Naohiro Shiragami

Introduction of foreign DNA into Chlorella saccharophila by electroporation

Plasmid, pBI221, was introduced into protoplasts of Chlorella saccharophila c-211-1a prepared from the cells in the stationary phase by electroporation. Transient expression of the introduced plasmid was observed under a field strength of between 600 and 900 V/cm, and a pulse duration of around 400 ms, where high membrane permeability to 70-kDa FITC-dextran was ascertained.

Effect of shear stress on activity of cellular enzyme in animal cell

The effect of shear stress on the activity of cellular enzyme in an animal cell was discussed by using a flow channel. The activity of lactate dehydrogenase (LDH) in cells exposed to a shear stress of 0.5 Pa for 12 h was about 4-fold greater than in the cells without exposure to shear stress. The relative LDH activity was correlated with the dissipation energy density of the flowing medium. A good correlation was obtained and it was found that the dependency of cellular enzyme activity on the shear stress and the exposure time was related to the transmission of the energy from the flowing medium to attached cells.

Anchorage-dependent animal cell culture by using a porous microcarrier

CHO-K1 cells were cultured by using a porous microcarrier. The effects of microcarrier concentration and agitation rate on cell growth in porous microcarrier cultures were investigated. The specific growth rate of 0.041 h−1 in porous microcarrier cultures was independent of both microcarrier concentration and agitation rate. By estimating the total surface area occupied by cells from the maximum cell number, it was found that not all the surface area of the porous microcarrier was utilizable for cell growth.The maximum cell number decreased with increasing the microcarrier concentration and the agitation rate. From this result, it was also found that not all the cells grown on the interior surface of the porous microcarrier were protected against mechanical damage due to agitation. The protection capacity of the porous microcarrier was estimated to be 300 cells/carrier. The direct gas sparging into the culture broth in porous microcarrier cultures improved the cell density without mechanical damage to animal cells.

A new type porous carrier and its application to culture of suspension cells

A new type porous carrier was fabricated from a mixture of sodium alginate, bovine serum albumin and sodium bicarbonate. The porous space of the carrier is an assembly of void spaces. The carrier was successfully applied to the cultivation of suspension animal cells. In the culture, while both cells and carriers were held in suspension, the cells were entrapped hydrodynamically into the void spaces in the carriers. A culture of hybridoma cells using this carrier resulted in a cell density up to 5.7×107 cells per ml-carrier.

MASS-PRODUCTION OF BIOCATALYST-ENTRAPPING ALGINATE GEL PARTICLES BY A FORCED OSCILLATION METHOD

Abstract A forced oscillation method applying reverse-piezo electric effect has been proposed for mass-production of biocatalyst-entrapping polymer gel particles. A jet of aqueous alginate solution flowing out through an orifice was disintegrated into droplets by an oscillating pressure which was exerted by a ceramic plate vibrated by the reverse-piezo electric effect. The droplets formed were immediately solidified into gel particles in calcium chloride solution. The particle production rate by the method was up to 270 particles s−1 or 1 × 10−6m3.s−1 The size of the particles produced were down to I mm depending on the applied frequency. The smallest matrix size of the gels polymer network was estimated to be around 5 nm. The method was applied for immobilization of yeast cells. The diameter of immobilized particles was 2.5 mm. The immobilized yeast grew well in the particle. The damage of yeast cells caused by the immobilization was not remarkable

Simulated-microcarrier motion and its effect on radial medium transfer inside a horizontally rotating cylindrical bioreactor (HRCB) for animal cell culture

The motion and distribution of microcarriers inside a horizontally rotating cylindrical bioreactor were simulated using spherical ion exchange resin particles. Particle motion deviated much from that expected under ideal conditions. The possible mass transfer enhancing effects of particle translation and cluster rotation are discussed.

Enhancement of autotrophic growth rate ofAlcaligenes eutrophus in a medium containing perfluorocarbon under low oxygen partial pressure

SummaryUnder low oxygen partial pressure,Alcaligenes eutrophus was cultivated autotrophically in a medium containing perfluorocarbon (PFC) as an oxygen-carrier. In the presence of PFC of more than 70% of the medium volume, enhancement of the growth rate was observed. At 90% PFC, the rate was about 18 times greater than that at 0% PFC.

Production of rG-CSF by CHO cell in aggregate microbeads culture

Chinese Hamster ovary (CHO) cells which produce granulocyte colony stimulating factor (G-CSF) were cultivated in a spinner flask containing dextran microbeads of about 50 μm in average diameter. After inoculation, cell attachment to the microbeads and formation of the aggregate between cells and microbeads were observed. After 5 days cultivation, some of the aggregates of cells and microbeads became over 200 μm in diameter. One aggregate was composed of about fifteen or more microbeads. The exchange of culture medium was done by settling the aggregates. By the aggregate microbeads culture method, continuous cultivation of CHO cells and production of G-CSF were performed.

A model for microcarrier motion inside a horizontally rotating bioreactor for animal cell culture

The motion of microcarriers inside the horizontally rotating bioreactor was simulated in order to obtain some insight as to how particle motion can affect radial mass transfer. Fluid motion was modeled taking into account momentum transfer induced by particle motion. The force balance on the particle included the viscous drag, inertia, gravitational and buoyant forces. The main characteristics of observed particle motion under conditions of low particle concentration were reproduced by the model. Some implications of particle motion to mass transfer are discussed.

Stimulation of Animal Cell Metabolism by Shear Stress

The effects of shear stress on cell viability and on the activities of intracellular enzymes were discussed. The cell viability decreased as exposure to shear stress continued but recovered to the previous level after a long exposure time. In cells exposed to a shear stress of 0.5 Pa for 12 h, the activities of lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) were about 4-fold and 3-fold greater than in the cells without exposure to shear stress, respectively. The relationship between the cell viability and the LDH activity was also investigated.