Determination of the Membrane Permeability to Water of Human Vaginal Mucosal Immune Cells at Subzero Temperatures Using Differential Scanning Calorimetry

Abstract

To study mucosal immunity and conduct HIV vaccine trials, it is important to be able to cryopreserve mucosal specimens and recover them in functional viable form. Obtaining a good recovery depends, in part, on cooling the cells at the appropriate rate, which is determined by the rate of water transport across the cell membrane during the cooling process. In this study, the cell membrane permeabilities to water at subzero temperatures of human vaginal mucosal T cells and macrophages were measured using the differential scanning calorimetry method proposed by Devireddy et al. in 1998. Thermal histograms were measured before and after cell lysis using a Slow-Fast-Fast-Slow cooling program. The difference between the thermal histograms of the live intact cells and the dead lysed cells was used to calculate the temperature-dependent cell membrane permeability at subzero temperatures, which was assumed to follow the Arrhenius relationship, [Formula: see text], where Lpg is the permeability to water at the reference temperature (273.15 K). The results showed that Lpg = 0.0209 ± 0.0108 μm/atm/min and Ea = 41.5 ± 11.4 kcal/mol for T cells and Lpg = 0.0198 ± 0.0102 μm/atm/min and Ea = 38.2 ± 10.4 kcal/mol for macrophages, respectively, in the range 0°C to -40°C (mean ± standard deviation). Theoretical simulations predicted that the optimal cooling rate for both T cells and macrophages was about -3°C/min, which was proven by preliminary immune cell cryopreservation experiments.

FIG. 1.

Simulation of L_pg and E_a by nonlinear regression curve fitting of the normalized cell volume fraction changes during cooling [V(T)/V₀].

FIG. 2.

Simulation of intracellular residual water content normalized to initial water content for the prediction of the optimal cooling rates. (A) Simulation for T cells. (B) Simulation for macrophages. Cooling rate B in unit: °C/min.

FIG. 3.

Experimental confirmation of the predicted optimal cooling rate. Human vaginal CD3⁺ T cells (left) and CD14⁺ macrophages (right) were cryopreserved at 1°C/min and 2.5°C/min, 3.5°C/min, or 10°C/min. Each gray dot represents the average of duplicate cryovials frozen from a single donor and gray lines indicate pairing. Black symbols indicate the mean of the experiments at that temperature and the black lines represent the 95% confidence interval (omitted for the 10°C/min case where n = 2). (A) Cell recovery rates at different cooling rates. (B) Recovery rates relative to that at 1°C/min.