Biomedical subjects

M L Shuler

Publications and source records attributed to M L Shuler.

61 records · Page 4Linked to original sources

PubMedDate not supplied

Optimization of an assay for baculovirus titer and design of regimens for the synchronous infection of insect cells.

We have previously described a quantitative model of the trafficking of baculovirus in insect cells that considers the various infection steps such as attachment, internalization, endosomal fusion, and nuclear accumulation. Concepts from the model were used to design synchronous infection regimens for various cell lines, to analyze the inherent inefficiency of existing assays for virus titer, and to develop a modified end-point dilution assay optimized to measure more completely the concentration of intrinsically infectious particles in a virus inoculum. The titer obtained from existing assays incompletely counts infectious virus particles due primarily to the incomplete adsorption of the virus during the short, standard 1-h incubation period. For representative assays, the calculated bound virus is generally about 10% of the added virus, but could be as low as 1.4%, underestimating actual titers by 3-70-fold. A modified end-point dilution assay involving centrifugation has been developed from both quantitative and qualitative analyses. The ratio of particle to plaque-forming unit with the optimized assay was 4-6 compared to 100-300 for typical assays, representing a significant improvement in the measurement of total infectious virus concentration.

Adsorption↗

PubMedDate not supplied

Effects of long-term passaging of BTI-Tn5B1-4 insect cells on growth and recombinant protein production.

The BTI-Tn-5B1-4 insect cell line from Trichopulsi ni has enabled better secretion and higher productivity of recombinant proteins in the baculovirus expression system. An early passage stock of this cell line is compared here to the commercially available "High Five" cell line stock. The early passage Tn5B1-4 cells grew to over 11 x 10(6) cells/mL and expressed 2.5-fold more recombinant secreted alkaline phosphatase (SEAP) than the High Five cells. While the specific productivity on a cell number basis was only slightly higher, the early passage Tn5B1-4 cells maintained this productivity at higher cell densities, increasing their volumetric productivity to 50 microg/mL under elevated oxygen conditions. We propose that the difference in these two stocks of cells is the length of time they have been cultured, 130 total passages for the early passage versus 360 passages for the High Five cells. This conclusion is supported by the facts that the early passage Tn5B1-4 cells are smaller in diameter by 4 micron, smaller in cellular protein content (206 versus 270 microg of protein/10(6) cells), have an increased propensity to clump in suspension culture, and are less susceptible to dispersion of these clumps by dextran sulfate compared to the High Five cells.

Alkaline Phosphatase↗

PubMedDate not supplied

Low-cost serum-free medium for the BTI-Tn5B1-4 insect cell line.

The BTI-Tn5B1-4 insect cell line, commercially available as the High Five cell line (Invitrogen), supports higher levels of recombinant protein production compared to existing insect cell lines. Proprietary serum-free media such as ExCell 405 (JRH Biosciences), Express Five (Life Technologies), IS BAC (Irvine Scientific), and CCM3 (HyClone) are available which were developed specifically for a suspension culture of High Five cells. While these media are highly optimized, a lower cost alternative is desirable for large-scale protein production which is also serum-free and supports good cell growth (>5 x 10(6) cells/mL) and recombinant protein production (>50 mg/L of secreted protein). The amino acid and carbohydrate metabolism of the Tn5B1-4 cells was first examined. It was found that asparagine was nearly depleted during batch growth in Ex-Cell 405, without limitations in glutamine, other amino acids, or glucose. Alanine also accumulated to about 35 mM during growth. We then extended the formulation techniques for medium development used for Spodoptera cell lines to the Tn5B1-4 cell line. A medium based on IPL-41 basal medium, Hy-Soy protein hydrolysate (Quest, International), yeastolate ultrafiltrate, a lipid-sterol emulsion, and Pluronic F-68 was developed. Dextran sulfate (100 microg/mL) was used to induce a single cell suspension culture. This medium is denoted as ISYL and performs best when supplemented with a 2.5% lipid-Pluronic F-68 mixture. Supplementation with additional aspargine in a 1.5% lipid-Pluronic F-68 mixture did not improve growth, suggesting that a lipid was growth-limiting and not an amino acid. Ex-Cell 405 and ISYL with 2.5% lipid-Pluronic F-68 supplement supported virtually identical growth rates, extent of growth (ca. 6.0 x 10(6) cells/mL) in an 80% oxygen atmosphere, and supported production of SEAP (secreted human alkaline phosphatase) at a volumetric level of about 65-70 mg/L. Thus, the less expensive ISYL medium can deliver acceptable performance and may be suitable for large-scale insect cell cultures.

Animals↗

PubMedDate not supplied

Hg2+ removal by genetically engineered Escherichia coli in a hollow fiber bioreactor.

Escherichia coli cells engineered to express an Hg2+ transport system and metallothionein accumulated Hg2+ effectively over a concentration range of 0.2-4 mg/L in batch systems. Bioaccumulation was selective against other metal ions and resistant to changes in ambient conditions such as pH, ionic strength, and the presence of common metal chelators or complexing agents (Chen, S.-L.; Wilson, D. B. Appl. Environ. Microbiol. 1997, 63, 2442-2445; Biodegradation 1997, 8, 97-103). Here we report the characterization of the bioaccumulation system based on its kinetics and an isotherm. Bioaccumulation was rapid and followed Michaelis-Menten kinetics. A hollow fiber bioreactor was constructed to retain the genetically engineered cells. The bioreactor was capable of removing and recovering Hg2+ effectively at low concentrations, reducing a 2 mg/L solution to about 5 microgram/L. A mathematical equation that quantitatively described Hg2+ removal by the bioreactor provides a basis for the optimization and extrapolation of the bioreactor. The genetically engineered E. colicells and the bioreactor system have excellent properties for bioremediation of Hg2+-contaminated environments.

