Evaluation of drug toxicity with hepatocytes cultured in a micro-space cell culture system

A micro-space cell culture system was recently developed in which cells such as hepatocytes can be cultured and formed into a multicellular three-dimensional (3D) architecture. In this study, we assessed the performance of HepG2 cells cultured in this micro-space cell culture system in a drug toxicity test, and evaluated the effects of micro-space culture on their hepatocyte-specific functions. The micro-space cell culture facilitated the formation of 3D HepG2 cell architecture. HepG2 cells cultured in a micro-space culture plate exhibited increased albumin secretion and enhanced mRNA expression levels of cytochrome P450 (CYP) enzyme compared to those cultured in a monolayer culture. When the cells were exposed to acetaminophen, a hepatotoxic drug, the damage to the HepG2 cells grown in micro-space culture was greater than the damage to the HepG2 cells grown in monolayer culture. In addition, human primary hepatocytes grown in micro-space culture also exhibited increased albumin secretion, enhanced CYP mRNA expression levels and increased sensitivity to acetaminophen compared to those grown in monolayer culture. These results suggest that this micro-space culture method enhances the hepatocyte-specific functions of hepatocytes, including drug-metabolizing enzyme activities, making hepatocytes grown in the micro-space culture system a useful tool for evaluating drug toxicity in vitro.     

Characterization of N-glycan structures and biofunction of anti-colorectal cancer monoclonal antibody CO17-1A produced in baculovirus-insect cell expression system

Advantages of the baculovirus insect cell expression system for production of recombinant proteins include high capacity, flexibility, and glycosylation capability. In this study, this expression system was exploited to produce anti-cancer monoclonal antibody (mAb) CO17-1A, which recognizes the antigen GA733. The heavy chain (HC) and light chain (LC) genes of mAb CO17-1A were cloned under the control of P₁₀ and Polyhedrin promoters in the pFastBac™ dual vector, respectively. Gene expression cassettes carrying the HC and LC genes were transposed into a bacmid in Escherichia coli (DH10Bac). The transposed bacmid was transfected to Sf9 insect cells to generate baculovirus expressing mAb CO17-1A. Confocal immunofluorescence and Western blot analyses confirmed expression of mAb CO17-1A in baculovirus-infected insect cells. The optimum conditions for mAb expression were evaluated at 24, 48, and 72 h after the virus infection at an optimum virus multiplicity of infection of 1. Expression of mAb CO17-1A in insect cells significantly increased at 72 h after infection. HPLC analysis of glycosylation status revealed that the insect-derived mAb (mAb^I) CO17-1A had insect specific glycan structures. ELISA showed that the purified mAb^I from cell culture supernatant specifically bound to SW948 human colorectal cancer cells. Fluorescence-activated cell sorting analysis showed that, although mAb^I had insect specific glycan structures that differed from their mammalian counterparts, mAb^I similarly interacted with CD64 (FcγRI) and Fc of IgG, compared to the interactions of mammalian-derived mAb. These results suggest that the baculovirus insect cell expression system is able to express, assemble, and secrete biofunctional full size mAb.     

Visualization of a recombinant gene protein in the baculovirus expression vector system using confocal scanning laser microscopy

Expression of the recombinant protein beta-galactosidase in the Spodoptera frugiperda Sf-9 insect cell line infected by the Autographa californica nuclear polyhedrosis virus expressing beta-galactosidase (AcNPV-betagal) was visualized using confocal scanning laser microscopy with fluorescent staining of both the recombinant protein and the cell nucleus. The average size of the insect cells and the intracellular DNA concentration both increased markedly, respectively reading 3.8- and 2.3-fold the values before infection. The average beta-galactosidase activity began to increase at 20-24 h post infection and finally reached 1.9 x 10(4) units/ml. As the post infection time increased, the stained nucleus images expanded and spread broadly. Beta-galactosidase was first identified by fluorescent staining at 12 h post-infection, filled the cell at 27 h, began to be released at 36 h, and finally spread out of the cell. The locations of the nucleus and expressed beta-galactosidase were identified from computerized tomograms and 3-dimensional images.     

