In situ FTIR assessment of dried Lactobacillus bulgaricus: KBr disk formation affects physical properties

Abstract. The overall protein secondary structure, heat-induced protein denaturation, membrane phase behaviour, and glassy behaviour of Lactobacillus bulgaricus dried with sucrose were studied by Fourier transform infrared spectroscopy (FTIR) using two sample preparation methods. Samples for FTIR analysis were either prepared by mixing dried sample with KBr and compression into disks or by air-drying of cell/sucrose suspensions on CaF 2 windows. The results show that KBr compression causes protein unfolding and affects the thermo-physical properties of dried cells when compared with cells that were dried on CaF 2 windows and directly used for FTIR analysis without further manipulation. The protein denaturation temperature of samples prepared in KBr disks was decreased by more than 70 • C compared to that of samples dried on CaF 2 windows. In addition, hydrogen bonding interactions of the glassy cell/sucrose matrix were drastically affected by KBr compression. In contrast with samples dried on CaF 2 windows, samples in KBr disks were not in a glassy state at room temperature. The membrane phase behaviour of the dried cells was also affected by preparation of the sample into KBr disks. We conclude that the KBr compression method for preparing samples for FTIR analysis affects conformation and physical properties of biomolecules in the dried state

Loading equine oocytes with cryoprotective agents captured with a finite element method model

Cryopreservation can be used to store equine oocytes for extended periods so that they can be used in artificial reproduction technologies at a desired time point. It requires use of cryoprotective agents (CPAs) to protect the oocytes against freezing injury. The intracellular introduction of CPAs, however, may cause irreversible osmotic damage. The response of cells exposed to CPA solutions is governed by the permeability of the cellular membrane towards water and the CPAs. In this study, a mathematical mass transport model describing the permeation of water and CPAs across an oocyte membrane was used to simulate oocyte volume responses and concomitant intracellular CPA concentrations during the exposure of oocytes to CPA solutions. The results of the analytical simulations were subsequently used to develop a phenomenological finite element method (FEM) continuum model to capture the response of oocytes exposed to CPA solutions with spatial information. FEM simulations were used to depict spatial differences in CPA concentration during CPA permeation, namely at locations near the membrane surface and towards the middle of the cell, and to capture corresponding changes in deformation and hydrostatic pressure. FEM simulations of the multiple processes occurring during CPA loading of oocytes are a valuable tool to increase our understanding of the mechanisms underlying cryopreservation outcome. © 2021, The Author(s)

Storage stability of liposomes stored at elevated subzero temperatures in DMSO/sucrose mixtures.

Cryopreservation of biological materials is predominantly done using liquid nitrogen, and its application involves high maintenance costs and the need for periodical refilling of liquid nitrogen. Stable storage in mechanical freezers at -80°C would eliminate these issues and allow for shipment of frozen specimens using dry ice. In this work, the possibility of increasing the storage temperature of cryopreserved samples to -80°C by using combinations of DMSO and sucrose has been studied. Preservation efficacy was studied by measuring stability of liposomes encapsulated with carboxyfluorescein during storage at -150, -80 and -25°C for up to three months. Thermal and molecular mobility properties of the different DMSO-sucrose formulations were measured using differential scanning calorimetry, whereas hydrogen bonding interactions of the formulations were probed by Fourier transform infrared spectroscopy. It was found that addition of sucrose to DMSO solutions increases the Tg, and decreases molecular mobility in the glassy state at a particular temperature. Although it was expected that storage above or close to Tg at -80°C would affect liposome stability, stability was found to be similar compared to that of samples stored at -150°C. Higher molecular mobility in the glassy state could not be associated with faster CF-leakage rates. Distinct differences in storage stability at -25°C, far above Tg, were found among the sucrose/DMSO formulations, which were explained by the differences in permeability of sucrose and DMSO resulting in different levels of osmotic stress in the formulations

Osmotic properties of stallion sperm subpopulations determined by simultaneous assessment of cell volume and viability

The aim of this study was to determine the osmotic tolerance limits of stallion sperm as well as the osmotic behavior of different sperm subpopulations, including viable and non-viable cells as well as viable cells of different average sizes. A flow cytometric approach was used for simultaneous assessment of cell volume and permeability of the plasma membrane for the fluorescent dye propidium iodide while exposing the cells to media with different solute concentrations. Equine spermatozoa have limited osmotic tolerance limits: exposure to hypotonic conditions below approximately 240 mOsm kg(-1) already results in an increase in plasma membrane damaged cells, increasing up to 50% at an osmolality of 136 mOsm kg(-1). Plasma membrane damaged stallion sperm do not show an osmotic response after 10 min incubation in hypotonic conditions, and their volume is smaller as compared to viable cells. It is shown that inclusion or exclusion of different subpopulations greatly affects Boyle van 't Hoff behavior and therewith determination of the osmotic inactive volume. Osmotic inactive volumes were determined to be 76% and 46% of the isotonic volume for the whole sperm population and the plasma membrane intact viable cells, respectively. In addition, viable subpopulations with different average cell volumes also show different osmotic behavior. The main subpopulation of viable cells increased up to 1.6 times its isotonic volume upon exposure to 150 mOsm kg(-1), and exhibited an osmotic inactive volume of 79%

Increasing storage stability of freeze-dried plasma using trehalose.

