Recrystallization and Water Absorption Properties of Vitrified Trehalose Near Room Temperature.

PURPOSE: To provide the physicochemical properties of vitrified trehalose for predicting its recrystallization. METHODS: Thin films of vitrified trehalose solutions were prepared at room temperature and exposed to various humid and temperature atmospheres. The in-situ amount of retained water in the vacuum-dried trehalose thin film during exposure was determined using its FTIR spectrum by quantifying the extremely infinitesimal amount of retained water in the trehalose solution. Recrystallization of the sample was also assessed by the FTIR spectrum of trehalose dihydrate. RESULTS: The effective water absorption coefficient, h meff , exponentially increased to the water activity of the trehalose sample, A w , at 25°C and 40°C at which the increasing rates are comparable. The surface energy of trehalose dihydrate, γ, was found to be lower than the value calculated from the reported equation, neglecting the effects of the activity of the solute and solvent water. CONCLUSIONS: The retained water in trehalose considerably increases its affinity for water vapor, and the change in this affinity with regard to the water activity is nearly independent of temperature. The dihydrate nucleation rate of trehalose-water system is maximal when trehalose weight ratio is ~0.8 at 25°C and is slightly higher (~0.85) at 40°C.

Slow water dynamics in dehydrated human Jurkat T cells evaluated by dielectric spectroscopy with the Bruggeman-Hanai equation.

The picosecond orientational dynamics of intracellular water was measured by dielectric spectroscopy, with the aim of revealing the effects of cryoprotective agents (CPAs) on biological cells. As a first step, Jurkat cells (human T lymphocyte cells) suspended in aqueous sucrose solutions of different concentrations ranging from 0.3 M (isotonic) to 0.9 M (hypertonic) were examined at 25 °C with a frequency range up to 43.5 GHz. The Bruggeman-Hanai equation was employed to obtain a cellular dielectric spectrum without extracellular contributions from the measured complex permittivity of the cell suspensions. By analyzing the γ process around 1010 Hz based on the Debye relaxation function, two types of water (bulk-like water and hydration water with slower molecular dynamics) were observed. An increase in the fraction of intracellular slower water was observed in the dehydrated cells which had a highly concentrated environment of biomolecules.

Low O2 metabolism of HepG2 cells cultured at high density in a 3D microstructured scaffold.

Among the features of in vivo liver cells that are rarely mimicked in vitro, especially in microchips, is the very high cell density. In this study, we have cultured HepG2 in a plate-type PDMS scaffold with a three-dimensional ordered microstructure optimally designed to allow cells to attach at a density of 10(8) cells/mL. After the first step of static open culture, the scaffold was sealed to simulate the in vivo oxygen supply, which is supplied only through the perfusion of medium. The oxygen consumption rate at various flow rates was measured. An average maximal cellular oxygen consumption rate of 3.4 x 10(-17) mol/s/cell was found, which is much lower than previously reported values for hepatocytes. Nevertheless, the oxygen concentration in the bulk stream was not the limiting factor. It has been further confirmed by the reported numerical model that the mass transport resistance on the surface of a cell that limits the oxygen supply to the cell. These results further emphasize that access to a sufficient quantity of oxygen, especially through the diffusion-limited layer on the surface of a cell, is very important for the metabolism of hepatocytes at such a high density.

A method for the design of 3D scaffolds for high-density cell attachment and determination of optimum perfusion culture conditions.

The application of in vitro cultured cells in tissue engineering or drug screening, aimed at complex soft tissues such as liver, requires in vivo physiological function of the cultured cells. For this purpose, the scaffold in which cells are cultured should provide a microenvironment similar to an in vivo one with a three-dimensional extracellular matrix, a high supply capacity of O(2) and nutrients, and high cell density. In this paper, we propose a method to design (1) the geometry of the scaffold, with a surface/volume ratio optimized to allow high-density (5 x 10(7)cells/mL) cell culture and (2) culture conditions that will supply optimal quantities of oxygen and nutrients. CFD modeling of mass transport was used to determine the shear stress as well as O(2) and glucose metabolism in the scaffold (20 mm width-35 mm length) for various flow rates. Validation of the model was done through comparison with flow resistance and micro-PIV experiments. CFD analysis showed the maximum metabolic rate densities for this scaffold are 6.04 x 10(-3)mol/s/m(3) for O(2) at 0.71 mL/min and 1.91 x 10(-2)mol/s/m(3) for glucose at 0.35 mL/min.