Control of specific growth rate to enhance the production of a novel disintegrin,saxatilin, in recombinant Pichia pastoris

The methylotrophic yeast Pichia pastoris is one of the best hosts for the production of foreign proteins because of the presence of a strong alcohol oxidase 1 (AOX1) promoter that can be induced by methanol. Feeding the yeast, methanol induces protein production and provides an energy source for the host cells. However, excessive levels of methanol inhibit the growth of host cells, and insufficient methanol levels lead to poor growth and protein production. We have used various methanol feeding strategies to enhance the production of saxatilin. Saxatilin is a novel snake venom-derived disintegrin that inhibits tumor angiogenesis and metastasis and has been shown to suppress ovarian cancer cell invasion. A two-step increase feeding strategy to control the specific growth rate led to the best results in terms of specific protein production rates and final saxatilin amounts within the limited fermentation time.     

Comparison of the National Research Council-2001 model with the Dutch system (DVE/OEB) in the prediction of nutrient supply to dairy cows from forages

The objective of this study was to compare the DVE/OEB system (DVE = truly absorbed protein in the small intestine; OEB = degraded protein balance) and the NRC-2001 model in the prediction of supply of protein to dairy cows from selected forages: alfalfa (Medicago sativa L. cv. Pioneer and Beaver) and timothy (Phleum pratense L. cv. Climax and Joliette). Comparisons were made in terms of 1) ruminally synthesized microbial CP, 2) truly absorbed protein in the small intestine, and 3) degraded protein balance. In addition, the effects of variety and cutting stage of the selected forages on the potential nutrient supply to dairy cows were also studied. The results showed that the predicted values from the DVE/OEB system and the NRC-2001 model had significant correlations with high R (>0.96) values. However, using the DVE/OEB system, the overall average microbial protein supply based on available energy was 12% higher, and the truly absorbed protein in the small intestine was 15% lower than that predicted by the NRC-2001 model. The difference was also found in the prediction of the degraded protein balances, which was 11% higher based on data from the NRC-2001 model. These differences are due to considerably different factors used in calculations in the two models, although both are based on similar principles. This indicates that a further refinement is needed for a modern protein evaluation and prediction system. In addition, this study showed that the two alfalfa varieties studied (Pioneer vs. Beaver) had no effect, but cutting stage had a profound influence on ruminally synthesized microbial CP (93, 96, 86 g/kg DM at stage of early bud, late bud, and early bloom, respectively) and truly absorbed intestinal protein predicted by the DVE/OEB system (80, 79, 67 g/kg DM at stage of early bud, late bud, and early bloom, respectively). With timothy, both variety (Climax vs. Joliette) and cutting stage had significant impacts on the potential protein supply predicted by both models. The potential protein supply (DVE or MP) to the dairy cow from Climax timothy was higher than that from Joliette timothy (DVE: 46 vs. 32 g/kg DM; MP: 61 vs. 38 g/kg DM). With increasing stage of cutting, the potential protein supply (DVE or MP) was reduced (DVE: 53, 39, 25 g/kg DM; MP: 62, 51, 36 g/kg DM at stage of joint, prebloom head, and full head, respectively).     

Efficiency of modified skimmed milk base media to achieve high exopolysaccharide/cell ratios by Lactobacillus delbrueckii subsp. bulgaricus SZ2 in optimised conditions defined by the response surface methodology

Exopolysaccharide (EPS) production by Lactobacillus delbrueckii subsp. bulgaricus SZ2 was optimised in modified MRS (M‐MRS) using the response surface methodology (RSM). Maximum EPS production was 74.3 ± 2 mg/L, and the optimised values of the three variables predicted for maximum EPS production included a temperature of 38.7 °C, Bacto‐casitone and glucose concentrations of 24.5 and 29.6 g/L, respectively. To compare EPS production in MRS and skimmed milk (SM), the kinetics of EPS formation and growth were monitored in M‐MRS, SM, skimmed milk plus 2% additional sucrose (Suc‐SM) and skimmed milk containing Bacto‐casitone (20 g/L) and yeast nitrogen base (5 g/L) (BY‐SM). EPS production in all the media tested seemed to be growth‐related. The EPS/cell ratios were determined to be 3.12 × 10^?10, 1.43 × 10^?10, 4.42 × 10^?11 and 3.16 × 10^?11 mg/cell, in Suc‐SM, SM, M‐MRS and BY‐SM, respectively, clearly indicating the greater effect of C/N ratio when cell behaviour in EPS production is considered.