Development of a microfluidic device for determination of cell osmotic behavior and membrane transport properties.

An understanding of cell osmotic behavior and membrane transport properties is indispensable for cryobiology research and development of cell-type-specific, optimal cryopreservation conditions. A microfluidic perfusion system is developed here to measure the kinetic changes of cell volume under various extracellular conditions, in order to determine cell osmotic behavior and membrane transport properties. The system is fabricated using soft lithography and is comprised of microfluidic channels and a perfusion chamber for trapping cells. During experiments, rat basophilic leukemia (RBL-1 line) cells were injected into the inlet of the device, allowed to flow downstream, and were trapped within a perfusion chamber. The fluid continues to flow to the outlet due to suction produced by a Hamilton Syringe. Two sets of experiments have been performed: the cells were perfused by (1) hypertonic solutions with different concentrations of non-permeating solutes and (2) solutions containing a permeating cryoprotective agent (CPA), dimethylsulfoxide (Me(2)SO), plus non-permeating solute (sodium chloride (NaCl)), respectively. From experiment (1), cell osmotically inactive volume (V(b)) and the permeability coefficient of water (L(p)) for RBL cells are determined to be 41% [n=18, correlation coefficient (r(2)) of 0.903] of original/isotonic volume, and 0.32+/-0.05 microm/min/atm (n=8, r(2)>0.963), respectively, for room temperature (22 degrees C). From experiment (2), the permeability coefficient of water (L(p)) and of Me(2)SO (P(s)) for RBL cells are 0.38+/-0.09 microm/min/atm and (0.49+/-0.13) x 10(-3)cm/min (n=5, r(2)>0.86), respectively. We conclude that this device enables us to: (1) readily monitor the changes of extracellular conditions by perfusing single or a group of cells with prepared media; (2) confine cells (or a cell) within a monolayer chamber, which prevents imaging ambiguity, such as cells overlapping or moving out of the focus plane; (3) study individual cell osmotic response and determine cell membrane transport properties; and (4) reduce labor requirements for its disposability and ensure low manufacturing costs.