Ryo Shirakashi

Intracellular Delivery of Trehalose into Mammalian Cells by Electropermeabilization

The disaccharide trehalose is increasingly being used as a very efficient stabilizer of cells, membranes and macromolecules during cryo- and lyoconservation. Although extracellular trehalose can reduce cryo- and lyodamage to mammalian cells, the sugar is required on both sides of the plasma membrane for maximum protection efficiency. In the present study, mouse myeloma cells were loaded with the disaccharide by means of reversible electropermeabilization in isotonic trehalose-substituted medium, which contained 290 mM trehalose as the major solute. By using the membrane-impermeable fluorescent dye propidium iodide as the reporter molecule, optimum electropulsing conditions were found, at which most permeabilized cells survived and recovered (i.e., resealed) their original membrane integrity within a few minutes after electric treatment. Microscopic examination during the resealing phase revealed that electropulsed cells shrank gradually to about 60% of their original volume. The kinetics of the dye uptake and the volumetric response of cells to electropulsing were analyzed using a theoretical model that relates the observed cell volume changes to the solute transport across the transiently permeabilized cell membrane. From the best fit of the model to the experimental data, the intracellular trehalose concentration in electropulsed cells was estimated to be about 100 mM. This loading efficiency compares favorably to other methods currently used for intracellular trehalose delivery. The results presented here point toward application of the electropermeabilization technique for loading cells with membrane-impermeable bioprotectants, with far-reaching implications for cryo- and lyopreservation of rare and valuable mammalian cells and tissues.

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.

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 108 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 × 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.

Pore size of swelling-activated channels for organic osmolytes in Jurkat lymphocytes, probed by differential polymer exclusion

The present study explores the impact of the molecular size on the permeation of low-molecular-weight polyethylene glycols (PEG200-1500) through the plasma membrane of Jurkat cells under iso- and hypotonic conditions. To this end, we analyzed the cell volume responses to PEG-substituted solutions of different osmolalities (100-300 mOsm) using video microscopy. In parallel experiments, the osmotically induced changes in the membrane capacitance and cytosolic conductivity were measured by electrorotation (ROT). Upon moderate swelling in slightly hypotonic solutions (200 mOsm), the lymphocyte membrane remained impermeable to PEG300-1500, which allowed the cells to accomplish regulatory volume decrease (RVD). During RVD, lymphocytes released intracellular electrolytes through the swelling-activated pathways, as proved by a decrease of the cytosolic conductivity measured by electrorotation. RVD also occurred in strongly hypotonic solutions (100 mOsm) of PEG600-1500, whereas 100 mOsm solutions of PEG300-400 inhibited RVD in Jurkat cells. These findings suggest that extensive hypotonic swelling rendered the cell membrane highly permeable to PEG300-400, but not to PEG600-1500. The swelling-activated channels conducting PEG300-400 were inserted into the plasma membrane from cytosolic vesicles via swelling-mediated exocytosis, as suggested by an increase of the whole cell capacitance. Using the hydrodynamic radii R(h) of PEGs (determined by viscosimetry), the observed size-selectivity of membrane permeation yielded an estimate of approximately 0.74 nm for the cut-off radius of the swelling-activated channel for organic osmolytes. Unlike PEG300-1500, the smallest PEG (PEG200, R(h)=0.5 nm) permeated the lymphocyte membrane under isotonic conditions thus leading to a continuous isotonic swelling. The results are of interest for biotechnology and biomedicine, where PEGs are widely used for cryopreservation of cells and tissues.

Hypotonic Activation of the Myo-Inositol Transporter SLC5A3 in HEK293 Cells Probed by Cell Volumetry, Confocal and Super-Resolution Microscopy

Swelling-activated pathways for myo-inositol, one of the most abundant organic osmolytes in mammalian cells, have not yet been identified. The present study explores the SLC5A3 protein as a possible transporter of myo-inositol in hyponically swollen HEK293 cells. To address this issue, we examined the relationship between the hypotonicity-induced changes in plasma membrane permeability to myo-inositol P ino [m/s] and expression/localization of SLC5A3. P ino values were determined by cell volumetry over a wide tonicity range (100–275 mOsm) in myo-inositol-substituted solutions. While being negligible under mild hypotonicity (200–275 mOsm), P ino grew rapidly at osmolalities below 200 mOsm to reach a maximum of ∼3 nm/s at 100–125 mOsm, as indicated by fast cell swelling due to myo-inositol influx. The increase in P ino resulted most likely from the hypotonicity-mediated incorporation of cytosolic SLC5A3 into the plasma membrane, as revealed by confocal fluorescence microscopy of cells expressing EGFP-tagged SLC5A3 and super-resolution imaging of immunostained SLC5A3 by direct stochastic optical reconstruction microscopy (dSTORM). dSTORM in hypotonic cells revealed a surface density of membrane-associated SLC5A3 proteins of 200–2000 localizations/μm2. Assuming SLC5A3 to be the major path for myo-inositol, a turnover rate of 80–800 myo-inositol molecules per second for a single transporter protein was estimated from combined volumetric and dSTORM data. Hypotonic stress also caused a significant upregulation of SLC5A3 gene expression as detected by semiquantitative RT-PCR and Western blot analysis. In summary, our data provide first evidence for swelling-mediated activation of SLC5A3 thus suggesting a functional role of this transporter in hypotonic volume regulation of mammalian cells.

