Showing papers in "Biotechnology and Bioengineering in 2004"

Toward an aggregated understanding of enzymatic hydrolysis of cellulose: noncomplexed cellulase systems.

Abstract: Information pertaining to enzymatic hydrolysis of cellulose by noncomplexed cellulase enzyme systems is reviewed with a particular emphasis on development of aggregated understanding incorporating substrate features in addition to concentration and multiple cellulase components. Topics considered include properties of cellulose, adsorption, cellulose hydrolysis, and quantitative models. A classification scheme is proposed for quantitative models for enzymatic hydrolysis of cellulose based on the number of solubilizing activities and substrate state variables included. We suggest that it is timely to revisit and reinvigorate functional modeling of cellulose hydrolysis, and that this would be highly beneficial if not necessary in order to bring to bear the large volume of information available on cellulase components on the primary applications that motivate interest in the subject.

Establishment of FUT8 knockout Chinese hamster ovary cells: an ideal host cell line for producing completely defucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity.

Abstract: To generate industrially applicable new host cell lines for antibody production with optimizing antibody-dependent cellular cytotoxicity (ADCC) we disrupted both FUT8 alleles in a Chinese hamster ovary (CHO)/DG44 cell line by sequential homologous recombination. FUT8 encodes an alpha-1,6-fucosyltransferase that catalyzes the transfer of fucose from GDP-fucose to N-acetylglucosamine (GlcNAc) in an alpha-1,6 linkage. FUT8(-/-) cell lines have morphology and growth kinetics similar to those of the parent, and produce completely defucosylated recombinant antibodies. FUT8(-/-)-produced chimeric anti-CD20 IgG1 shows the same level of antigen-binding activity and complement-dependent cytotoxicity (CDC) as the FUT8(+/+)-produced, comparable antibody, Rituxan. In contrast, FUT8(-/-)-produced anti-CD20 IgG1 strongly binds to human Fcgamma-receptor IIIa (FcgammaRIIIa) and dramatically enhances ADCC to approximately 100-fold that of Rituxan. Our results demonstrate that FUT8(-/-) cells are ideal host cell lines to stably produce completely defucosylated high-ADCC antibodies with fixed quality and efficacy for therapeutic use.

Effect of Xylan and Lignin Removal by Batch and Flowthrough Pretreatment on the Enzymatic Digestibility of Corn Stover Cellulose

Abstract: Compared with batch systems, flowthrough and countercurrent reactors have important potential advan- tages for pretreating cellulosic biomass, including higher hemicellulose sugar yields, enhanced cellulose digestibility, and reduced chemical additions. Unfortunately, they suffer from high water and energy use. To better understand these trade-offs, comparative data are reported on xylan and lignin removal and enzymatic digestibility of cellu- lose for corn stover pretreated in batch and flowthrough reactors over a range of flow rates between 160j and 220jC, with water only and also with 0.1 wt% sulfuric acid. Increasing flow with just water enhanced the xylan dissolution rate, more than doubled total lignin removal, and increased cellulose digestibility. Furthermore, adding dilute sulfuric acid increased the rate of xylan removal for both batch and flowthrough systems. Interestingly, adding acid also increased the lignin removal rate with flow, but less lignin was left in solution when acid was added in batch. Although the enzymatic hydrolysis of pretreated cellulose was related to xylan removal, as others have shown, the digestibility was much better for flowthrough compared with batch systems, for the same degree of xylan removal. Cellulose digestibility for flowthrough reactors was related to lignin removal as well. These results suggest that altering lignin also affects the enzymatic digestibility of corn stover. B 2004 Wiley Periodicals, Inc.

Preparation, optimization, and structures of cross-linked enzyme aggregates (CLEAs).

Abstract: The broad applicability of the cross-linking of enzyme aggregates to the effective immobilisation of enzymes is demonstrated and the influence of many parameters on the properties of the resulting CLEAs is determined. The relative simplicity of the operation ideally lends itself to high-throughput methodologies. The aggregation method was improved up to 100% activity yield for any enzyme. For the first time, the physical structures of CLEAs are elucidated.

Mimicking the Escherichia coli cytoplasmic environment activates long‐lived and efficient cell‐free protein synthesis

Abstract: Cell-free translation systems generally utilize high-energy phosphate compounds to regenerate the adenosine triphosphate (ATP) necessary to drive protein synthesis. This hampers the widespread use and practical implementation of this technology in a batch format due to expensive reagent costs; the accumulation of inhibitory byproducts, such as phosphate; and pH change. To address these problems, a cell-free protein synthesis system has been engineered that is capable of using pyruvate as an energy source to produce high yields of protein. The "Cytomim" system, synthesizes chloramphenicol acetyltransferase (CAT) for up to 6 h in a batch reaction to yield 700 microg/mL of protein. By more closely replicating the physiological conditions of the cytoplasm of Escherichia coli, the Cytomim system provides a stable energy supply for protein expression without phosphate accumulation, pH change, exogenous enzyme addition, or the need for expensive high-energy phosphate compounds.

