Determination of the permeability of human lymphocytes with a microscope diffusion chamber

Abstract

A diffusion chamber similar to that proposed by J. J. McGrath ( J. Microsc., in press) was constructed which allows microscopic observation of osmotically induced volume changes of individual cells in small (microliter) sample volumes. The cells are kept fixed in position in the upper compartment of the chamber by means of a highly permeable membrane and exposed to a step-like change in concentration generated in the lower compartment. An electrical conductivity probe in the upper compartment was used to monitor the temporal change of salt concentration as experienced by the cells. The rise from isotonic to hypertonic can be approximated by an exponential function. Its time constant of τ = 2.08 sec seems to be mainly determined by the change in flushing solution as τ = 1.48 sec was measured with no membrane installed. With human lymphocytes, no loss of cell volume was detected before 5 sec, i.e., when 95% of the final concentration was reached extracellularly. A step change can hence be assumed when modeling exosmosis for determining the lymphocyte membrane permeability. The equations for coupled transport of water and salt were solved numerically and fitted to the experimental data. The results were also compared to various other transport models described in the literature. Human lymphocytes are almost ideally semipermeable with a hydraulic reference permeability of L p = 4.23 × 10 −4 cm/sec (3.13 × 10 −3 μm · atm −1 · sec −1) at T = 23 °C. The temperature and concentration dependence are described by an activation energy E a = 14.3 kJ/mol and a concentration coefficient α 2 = 0.261 osmol/kg. An osmotically inactive volume fraction of 36.9% was determined from the final cell volumes reached asymptotically after shrinkage.