Antibody‐immobilized column for quick cell separation based on cell rolling

Abstract

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.

Separation technologies for stem cell bioprocessing

“…These differential interactions were already used for the separation of primitive populations of HSPC from adult BM and fetal liver (CD34 + and CD34 + CD38 − ) from more differentiated cells (CD34 − and CD34 + CD38 + ) since the CD34 + and the CD34 + CD38 − cells were found to roll slowly especially on P‐selectin and L‐selectin immobilized in a parallel plate flow chamber when compared to more differentiated CD34 − and CD34 + CD38 + cells (Greenberg et al, 2000). The same basic principle was applied for developing an anti‐CD34 antibody‐immobilized cell‐rolling column that can separate cells according to CD34 density on their surface (Mahara and Yamaoka, 2010a). This strategy was already applied with success for the separation of different stem cell populations from BM namely MSC with distinct osteoblastic differentiation potential (Mahara and Yamaoka, 2010b).…”

Stem cells in microfluidics

“…Many processing tools for HSCs are introduced and reviewed in this section including purification and separation, 145,[154][155][156][157][158] signaling analysis, [159][160][161][162][163][164] microchip electrophoresis assays, and microfluidicbased, digital, reverse transcription-polymerase chain reaction ͑RT-PCR͒ assays. [165][166][167] …”

“…Furthermore, an anti-CD34 antibody-immobilized, cell-rolling column has been developed with the ability to separate CD34+ cells by the CD34 density of the cell surfaces. 158 This device consisted of a tubular column with a continuous surface for cell separation such that the anti-CD34 antibody was immobilized at a high density on the surface. Then the injected cells were rolled onto the inner surface of the column to sheer force of the medium flow.…”

Isolation of Mesenchymal Stem Cells from Bone Marrow Aspirate

“…A method of separating cell populations has been described by Mahara & Yamoaka, whereby CD34-positive cells were bound to a cell rolling column containing immobilized anti-CD34-antibodies [64].…”

“…Phosphate-buffered saline (PBS) was also washed through the column to promote cell rolling. The authors of the study claim the system to be superior to MACS in that it is capable of separating cells based on their surface marker density, due to the additional cell rolling [64]. They also indicated their belief that this system would prove quicker and less damaging than other separation techniques, as well as producing a highly pure population of cells.…”