Physical Studies on Cell Immobilization Using Calcium Alginate Gels

Columns of calcium alginate gel pellets have excellent physical properties when used as a cell immobilization support. Columns of pellets were very resistant to compression and abrasion during passage of high concentrations of sucrose at high flow rates, but if the pellets were formed using low alginate and Ca²⁺ concentrations, compression occurred and flow out of the column was reduced and pressure built up. Transfer of sucrose into the pellets was controlled by internal diffusion, the rate of diffusion being increased by reductions in the alginate and Ca²⁺ concentrations used for immobilization and by the presence of entrapped active cells. Some leakage of cells occurred during use especially when cell division of the entrapped cells took place, but leakage could be minimized by using more highly polymerized pellets. Therefore, immobilization conditions can be chosen so as to form strong pellets, possessing high substrate transfer rates and low rates of cell leakage.     

Isomaltulose production using immobilized cells

Three strains of Erwinia rhapontici especially suitable for use in the form of nongrowing immobilized cells were selected by screening strains of cells for high activity and operational stability in an immobilized form. Immobilization in calcium alginate gel pellets was easily the best method of immobilizing E. rhapontici. Much greater operational stabilities were obtained than when other immobilization methods were used. Conditions of operation which optimize the activity, stability, and yield and the ease of operation of the immobilized cell columns working in a steady state are described. These include the effects of substrate concentration, diffusional restrictions and water activity, the concentration of cells immobilized, and the type of reactor used. Thus, the immobilized cells produce about 1500 times their own weight of isomaltulose during one half-life of use (ca. 1 year). Loss of activity was most closely correlated with the volume of substrate processed and so presumably is due to the presence of low concentrations of a cummulative inhibitor in the substrate. Methods for regenerating the activity of the immobilized cells by the periodic administration of nutrients, of forming isomaltulose by continuously supplying nutrients to growing immobilized cells, and of crystallizing isomaltulose from the column eluate are also described.     

Influence of chitosan type on the properties of extruded pellets with low amount of microcrystalline cellulose

The purpose of this research was to study the influence of type of chitosan with different molecular weights, ie, 190 and 419 kDa, on properties of pellets prepared by extrusion/ spheronization. The formulations, consisting of acetaminophen as model drug, chitosan, microcrystalline cellulose (MCC), and dibasic calcium phosphate dihydrate with/without sodium alginate, were extruded using a twin-screw extruder and water as the granulating liquid. With 30% wt/wt MCC and no added sodium alginate, spherical pellets were produced containing low and high molecular weight chitosan at a maximum amount of 60% and 40% wt/wt, respectively. With sodium alginate (2.5% wt/wt), pellets with either type of chitosan (60% wt/wt), MCC (17.5% wt/wt), and acetaminophen (20% wt/wt) could be produced indicating an improved pelletforming ability. Type and amount of chitosan and added sodium alginate affected physical properties of pellets including size, roundness, crushing force, and drug release. Low molecular weight chitosan produced pellets with higher mean diameter, sphericity, and crushing force. Additionally, the pellets made of low molecular weight chitosan and added sodium alginate showed faster drug release in 0.1 N HCl but had slower drug release in pH 7.4 phosphate buffer. This indicated that drug release from pellets could be modified by the molecular weight of chitosan. In conclusion, the molecular weight of chitosan had a major influence on formation, physical properties, and drug release from the obtained pellets. Published: August 10, 2007     

A framework for the prediction of scale-up when using compressible chromatographic packings

Experimental data are given for the solid pressure distributions in chromatography columns of various column aspect ratios packed with four types of agarose-based resin. The loss of column wall support at large scales can result in unexpectedly high pressures caused by the compression of the matrix via drag forces exerted by fluid flow through the bed. The need for an accurate model to predict flow conditions at increasing scale is essential for the scaling-up of chromatographic processes and for avoiding bed compression during operation. Several studies have generated correlations that allow for the prediction of column pressure drops, but they either are mathematically complex, which impairs their practical use, or require a large number of experiments to be performed before they can be used. In this study an empirical correlation was developed based on a previously proposed model, which links the critical velocity of operation of a chromatographic system (microcrit), to the gravity-settled bed height (L0), the column diameter (D), the feed viscosity (micro), and the compressibility of the chromatographic media used (micro 10%). The methodology developed in this study is straightforward to use and significantly reduces the burden of preceding laboratory-scale experimentation. The approach can be used to predict the critical velocity of any chromatographic system and will be useful in the development of chromatographic operations and for column sizing.     