Differential effects of equiaxial and uniaxial strain on mesenchymal stem cells.

Abstract: Bone marrow mesenchymal stem cells (MSCs) can differentiate into a variety of cell types, including vascular smooth muscle cells (SMCs), and have tremendous potential as a cell source for cardiovascular regeneration. We postulate that specific vascular environmental factors will promote MSC differentiation into SMCs. However, the effects of the vascular mechanical environment on MSCs have not been characterized. Here we show that mechanical strain regulated the expression of SMC markers in MSCs. Cyclic equiaxial strain downregulated SM alpha-actin and SM-22alpha in MSCs on collagen- or elastin-coated membranes after 1 day, and decreased alpha-actin in stress fibers. In contrast, cyclic uniaxial strain transiently increased the expression of SM alpha-actin and SM-22alpha after 1 day, which subsequently returned to basal levels after the cells aligned in the direction perpendicular to the strain direction. In addition, uniaxial but not equiaxial strain induced a transient increase of collagen I expression. DNA microarray experiments showed that uniaxial strain increased SMC markers and regulated the expression of matrix molecules without significantly changing the expression of the differentiation markers (e.g., alkaline phosphatase and collagen II) of other cell types. Our results suggest that uniaxial strain, which better mimics the type of mechanical strain experienced by SMCs, may promote MSC differentiation into SMCs if cell orientation can be controlled. This study demonstrates the differential effects of equiaxial and uniaxial strain, advances our understanding of the mechanical regulation of stem cells, and provides a rational basis for engineering MSCs for vascular tissue engineering and regeneration.

Optimization of polyhydroxybutyrate production by mixed cultures submitted to aerobic dynamic feeding conditions.

Abstract: Activated sludge submitted to aerobic dynamic feeding conditions showed a good and stable capacity to store polyhydroxybutyrate (PHB). The system, working for 2 years, selected a microbial population with a high PHB storage capacity. The influence of carbon and nitrogen concentrations on the PHB accumulation yield was studied in a range of 15-180 Cmmol/l for acetate and 0-2.8 Nmmol/l for ammonia. Low ammonia concentrations favored PHB accumulation. The maximum PHB content, 67.5%, was obtained for 180 Cmmol/l of acetate supplied in one pulse. However, such high substrate concentration proved to be inhibitory for the storage mechanism, causing a slowdown of the specific PHB storage rate. In order to avoid substrate inhibition, 180 Cmmol/l of acetate was supplied in different ways: continuously fed and in three pulses of 60 Cmmol/l each. In both cases the specific PHB storage rate increased and the PHB content obtained were 56.2% and 78.5%, respectively. The latter value of PHB content is similar to that obtained by pure cultures and was never reported for mixed cultures. Addition of acetate by pulses controlled by the oxygen concentration was kept for 16 days, the PHB content being always above 70% of cell dry weight.

Oxygen gradients in tissue-engineered PEGT/PBT cartilaginous constructs: measurement and modeling.

Abstract: The supply of oxygen within three-dimensional tissue-engineered (TE) cartilage polymer constructs is mainly by diffusion. Oxygen consumption by cells results in gradients in the oxygen concentration. The aims of this study were, firstly, to identify the gradients within TE cartilage polymer constructs and, secondly, to predict the profiles during in vitro culture. A glass microelectrode system was adapted and used to penetrate cartilage and TE cartilaginous constructs, yielding reproducible measurements with high spatial resolution. Cartilage polymer constructs were cultured for up to 41 days in vitro. Oxygen concentrations, as low as 2-5%, were measured within the center of these constructs. At the beginning of in vitro culture, the oxygen gradients were steeper in TE constructs in comparison to native tissue. Nevertheless, during the course of culture, oxygen concentrations approached the values measured in native tissue. A mathematical model was developed which yields oxygen profiles within cartilage explants and TE constructs. Model input parameters were assessed, including the diffusion coefficient of cartilage (2.2 × 10-9) + (0.4 × 10-9 m2 s-1), 70% of the diffusion coefficient of water and the diffusion coefficient of constructs (3.8 × 10-10 m2 s-1). The model confirmed that chondrocytes in polymer constructs cultured for 27 days have low oxygen requirements (0.8 × 10-19 mol m-3 s-1), even lower than chondrocytes in native cartilage. The ability to measure and predict local oxygen tensions offers new opportunities to obtain more insight in the relation between oxygen tension and chondrogenesis.

Encapsulating chondrocytes in degrading PEG hydrogels with high modulus: engineering gel structural changes to facilitate cartilaginous tissue production.

