Erythrocyte enrichment in hematopoietic progenitor cell cultures based on magnetic susceptibility of the hemoglobin

Using novel media formulations, it has been demonstrated that human placenta and umbilical cord blood-derived CD34+ cells can be expanded and differentiated into erythroid cells with high efficiency. However, obtaining mature and functional erythrocytes from the immature cell cultures with high purity and in an efficient manner remains a significant challenge. A distinguishing feature of a reticulocyte and maturing erythrocyte is the increasing concentration of hemoglobin and decreasing cell volume that results in increased cell magnetophoretic mobility (MM) when exposed to high magnetic fields and gradients, under anoxic conditions. Taking advantage of these initial observations, we studied a noninvasive (label-free) magnetic separation and analysis process to enrich and identify cultured functional erythrocytes. In addition to the magnetic cell separation and cell motion analysis in the magnetic field, the cell cultures were characterized for cell sedimentation rate, cell volume distributions using differential interference microscopy, immunophenotyping (glycophorin A), hemoglobin concentration and shear-induced deformability (elongation index, EI, by ektacytometry) to test for mature erythrocyte attributes. A commercial, packed column high-gradient magnetic separator (HGMS) was used for magnetic separation. The magnetically enriched fraction comprised 80% of the maturing cells (predominantly reticulocytes) that showed near 70% overlap of EI with the reference cord blood-derived RBC and over 50% overlap with the adult donor RBCs. The results demonstrate feasibility of label-free magnetic enrichment of erythrocyte fraction of CD34+ progenitor-derived cultures based on the presence of paramagnetic hemoglobin in the maturing erythrocytes. © 2012 Jin et al

Donor blood oxyHb RBC settling velocity as a control for HSC culture analysis.

<p>The blood settling velocity histogram and its cumulative frequency distribution are shown. The cut-off settling velocity was set at 0.995 cumulative frequency for the subsequent HSC culture analysis.</p

Magnetic separation of HSC cultures increases concentration of fast sedimenting cells.

<p>Cell volume distributions by settling velocity measurements with CTV: the unsorted cells (A), “positive” cell fraction (B), and the “negative” cell fraction (C) against the donor RBC control. The comparison shows small increase in the fast sedimenting cell fraction in the “positive” fraction, in agreement with the Coulter counter results shown in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039491#pone.0039491.s006" target="_blank">Figure S4</a>.</p

Conversion to methemoglobin by oxidative treatment of HSC culture increases the magnetic cell fractional concentration.

<p>MM histograms (A) and dot plots (B) of HSC culture before and after the oxidative treatment used for conversion of Hb to paramagnetic metHb and used to determine the fractional concentration of maturing RBCs in culture.</p

MCHC distribution of the magnetically enriched cells falls within the range expected of the maturing RBCs (compare with Figure S1).

<p>MCHC distribution of the magnetically enriched cells falls within the range expected of the maturing RBCs (compare with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039491#pone.0039491.s003" target="_blank">Figure S1</a>).</p

Magnetic separation of HSC cultures improves viscoelastic properties of the cells.

<p>Deformability of cells in positive fraction (A) and negative fraction (B). Note increased frequency of dark, elongated objects, associated with hemoglobin containing deformable cells characteristic of mature RBC, in panel A.</p

Strong magnetic field gradient deflects sedimentation trajectories of high spin hemoglobin RBCs.

<p>Examples of the computer screen output of the CTV software showing the trajectories of A) oxygenated RBCs (low spin hemoglobin) and B) methemoglobin-conatining RBCs (high spin hemoglobin). Note the horizontal component of the cell trajectory due to cell magnetophoresis along the horizontal lines of the magnetic force. The cell magnetophoretic mobility, <i>m</i>, was defined as the horizontal distance traveled by the cell divided by the time of the image sequence acquisition and by the local magnetic energy density gradient (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039491#pone.0039491.e001" target="_blank">Eqn 1</a> in text).</p

Magnetic separation of HSC cultures increases concentration of the hemoglobin containing cells.

<p>Dot plots of sorted HSC culture under anoxic conditions (in N2 atmosphere). (A) Positive fraction, (B) negative fraction showing shift towards more magnetic, smaller cells (putative maturing RBCs) in the “positive” fraction.</p