5. Culturing

In order to avoid patient morbidity, the amount of MSCs that could be isolated from BM aspirate should be too small [12]. Therefore, they should be cultured in vitro to enable the expansion of MSCs to generate millions of cells which can be used for further therapeutic applications [39]. It was stated that MSCs retain more potential to differentiate after the third passage (P) [41]. In addition, over 70% of clinical trials used MSCs from 1 to 5 passages [42]. Moreover, a study reported that MSCs from 7 to 9 passages underwent osteogenic differentiation more than cells of later passages. Moreover, recent data indicated that reactive oxygen specieshandling mechanisms (i.e., antioxidative activity/reduction potential) become disrupted in later passages, a condition, which was not observed in the lower passage [43].

Although several researchers [41] showed that with the long-term expansion of MSCs and with several sub-culturing, the cells lose their differentiating ability, a study performed [44] reported that no change at the level of genetic expression or differentiation capability of longterm cultured MSCs. Furthermore, MSCs have a stable phenotype over many generations in vitro [45]. Another study [46] reported that MSCs retained their multilineage differentiation potential till passage 10 (P10) and maintain high levels of telomerase activity and long telomere length up to P10, but steady decline in the efficiency of proliferation in all cell populations after P10. Furthermore, MSCs showed a marked increase in the time required for cell doubling and showed an enlarged, flattened cellular morphology at P15, after which they ceased to undergo cell division but remained viable in culture. Thus, cells from passage 9 were used for differentiation as it was needed to obtain sufficient cell numbers for use through extensive cell quantity amplification and later passages were avoided [47].

revealed that the cells were adherent to the surface of tissue culture plastic flask. Furthermore, the cells were spindle in shape; which is considered as a second important characteristic of mesenchymal cell morphology. Researchers [38] described a population of adherent cells in culture till P5 (Figure 2). Most of the cells exhibited fibroblast-like spindle shape and showed vesicular nuclei with prominent nucleoli. Moreover, in P9, the adherent cells remained attached to the surface with their characteristic spindle shape (Figure 3). The cells exhibited

Stromal Cell Ultrastructure

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http://dx.doi.org/10.5772/intechopen.76870

The two-dimensional morphology of MSCs demonstrated by scanning electron microscope (SEM) [38] showed the spindle-shaped cells with eccentric nuclei and several thin cytoplasmic processes extending from the edge of the cell surface in P5 and P9. In addition, cells in P 9

Figure 2. Cultured human bone marrow derived stromal cell from passage 5, showing adherent cells with their charac-

Figure 3. Cultured human bone marrow derived stromal cell from passage 9, showing adherent cells with their charac-

maintained their spindle shape (Figure 4). These SEM results were also reported [49].

vesicular nucleus, prominent nucleolus and multiple processes [38].

6.3. Electron microscope

6.3.1. Scanning electron microscope (SEM)

teristic spindle shape (arrow) [38]. Scale bar 200 μm.

teristic spindle shape (arrow) [38]. Scale bar 200 μm.