Abstract: A major challenge when designing cell scaffolds for chondrocyte delivery in vivo is creating scaffolds with sufficient mechanical properties to restore initial function while simultaneously controlling temporal changes in the gel structure to facilitate tissue formation. To address this design challenge, degradable photocrosslinked hydrogels based on poly(ethylene glycol) were investigated. To alter the gel's initial mechanical properties, hydrogels were fabricated by varying the initial macromer concentration from 10% to 15% to 20%. A twofold increase in macromer concentration resulted in an eightfold increase in the initial compressive modulus from 60 to 500 kPa. Gel degradation was tailored by incorporating fast-degrading crosslinks that enable maximal extracellular matrix (ECM) diffusion with time and a minimal number of nondegrading (or slowly degrading) crosslinks to maintain scaffold integrity and prevent complete gel erosion during tissue formation. Chondrocytes encapsulated in these gels produced cartilaginous tissue rich in glycosaminoglycans and collagen as seen biochemically and histologically. Interestingly, mass loss appeared to more closely match tissue secretion in gels fabricated from a 15% macromer concentration. However, the spatial ECM distribution was grossly similar in all three gels. By tailoring gel degradation and controlling network evolution during degradation, gels with optimal properties can be fabricated to support initially physiologic compressive loads while simultaneously supporting the formation of a neotissue.

Modeling mass transfer in hepatocyte spheroids via cell viability, spheroid size, and hepatocellular functions.

Abstract: Hepatocyte aggregation into spheroids attributes to their increased activity, but in the absence of a vascular network the cells in large spheroids experience mass transfer limitations. Thus, there is a need to define the spheroid size which enables maximal cell viability and productivity. We developed a combined theoretical and experimental approach to define this optimal spheroid size. Hepatocyte spheroids were formed in alginate scaffolds having a pore diameter of 100 microm, in rotating T-flasks or spinners, to yield a maximal size of 100, 200, and 600 microm, respectively. Cell viability was found to decrease with increasing spheroid size. A mathematical model was constructed to describe the relationship between spheroid size and cell viability via the oxygen mass balance equation. This enabled the prediction of oxygen distribution profiles and distribution of viable cells in spheroids with varying size. The model describes that no oxygen limitation will take place in spheroids up to 100 microm in diameter. Spheroid size affected the specific rate of albumin secretion as well; it reached a maximal level, i.e., 60 microg/million cells/day in 100-microm diameter spheroids. This behavior was depicted in an equation relating the specific albumin secretion rate to spheroid size. The calculated results fitted with the experimental data, predicting the need for a critical number of viable hepatocytes to gain a maximal albumin secretion. Taken together, the results on mass transport in spheroids and its effects on cell viability and productivity provide a useful tool for the design of 3D scaffolds with pore diameters of 100 microm.

Engineering cartilage‐like tissue using human mesenchymal stem cells and silk protein scaffolds

Abstract: Human mesenchymal stem cells (hMSC) derived from bone marrow aspirates can form the basis for the in vitro cultivation of autologous tissue grafts and help alleviate the problems of immunorejection and disease transmission associated with the use of allografts. We explored the utility of hMSC cultured on protein scaffolds for tissue engineering of cartilage. hMSC were isolated, expanded in culture, characterized with respect to the expression of surface markers and ability for chondrogenic and osteogenic differentiation, and seeded on scaffolds. Four different scaffolds were tested, formed as a highly porous sponge made of: 1) collagen, 2) cross-linked collagen, 3) silk, and 4) RGD-coupled silk. Cell-seeded scaffolds were cultured for up to 4 weeks in either control medium (DMEM supplemented with 10% fetal bovine serum) or chondrogenic medium (control medium supplemented with chondrogenic factors). hMSC attachment, proliferation, and metabolic activity were markedly better on slowly degrading silk than on fast-degrading collagen scaffolds. In chondrogenic medium, hMSC formed cartilaginous tissues on all scaffolds, but the extent of chondrogenesis was substantially higher for hMSC cultured on silk as compared to collagen scaffolds. The deposition of glycosaminoglycan (GAG) and type II collagen and the expression of type II collagen mRNA were all higher for hMSC cultured on silk than on collagen scaffolds. Taken together, these results suggest that silk scaffolds are particularly suitable for tissue engineering of cartilage starting from hMSC, presumably due to their high porosity, slow biodegradation, and structural integrity.

Effects of gene dosage, promoters, and substrates on unfolded protein stress of recombinant Pichia pastoris.

Abstract: The expression of heterologous proteins may exert severe stress on the host cells at different levels. Depending on the specific features of the product, different steps may be rate-limiting. For the secretion of recombinant proteins from yeast cells, folding and disulfide bond formation were identified as rate-limiting in several cases and the induction of the chaperone BiP (binding protein) is described. During the development of Pichia pastoris strains secreting human trypsinogen, a severe limitation of the amount of secreted product was identified. Strains using either the AOX1 or the GAP promoter were compared at different gene copy numbers. With the constitutive GAP promoter, no effect on the expression level was observed, whereas with the inducible AOX1 promoter an increase of the copy number above two resulted in a decrease of expression. To identify whether part of the product remained in the cells, lysates were fractionated and significant amounts of the product were identified in the insoluble fraction containing the endoplasmic reticulum, while the soluble cytosolic fraction contained product only in clones using the GAP promoter. An increase of BiP was observed upon induction of expression, indicating that the intracellular product fraction exerts an unfolded protein response in the host cells. A strain using the GAP promoter was grown both on glucose and methanol and trypsinogen was identified in the insoluble fractions of both cultures, but only in the soluble fraction of the glucose grown cultures, indicating that the amounts and distribution of intracellularly retained product depends on the culture conditions, especially the carbon source.

