Combined use of micropipette aspiration and perifusion for studying red blood cell volume regulation

Abstract

We combined micropipette aspiration with a new technique of perifusion to study volume behavior of single red blood cells (RBCs) during anisotonic conditions. Other techniques have suggested that RBC volume changes are complete within 1 s, but our pipette data indicate a slower volume response of 5-10 s. This difference appears to be due to the partial aspiration of the cell into the pipette, which in part prevents exposure of the membrane area to the anisotonic medium. However, we found that medium will pass the aspirated RBC (220 mm H 2 0 aspiration pressure) and enter the pipette at a volume rate of 0.3 mm 3 /s; this rate was measured by introducing a flow marker, a separate RBC or a cell fragment, into the pipette. The osmotic balance across the RBC also affected the flow: if the outside fluid was made hypertonic, the pipette flow decreased, but at hypotonic conditions, the flow increased and contributed to the exposure of the aspirated portions of the RBC. However, after the initial 10 s, the RBC geometry can be precisely monitored; at 200 mOsm, the RBC swelled by 41.0 6 0.9% and then demonstrated a small but significant (P F 0.001) regulatory volume decrease. We suggest that this technique is very precise in measuring the dynamics of geometric regulation, although acute changes are affected by the partial cell aspiration and pipette flow.

Nanoscale Intracellular Organization and Functional Architecture Mediating Cellular Behavior

“…Over the past several decades, microtechnology such as micropipette aspiration devices has improved the understanding of membrane mechanics including: the interconnection between the membrane of the outer hair cell with its cortical lattice; the cell-cycle-regulated viscoelastic properties of hepatocellular carcinoma cells; and the volume differential behavior of red blood cells under an anisotonic environment, which has direct and indirect links to the cytoskeleton. 19,71,113 Fluid shearing devices have helped uncover protein-specific attachment sites and the contributions of the cytoskeleton to them. 17,49,61,87,106,110 Alternately, other methods can examine local domains.…”

Narrow-pore Flow Behavior of Blood Cells in Cardioplegic Suspension

“…The increased osmolality of the cardioplegia alone can be expected to influence the ability of the RBC to pass through the capillaries. Increased osmolality leads to shrinkage of the RBC, with increase in internal viscosity in a way that is known to reduce cellular deformability (6,17). On the other hand, RBC shrinkage is paralleled by an increased surface area-to-cell volume ratio, which is known to improve deformability (17).…”