Selection of Cell Populations with High or Low Surface Marker Expression Using Magnetic Sorting

Abstract

Magnetic cell sorting technology stands out because of its speed, simplicity, and ability to process large cell numbers. However, it also suffers from a number of drawbacks, in particular low discrimination power, which results in all-or-none selection outcomes limited to a bulk separation of cell populations into positive and negative fractions, as well as the modest purity of the selected cells and the inability to select subpopulations of cells with high expression of a surface marker. In the present study, we developed a simple solution to this problem and confirmed the effectiveness of this approach by multiple experiments with the magnetic selection of transduced cell populations. Murine NIH 3T3 cells were transduced with the bicistronic retroviral vector constructs co-expressing fluorescent reporter proteins EGFP (enhanced green fluorescent protein) or DsRed-Express 2 and LNGFR (low-affinity nerve growth factor receptor) as surface selection markers. The effects of the magnetic selection of transduced cells with anti-LNGFR Micro Bead (MB) doses ranging from 0.5 to 80 µL have been assessed. Low doses of MBs favored the depletion of weakly positive cells from the population, resulting in the higher expression levels of EGFP or DsRed-Express2 reporters in the selected cell fractions. Low MB doses also contributed to the increased purity of the selected population, even for samples with a low initial percentage of positive cells. At the same time, high MB doses resulted in the increased yield and a more faithful representation of the original expression profiles following selection. We further demonstrate that for populations with fairly narrow distribution of expression levels, it is possible to achieve separation into high- and low-expressing subsets using the two-stage selection scheme based on the sequential use of low and high doses of MBs. For populations with broad expression distribution, a one-stage selection with low or high doses of MBs is sufficient for a clear separation of low- and high-expressing subsets in the column-retained and flow-through fractions, respectively. This study substantially extends the potential of magnetic cell sorting, and may open new possibilities in a number of biomedical applications.

CD52/FLAG and CD52/HA Fusion Proteins as Novel Magnetic Cell Selection Markers

“…The two variants of the human CD52 open reading frame (ORF) containing embedded FLAG or HA epitope tags were amplified from the plasmids pUCHR-mClover-sAID_CD5Flag2 and pUCHR-mClover-sAID_CD5HA2 (kindly provided by Dr. Mazurov), and cloned into pTZ57R/T plasmid vector, followed by insert sequence verification. The hCD52/HA and hCD52/FLAG ORFs were excised by BspH I/Sal I digestion and re-cloned into the 2 nd position of the bucistronic MigR1ad1 retroviral expression vector (described in [12]) via Nco I/Sal I sites to obtain MigRCD52HA and MigRCD52Fl vectors.…”

“…The final constructs used in this study, namely constructs pMigRCD52H-EGFP and pMigRCD52Fl-EGFP, were prepared by re-cloning the EGFP ORF from the pMigLNR2-EGFP construct [12] into the 1 st position of MigRCD52HA and MigRCD52Fl vectors, respectively, via BamH I/EcoR I sites, while constructs pMigRCD52H-DsRex and pMigRCD52Fl-DsRex were similarly prepared by the transfer of the DsRedExpress2 ORF from the pMigLNR2-DsRex construct via BamH I/EcoR I sites of the above vectors.…”

“…The preparation of bicistronic retroviral construct with DsRedExpress2 reporter in the 1st position and hLNGFR selection marker in the 2nd position (pMigLNR2-DsRex) was described earlier [12].…”

“…The unquestionable advantages of magnetic selection include the simplicity of the procedure and its ability to process large numbers of cells in a short time [7][8][9]. In addition, this approach makes it possible to select rare populations [10,11], as well as populations with high and low levels of surface marker expression [12]. For magnetic selection of genetically modified cells, approaches based on the use of streptavidin-biotin systems might be used, including biotinylation of the surface tag via coexpression in cells of biotin ligase BirA [13], expression of streptavidin [14] or streptavidin-binding peptide on the cell surface [15].…”

“…For magnetic selection of genetically modified cells, approaches based on the use of streptavidin-biotin systems might be used, including biotinylation of the surface tag via coexpression in cells of biotin ligase BirA [13], expression of streptavidin [14] or streptavidin-binding peptide on the cell surface [15]. These approaches, however, have not become widespread; instead, certain surface markers naturally expressed by cells are primarily used for magnetic selection of modified cells, in particular CD4 [16,17] and LNGFR [10,12,18]. Commercial kits (Miltenyi Biotec) use LNGFR, CD4 and H-2Kk markers for magnetic selection of transiently modified cell populations.…”