A new, mild cross-linking methodology to prepare cross-linked enzyme aggregates.

Abstract: Cross-linked enzyme aggregates (CLEAs) were prepared from several enzymes (penicillin G acylase, hydroxynitrile lyase, alcohol dehydrogenase, and two different nitrilases) by precipitation and subsequent cross-linking using dextran polyaldehyde. In most cases, higher immobilization yields were obtained using the latter cross-linker as compared with the commonly used glutaraldehyde. Active site titration of penicillin acylase CLEAs showed that the higher activity originated from a significantly lower loss in active sites using dextran polyaldehyde as a cross-linking agent. It is proposed that macromolecular cross-linkers are too large to penetrate the protein active site and react with catalytically essential amino acid residues.

Fermentation of biomass-generated producer gas to ethanol.

Abstract: The development of low-cost, sustainable, and renewable energy sources has been a major focus since the 1970s. Fuel-grade ethanol is one energy source that has great potential for being generated from biomass. The demonstration of the fermentation of biomass-generated producer gas to ethanol is the major focus of this article in addition to assessing the effects of producer gas on the fermentation process. In this work, producer gas (primarily CO, CO(2), CH(4), H(2), and N(2)) was generated from switchgrass via gasification. The fluidized-bed gasifier generated gas with a composition of 56.8% N(2), 14.7% CO, 16.5% CO(2), 4.4% H(2), and 4.2% CH(4). The producer gas was utilized in a 4-L bioreactor to generate ethanol and other products via fermentation using a novel clostridial bacterium. The effects of biomass-generated producer gas on cell concentration, hydrogen uptake, and acid/alcohol production are shown in comparison with "clean" bottled gases of similar compositions for CO, CO(2), and H(2). The successful implementation of generating producer gas from biomass and then fermenting the producer gas to ethanol was demonstrated. Several key findings following the introduction of producer gas included: (1) the cells stopped growing but were still viable, (2) ethanol was primarily produced once the cells stopped growing (ethanol is nongrowth associated), (3) H(2) utilization stopped, and (4) cells began growing again if "clean" bottled gases were introduced following exposure to the producer gas.

MIRACLE: mass isotopomer ratio analysis of U-13C-labeled extracts. A new method for accurate quantification of changes in concentrations of intracellular metabolites.

Abstract: First, we report the application of stable isotope dilution theory in metabolome characterization of aerobic glucose limited chemostat culture of S. cerevisiae CEN.PK 113-7D using liquid chromatography-electrospray ionization MS/MS (LC-ESI-MS/MS). A glucose-limited chemostat culture of S. cerevisiae was grown to steady state at a specific growth rate (mu)=0.05 h(-1) in a medium containing only naturally labeled (99% U-12C, 1% U-13C) carbon source. Upon reaching steady state, defined as 5 volume changes, the culture medium was switched to chemically identical medium except that the carbon source was replaced with 100% uniformly (U) 13C labeled stable carbon isotope, fed for 4 h, with sampling every hour. We observed that within a period of 1 h approximately 80% of the measured glycolytic metabolites were U-13C-labeled. Surprisingly, during the next 3 h no significant increase of the U-13C-labeled metabolites occurred. Second, we demonstrate for the first time the LC-ESI-MS/MS-based quantification of intracellular metabolite concentrations using U-13C-labeled metabolite extracts from chemostat cultivated S. cerevisiae cells, harvested after 4 h of feeding with 100% U-13C-labeled medium, as internal standard. This method is hereby termed "Mass Isotopomer Ratio Analysis of U-13C Labeled Extracts" (MIRACLE). With this method each metabolite concentration is quantified relative to the concentration of its U-13C-labeled equivalent, thereby eliminating drawbacks of LC-ESI-MS/MS analysis such as nonlinear response and matrix effects and thus leads to a significant reduction of experimental error and work load (i.e., no spiking and standard additions). By coextracting a known amount of U-13C labeled cells with the unlabeled samples, metabolite losses occurring during the sample extraction procedure are corrected for.

Bioreactor cultivation enhances the efficiency of human embryoid body (hEB) formation and differentiation

Abstract: The promise of human embryonic stem cells (hESCs) to provide an unlimited supply of cells for cell therapy and tissue engineering depends on the availability of a controllable bioprocess for their expansion and differentiation. We describe for the first time the formation of differentiating human embryoid bodies (hEBs) in rotating bioreactors to try and control their agglomeration. The efficacy of the dynamic process compared to static cultivation in Petri dishes was analyzed with respect to the yield of hEB formation and differentiation. Quantitative analyses of hEBs, DNA and protein contents, and viable cell concentration, as measures for culture cellularity and scale-up, revealed 3-fold enhancement in generation of hEBs compared to the static culture. Other metabolic indices such as glucose consumption, lactic acid production, and pH pointed to efficient cell expansion and differentiation in the dynamic cultures. The type of rotating vessel had a significant impact on the process of hEB formation and agglomeration. In the slow turning lateral vessel (STLV), hEBs were smaller in size and no large necrotic centers were seen, even after 1-month cultivation. In the high aspect rotating vessel (HARV), hEB agglomeration was massive. The appearance of representative tissues derived from the three germ layers as well as primitive neuronal tube organization, blood vessel formation, and specific-endocrine secretion indicated that the initial developmental events are not altered in the dynamically formed hEBs. Collectively, our study defines the culture conditions in which control over the aggregation of differentiating hESCs is obtained, thus enabling scaleable cell production for clinical and industrial applications.

