Development of sensors for direct detection of organophosphates. Part I: Immobilization, characterization and stabilization of acetylcholinesterase and organophosphate hydrolase on silica supports

Biosensors for organophosphates in solution may be constructed by monitoring the activity of acetylcholinesterase (AChE) or organophosphate hydrolase (OPH) immobilized to a variety of microsensor platforms. The area available for enzyme immobilization is small (< 1 mm2) for microsensors. In order to construct microsensors with increased surface area for enzyme immobilization, we used a sol-gel process to create highly porous and stable silica matrices. Surface porosity of sol-gel coated surfaces was characterized using scanning electron microscopy; pore structure was found to be very similar to that of commercially available porous silica supports. Based upon this analysis, porous and non-porous silica beads were used as model substrates of sol-gel coated and uncoated sensor surfaces. Two different covalent chemistries were used to immobilize AChE and OPH to these porous and non-porous silica beads. The first chemistry used amine-silanization of silica followed by enzyme attachment using the homobifunctional linker glutaraldehyde. The second chemistry used sulfhydryl-silanization followed by enzyme attachment using the heterobifunctional linker N-gamma-maleimidobutyryloxy succinimide ester (GMBS). Surfaces were characterized in terms of total enzyme immobilized, total and specific enzyme activity, and long term stability of enzyme activity. Amine derivitization followed by glutaraldehyde linking yielded supports with greater amounts of immobilized enzyme and activity. Use of porous supports not only yielded greater amounts of immobilized enzyme and activity, but also significantly improved long term stability of enzyme activity. Enzyme was also immobilized to sol-gel coated glass slides. The mass of immobilized enzyme increased linearly with thickness of coating. However, immobilized enzyme activity saturated at a porous silica thickness of approximately 800 nm.     

Non-porous magnetic supports for cell immobilization

A new method for covering magnetic particles with a stable non-porous layer of a material like zeolite or activated carbon was used for the preparation of support materials with good properties for the immobilization of yeast Saccharomyces cerevisiae cells. The immobilized cells can be used in batch and continuous alcoholic fermentation. A productivity of 35.6 g ethanol/l · h was reached. The adsorption isotherms of the immobilized yeast cells were determined. Yeast cell immobilization on non-porous magnetic supports obeyed the Langmuir isotherm equation. Satisfactory results were obtained also from repeated batch fermentations with fixed cells on supports additionally treated with glutaraldehyde or by simple adsorption.     

Responses from freshwater sediment during electricity generation using microbial fuel cells

In a two-electrode system, freshwater sediment was used as a fuel to examine the relationship between current generation and organic matter consumption with different types of electrode. Sediment microbial fuel cells using porous electrodes showed a superior performance in terms of generating current when compared with the use of non-porous electrodes. The maximum current densities with thicker and thin porous electrodes were 45.4 and 37.6 mA m⁻², respectively, whereas the value with non-porous electrodes was 13.9 mA m⁻². The amount of organic matter removed correlated with the current produced. The redox potential in the anode area under closed-circuit conditions was +246.3 ± 67.7 mV, while that under open-circuit conditions only reached −143.0 ± 7.18 mV. This suggests that an application of this system in organic-rich sediment could provide environmental benefits such as decreasing organic matter and prohibiting methane emission in conjunction with electricity production via an anaerobic oxidation process.     

Effects of agitation rate on aggregation during beads-to-beads subcultivation of microcarrier culture of human mesenchymal stem cells

With the aim to utilize human mesenchymal stem cells (hMSCs) grown in large scale for regenerative medicine, effects of agitation rate on aggregation during beads-to-beads subcultivation of microcarrier culture of hMSCs were studied. hMSCs could attach and grew on surface-type microcarriers of Cytodex 1, whereas almost no cell elongation and growth were observed on porous type microcarriers of Cytopores. The percentages of aggregated Cytodex 1 microcarriers at an agitation rate of 60 and 90 rpm were lower than that at 30 rpm, which was the lowest agitation rate necessary for the suspension of Cytodex 1 microcarriers, and the cells grew fastest at 60 rpm. hMSC could be subcultivated on Cytodex 1 by the beads-to-beads method at both 30 and 60 rpm without trypsinization. However, agitation at 60 rpm resulted in a markedly lower percentage of aggregated microcarriers not only before but also after subcultivation. The percentages of CD90- and CD166-positive cells among cells grown on Cytodex 1 at 60 rpm (91.5 and 87.6 %) were comparable to those of cells grown in the pre-culture on dishes. In conclusion, hMSCs could be subcultivated on Cytodex 1 by beads-to-beads method maintaining the expressions of the cell surface antigens CD90 and CD166, while adjusting agitation rate could decrease the microcarrier aggregation.     

