separately. The combined methods of adhering

immunomagnetic microbeads

and serial subcultivation is effective in vitro in retrieve mMSCs.