Inhibition of the Trichoderma reesei cellulases by cellobiose is strongly dependent on the nature of the substrate.

Abstract: The inhibition effect of cellobiose on the initial stage of hydrolysis when cellobiohydrolase Cel 7A and endoglucanases Cel 7B, Cel 5A, and Cel 12A from Trichoderma reesei were acting on bacterial cellulose and amorphous cellulose that were [(3)H]- labeled at the reducing end was quantified. The apparent competitive inhibition constant (K(i)) for Cel 7A on [(3)H]-bacterial cellulose was found to be 1.6 +/- 0.5 mM, 100-fold higher than that for Cel 7A acting on low-molecular-weight model substrates. The hydrolysis of [(3)H]-amorphous cellulose by endoglucanases was even less affected by cellobiose inhibition with apparent K(i) values of 11 +/- 3 mM and 34 +/- 6 mM for Cel 7B and Cel 5A, respectively. Contrary to the case for the other enzymes studied, the release of radioactive label by Cel 12A was stimulated by cellobiose, possibly due to a more pronounced transglycosylating activity. Theoretical analysis of the inhibition of Cel 7A by cellobiose predicted an inhibition analogous to that of mixed type with two limiting cases, competitive inhibition if the prevalent enzyme-substrate complex without inhibitor is productive and conventional mixed type when the prevalent enzyme-substrate complex is nonproductive.

High solid simultaneous saccharification and fermentation of wet oxidized corn stover to ethanol.

Abstract: In this study ethanol was produced from corn stover pretreated by alkaline and acidic wet oxidation (WO) (195°C, 15 min, 12 bar oxygen) followed by nonisothermal simultaneous saccharification and fermentation (SSF). In the first step of the SSF, small amounts of cellulases were added at 50°C, the optimal temperature of enzymes, in order to obtain better mixing condition due to some liquefaction. In the second step more cellulases were added in combination with dried baker's yeast (Saccharomyces cerevisiae) at 30°C. The phenols (0.4–0.5 g/L) and carboxylic acids (4.6–5.9 g/L) were present in the hemicellulose rich hydrolyzate at subinhibitory levels, thus no detoxification was needed prior to SSF of the whole slurry. Based on the cellulose available in the WO corn stover 83% of the theoretical ethanol yield was obtained under optimized SSF conditions. This was achieved with a substrate concentration of 12% dry matter (DM) acidic WO corn stover at 30 FPU/g DM (43.5 FPU/g cellulose) enzyme loading. Even with 20 and 15 FPU/g DM (corresponding to 29 and 22 FPU/g cellulose) enzyme loading, ethanol yields of 76 and 73%, respectively, were obtained. After 120 h of SSF the highest ethanol concentration of 52 g/L (6 vol.%) was achieved, which exceeds the technical and economical limit of the industrial-scale alcohol distillation. The SSF results showed that the cellulose in pretreated corn stover can be efficiently fermented to ethanol with up to 15% DM concentration. A further increase of substrate concentration reduced the ethanol yield significant as a result of insufficient mass transfer. It was also shown that the fermentation could be followed with an easy monitoring system based on the weight loss of the produced CO2. © 2004 Wiley Periodicals, Inc.

Mechanisms of biosorption of different heavy metals by brown marine macroalgae

Abstract: The biosorption mechanisms of different heavy metallic cations (Cd, Ni, Pb) to active chemical groups on the cell wall matrix of the nonliving brown marine macro- alga, Sargassum vulgaris in its natural form, were examined by the following instrumental and chemical techniques: Fourier-transform infrared (FTIR) analysis, X-ray photoelec- tron spectroscopy (XPS), scanning electron microscopy (SEM), and extraction of alginic acid and sulfated poly- saccharides, which act as metal-binding moieties present in cell wall. From the different techniques used and the known chemical composition of the algal cell wall, it was observed that biosorption of the metallic cations to the algal cell wall component was a surface process. The binding capacities of the different metal cations were be- tween 1 and 1.2 mmol metal/g on a dry weight basis. The main chemical groups involved in the metallic cation bio- sorption were apparently carboxyl, amino, sulfhydryl, and sulfonate. These groups were part of the algal cell wall structural polymers, namely, polysaccharides (alginic acid, sulfated polysaccharides), proteins, and peptidoglycans. The main cadmium cation sequestration mechanism by the algal biomass was apparently chelation, while the nickel cation sequestration mechanism was mainly ion exchange. Lead cations exhibit higher affinity to the algal biomass, and their binding mechanism included a combination of ion exchange, chelation, and reduction reactions, accompa- nied by metallic lead precipitation on the cell wall matrix. During the ion exchange process, calcium, magnesium, hydrogen cations, and probably other cations (sodium and potassium) in the algal cell wall matrix were replaced by the tested heavy metals. B 2004 Wiley Periodicals, Inc.

