**7. Potential merits of hemolysis treatment or density gradient centrifugation of bone marrow to isolate BMSCs**

Although hemolysis treatment of bone marrow with ammonium chloride primarily removes only erythrocytes from bone marrow, *in vivo* transplantation experiments indicated that some of the committed osteogenic cells contained in bone marrow are lost or damaged during the hemolysis treatment (Figure 1A and 1B). Thus, we hypothesized that BMSCs grown from hemolysed bone marrow might contain lower numbers of committed osteogenic cells and their cellular composition would differ from that of normal BMSCs (BMSCs grown from untreated bone marrow). However, contrary to the hypothesis, flow cytometric analyses revealed that these BMSCs contained equivalent numbers of committed osteogenic cells (Figure 3A). Since these BMSCs showed similar responses to osteogenic induction medium (Figure 4A), they seem to contain similar numbers of uncommitted stem cells as well. Therefore, it is likely that the cellular composition of BMSCs grown from hemolysed bone marrow is relatively close to that of normal BMSCs. As the cell yield in primary culture (harvested cell number after primary culture/ days of primary culture/ initially seeded cell number) was greater in the hemolysis group (0.52 in the hemolysed group and 0.44 in the untreated group), it can be concluded that hemolysis treatment of bone marrow is an efficient approach to the isolation of BMSCs.

After centrifugation over Ficoll®, bone marrow is separated into several fractions such as plasma, mononuclear cells, granulocytes, and erythrocytes. Since BMSCs belong to the mononuclear cell fraction in the bone marrow, it is likely that BMSCs are efficiently enriched in this fraction even though this isolate contains significantly lower cell numbers than untreated or hemolysed bone marrow (Figure 5).

**Figure 5.** Rat bone marrow was divided into three portions and the suspensions were either hemolyzed, or subjected to Ficoll fractionation, or left without treatment (untreated). Significant differences were observed in the average numbers of cells isolated among the groups. Data are presented as the mean ± standard deviation (n=6). \*\*: p < 0.01 (Modified from Agata et al., 2012 [13] with permission).

**7. Potential merits of hemolysis treatment or density gradient**

Although hemolysis treatment of bone marrow with ammonium chloride primarily removes only erythrocytes from bone marrow, *in vivo* transplantation experiments indicated that some of the committed osteogenic cells contained in bone marrow are lost or damaged during the hemolysis treatment (Figure 1A and 1B). Thus, we hypothesized that BMSCs grown from hemolysed bone marrow might contain lower numbers of committed osteogenic cells and their cellular composition would differ from that of normal BMSCs (BMSCs grown from untreated bone marrow). However, contrary to the hypothesis, flow cytometric analyses revealed that these BMSCs contained equivalent numbers of committed osteogenic cells (Figure 3A). Since these BMSCs showed similar responses to osteogenic induction medium (Figure 4A), they seem to contain similar numbers of uncommitted stem cells as well. Therefore, it is likely that the cellular composition of BMSCs grown from hemolysed bone marrow is relatively close to that of normal BMSCs. As the cell yield in primary culture (harvested cell number after primary culture/ days of primary culture/ initially seeded cell number) was greater in the hemolysis

**Figure 4.** Differences in the responses to osteogenic induction medium among BMSCs isolated from untreated, hemo‐ lysed, or Ficoll-treated bone marrow. (A) ALP activities in osteogenic induction medium. (B) Gene expression of osteo‐ pontin in non-induction medium. (C) Gene expression of osteopontin in osteogenic induction medium. (D) Gene expression of *Cbfa-1* in non-induction medium. (E) Gene expression of *Cbfa-1* in osteogenic induction medium. Data are presented as the means ± standard deviation (n = 3). \*: P < 0.05, \*\*: P < 0.01. (Modified from Agata et al*.,* 2012 [13]

**centrifugation of bone marrow to isolate BMSCs**

with permission)

44 Regenerative Medicine and Tissue Engineering

However, in contrast to expectations, the cell yield in primary culture was the lowest in this group (0.13 in the Ficoll-treated group). In addition, the cellular composition of this group's BMSCs seemed to be different from that of normal BMSCs, because these BMSCs showed significant differences in the percentage of cell-surface ALP-positive cells and the responses to osteogenic induction medium (Figure 3A and 4A), though they showed similarities in the morphologies and the expression of cell-surface CD54 and CD90 (Figure 2). Therefore, it can be concluded that density gradient centrifugation of bone marrow is not an efficient approach to the isolation of BMSCs that possess normal characteristics. However, this technique may be useful for the isolation of more potent (more primitive) BMSCs because BMSCs grown from Ficoll-treated bone marrow seem to contain greater numbers or higher concentrations of uncommitted stem cells.

### **8. Conclusion**

As the cellular composition of BMSCs varies significantly with the isolation technique, it is important to select an appropriate isolation technique for the purpose that is intended. For example, if BMSCs are used for bone tissue engineering, it might be better to isolate BMSCs by hemolysis, because BMSCs that contain greater numbers of committed osteogenic cells are efficiently obtained by this technique. On the contrary, if BMSCs are used for the stem cell therapies of non-bone diseases such as stroke, it might be better to isolate BMSCs by density gradient centrifugation, because BMSCs obtained by this technique contain greater numbers of uncommitted stem cells. Flow cytometric or magnetic cell sorting with antibodies might also be useful for the isolation of BMSCs for use in stem cell therapies because BMSCs isolated by this technique possess greater multi-lineage potency. However, most of the current clinical studies still use the conventional adherence technique for the isolation of BMSCs because the fact that the characteristics of BMSCs varies with the isolation techniques remains largely unknown. Since the results of clinical studies are greatly affected by the potentials of the BMSCs used, selection of an appropriate isolation technique may lead to a better outcome. Nonethe‐ less, further investigations are required to use these new techniques in clinical studies because available information concerning the safety, feasibility, and efficacy of these techniques is still limited. Furthermore, the cost effectiveness of these techniques should be investigated, since the conventional technique does not require any special reagents. Continuing investigations are important for the establishment of truly reliable new therapies using BMSCs.
