Osmotic properties of T cells determined by flow imaging microscopy in comparison to electrical sensing zone analysis

Abstract

To develop cryopreservation methods for cell-based medicinal products it is important to understand osmotic responses of cells upon immersion into solutions with cryoprotective agents (CPAs) and during freezing. The aim of this study was to assess the osmotic response of T cells by using flow imaging microscopy (FIM) as a novel cell-sizing technique, and to corroborate the findings with electrical impedance measurements conducted on a Coulter counter. Jurkat cells were used as a potential model cell line for primary T cells. Cell volume responses were used to derive important cell parameters for cryopreservation such as the osmotically inactive cell volume V_b and the membrane permeability towards water and various CPAs. Unlike Coulter counter measurement, FIM, combined with Trypan blue staining can differentiate between viable and dead cells, which yields a more accurate estimation of V_b. Membrane permeabilities to water, dimethyl sulfoxide (Me₂SO) and glycerol were measured for Jurkat cells at different temperatures. The permeation of Me₂SO into the cells was faster in comparison to glycerol. CPA permeation decreased with decreasing temperature following Arrhenius behavior. Moreover, membrane permeability to water decreased in the presence of CPAs. V_b of Jurkat cells was found to be 49% of the isotonic volume and comparable to that of primary T cells. FIM proved to be a valuable tool to determine the membrane permeability parameters of mammalian cells to water and cryoprotective agents, which in turn can be used to rationally design CPA loading procedures for cryopreservation.

Keywords: Cell viability; Cryopreservation; Cryoprotectant loading; Cryoprotective agent; Flow imaging microscopy; Membrane permeability; Osmotic tolerance limit.