Preservation of blood plasma in the dried state would facilitate long-term storage and transport at ambient temperatures, without the need of to use liquid nitrogen tanks or freezers. The aim of this study was to investigate the feasibility of dry preservation of human plasma, using sugars as lyoprotectants, and evaluate macromolecular stability of plasma components during storage. Blood plasma from healthy donors was freeze dried using 0-10% glucose, sucrose, or trehalose, and stored at various temperatures. Differential scanning calorimetry was used to measure the glass transition temperatures of freeze-dried samples. Protein aggregation, the overall protein secondary structure, and oxidative damage were studied under different storage conditions. Differential scanning calorimetry measurements showed that plasma freeze-dried with glucose, sucrose and trehalose have glass transition temperatures of respectively 72±3.4°C, 46±11°C, 15±2.4°C. It was found that sugars diminish freeze-drying induced protein aggregation in a dose-dependent manner, and that a 10% (w/v) sugar concentration almost entirely prevents protein aggregation. Protein aggregation after rehydration coincided with relatively high contents of β-sheet structures in the dried state. Trehalose reduced the rate of protein aggregation during storage at elevated temperatures, and plasma that is freeze- dried plasma with trehalose showed a reduced accumulation of reactive oxygen species and protein oxidation products during storage. In conclusion, freeze-drying plasma with trehalose provides an attractive alternative to traditional cryogenic preservatio

Enthalpy relaxation behavior of DMSO/sucrose solutions (S1–4) at different temperatures below the T_g of the solutions.

<p>DSC pans with solution were maintained ~0–15°C below T_g for up to 300 min, after which thermograms were recorded (A–D). Enthalpy relaxation is evident as an endothermic event (oriented upwards) on top of the glass transition event, increasing with storage duration while decreasing if further from T_g. The area of this event, ΔH_relaxation, was determined and plotted versus the storage duration (E–G), for the indicated storage temperatures. Solution S1 was composed of 5% v/v DMSO, 1 M sucrose (A,E), S2 of 10% v/v DMSO, 1 M sucrose (B,F), S3 of 5% DMSO, 0.5 M sucrose (C,G), and S4 of 10% v/v DMSO with 0.5 M sucrose D,H).</p

CF leakage rates of PC liposomes with trapped CF in DMSO/sucrose solutions at different subzero temperatures.

<p>The difference between the storage temperature and T_g was plotted against the CF leakage rate. Cryoprotective solutions that were tested: S1 (5% DMSO, 1 M sucrose, white circles), S2 (10% DMSO, 1 M sucrose, black circles), S3 (5% DMSO with 0.5 M sucrose, white triangles), and S4 (10% DMSO with 0.5 M sucrose, black triangles).</p

Storage stability of PC liposomes with trapped CF, frozen in DMSO/sucrose solutions and stored at different temperatures.

<p>Cryoprotective solutions tested were: S1 (5% DMSO, 1 M sucrose, white circles), S2 (10% DMSO, 1 M sucrose, black circles), S3 (5% DMSO with 0.5 M sucrose, white triangles), and S4 (10% DMSO with 0.5 M sucrose, black triangles) HEPES buffered solution without further supplements (grey squares) served as a control. Samples were stored at −150°C (A), −80°C (B) and −25°C (C) for up to 3 months. As a measure for storage stability, CF-retention (i.e. protection against membrane leakiness) was assessed and plotted versus the storage duration. The insets in the panels (A) and (B) show the CF-retention after 90 d at −150°C and −80°C, respectively (no significant differences in CF-retention were found among the formulations). Data points representing mean values ± standard deviations were calculated from four measurements (control samples were measured once).</p

Glass transition temperatures of solutions composed of different contents of DMSO and sucrose.

<p>The onset temperature of glass transition was determined using DSC. This was done for 0–20% (v/v) DMSO solutions without supplements (white circles), as well as supplemented with 0.5 M sucrose (grey circles) or 1 M sucrose (black circles). For liposome storage experiments, performed at −80°C (dotted line), four different formulations were selected (labeled S1–4). Solution S1 was composed of 5% DMSO with 1 M sucrose (T_g: −68°C), S2 of 10% DMSO with 1 M sucrose (T_g: −77°C), S3 of 5% DMSO with 0.5 M sucrose (T_g: −84°C) and S4 of 10% DMSO with 0.5 M sucrose (T_g: −101°C).</p

Water fraction distributions in solutions composed of DMSO and sucrose, determined from the OH-stretching band in FTIR spectra.

<p>In panel A–B, the 3900–2700 cm⁻¹ spectral region is shown for two DMSO/sucrose solutions (S4: 10% v/v DMSO with 0.5 M sucrose, S2: 10% v/v DMSO with 1 M sucrose). Contributions of different water fractions were fitted, as Gaussian profiles centered initially at 3139 cm⁻¹ (fully hydrogen bonded water), 3241 cm⁻¹ (symmetrically hydrogen bonded water), 3389 cm⁻¹ (asymmetrically hydrogen bonded water) and 3533 cm⁻¹ (weakly hydrogen bonded water). The relative shifts in peak positions (C,D) and relative band areas (E,F) of these contributions were determined as a function of the DMSO concentration, in combination with either 0.5 M sucrose (C,E) or 1 M sucrose (D,F).</p