Synthetic extracellular matrices for in situ tissue engineering.

Abstract: Cell interactions with the extracellular matrix play important roles in guiding tissue morphogenesis. The matrix stimulates cells to influence such things as differentiation and the cells actively remodel the matrix via local proteolytic activity. We have designed synthetic hydrogel networks that participate in this interplay: They signal cells via bound adhesion and growth factors, and they also respond to the remodeling influence of cell-associated proteases. Poly(ethylene glycol)-bis-vinylsulfone was crosslinked by a Michael-type addition reaction with a peptide containing three cysteine residues, the peptide sequence being cleavable between each cysteine residue by the cell-associated protease plasmin. Cells were able to invade gel networks that contained adhesion peptides and were crosslinked by plasmin-sensitive peptides, while materials lacking either of these two characteristics resisted cell infiltration. Incorporated bone morphogenetic protein-2 (BMP-2) induced bone healing in a rat model in materials that were both adhesive and plasmin-sensitive, while materials lacking plasmin sensitivity resisted formation of bone within the material. Furthermore, when a heparin bridge was incorporated as a BMP-2 affinity site, mimicking yet another characteristic of the extracellular matrix, statistically improved bone regeneration was observed.

Arabidopsis glycosyltransferases as biocatalysts in fermentation for regioselective synthesis of diverse quercetin glucosides.

Abstract: Regioselectivity of glycosyltransferases offers an important means to overcome the limitations of chemical synthesis of small molecule glycosides. In this study we explore a large multigene family of UDP-glucose:glycosyltransferases of Arabidopsis for their potential as novel biocatalysts for in vitro synthesis and whole-cell catalysis. We used quercetin as a substrate for this study because the flavonol and its glycosides have important medicinal properties and the metabolite provides a complex structure for regioselective glucosylation. We analyzed the activity of 91 recombinant enzymes for in vitro activity toward quercetin and discovered 29 that are capable of glucosylating the substrate. We demonstrate the first enzymic synthesis of a range of glucosides in vitro, including the 3-O-, 7-O-, 3'-O-, and 4'-O-monoglucosides, 3,7-di-O-glucoside, and 7,3'-di-O-glucoside. We also show that the regioselectivity of glucosylation can be maintained when the enzymes are used as whole-cell biocatalysts in Escherichia coli.

Volumetric measurements of bacterial cells and extracellular polymeric substance glycoconjugates in biofilms

Abstract: In this study an enrichment culture developed from activated sludge was used to investigate the architecture of fully hydrated multispecies biofilms. The assessment of biofilm structure and volume was carried out using confocal laser scanning microscopy (CLSM). Bacterial cell distribution was determined with the nucleic acid-specific stain SYTO 60, whereas glycoconjugates of extracellular polymeric substances (EPS) were stained with the Alexa-488-labeled lectin of Aleuria aurantia. Digital image analysis was employed for visualization and quantification of three-dimensional CLSM data sets. The specific volumes of the polymeric and cellular biofilm constituents were quantified. In addition, gravimetric measurements were done to determine dry mass and thickness of the biofilms. The data recorded by the CLSM technique and the gravimetric data were then compared. It was shown that the biofilm thicknesses determined with both methods agree well for slow-growing hetorotrophic and chemoautotrophic biofilms. In addition, for slow-growing biofilms, the volumes and masses calculated from CLSM and the biomass calculated from gravimetric measurements were also comparable. For fast-growing heterotrophic biofilms cultivated with high glucose concentrations the data sets fit to a lesser degree, but still showed the same common trend. Compared with traditional gravimetric measurements, CLSM allowed differential recording of multiple biofilm parameters with subsequent three-dimensional visualization and quantification. The quantitative three-dimensional results recorded by CLSM are an important basis for understanding, controlling, exploiting, and modeling of biofilms. © 2004 Wiley Periodicals, Inc.

Differential gene expression shows natural brominated furanones interfere with the autoinducer-2 bacterial signaling system of Escherichia coli.