Decreased brain weights in rats after long-term barbital treatments

To study dependence on barbiturates, rats were in three experiments given a solution of barbital as their only drinking fluid for periods around 30 weeks. The animals were sacrificed after abstinence periods of up to 30 days and the brains were weighed. Compared with untreated controls the barbital treated animals in these experiments consistently had reduced wet brain weights. This reduction was not restituted after an abstinent period of 30 days. The decrease did not seem to be secondary to a reduced body weight. The decrease in brain weight found throughout the abstinence period was not influenced by changes in water intake.In a 4th experiment 12 weeks of barbital treatment caused a similar slightly smaller reduction in wet brain weight. Since there was no difference in water content the dry weight was also reduced. The possibility that this finding is related to the brain atrophy found in humans after long-term ethanol abuse is pointed out.     

Interaction during fibril formation of soluble collagen with cartilage proteinpolysaccharide

Precipitation of soluble forms of collagen from solutions containing the soluble protein-polysaccharide (PP-L) of bovine nasal cartilage, followed by centrifugation at 100,000 g, resulted in the formation of coherent elastic pellets whose wet weights increased with the concentration of PP-L in the initial solution. Dry weights and uronic acid contents of these pellets showed that the amount of water held in the wet pellet was nearly constant for any one kind and concentration of collagen, and ranged from 20 to 100 mg./mg. PP-L in the pellet. Soluble collagens from four different sources and PP-L from three kinds of cartilage showed similar effects. Precipitation of soluble collagen in the presence of hyaluronate or dextran yielded pellets of much smaller size than those formed in the presence of PP-L. The presence of chondroitin sulfate had only a slight effect on wet pellet weights. Wet weights of pellets formed in the presence of PP-L decreased with increasing ionic strength. A model involving entanglement between insoluble collagen fibrils and the relatively stiff chondroitin sulfate chains of branched PP-L seems qualitatively capable of accounting for these results.     

Tissue fluid pressure and flow during pneumatic compression in lymphedema of lower limbs

Background: Physiotherapy of edema in cases with obstructed main lymphatics of lower limbs requires knowledge of how high external pressures should be applied manually or set in compression devices in order to generate tissue pressures high enough to move tissue fluid to nonswollen regions and to measure its flow rate. Methods: We measured tissue fluid pressure and flow in subcutaneous tissue of lymphedematous limbs stages II to IV at rest and during pneumatic compression under various pressures and inflation timing. An 8-chamber sequential compression device inflated to pressures 50-120?mmHg, for 50 sec each chamber, with no distal deflation, was used. Pressures were measured using a wick-in-needle and electronic manometer. Fluid flow was calculated from continuously recorded changes in limb circumference using strain gauge plethysmography. Results: Before massage, in all stages of lymphedema, stagnant tissue fluid pressures in subcutaneous tissue ranged between -1 and +10 mmHg and did not differ from those measured in normal subjects. Pressures generated in tissue fluid by pneumatic compression reached 40-100 mmHg and were lower than those in inflated chambers. High pressure gradient through the skin was caused by its rigidity (fibrosis) and dissipation of applied compression force to proximal noncompressed limb regions. The calculated volumes of displaced tissue fluid ranged from 10 to 30 ml per compression cycle, to reach in some cases 100 ml in the groin region. Conclusions: Tissue fluid pressures generated by a pneumatic device were found lower than in the compression chambers. The obtained results point to the necessity of applying high pressures and longer compression times to generate effective tissue fluid pressures and to provide enough time for moving the stagnant fluid.     

