A study of the osmotic characteristics, water permeability, and cryoprotectant permeability of human vaginal immune cells

Abstract

Cryopreservation of specimens taken from the genital tract of women is important for studying mucosal immunity during HIV prevention trials. However, it is unclear whether the current, empirically developed cryopreservation procedures for peripheral blood cells are also ideal for genital specimens. The optimal cryopreservation protocol depends on the cryobiological features of the cells. Thus, we obtained tissue specimens from vaginal repair surgeries, isolated and flow cytometry-purified immune cells, and determined fundamental cryobiological characteristics of vaginal CD3(+) T cells and CD14(+) macrophages using a microfluidic device. The osmotically inactive volumes of the two cell types (Vb) were determined relative to the initial cell volume (V0) by exposing the cells to hypotonic and hypertonic saline solutions, evaluating the equilibrium volume, and applying the Boyle van't Hoff relationship. The cell membrane permeability to water (Lp) and to four different cryoprotective agent (CPA) solutions (Ps) at room temperature were also measured. Results indicated Vb values of 0.516 V0 and 0.457 V0 for mucosal T cells and macrophages, respectively. Lp values at room temperature were 0.196 and 0.295 μm/min/atm for T cells and macrophages, respectively. Both cell types had high Ps values for the three CPAs, dimethyl sulfoxide (DMSO), propylene glycol (PG) and ethylene glycol (EG) (minimum of 0.418 × 10(-3) cm/min), but transport of the fourth CPA, glycerol, occurred 50-150 times more slowly. Thus, DMSO, PG, and EG are better options than glycerol in avoiding severe cell volume excursion and osmotic injury during CPA addition and removal for cryopreservation of human vaginal immune cells.

Keywords: Cryobiological characteristics; Cryopreservation; Human vaginal mucosa; Macrophage; Microfluidic perfusion channel; T cell.

Fig. 1

Cell volume excursion during perfusion and image processing. T cells were perfused by 10% DMSO in 0.9% NaCl.

Fig. 2

Determination of the osmotically inactive cell volume V_b for human vaginal mucosal immune cells. Results are presented as mean ± standard deviation (7–8 cells from 4 donors for each data point). (A) Linear curve fitting for T cells. (B) Linear curve fitting for macrophages.

Fig. 3

Cell volume excursion during perfusion by hypertonic solutions. (a) T cell volume excursion when perfused by a hypertonic saline solution (2× PBS). (b) T cell volume excursion when perfused by a hypertonic CPA solution (10% DMSO in 0.9% NaCl).

Fig. 4

CPA exposure tolerance tests for human vaginal mucosal T cells and macrophages. The x-axis shows the final CPA concentration (v/v, %) after addition into the cell suspensions. The cell viabilities were tested with flow cytometry after CPA addition and removal, and the results were normalized to the viability of fresh cells (mean ± STD, n = 4–5).