Abstract: The quorum sensing disrupter (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone (furanone) of the alga Delisea pulchra was previously found by us (Environ Microbiol 3:731-736, 2001) to inhibit quorum sensing in Escherichia coli via autoinducer-2 (AI-2, produced by LuxS). In this study, DNA microarrays were used to study the genetic basis of this natural furanone inhibition of AI-2 signaling (significant values with p < 0.05 are reported). Using DNA microarrays, the AI-2 mutant Escherichia coli DH5alpha was compared with the AI-2 wild-type strain, E. coli K12, to determine how AI-2 influenced gene expression. Escherichia coli K12 was also grown with 0 and 60 microg/mL furanone to study the inhibition of quorum sensing gene expression. It was found that 166 genes were differentially expressed by AI-2 (67 were induced and 99 were repressed) and 90 genes were differentially expressed by furanone (34 were induced and 56 were repressed). Importantly, 79% (44 out of 56) of the genes repressed by furanone were induced by AI-2, which indicated that furanone inhibited AI-2 signaling and influenced the same suite of genes as a regulon. Most of these genes have functions of chemotaxis, motility, and flagellar synthesis. Interestingly, the aerotaxis genes aer and tsr were discovered to be induced by AI-2 and repressed by furanone. Representative microarray results were confirmed by RNA dot blotting. Furthermore, the E. coli air-liquid interface biofilm formation was repressed by furanone, supporting the results that taxis and flagellar genes were repressed by furanone. The autoinducer bioassay indicated that 100 microg/mL furanone decreased the extracellular concentration of AI-2 2-fold, yet luxS and pfs transcription were not significantly altered. Hence, furanone appeared to alter AI-2 signaling post-transcriptionally.

Membrane‐aerated microbioreactor for high‐throughput bioprocessing

Abstract: A microbioreactor with a volume of microliters is fabricated out of poly(dimethylsiloxane) (PDMS) and glass. Aeration of microbial cultures is through a gas-permeable PDMS membrane. Sensors are integrated for on-line measurement of optical density (OD), dissolved oxygen (DO), and pH. All three parameter measurements are based on optical methods. Optical density is monitored via transmittance measurements through the well of the microbioreactor while dissolved oxygen and pH are measured using fluorescence lifetime-based sensors incorporated into the body of the microbioreactor. Bacterial fermentations carried out in the microbioreactor under well-defined conditions are compared to results obtained in a 500-mL bench-scale bioreactor. It is shown that the behavior of the bacteria in the microbioreactor is similar to that in the larger bioreactor. This similarity includes growth kinetics, dissolved oxygen profile within the vessel over time, pH profile over time, final number of cells, and cell morphology. Results from off-line analysis of the medium to examine organic acid production and substrate utilization are presented. By changing the gaseous environmental conditions, it is demonstrated that oxygen levels within the microbioreactor can be manipulated. Furthermore, it is demonstrated that the sensitivity and reproducibility of the microbioreactor system are such that statistically significant differences in the time evolution of the OD, DO, and pH can be used to distinguish between different physiological states. Finally, modeling of the transient oxygen transfer within the microbioreactor based on observed and predicted growth kinetics is used to quantitatively characterize oxygen depletion in the system.

Microplate‐based filter paper assay to measure total cellulase activity

Abstract: The standard filter paper assay (FPA) published by the International Union of Pure and Applied Chemistry (IUPAC) is widely used to determine total cellulase activity. However, the IUPAC method is not suitable for the parallel analyses of large sample numbers. We describe here a microplate-based method for assaying large sample numbers. To achieve this, we reduced the enzymatic reaction volume to 60 microl from the 1.5 ml used in the IUPAC method. The modified 60-microl format FPA can be carried out in 96-well assay plates. Statistical analyses showed that the cellulase activities of commercial cellulases from Trichoderma reesei and Aspergillus species determined with our 60-microl format FPA were not significantly different from the activities measured with the standard FPA. Our results also indicate that the 60-microl format FPA is quantitative and highly reproducible. Moreover, the addition of excess beta-glucosidase increased the sensitivity of the assay by up to 60%.

Fluid mechanics of a spinner-flask bioreactor

Abstract: Spinner-flask bioreactors have been used for the production of articular cartilage in vitro. The dynamic environment within bioreactors is known to significantly affect the growth and development of the tissue. The present research focuses on the experimental and numerical characterization of the flow field within a spinner flask operating under conditions used to produce cartilage. Laboratory experiments carried out in a scaled-up model bioreactor employ particle-image velocimetry (PIV) to determine velocity and shear-rate fields in the vicinity of the construct closest to the stir bar, in addition to turbulence properties. Numerical computations calculated using FLUENT, a commercial software package, simulate the flow field in the same model bioreactor under similar operating conditions. In the computations, scaffolds were modeled as both solid and porous media with different permeabilities and flow rates through various faces of the construct nearest the stir bar were examined.

Engineering Chinese hamster ovary cells to maximize effector function of produced antibodies using FUT8 siRNA

Abstract: We explored the possibility of converting established antibody-producing cells to cells producing high antibody-dependent cellular cytotoxicity (ADCC) antibodies. The conversion was made by constitutive expression of small interfering RNA (siRNA) against alpha1,6 fucosyltransferase (FUT8). We found two effective siRNAs, which reduce FUT8 mRNA expression to 20% when introduced into Chinese hamster ovary (CHO)/DG44 cells. Selection for Lens culinaris agglutinin (LCA)-resistant clones after introduction of the FUT8 siRNA expression plasmids yields clones producing highly defucosylated (approximately 60%) antibody with over 100-fold higher ADCC compared to antibody produced by the parental cells (approximately 10% defucosylated). Moreover, the selected clones remain stable, producing defucosylated antibody even in serum-free fed-batch culture. Our results demonstrate that constitutive FUT8 siRNA expression can control the oligosaccharide structure of recombinant antibody produced by CHO cells to yield antibodies with dramatically enhanced ADCC.