Nikkomycin production in pellets of Streptomyces tendae.

In previous submerged fermentation experiments mycelial suspensions of Streptomyces tendae were viscous and availability of oxygen limited the yield of nikkomycins (Nk), a complex of secondary metabolites which includes nucleoside-peptides with antibiotic activity. Increasing agitation improved oxygen transfer but consumed considerable power and shear-damaged cells. In this paper, cellular aggregates (pellets) were used to reduce viscosity and protect cells from shear. Under the conditions tested, specific productivity of S. tendae pellets increased with increasing size up to 1.4 mm diameter and then decreased. The maximal specific productivity of S. tendae pellets (44 mg Nk/g dry weight/h) occurred at a very low cell concentration. Pellet formation or high biomass concentration was required for the production of bioactive dipeptide and tripeptide Nks. It is speculated that accumulation of intermediates in large pellets activates the production of mature secondary metabolites.     

CONTINUOUS ALCOHOL/MALOLACTIC FERMENTATION OF GRAPE MUST IN A BIOREACTOR SYSTEM USING IMMOBILIZED CELLS

Three cultures immobilized by entrapping within alginate gel beads and packed in near-horizontal acrylic columns (15.0° angle) were used for alcohol/malolactic fermentation of grape must. Immobilized cells of Saccharomyces cerevisiae spp. chablis were placed in the 1st column, S. cerevisiae cells (an alcohol-sucrose-tolerant yeast) in the 2nd and the Lactobacillus delbrueckii cells in the 3rd column. Grape must with different levels of sugar(s), were each fed to the bioreactor columns at dilution rate of 0.74 h⁻¹ and recycled at 37.0C. The percent fermentation efficiency and yield using the 1st and 2nd columns for grape must containing 33.3% sugar(s) were 92.9 and 91.5%, respectively, and the wine had 15.5% alcohol after 23 cycles (∼ 50 h fermentation). The viability of the immobilized yeast cells in the alginate gel-bead was 84%± 4.0. Immobilized Lactobacillus delbrueckii cells were then added to the 3rd column (in series 37.0C) and the three cultures resulted in alcohol/malolactic fermentation of the grape must, evidenced by the high level of alcohol formed and simultaneous transformation of malic to lactic acid. Sensory evaluation of the wine scored high (7.8 ± 2.0 based on a value of 10.0) and indicated the potential of using multiple immobilized cells of two specific yeast cultures and a malolactic Lactobacillus for wine production.     

Nikkomycin production in pellets of Streptomyces tendae

S.E. VECHT-LIFSHITZ, Y. SASSON AND S. BRAUN. 1992. In previous submerged fermentation experiments mycelial suspensions of Streptomyces tendae were viscous and availability of oxygen limited the yield of nikkomycins (Nk), a complex of secondary metabolites which includes nucleoside-peptides with antibiotic activity. Increasing agitation improved oxygen transfer but consumed considerable power and shear-damaged cells. In this paper, cellular aggregates (pellets) were used to reduce viscosity and protect cells from shear. Under the conditions tested, specific productivity of S. tendae pellets increased with increasing size up to 1.4 mm diameter and then decreased. The maximal specific productivity of S. tendae pellets (44 mg Nk/g dry weight/h) occurred at a very low cell concentration. Pellet formation or high biomass concentration was required for the production of bioactive dipeptide and tripeptide Nks. It is speculated that accumulation of intermediates in large pellets activates the production of mature secondary metabolites.     

