Dept. of Biomedical Engineering, Advanced Medical Engineering Center, National Cardiovascular Center Research Institute, Suita, Osaka, Japan.
Biotechnol Prog. 2010 Mar-Apr;26(2):441-7. doi: 10.1002/btpr.354.
Cell separation using methodological standards that ensure high purity is a very important step in cell transplantation for regenerative medicine and for stem cell research. A separation protocol using magnetic beads has been widely used for cell separation to isolate negative and positive cells. However, not only the surface marker pattern, e.g., negative or positive, but also the density of a cell depends on its developmental stage and differentiation ability. Rapid and label-free separation procedures based on surface marker density are the focus of our interest. In this study, we have successfully developed an antiCD34 antibody-immobilized cell-rolling column, that can separate cells depending on the CD34 density of the cell surfaces. Various conditions for the cell-rolling column were optimized including graft copolymerization, and adjustment of the column tilt angle, and medium flow rate. Using CD34-positive and -negative cell lines, the cell separation potential of the column was established. We observed a difference in the rolling velocities between CD34-positive and CD34-negative cells on antibody-immobilized microfluidic device. Cell separation was achieved by tilting the surface 20 degrees and the increasing medium flow. Surface marker characteristics of the isolated cells in each fraction were analyzed using a cell-sorting system, and it was found that populations containing high density of CD34 were eluted in the delayed fractions. These results demonstrate that cells with a given surface marker density can be continuously separated using the cell rolling column.
使用确保高纯度的方法学标准进行细胞分离是再生医学和干细胞研究中细胞移植的非常重要的步骤。使用磁性珠的分离方案已广泛用于细胞分离，以分离阴性和阳性细胞。然而，不仅细胞的表面标记模式（例如阴性或阳性），而且细胞的密度还取决于其发育阶段和分化能力。基于表面标记密度的快速和无标记分离程序是我们关注的焦点。在这项研究中，我们成功开发了一种抗 CD34 抗体固定化的细胞滚动柱，可根据细胞表面的 CD34 密度分离细胞。优化了细胞滚动柱的各种条件，包括接枝共聚和调整柱倾斜角度以及介质流速。使用 CD34 阳性和阴性细胞系，确立了该柱的细胞分离潜力。我们观察到在抗体固定化微流控装置上，CD34 阳性和 CD34 阴性细胞之间的滚动速度存在差异。通过倾斜表面 20 度并增加介质流速来实现细胞分离。使用细胞分选系统分析了每个馏分中分离细胞的表面标记特征，发现具有高 CD34 密度的群体在延迟馏分中洗脱。这些结果表明，可以使用细胞滚动柱连续分离具有给定表面标记密度的细胞。
