Total cell pooling in vitro: an effective isolation method for bone marrow-derived multipotent stromal cells.

In vitro cellular proliferation and the ability to undergo multilineage differentiation make bone marrow-derived multipotent stromal cells (MSCs) potentially useful for clinical applications. Several methods have been described to isolate a homogenous bone marrow-derived MSCs population; however, none has been proven most effective, mainly due to their effects on proliferation and differentiation capability of the isolated cells. It is hypothesized that our newly established total cell pooling method may provide a better alternative as compared to the standard isolation method (density gradient centrifugation method). For the total cell pooling method, MSCs were isolated from rabbit bone marrow and were subsequently cultured in the growth medium without further separation as in the standard isolation method. The total cell pooling method was 65 min faster than the standard isolation method in completing cell isolation. Nevertheless, both methods did not differ significantly in the number of primary viable cells and population doubling time in the cultures (p > 0.05). The isolated cells from both methods expressed CD29 and CD44 markers, but not CD45 markers. Furthermore, they displayed multilineage differentiation characteristics of chondroblasts, osteoblasts, and adipocytes. In conclusion, both methods provide similar efficiency in the isolation of rabbit bone marrow-derived MSCs; however, the total cell pooling method is technically simpler and more cost effective than the standard isolation method.