Preservative fluid and storage conditions alter body mass estimation in a terrestrial insect

Body mass is a frequently used trait in ecological and evolutionary research. In the present study, I demonstrate that sampling and storage conditions affect wet and dry weights in an insect predator, Anchomenus dorsalis (Pontoppidan) (Coleoptera: Carabidae). Live beetles were placed in one of five preservative fluids for 1 month to simulate sampling by pitfall traps. Sodium chloride solution, ethylene glycol, ethylene glycol + detergent, and propylene glycol caused significant increases in both wet and dry weights compared with control (short‐term frozen) specimens, whereas formaldehyde did not. In a separate experiment, four methods of long‐term (6 months) sample storage (freezing, ethanol, propylene glycol, and ethyl acetate vapour) all caused significant changes in wet weight compared with the control treatment. The dry weight of the specimens preserved in ethanol decreased significantly in contrast to the long‐term frozen specimens and those in propylene glycol and ethyl acetate vapour, whose dry weight did not differ significantly from the control specimens. The combination of formaldehyde as the preservative fluid and freezing as the storage method thus appears to be an optimal combination for studies in which the body mass of dead insects is considered.     

Role of pericellular matrix in development of a mechanically functional neocartilage

The role of the chondrocyte pericellular matrix (PCM) was examined in a three-dimensional chondrocyte culture system to determine whether retention of the native pericellular matrix could stimulate collagen and proteoglycan accumulation and also promote the formation of a mechanically functional hyaline-like neocartilage. Porcine chondrocytes and chondrons, consisting of the chondrocyte with its intact pericellular matrix, were maintained in pellet culture for up to 12 weeks. Sulfated glycosaminoclycans and type II collagen were measured biochemically. Immunocytochemistry was used to examine collagen localization as well as cell distribution within the pellets. In addition, the equilibrium compressive moduli of developing pellets were measured to determine whether matrix deposition contributed to the mechanical stiffness of the cartilage constructs. Pellets increased in size and weight over a 6-week period without apparent cell proliferation. Although chondrocytes quickly rebuilt a PCM rich in type VI collagen, chondron pellets accumulated significantly more proteoglycan and type II collagen than did chondrocyte pellets, indicating a greater positive effect of the native PCM. After 5 weeks in chondron pellets, matrix remodeling was evident by microscopy. Cells that had been uniformly distributed throughout the pellets began to cluster between large areas of interterritorial matrix rich in type II collagen. After 12 weeks, clusters were stacked in columns. A rapid increase in compressive strength was observed between 1 and 3 weeks in culture for both chondron and chondrocyte pellets and, by 6 weeks, both had achieved 25% of the equilibrium compressive stiffness of cartilage explants. Retention of the in vivo PCM during chondrocyte isolation promotes the formation of a mechanically functional neocartilage construct, suitable for modeling the responses of articular cartilage to chemical stimuli or mechanical compression.     

Hyaluronate-alginate gel as a novel biomaterial: mechanical properties and formation mechanism

With the aim of producing a biomaterial for surgical applications, the alginate-hyaluronate association has been investigated to combine the gel-forming properties of alginate with the healing properties of hyaluronate. Gels were prepared by diffusion of calcium into alginate-hyaluronate mixtures, with an alginate content of 20 mg/mL. The hyaluronate source was shown to have significant effect on the aspect and the properties of the gels. The gels have viscoelastic behaviour and the transient measurements carried out in creep mode could be interpreted through a Kelvin-Voigt generalised model: experimental data led to the steady state hardness and a characteristic viscosity of the gel. Gels prepared from Na rooster comb hyaluronate with weight ratio up to 0.50 have satisfactory mechanical properties, and fully stable gels are obtained after a few days; on the contrary, use of lower molecular weight hyaluronate led to loose gels for hyaluronate contents over 0.25. Gel formation was investigated by measurements of the exchange fluxes between the calcium chloride solution and the forming gel, which allowed thorough investigations of the occuring diffusion phenomena of water, calcium ion and hyaluronate. Strong interactions of water with hyaluronate reduce significantly the rate of weight loss from the gel beads and allows higher water content in steady-state gels. Calcium content in the gel samples could be correlated to the actual alginate concentration, whatever the nature and the weight ratio of hyaluronate.     