Bioreactors↗

PubMedDate not supplied

Use of mannosamine for inducing the addition of outer arm N-acetylglucosamine onto N-linked oligosaccharides of recombinant proteins in insect cells.

Glycosylation is a cellular process accomplished by a series of sequential enzymatic processing steps through the endoplasmic reticulum and Golgi apparatus with vesicle transport between the membranous organelles. The capacity for complex glycosylation is considered to be conferred by cell genetics, while the roles of nongenetic factors in protein processing are often ignored. It was hypothesized that the glycosyltransferase reactions in the insect cell-baculovirus system were limited by the small supply of sugar donor cosubstrates. By adding mannosamine, the glycosylation of a human secreted alkaline phosphatase in Spodoptera frugiperda (Sf-21) cells was extended to include terminal N-acetylglucosamine structures which were not seen in control cultures, and in Trichoplusic ni (BTI-Tn5B1-4) cells the amount of terminal N-acetylglucosamine structures was increased.

Acetylglucosamine↗

PubMedDate not supplied

Use of in vitro data for construction of a physiologically based pharmacokinetic model for naphthalene in rats and mice to probe species differences.

A physiologically based pharmacokinetic (PBPK) model with five tissue groups (lung, liver, fat, richly perfused, and poorly perfused tissues plus venous and arterial blood compartments) has been developed from in vitro data and models of primary cell cultures for naphthalene toxicity in mice and rats. It extends a previous naphthalene PBPK model (Sweeney et al., 1996) and demonstrates a possible approach to a predictive mathematical model that requires minimal animal data. Naphthalene metabolism was examined after four exposure routes (intraperitoneal injection (ip), intravenous injection (iv), ingestion (po), and inhalation). Naphthalene and its primary metabolite, naphthalene oxide, are consumed by enzymes in pulmonary and hepatic tissues (cytochrome P450 monooxygenases, epoxide hydrolase, and glutathione-S-transferase). Additionally, the nonenzymatic reactions of naphthalene oxide in all tissues and in blood are included in the model. Kinetic constants for the model were derived primarily from cell fraction and primary cell culture incubations presented in the literature. The mouse model accurately predicts glutathione (GSH) and covalent naphthalene oxide-protein binding levels after a range of ip doses, and the rat model provides excellent estimates for mercapturate excretion following po doses; but neither model simulates well naphthalene blood concentrations after low iv doses. Good prediction of in vivo response using only in vitro data for parameter estimation (except for epoxide-protein binding rates) suggests that the assumed molecular description is a plausible representation of the underlying mechanisms of toxicity. Mice and rats show significant species differences in response to naphthalene. The model results suggest that species differences in toxicity may be explained, in part, by the lower overall rate of enzyme activities in the rat cells. Lower enzyme activities in the rat result in out-of-phase GSH minima in hepatic and lung compartments, while the simultaneous occurrence of these minima in mice results in higher naphthalene oxide concentrations, thereby allowing formation of more metabolites (e.g., covalent binding to proteins) that may be toxic.

Adipose Tissue↗

PubMedDate not supplied

Combining cell culture analogue reactor designs and PBPK models to probe mechanisms of naphthalene toxicity.

An alternative method of evaluating the toxicology of a chemical is to use cultured mammalian cells in a novel cell culture analogue reactor (CCA) together with a corresponding physiologically based pharmacokinetic model (PBPK). The PBPK is a mathematical model that divides the body into compartments representing organs, integrating the kinetic, thermodynamic, and anatomical parameters of the animal. The bioreactor is a physical replica of the PBPK; where the PBPK specifies an organ or tissue compartment, the bioreactor contains compartments with a corresponding cell type. The device is a continuous, dynamic system composed of multiple cell types that interact through a common circulating cell culture medium. The bioreactor and the model are coupled to evaluate the plausibility of the molecular mechanism that is input into the model. This concept is tested with naphthalene as a model of PAH (polycyclic aromatic hydrocarbons) toxicants. Two physically different CCA reactors were tested with naphthalene, and different results were observed. In the prototype system using cells attached to glass dilution bottles, naphthalene dosing resulted in generation of a circulating metabolite from the "liver" compartment (based on H4IIE cells from a rat hepatoma) that caused cell death in the "lung" compartment (L2 cells from a rat lung), as well as depletion of glutathione in the L2 cells. An improved CCA using packed bed reactors of microcarrier cultured cells did not show differences between naphthalene-dosed and nondosed controls. To explain the different responses of the two CCA designs, PBPKs of the two reactors were tested with variations in physical and kinetic parameters, and toxic mechanism. When the toxic metabolite of naphthalene was naphthoquinone rather than naphthalene epoxide as initially assumed, the PBPK results were consistent with the results of the two CCA designs. This result indicates that the mechanism of naphthalene toxicity in the CCAs may be mediated through naphthoquinone formation. The CCA-PBPK concept is demonstrated to be applicable to the study of toxic mechanisms. In particular, use of this approach suggests that in vitro naphthalene toxicity is mediated through the naphthoquinone metabolite.

Animals↗