BACKGROUND: PA6 cells are bone marrow stromal stem cells from the mouse skull, and scientists have found that PA6 cells co-cultured with some kinds of stem cells have shown neural differentiation, based on which, PA6 cells can be used for repair of nerve injury. Therefore, researchers give more and more attentions to PA6, but few studies have addressed isolation and culture methods of bone marrow stromal stem cells from the skull.
OBJECTIVE: To isolate and culture bone marrow stromal stem cells from the skull of Sprague-Dawley rats and to observe cellular morpholopy in vitro and perform immunofluorescence identification.
METHODS: Under sterile conditions, newborn Sprague-Dawley rat’s skull was cut into pieces. Small skull pieces were washed using Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum. Single cell suspension was made, and placed in culture flask. The culture medium was changed many times for cell purification. The second passage of cells was obtained for morphology observation under an inverted microscope. Cell surface markers were detected by using immunofluorescence staining.
RESULTS AND CONCLUSION: After the primary culture for 24 hours, cells exhibited adherent growth; after 3 days, cells were increased in number, and presented with irregular shapes, such as polygon, triangle, spindle and flat shape. After passage, cells were uniform in morphology, arranged in radial and bunch patterns, and exhibited strong adherent ability. Some cells grew in cluster, and proliferated faster than primary cells. Immunofluorescence
staining showed that cells were positive for CD105, CD73, CD44, CD90, but negative for CD45, D34,CD14, HLA-DR. Results indicate that this method can obtain bone marrow stromal stem cells.