Non-porous magnetic micro-particles: comparison to porous enzyme carriers for a diffusion rate-controlled enzymatic conversion

In this study the kinetics of conversion of a low-soluble substrate by an immobilized enzyme was investigated with respect to the diffusion limitation within porous and non-porous carriers. Non-porous micro-magnetic beads in comparison to conventional porous supports like Eupergit and Sepharose were tested. Due to their small diameters and their magnetic properties, micro-magnetic beads are especially applicable in diffusion rate-controlled processes in biological suspensions. The enzymatic reaction studied was the conversion of emulsified dirhamnolipid by immobilized Naringinase from Penicillium decumbens to monorhamnolipid and L-rhamnose. Taking into account mass transfer phenomena, the variation of the reaction effectiveness factor with increasing enzyme loading was estimated and compared with experimental efficiencies utilizing different enzyme loaded immobilized preparations. For comparison, carrier activities were also determined with the model substrate p-nitro-phenyl-rhamnoside. Intrinsic enzyme activities were thereby evaluated for porous supports. Highest specific activities were obtained with the micro-magnetic beads. These non-porous micro-beads demonstrated to be the most suitable carrier for bioconversion of a low-soluble substrate like rhamnolipids, where mass diffusional resistances in the three-phase reaction process are completely overcome. However, the smaller particle surface available limited the specific activity obtained at high protein loadings.     

Operational optimization of a turbine blade reactor using macroporous support and its application to rice callus regeneration

Optimal operation condition was investigated for immobilized rice callus culture using a turbine blade reactor (TBR²) with polyurethane foam supports. By using polyurethane foam block as immobilization support, the inhibition of cell growth at a high agitation speed was avoided because the hydrodynamic stress against immobilized cell was probably reduced. Experimental results in each operational condition were assessed by means of rice callus growth, immobilization ratio in TBR and those regeneration frequencies in regeneration culture using solid medium. Concerning with pore size of polyurethane foam and support size, three-millimeter cube support of polyurethane foam with an average pore size of 1.3 mm was the most suitable support. The maximum immobilization ratio was 50% under 5% support volume by volume of growth medium. For improving the immobilization ratio of rice callus in the TBR, the optimum TBR operation and modification were investigated further. By repeating a periodic operation 3 times (agitating at 300 rpm for 5 min and then 50 rpm for 2 min, and then 200 rpm of constant agitation speed during the remaining time), almost all supports could entrap rice callus and homogeneous immobilization was attained. The immobilization ratio was improved as compared with that using a constant operation at 200 rpm. Next, the TBR was modified by setting an air sparger inside the stainless mesh cylinder. In the modified TBR, the floating support by air bubbles was reduced, and the immobilization ratio increased further and reached 86.3% when we increased the support volume to 15% under periodic operation on a daily basis. The regeneration frequency of immobilized callus was also slightly increased by periodic operation and modification of the TBR.     

Two-dimensional versus three-dimensional culture systems: Effects on growth and productivity of BHK cells

The influence of surface growth (two-dimensional microcarriers) and three-dimensional growth (aggregates and macroporous supports) in agitated, suspended batch culture systems upon growth and productivity of BHK was compared. Cultures using three porous microcarriers (CultiSpher G, Cellsnow EX, and Cytocell), one nonporous microcarrier (Cytodex 3) and natural aggregates were performed in stirred tanks using two different agitation rates (60 and 100 RPM). With the exception of Cytocell, cell growth, viability, and productivity were similar when three-dimensional structures (porous microcarriers and aggregates) were used. Nonporous microcarriers only compared well at 60 RPM as growth ceased under overagitation. These results suggest that cultures less susceptible to fluid shear are advantageous for scale-up. (c) 1996 John Wiley & Sons, Inc.     

Spatial development of the cultivation of a bone marrow stromal cell line in porous carriers

The spatial development of the cultivation of a bone marrow stromal cell line (SR-4987) in porous carriers was investigated in order to construct a three-dimensional hematopoietic culture system. Low-rate continuous agitation, 20 rpm, was an appropriate method to achieve initial adhesion of cells onto a cellulose porous beads (CPB, 100 μm pore diameter) in a spinner bottle, compared with other methods such as centrifugation and intermittent agitation. Cell growth with continuous agitation at 70 rpm after initial cell adhesion was not inferior to that at 20 rpm. A 2- and 10-fold increase in the inoculum cell concentration for CPB and another type of porous cellulose beads (Micro-cube (MC), 500 μm pore diameter) resulted in a 1.2- and 2-fold increase in final cell concentrationm, respectively. Cells attached to the MC beads and a polyester nonwoven dic (Fibra-cell (FC)) could grow and spread well on the carriers and a fibroblast-like shape was observed under scanning electron microscopy while the cells on CPB were globular. The flatness and inner surface area of these carriers may be the reason for the differences in cell morphology. This revised version was published online in July 2006 with corrections to the Cover Date.     

