Pi-Chao Wang

Rapid nitrification with immobilized cell using macro-porous cellulose carrier

Abstract Macro-porous cellulose carrier (AQUACEL) was applied for immobilization of nitrifying bacteria to develop a practical nitrogen removal system. The results of nitrification experiments revealed that both particle and pore sizes of the carrier significantly influenced the nitrification process. When the optimized carrier (particle size of 1 mm and pore size of 500 μm) was employed, the complete ammonium oxidation was attained at a high ammonium loading rate of 12 kg-N·m−3-carrier·d−1 at 25°C. In the nitrification using 5 mm cubic carrier, the accumulation of ammonium and nitrite ions resulting from complete inhibition of nitrite oxidation occurred at an ammonium loading rate of 10 kg-N·m−3-carrier·d−1. By measuring oxygen effectiveness factor of immobilized cells, the oxygen transfer inside the macro-porous carriers was found to be enhanced by liquid convection.

Effect of Shear Stress on Recombinant Chinese Hamster Ovary Cells

Abstract In a previous study, we speculated that shear stress might enhance the renin productivity of recombinant Chinese hamster ovary (rCHO) cells cultivated in a radial-flow reactor (Motobu et al., J. Ferment, Bioeng., 83, 443–450, 1997). In order to clarify the effect of shear stress on cell function, we designed a shear stress apparatus and investigated the cell morphology, cell cycle, renin productivity, and mRNA expression. In the case of subconfluent cells, which were mostly observed in the radial-flow reactor, it was found that after 24 h of shear stress exposure at 0.02 and 0.082 N/m2, the cell morphology became longer and slimmer, and cell growth in the subconfluent monolayer was inhibited as compared to that in a static culture. The results also showed that the cells were arrested in the G 0 G 1 phase of the cell cycle. However, renin productivity was found not to change under shear stress at 0.02 and 0.082 N/m2 within 24 h, although the expression of renin mRNA under the shear stress condition was increased. With regard to confluent cells, it was surprising to find that shear stress did not influence the cell growth, cell cycle, renin productivity, or expression of mRNA within 24 h. Shear stress thus appears to affect subconfluent cells rather than confluent ones.

A novel simple method to purify recombinant soluble human complement receptor type 1 (sCR1) from CHO cell culture

The human complement receptor type 1 (CR1, C3 b/C4b receptor) is a polymorphic membrane glycoprotein expressed on human erythrocytes, peripheral leukocytes, plasma and renal glomerular podocytes, which consists of transmembrane and cytoplasmic domains with 30 repeating homologous protein domains knows as short consensus repeats (SCR). CR1 has been used as an inhibitor for inflammatory and immune system for the past several years. Recently, it is reported that CR1 was found to suppress the hyper-acute rejection in xeno-transplantation and can be used to cure autoimmune diseases. A soluble form of CR1, called sCR1, is a recombinant CR1 by cleaving the transmembrane domain at C-terminus and has been expressed in Chinese Hamster Ovary (CHO) cells. Several purification methods for sCR1 from CHO cells have been reported, but most of them require complicated steps at high cost. Moreover, such methods are mostly performed under the pH condition apt to denaturing sCR1 and causes sCR1 losing its activity. We here report a rapid and efficient method to purify sCR1 from CHO cell. The new method consists of a two-stage of cell culture by cultivating cells in serum medium followed by serum-free medium, and a two-stage of column purification by means of heparin and gel filtration column chromatography. By using this novel method, sCR1 can be purified in a simple and effective way with high yield and purity. Furthermore, the purified sCR1 was confirmed to retain its activity to suppress the complement activationin vivo andex vivo.

Development of bioreactors for denitrification with immobilized cells

Abstract A macro-porous cellulose carrier (AQUACEL TM ) was used for immobilization of denitrifying bacteria to develop a practical, high-performance nitrogen removal system. When the immobilized cells were used for denitrification at a high nitrogen loading rate, flotation of carriers caused by the evolution of nitrogen gas occurred. For countering the problem of carrier flotation, new reactors in which hydrodynamic jet flow and centrifugal force are used. In these new reactors the floating carriers were distributed homogeneously and complete denitrification was achieved even at high loading rate of 20 kg-N/m 3 -carrier/d. This AQUACEL system was used for efficient denitrification of wastewater discharged from an electroplating factory.

High renin productivity of rCHO cells cultivated in radial-flow nonwoven fabric mat packed-bed reactor with increasing circulating flow rate

Radial-flow bioreactors are effective for reducing concentration gradients, because the flow area is large and the path for circulating culture broth is short. In this study, a polyester nonwoven fabric mat was employed as a packing material in a radial-flow reactor, and the renin production by recombinant Chinese hamster ovary (rCHO) cells in the bioreactor was investigated. The renin productivity of the cells was enhanced by use of an increasing circulating flow rate in the radial-flow reactor. It was also observed that the cells in the case of the increasing circulating flow rate formed aggregates, and the specific renin production rate was 6 times higher than that in the case of a constant circulating flow rate. This may imply that the cell aggregation which occurred on the nonwoven fabric mat in the case of the increasing circulating flow rate may have been due in part to the enhancement of the renin productivity.

