Enrichment of live unlabelled cardiomyocytes from heterogeneous cell populations using manipulation of cell settling velocity by magnetic field

Abstract

The majority of available cardiomyocyte markers are intercellular proteins, limiting our ability to enrich live cardiomyocytes from heterogeneous cell preparations in the absence of genetic labeling. Here, we describe enrichment of live cardiomyocytes from the hearts of adult mice in a label-free microfluidic approach. The separation device consisted of a vertical column (15 mm long, 700 μm diameter), placed between permanent magnets resulting in a field strength of 1.23 T. To concentrate the field at the column wall, the column was wrapped with 69 μm diameter nickel wire. Before passing the cells through the column, the cardiomyocytes in the cell suspension had been rendered paramagnetic by treatment of the adult mouse heart cell preparation with sodium nitrite (2.5 mM) for 20 min on ice. The cell suspension was loaded into the vertical column from the top and upon settling, the non-myocytes were removed by the upward flow from the column. The cardiomyocytes were then collected from the column by applying a higher flow rate (144 μl/min). We found that by applying a separation flow rate of 4.2 μl/min in the first step, we can enrich live adult cardiomyocytes to 93% ± 2% in a label-free manner. The cardiomyocytes maintained viability immediately after separation and upon 24 h in culture.

Figure 1

Mechanism of action for cell separation. (a) Cell suspension is loaded into a vertical column placed in a magnetic field. (b) Paramagnetic cells are attracted to the column wall where magnetic field is concentrated due to the circumferentially positioned nickel wire. (c) Non-paramagnetic cells are rinsed out of the column by application of the flow at the bottom inlet to the column.

Figure 2

Microfluidic device schematics and forces acting on the paramagnetic particle in the column. (a) Typical dimensions of the vertical column are: length of 15 mm and diameter of 700 μm. The nickel wire diameter was 69 μm. Trajectory of the paramagnetic particle in the vertical column is determined by the balance of the gravitational force (F_g), drag (F_D), buoyancy (F_b), and magnetic force (F_mr, F_mθ). (b) The column is placed vertically in the field generated by four permanent nickel-iron-boron magnets on each side of the column.

Figure 3

Myoglobin quantification in cells by ELISA. m-FB, neonatal mouse fibroblasts; m-neo CM, neonatal mouse cardiomyocyte; m-adult CM, adult mouse cardiomyocyte.

Figure 4

Metmyoglobin induction as a function of NaNO₂ treatment in cardiomyocytes. Treatment of adult cardiomyocytes with 2 mM NaNO₂ indicates maximal induction of metmyoglobin after 45 min.

Figure 5

Mathematical modeling results of hydrodynamic and magnetic force effects on a paramagnetic particle trajectory in the vertical separation column. (a) Theoretical velocity profile of the fluid flow at $4.2\hspace{0.17em}\mu \mathrm{l}/\text{min}$ within a $700\hspace{0.17em}\mu \mathrm{m}$ diameter vertical column. (b) Balance of magnetic and gravitational forces in the vertical separation column as a function of radial position in the column. (c) Trajectories of a paramagnetic particle initially placed in the center at the bottom of the column as a function of fluid flow rate (4.2, 9.5, or 10 μl/min). Column length of 15 mm is assumed.

Figure 6

Correlation between input cardiomyocyte percentage and output percentage in the cell population during the separation at 4.2 μl/min. Application of magnetic field during separation enables consistently high enrichment efficiently between 90% and 100%.

Figure 7

Viability of adult mouse cardiomyocytes (a) after separation and (b) subsequent culture for 24 h. All values are normalized to the average cell viability of the control group, at the appropriate time point. The control was kept on ice during separation and was not treated with NaNO₂ (no treatment).