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

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.

Numerical Modeling of Temperature-Dependent Cell Membrane Permeability to Water Based on a Microfluidic System with Dynamic Temperature Control.

In order to describe temperature-dependent cell osmotic behaviors in a more reliable method, a novel mathematical mass transfer model coupled with dynamic temperature change has been established based on the combination of a time domain to temperature domain transformation equation and a constant temperature mass transfer model. This novel model is numerically simulated under multiple temperature changing rates and extracellular osmolarities. A microfluidic system that can achieve single-cell osmotic behavior observation and provide dynamic and swift on-chip temperature control was built and tested in this paper. Utilizing the temperature control system, the on-chip heating processes are recorded and then described as polynomial time-temperature relationships. These dynamic temperature changing profiles were performed by obtaining cell membrane properties by parameter fitting only one set of testing experimental data to the mathematical model with a constant temperature changing rate. The numerical modeling results show that predicting the osmotic cell volume change using selected dynamic temperature profiles is more suitable for studies concerning cell membrane permeability determination and cryopreservation process than tests using constant temperature changing rates.

Determination of the temperature-dependent cell membrane permeabilities using microfluidics with integrated flow and temperature control.

We developed an integrated microfluidic platform for instantaneous flow and localized temperature control. The platform consisted of a flow-focusing region for sample delivery and a cross-junction region embedded with a microheater for cell trapping and localized temperature control by using an active feedback control system. We further used it to measure the membrane transport properties of Jurkat cells, including the osmotically inactive cell volume (Vb) and cell membrane permeabilities to water (Lp) and to cryoprotective agent (CPA) solutions (dimethyl sulfoxide (DMSO) in this study) (PS) at various temperatures (room temperature, 30 °C, and 37 °C). Such characteristics of cells are of great importance in many applications, especially in optimal cryopreservation. With the results, the corresponding activation energy for water and CPA transport was calculated. The comparison of the results from the current study with reference data indicates that the developed platform is a reliable tool for temperature-dependent cell behavior study, which provides valuable tools for general cell manipulation applications with precise temperature control.

Temperature controlled tensiometry using droplet microfluidics.

We develop a temperature controllable microfluidic device for the accurate measurement of temperature dependent interfacial tensions between two immiscible liquids. A localized temperature control system is integrated with the microfluidic platform to maintain an accurate temperature inside the device. The temperature uniformity and sensitivity are verified by both simulation and experimental results. Temperature dependent interfacial tensions are measured dynamically and rapidly, relying on quantitative analysis of the deformation and retraction dynamics of droplets under extensional flow. Our microfluidic tensiometry offers the capability of measuring temperature dependent interfacial tensions with precise and systematic temperature control in the range of room temperature to 70 °C, which is valuable for studying transient interfacial dynamics, interfacial reactions, and the surfactant adsorption process.

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

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 Leukocytes.

BACKGROUND: Understanding how leukocytes in the cervicovaginal and colorectal mucosae respond to pathogens, and how medical interventions affect these responses, is important for developing better tools to prevent HIV and other sexually transmitted infections. An effective cryopreservation protocol for these cells following their isolation will make studying them more feasible. METHODS AND FINDINGS: To find an optimal cryopreservation protocol for mucosal mononuclear leukocytes, we compared cryopreservation media and procedures using human vaginal leukocytes and confirmed our results with endocervical and colorectal leukocytes. Specifically, we measured the recovery of viable vaginal T cells and macrophages after cryopreservation with different cryopreservation media and handling procedures. We found several cryopreservation media that led to recoveries above 75%. Limiting the number and volume of washes increased the fraction of cells recovered by 10-15%, possibly due to the small cell numbers in mucosal samples. We confirmed that our cryopreservation protocol also works well for both endocervical and colorectal leukocytes. Cryopreserved leukocytes had slightly increased cytokine responses to antigenic stimulation relative to the same cells tested fresh. Additionally, we tested whether it is better to cryopreserve endocervical cells on the cytobrush or in suspension. CONCLUSIONS: Leukocytes from cervicovaginal and colorectal tissues can be cryopreserved with good recovery of functional, viable cells using several different cryopreservation media. The number and volume of washes has an experimentally meaningful effect on the percentage of cells recovered. We provide a detailed, step-by-step protocol with best practices for cryopreservation of mucosal leukocytes.