Reduced Lipopolysaccharide (LPS)-Induced Nitric Oxide Production in Peritoneal Macrophages and Inhibited LPS-Induced Lethal Shock in Mice by a Sugar Cane (Saccharum officinarum L.) Extract

A sugar cane extract (SCE) has been found to have an immunostimulating effect in several animals. Lipopolysaccharide (LPS) is known to induce endotoxin shock via the production of inflammatory modulators such as tumor necrosis factor (TNF)-α and nitric oxide (NO). We examined in the present study the effects of SCE on the TNF-α and NO production in LPS-stimulated mice peritoneal cells and the endotoxin shock in mice. The supplementation of SCE to peritoneal macrophages cultured with LPS resulted in a significant decrease in NO production. All the mice injected intraperitoneally with LPS and D-galactosamine (LPS+GalN) died within 24 h. However, a peritoneal injection, but no intravenous or oral administration, of SCE (500–1,000 mg/kg) at 3 to 48 h before the LPS+GalN-challenge resulted in a significantly improved survival rate. These results suggest that SCE had a protective effect on LPS-induced endotoxin shock via one of possible mechanisms involving the suppression of NO production in the mouse peritoneal cavity.

Effect of stimulators such as GLP-1, PACAP, and nicotinamide on glucose-stimulated insulin secretion from porcine pancreatic endocrine cells in long-term culture.

Introduction Transplantation of glucose-responsive insulin-secreting cells has the potential to result in a cure for diabetes. Aim To report the development of a model of adult porcine pancreatic endocrine cells (PE cells) exhibiting glucose-stimulated insulin secretion during a long-term culture period, in vitro. Methodology The PE cells were prepared by non-enzymatic digestion and purified by modifying a technique developed in our laboratory. The cells were first cultured for 7 days in RPMI-1640 medium containing 10% fetal bovine serum and 10 m M nicotinamide. On adhesion to the culture flasks, cells were collected by trypsinization, and then cultured in tissue culture dishes in medium with or without stimulators such as glucagon-like peptide-1 (GLP-1), pituitary adenylate cyclase-activating polypeptide (PACAP), and nicotinamide. The ability of the cells to respond to glucose-stimulated insulin secretion was also observed with and without stimulators. The immunocytochemical studies demonstrated pancreatic islets with well-preserved insulin and glucagon-containing cells. The morphologic integrity of cultured porcine cells was observed for up to 5–6 weeks after the purification. Results At a concentration of 3.3 m M glucose, PACAP and nicotinamide did not affect glucose-dependent insulin secretion, whereas 10 n M GLP-1 stimulated insulin secretion significantly. However, when glucose concentration was increased to 20 m M, 10 n M GLP-1 had no effect on insulin secretion. We also demonstrated that GLP-1 and PACAP could maintain insulin secretion better than control in the culture up to 5 weeks. Also GLP-1 and PACAP increased the number of insulin-secreting cells in culture. Conclusion These results demonstrate that GLP-1 and PACAP increased the number of pancreatic &bgr;-cells in culture.

Improved MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay for the measurement of viable animal cell number in porous cellulose carriers

A method to measure cell concentration in porous cellulose carriers was developed. Results of the comparison of 5 colorimetric assays showed that MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay by the solubilization of the formazan with DMSO(dimethyl sulfoxide) was the most suitable method for the measurement of cell concentration in porous cellulose carriers. The growth of CHO-K1 (Chinese hamster ovary)-immobilized in different types of carrier was evaluated.

Anionic C-Terminal Proregion of Nematode Antimicrobial Peptide Cecropin P4 Precursor Inhibits Antimicrobial Activity of the Mature Peptide

Recently, an anionic proregion was found to be conserved at the C terminus of the antimicrobial peptide, nematode cecropin. Our results suggest that the antimicrobial activity of mature peptide is suppressed by the proregion in its precursor and is released from inhibition after processing. Inhibition is not likely to be due to direct suppression of membrane disruption.

Production of human growth hormone by protein-free cultivation of anchorage-dependent cells with porous cellulose carrier

Abstract Verots S3 cells derived from Vero-317 cells which can grow in biotin-containing MEM medium were successfully cultured in a protein-free medium with a porous cellulose carrier. The growth of Verots S3 cells without a carrier was inhibited because they easily aggregated under excessive shear stress, but the cells grew very quickly when cultured with the porous cellulose carrier. This improvement in cell growth was thought to be due to the protection from fluid shear stress afforded by the porous structure of the carrier. Production of human growth hormone (hGH) by repeated batch cultivation of Verots S3 cells was much better with than without the carrier. Cultivation with a temperature shift from 39 to 33°C gave hGH production up to 400% higher in a spinner culture with the cellulose carrier. For large-scale cultivation, temperature sensitive Verots S3 cells were cultivated with the porous cellulose carrier in a rotary column bioreactor and the perfusion culture results showed that the cell density inside the carrier increased up to 7.9 × 10 8 cells·ml-carrier −1 in 2 weeks. No concentration gradient of the cells in the rotary column was observed.