Effect of the Grain Boundary of Ice Crystals in a Frozen Gelatin Solution on the Dielectric Properties at a Subzero Temperature

The effect of the grain boundary of ice crystals in a frozen gelatin solution on the dielectric properties was investigated by the combination of a dielectric spectrometer and image analysis. A micro-slicer image processing system (MSIPS) was applied to measure the grain boundary properties as the perimeter density and number density of ice crystals. The perimeter density and number density of the ice crystals increased with increasing freezing rate. The dielectric properties of the frozen gelatin solution at various freezing rates were measured in the frequency range of 100 Hz to 100 kHz at −40 °C. The relaxation time did not affect the grain boundary properties. The perimeter density and number density significantly affected dielectric parameter ε0−ε∞ and electrical conductivity σ0. These results indicate that the dielectric spectrometer could be used to estimate the grain boundary properties in a frozen gelatin solution.

Recrystallization and Water Absorption Properties of Vitrified Trehalose Near Room Temperature

PurposeTo provide the physicochemical properties of vitrified trehalose for predicting its recrystallization.MethodsThin 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.ResultsThe effective water absorption coefficient, hmeff, exponentially increased to the water activity of the trehalose sample, Aw, 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.ConclusionsThe 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.

Measurement of Cellular Adhesion Ratios Under Shear Flow on Various Substrates

Perfusion culture is an effective method to enhance the oxygen and nutrient mass transfer for the culture of highly metabolic cells and/or the culture at a high cell density. However, the flow rate of culture medium induces a shear stress that may lead to the death of cells if it is too high. In this study, we measured the cellular adhesion ratio on various materials coated with type-I collagen under Poiseuille flow with flow rates in the range 1–21 mL/min. Hepatoma cell line, HepG2 cells, attached better on a polystyrene plate for tissue culture coated with type-I collagen (with τ0.5 , the shear stress required to detach 50% of cells, equal to 42.2 Pa) followed by a collagen coated glass plate (τ0.5 of 40.5Pa), then a polystyrene plate for tissue culture without collagen coating (τ0.5 of 33.8Pa), and finally on a PDMS (τ0.5 of 24.8Pa) plate coated with collagen. The fluorescence staining of the collagen suggests that clumps of cells and collagen were detached from the surface, which implies that the cell-collagen bonds are stronger than collagen-substrate bonds. Accounting these results, it can be concluded that by reinforcing the bonds between collagen and substrate, it might be possible for the cellular monolayer to stay attached on the substrate until τ0.5 reaches ∼40Pa. This conclusion suggests the importance of carefully choosing the cell substrate, which has a strong binding with the coated extracellular matrix, for the cell culture under a high shear stress.Copyright

Fabrication of Microbioreactors with an Optimized Structure Designed for High Density Culture of Hepatocyte

In order to perform cell culture from soft tissue in good condition, i.e. maintaining high viability and keeping specific physiological function, scaffolds with microstructured surface mimicking in vivo environment is necessary. In this paper we proposed PDMS microbioreactors with high surface/volume ratio for hepatocyte culture. These PDMS microbioreactors were designed for cells to attach in high density (over 108 cells/cm3), and were characterized in terms of flow resistance, which is important for mass transport of nutrients and oxygen

Study of a Micro-miniature JT Cooler

The micro-miniature JT cooler is a promising cooling device as an element of the micro-thermal system for semiconductor devices, which dissipate an incredible amount of heat. In particular, the thermal management of laser diodes is a serious problem because they require a large amount of power but must maintain a fixed temperature. Considering such a situation, the performance of a micro-miniature JT cooler was calculated under the assumption of a countercurrent heat exchanger operating with a laminar gas flow, and the guidelines of a micro-miniature JT cooler for optimizing pump power and size were designed from the viewpoint of minimizing the cost. The authors present the results in this paper. A micro-miniature JT cooler was fabricated from Si wafer using a photolithographic process, which is available for operation with C2H6 from 3 MPa to 0.1 MPa. It is shown that a cooling power of 2.1 W was obtained at 239 K with a mass flow of 51.7 mg/s.