Study of conformational effects of recombinant interferon γ adsorbed on a non-porous reversed-phase silica support

Reversed-phase chromatography is a powerful method for separating recombinant interferon γ and one of its analogues differing only by a single amino acid residue. Structural differences of the proteins explain this separation ability as demonstrated from adsorption studies on a non-porous reversed-phase support. To reveal the structural differences occurring in the adsorbed state, two different and independent methods were employed. The variation of the retention with the slope of the linear gradient gave information about the molecular contact area of the protein with the support. For different experimental conditions, these data were correlated with the adsorbent capacities measured on an n-octadecyl-modified non-porous silica support. These supports are useful for these types of experiments because the protein is adsorbed exclusively at the external surface of the beads. Moreover, a small amount of protein is necessary to saturate the column, owing to its low capacity.     

Repurposing fed-batch media and feeds for highly productive CHO perfusion processes

Perfusion is a cell culture mode that is gaining popularity for the manufacture of monoclonal antibodies and their derivatives. The cell culture media supporting perfusion culture need to support higher cell densities than those used in fed-batch culture. Therefore, when switching from a fed-batch to a perfusion mode, a new medium need to be developed which supports high cell densities, high productivity, and favorable product quality. We have developed a method for deriving perfusion culture media based on existing fed-batch media and feeds. We show that we can obtain culture media that successfully support perfusion cultures in a single-use rocking bioreactor system at cell-specific perfusion rates below 25 pL⁻¹ cell⁻¹ day⁻¹. High productivities and favorable product quality are also achievable.     

Investigation of production of dextran and dextransucrase by Leuconostoc mesenteroides immobilized within porous stainless steel

Cells of Leuconostoc mesenteroides were immobilized within porus, stainless-steel (SS) supports and used for dextransucrase (DS) and dextran production. The pore size of the support significantly affected the dextran yields, which were greatest with average pore sizes of 2-5 mum. All immobilized-cell biocatalysts in porous stainless steel produced higher yields than free cells, with the exception of cells confined in submicrometer pores (0.5 mum). Coating supports of larger pore size (40 and 100 mum) with calcium alginate enhanced the cell-loading capacity of the supports and increased dextran and fructose yields in the cell-free broth. Controlled, fed-batch, DS production (activation), as a step preliminary to dextran production, significantly improved the subsequent dextran and fructose yields and shortened the time required to attain the maximum such yields. Scanning electron microscopy (SEM) of immobilized L. mesenteroides in stainless steel shows an irregular pattern of the microorganism inside the pores of the solid supports. Coating the porous solid supports with a cell-free calcium alginate layer led to an increase in the cell density inside the support. Cell growth inside the coated, porous stainless steel had no distinct growth form. (c) 1992 John Wiley & Sons, Inc.     

Movement of villi induces endocytosis of NK1 receptors in myenteric neurons from guinea-pig ileum

Agitation of villi evokes reflexes that affect the motility of the guinea-pig small intestine. NK1 receptor endocytosis was used to investigate the possible involvement of tachykinins acting on neuronal NK1 receptors in these reflexes. Segments of guinea-pig ileum were incubated at 37°C in Krebs physiological saline containing 3×10^–6 M nicardipine, with or without agitation of the villi by gas bubbles. Gut segments were fixed after 0–75 min and processed for immunohistochemistry to reveal the NK1 receptors, following which cells were imaged by confocal microscopy. Initially, receptors were located on the surface and in the cytoplasm of myenteric neurons. In gut incubated without movement of the villi, NK1 receptors returned to the cell surface. After 45 and 60 min, NK1 receptors were detected almost exclusively at the cell surface of 83% and 97% (respectively) of nerve cells that were immunoreactive for NK1 receptors and only 12%–13% of the NK1 receptor fluorescence was located in the cytoplasm. Following the return of receptor to the cell surface, agitation of the villi caused a new wave of endocytosis of the NK1 receptors in 70%–80% of the NK1 receptor-immunoreactive neurons. The percentage of the NK1 receptor fluorescence that was in the cytoplasm increased more than 2-fold to 27±2% after 15 min villous agitation. Action potential blockade by tetrodotoxin (3×10^–7 M) prevented the internalisation of the NK1 receptor in response to villous agitation. The degree of internalisation caused by bubbling was similar to that caused by 2×10^–9 M substance P. These results indicate that, when enteric reflex circuits are activated by villous movement, tachykinins are released and cause endocytosis of the NK1 receptor in a subpopulation of myenteric neurons.     

