Effect Comparison With The Different Separation Methods Of Mesenchymal Stem Cells From Blood Loss Of Vertebral Operation

Objective This study is compare the effect with the different separation methods of mesenchymal stem cells (MSCs) in bleeding of thoracolumbar vertebral operation. The aim is to explore for the ideal method of MSCs separating from bleeding of spinal operation. Methods 12 patients whith thoracolumbar fracture were included in this experiment. The 30ml blood specimens from bleeding of cancellous bone in thoracolumbar vertebral operation of every patient were divided into three equal parts randomly and marked as A, B,C three groups respectively with different isolating methods:A group by density gradient seperation, B group with methyl cellulose (MC) precipitate seperation, C group by two-step seperation,which combined density gradient separation with MC precipitate seperation. The obtained MNC were cultured in low level sugar DMEM medium containing 10% fetal bovine serum. All medium was changed wholely after 72 hours and changed partly medium on time after 3-5 days. MNC were trypsined and passaged when these cells reached confluence state. MNC number of adherent cells on the third day, the primary culture time, the third generation of cells with three methods of separation wre compared. MNC morphology and their growth behavior were observed with inverted microscope. Cell surface markers of these cells were identified with flow cytometry. Results MNC number with three kinds of separation method were (6.99±0.77)×106 in A group,(11.5±1.70)×106 in B group and (8.20±0.96)×106 in C group. There is significant differences between the three groups (P<0.05). Adherent cells number on the third day were 11.40±2.37 in A group,10.10±1.91 in B group and 24±3.27 in C group. There was no significant differences between A group and B group (P>0.05), but there is significant differences between C group and anohter two groups (P<0.05).The primary culture time were 28.20±1.69 days in A group,28.40±2.12 days in B group and 20.40±1.17 days in C group. There is no significant differences between A group and B group (P>0.05), but there is significant differences between C group and anohter two groups (P<0.05). Subcultured third generations of cells were (6.39±0.30)×106 in A group, (5.54±0.42)×106 in B group and (8.43±0.30)×106 in C group. There is no significant differences between A group and B group (P>0.05), but there is significant differences between C group and anohter two groups (P<0.05). The cell morphology of three groups were the same feature:adherent longer spindle or fibroblastoid under inverted microscope. Cell surface markers were identified by flow cytometry. Cells were first isolated have CD44 expression always, however, positive rate of CD34, CD45 was lower. These MSCs after passage culture have highly CD29, CD44, CD90 expression, and lower expression of CD14, CD34, CD45. Conclusion There are cell identified as MSCs in the bleeding samples of thoracolumbar vertebral cancellous operation, but the number is very fewer. Two-step separation is a better separation of monocytes method. MSCs extract from bleeding in vertebral operation can be cultured in vitro amplification effectively and will have a wide range of application.