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.

Cryopreservation of human mucosal tissues

“…Therefore, we believe that samples stored according to our protocols will remain stable indefinitely. This is the last of a series of papers reporting our studies designed to provide guidance on cryopreserving viable cells and tissues from mucosal sites of sexual pathogen transmission [1,17,18]. In the step-by-step protocols in S1 Text and the Supplement to [1], we aim to provide clear procedures to ensure that cryopreservation can be carried out in the best known manner.…”

Cryopreservation of Human Mucosal Leukocytes

“…Based on measurements of the cryobiological properties of vaginal T cells and macrophages [ 4 , 5 ], we hypothesized that dimethyl sulfoxide (DMSO), ethylene glycol (EG), and propylene glycol (PG) would be effective cryoprotective agents (CPA) for these cells, whereas glycerol would not be due to very limited cell membrane permeability to glycerol. Indeed, cryopreservation with 1.5M glycerol in FBS leads to considerably worse viability than either 10% DMSO or 1.5M PG for both T cells and macrophages ( Fig 1A ) .…”

“…We set out to develop an optimal procedure for the cryopreservation of mucosal leukocytes, including formulation of the cryopreservation medium. We isolated T cells and macrophages from the human vagina and measured their physical properties relevant to cryopreservation, as reported previously [ 4 , 5 ]. Based on these measurements, we conducted a series of cryopreservation studies to determine the protocol that leads to maximal recovery of live, functional cells.…”

A microfluidic perfusion approach for on-chip characterization of the transport properties of human oocytes

“…are commonly used for cell cryopreservation by controlled cooling rate freezing, 15, 39, 40 osmotic responses of human oocytes upon 0.56 M Trehalose, 1.5 M EG, or 1.5 M PG in the isotonic solution were performed for investigation of oocyte membrane permeability as with most of previous studies. 25, 41–46 The temperature dependence of the permeability (Arrhenius relationship 2, 47, 48 ) is another essential part of the oocyte membrane transport properties, which requires the permeability data at least two different temperatures. 1 We chose three typical temperatures (4, 15, and 25 °C) for this study because almost all the studies on this topic used two to four different temperatures ranging over −3 to 37 °C, 25, 41–46, 49 4 °C is the typical temperature commonly used for short-term preservation of biosamples, and 25 °C is the room temperature under which CPA loading/unloading is often done.…”