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.List of Symbols d m microcarrier diameter - d _i m impeller diameter - d _p m mean pore diameter - n _i s^–1 agitation rate - p Pa pressure difference - v m/s velocity of microcarrier - v _p m/s average velocity flowing through cyclinder - Pa · s viscosity of medium - angle measured from stagnant point - Pa average shear stress - Pa shear stress distribution     

Massive culture of human liver cancer cells in a newly developed radial flow bioreactor system: Ultrafine structure of functionally enhanced hepatocarcinoma cell lines

Summary With a view to initiating clinical trials, cell morphology and function for a newly developed artificial liver support system employing highly functional human liver cell line, FLC-7, cultured in a radial flow bioreactor were compared to cells grown in a conventional monolayer culture. The radial flow bioreactor consists of a vertically extended cylindrical matrix comprised of porous glass bead microcarriers through which liquid medium flows from the periphery in toward the central axis generating a beneficial concentration gradient of oxygen and nutrients, while preventing excessive shear stresses or buildup of waste products. The three-dimensional culture system supports high-density (1.1 × 10⁸ cells/ml-matrix), large scale cultures (4.4 × 10¹⁰ cells/400 ml-bioreactor) with long-term viability. Scanning and transmission electron microscopy (SEM and TEM) revealed that cells cultured in a monolayer system were flattened and extended with numerous cytoplasmic projections. Cells in the three-dimensional culture were spherical and covered with microvillilike processes resembling liver cells in vivo. The cells were solidly attached on the surfaces and within the pores of the microcarriers in highly dense colonies. The spherical cells remained in close contact with adjacent cells, while circulation of liquid medium flowed freely through spaces between cells. FLC-7 cells produced albumin at a rate of 6.41 μg/24 h/10⁶ cells. Alpha-fetoprotein (AFP) production dropped nearly threefold in comparison to monolayer cultures. Results demonstrated that the new artificial liver support systems (ALSS) provides a superior three-dimensional culture environment that allows cells to perform at naturally functioning levels.     

Approach toward an efficient inoculum preparation stage for suspension BHK-21 cell culture

Mammalian cells are the most frequently used hosts for biopharmaceutical proteins manufacturing. Inoculum quality is a key element for establishing an efficient bioconversion process. The main objective in inoculation expansion process is to generate large volume of viable cells in the shortest time. The aim of this paper was to optimize the inoculum preparation stage of baby hamster kidney (BHK)-21 cells for suspension cultures in benchtop bioreactors, by means of a combination of static and agitated culture systems. Critical parameters for static (liquid column height: 5, 10, 15 mm) and agitated (working volume: 35, 50, 65 mL, inoculum volume percentage: 10, 30 % and agitation speed: 25, 60 rpm) cultures were study in T-flask and spinner flask, respectively. The optimal liquid column height was 5 mm for static culture. The maximum viable cell concentration in spinner flask cultures was reached with 50 mL working volume and the inoculum volume percentage was not significant in the range under study (10–30 %) at 25 rpm agitation. Agitation speed at 60 rpm did not change the main kinetic parameters with respect to those observed for 25 rpm. These results allowed for a schedule to produce more than 4 × 10⁹ BHK-21 cells from 4 × 10⁶ cells in 13 day with 1,051 mL culture medium.     

Growth and death behaviour of anchorage-independent animal cells immobilized within porous support matrices

Summary Growth and death of anchorage-independent animal cells entrapped within porous biomass support particles (BSPs) in static or shake-flask cultures were evaluated by comparison of enzyme activity with non-immobilized cells grown under static culture using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and release of lactate dehydrogenase into the culture medium. Mouse myeloma MPC-11 (ATCC CCL 167) cells inoculated within porous polyvinyl formal resin BSPs (3 × 3 × 3 or 2 × 2 × 2 mm; mean pore diameter, 60 ) grew exponentially at a specific growth rate comparable to that of non-immobilized cells in the initial period of incubation. Entrapped cells then reached the stationary phase with a cell density over 10⁷ cells/cm³ BSP. The death rate of entrapped cells increased in response to the rise in viable cell density in the BSPs. Observation of viable cell distribution within the BSPs using MTT staining indicated that the cells concentrated within a thin outer shell of the BSPs with time. After the immobilized cells reached the stationary phase, penetration of cells into the outer shell ceased and heterogeneous distribution of cell density occurred in the viable cell layer in the shake-flask culture.     