Anaerobic hydrogen production with an efficient carrier-induced granular sludge bed bioreactor.

Abstract: A novel bioreactor containing self-flocculated anaerobic granular sludge was developed for high-performance hydrogen production from sucrose-based synthetic wastewater. The reactor achieved an optimal volumetric hydrogen production rate of ∼7.3 L/h/L (7,150 mmol/d/L) and a maximal hydrogen yield of 3.03 mol H2/mol sucrose when it was operated at a hydraulic retention time (HRT) of 0.5 h with an influent sucrose concentration of 20 g COD/L. The gas-phase hydrogen content and substrate conversion also exceeded 40 and 90%, respectively, under optimal conditions. Packing of a small quantity of carrier matrices on the bottom of the upflow reactor significantly stimulated sludge granulation that can be accomplished within 100 h. Among the four carriers examined, spherical activated carbon was the most effective inducer for granular sludge formation. The carrier-induced granular sludge bed (CIGSB) bioreactor was started up with a low HRT of 4–8 h (corresponding to an organic loading rate of 2.5–5 g COD/h/L) and enabled stable operations at an extremely low HRT (up to 0.5 h) without washout of biomass. The granular sludge was rapidly formed in CIGSB supported with activated carbon and reached a maximal concentration of 26 g/L at HRT = 0.5 h. The ability to maintain high biomass concentration at low HRT (i.e., high organic loading rate) highlights the key factor for the remarkable hydrogen production efficiency of the CIGSB processes. © 2004 Wiley Periodicals, Inc.

Microfabricated platform for studying stem cell fates.

Abstract: Platforms that allow parallel, quantitative analysis of single cells will be integral to realizing the potential of postgenomic biology. In stem cell biology, the study of clonal stem cells in multiwell formats is currently both inefficient and time-consuming. Thus, to investigate low-frequency events of interest, large sample sizes must be interrogated. We report a simple, versatile, and efficient micropatterned arraying system conducive to the culture and dynamic monitoring of stem cell proliferation. This platform enables: 1) parallel, automated, long-term (∼days to weeks), live-cell microscopy of single cells in culture; 2) tracking of individual cell fates over time (proliferation, apoptosis); and 3) correlation of differentiated progeny with founder clones. To achieve these goals, we used microfabrication techniques to create an array of ∼10,000 microwells on a glass coverslip. The dimensions of the wells are tunable, ranging from 20 to >500 μm in diameter and 10–500 μm in height. The microarray can be coated with adhesive proteins and is integrated into a culture chamber that permits rapid (∼min), addressable monitoring of each well using a standard programmable microscope stage. All cells share the same media (including paracrine survival signals), as opposed to cells in multiwell formats. The incorporation of a coverslip as a substrate also renders the platform compatible with conventional, high-magnification light and fluorescent microscopy. We validated this approach by analyzing the proliferation dynamics of a heterogeneous adult rat neural stem cell population. Using this platform, one can further interrogate the response of distinct stem cell subpopulations to microenvironmental cues (mitogens, cell–cell interactions, and cell–extracellular matrix interactions) that govern their behavior. In the future, the platform may also be adapted for the study of other cell types by tailoring the surface coatings, microwell dimensions, and culture environment, thereby enabling parallel investigation of many distinct cellular responses. © 2004 Wiley Periodicals, Inc.

Microbial population dynamics during start-up and overload conditions of anaerobic digesters treating municipal solid waste and sewage sludge.

Abstract: Microbial population dynamics were investigated during start-up and during periods of overload conditions in anaerobic co-digesters treating municipal solid waste and sewage sludge. Changes in community structure were monitored using ribosomal RNA-based oligonucleotide probe hybridization to measure the abundance of syntrophic propionate-oxidizing bacteria (SPOB), saturated fatty acid-beta-oxidizing syntrophs (SFAS), and methanogens. These changes were linked to traditional performance parameters such as biogas production and volatile fatty acid (VFA) concentrations. Digesters with high levels of Archaea started up successfully. Methanosaeta concilii was the dominant aceticlastic methanogen in these systems. In contrast, digesters that experienced a difficult start-up period had lower levels of Archaea with proportionally more abundant Methanosarcina spp. Syntrophic propionate-oxidizing bacteria and saturated fatty acid-beta-oxidizing syntrophs were present at low levels in all digesters, and SPOB appeared to play a role in stabilizing propionate levels during start-up of one digester. Digesters with a history of poor performance tolerated a severe organic overload event better than digesters that had previously performed well. It is hypothesized that higher levels of SPOB and SFAS and their methanogenic partners in previously unstable digesters are responsible for this behavior.