Objective: In all protocols for isolation of mesenchymal stem cells (MSCs), a few days after culture initiation, the medium were discarded along with its contents of non-adherent cells and the adherent cell population kept and expanded as MSCs population. In the present study, attempt was made to expand the cells suspended in removed medium of primary culture and compare them with the adherent cell population. Materials and Methods: Four days after rat's bone marrow culture initiation, medium

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... the culture was collected and its suspended cells were culture-expanded in parallel with adherent cells till passage 3. During the culture period, the cells from either group were statistically compared with respect of the time required for cell confluency (the stage in which cells cover the entire surfaces) as an index of growth rate. At the end, the cells from both cultures were evaluated in terms of their differentiation potential. Results: The primary culture of the cells from removed medium contained large colonies of spindle-shaped cells that reached into confluency after 5.36±0.5 days, while those from the adherent population possessed small colonies reaching into confluency in 8.09±0.70 days. According to the results, at all studied passages, the cells of removed medium were significantly (p<0.05) achieved confluency in shorter time than the adherent population. Moreover, the cells from either culture could easily differentiate into bone, cartilage and adipose cells. Conclusions: It seems that some cells from removed medium, usually discarded in medium substitution, are MSCs possessing more growth rate than the primarily adherent cell population.