Structured fiber supports for gas phase biocatalysis

Pseudomonas cepaciae lipase adsorbed onto non-porous structured fiber supports in the form of woven fabrics, was used to catalyze hydrolysis and transesterification reactions in the gas phase. The enzyme adsorbed onto carbon fiber support exhibited much higher catalytic activity compared to the enzyme immobilized onto glass fiber carrier. The effect of temperature and relative humidity on reactions catalyzed by P. cepaciae lipase adsorbed onto structured fiber carbon support was studied in the gas system. Under the conditions investigated (up to 60 °C and 80% relative humidity), the immobilized enzyme showed a high thermostability and could be efficiently used to catalyze hydrolytic and transesterification reactions in continuous mode. Structured fiber supports, with a high specific surface area and a high mechanical resistance, showed a low-pressure drop during the passage of reactants through a reactor. The approach proposed in this study could be suitable for immobilization of a wide variety of enzymes.     

Bacterial cellulose membrane – A new support carrier for yeast immobilization for ethanol fermentation

The aim of the work was to study the properties of the bacterial cellulose membrane (BCM) and the feasibility of using it as a new, environmentally friendly support carrier for yeast cell immobilization. It was observed that the morphology of BCM varied with different cultivation methods and the scanning electron microscopy (SEM) images confirmed that the yeast cells were entrapped in the porous network of BCM obtained from the static culture and stabilized by the cross-linked fibrils. Particularly, the research confirmed the effectiveness of yeast immobilization in BCM reflected by the high yield of alcohol (9.7% v/v, a 21.25% increase of those using free cells) and the high stability. The specific rate of ethanol production by the immobilized cells in BCM was 2.1 g g⁻¹ h⁻¹, 31.3% greater than that of the suspended cells. Results implied that applying BCM as the support carrier had little adverse effects on cell viability and proliferation. Instead, it facilitated the product leakage and nutrients transportation through the porous network.     

Glycosylated α-chymotrypsin as a catalyst for kyotorphin synthesis in water-organic media

-Chymotrypsin was covalently modified with cellobiose by chemical means. After adsorption on to a porous polyamide support, both the native and the glycosylated immobilized derivatives were used to synthesize a kyotorphin derivative (N-benzoyl-l-tyrosyl-l-argininamide) in acetonitrile/water. Glycosylated chymotrypsin gave a 125% increase in product formation (750 nmol mg^–1 catalyst in 3 h) at 60% (v/v) acetonitrile/water. Maximal protective effect of this glycosylation process was at 70% (v/v) acetonitrile/water, at which concentration the half-life of the glycosylated enzyme was 20-times longer than that of the native form (52 min and 2.8 min, respectively).     

Uniform tissues engineered by seeding and culturing cells in 3D scaffolds under perfusion at defined oxygen tensions

In this work, we assessed whether culture of uniformly seeded chondrocytes under direct perfusion, which supplies the cells with normoxic oxygen levels, can maintain a uniform distribution of viable cells throughout porous scaffolds several milimeters in thickness, and support the development of uniform tissue grafts. An integrated bioreactor system was first developed to streamline the steps of perfusion cell seeding of porous scaffolds and perfusion culture of the cell-seeded scaffolds. Oxygen tensions in perfused constructs were monitored by in-line oxygen sensors incorporated at the construct inlet and outlet. Adult human articular chondrocytes were perfusion-seeded into 4.5 mm thick foam scaffolds at a rate of 1 mm/s. Cell-seeded foams were then either cultured statically in dishes or further cultured under perfusion at a rate of 100 microm/s for 2 weeks. Following perfusion seeding, viable cells were uniformly distributed throughout the foams. Constructs subsequently cultured statically were highly heterogeneous, with cells and matrix concentrated at the construct periphery. In contrast, constructs cultured under perfusion were highly homogeneous, with uniform distributions of cells and matrix. Oxygen tensions of the perfused medium were maintained near normoxic levels (inlet congruent with 20%, outlet > 15%) at all times of culture. We have demonstrated that perfusion culture of cells seeded uniformly within porous scaffolds, at a flow rate maintaining a homogeneous oxygen supply, supports the development of uniform engineering tissue grafts of clinically relevant thicknesses.