Pressure-flow relationships for packed beds of compressible chromatography media at laboratory and production scale

Pressure drop across chromatography beds employing soft or semirigid media can be a significant problem in the operation of large-scale preparative chromatography columns. The shape or aspect ratio (length/diameter) of a packed bed has a significant effect on column pressure drop due to wall effects, which can result in unexpectedly high pressures in manufacturing. Two types of agarose-based media were packed in chromatography columns at various column aspect ratios, during which pressure drop, bed height, and flow rate were carefully monitored. Compression of the packed beds with increasing flow velocities was observed. An empirical model was developed to correlate pressure drop with the aspect ratio of the packed beds and the superficial velocity. Modeling employed the Blake-Kozeny equation in which empirical relationships were used to predict bed porosity as a function of aspect ratio and flow velocity. Model predictions were in good agreement with observed pressure drops of industrial scale chromatography columns. A protocol was developed to predict compression in industrial chromatography applications by a few laboratory experiments. The protocol is shown to be useful in the development of chromatographic methods and sizing of preparative columns.     

Production of mead by immobilized cells of Hansenula anomala

Summary Mead was produced by immobilized cells of Hansenula anomala in calcium alginate gels. The immobilized cell beads of 3 mm diameter packed in column reactors of dimensions 2.2x60, 4x40 and 8x80 cm, produced mead containing maximum concentrations of ethanol and ethyl acetate of 70 g/l and 730 mg/l, respectively at a dilution rate of 0.1 h^–1. The maximum alcohol productivity achieved was 23.1 g/l·h at a dilution rate of 0.33 h^–1. With intermittent regenerations of the cells the reactor operated continuously for 110 days. This process enables the quick production of matured mead by a single culture and the elimination of the traditionally used long aging periods.     

Characterization of purified Shiga toxin from Shigella dysenteriae 1

Shiga toxin was purified from the culture supernatant of Shigella dysenteriae 1 by ammonium sulfate fractionation, DEAE-cellulose column chromatography and repeated chromatofocusing column chromatography. About 1.6 mg of purified Shiga toxin was obtained from 15 liters of culture with a yield of about 27%. The molecular weight of purified Shiga toxin was estimated to be 62,000. The toxin consisted of A and B subunits with molecular weights of about 30,000 and 5,000-6,000, respectively. The isoelectric point of purified Shiga toxin was 7.0. Purified Shiga toxin showed the following biological activities: lethal toxicity to mice when injected intraperitoneally with an LD50 of 28 ng per mouse; cytotoxicity to Vero cells, killing about 50% of the cells at 1 pg and all of the cells at 10 pg; and fluid accumulation in rabbit ileal loops at a concentration of more than 1 microgram.     

Cells (MC3T3-E1)-Laden Alginate Scaffolds Fabricated by a Modified Solid-Freeform Fabrication Process Supplemented with an Aerosol Spraying

In this study, we propose a new cell encapsulation method consisting of a dispensing method and an aerosol-spraying method. The aerosol spray using a cross-linking agent, calcium chloride (CaCl(2)), was used to control the surface gelation of dispensed alginate struts during dispensing. To show the feasibility of the method, we used preosteoblast (MC3T3-E1) cells. By changing the relationship between the various dispensing/aerosol-spraying conditions and cell viability, we could determine the optimal cell-dispensing process: a nozzle size (240 μm) and an aerosol spray flow rate (0.93 ± 0.12 mL min(-1)), 10 mm s(-1) nozzle moving speed, a 10 wt % concentration of CaCl(2) in the aerosol solution, and 2 wt % concentration of CaCl(2) in the second cross-linking process. Based on these optimized process conditions, we successfully fabricated a three-dimensional, pore-structured, cell-laden alginate scaffold of 20 × 20 × 4.6 mm(3) and 84% cell viability. During long cell culture periods (16, 25, 33, and 45 days), the preosteoblasts in the alginate scaffold survived and proliferated well.     

Dedifferentiated chondrocytes cultured in alginate beads: Restoration of the differentiated phenotype and of the metabolic responses to Interleukin-1β

Chondrocytes cultivated in monolayer rapidly divide and lose their morphological and biochemical characteristics, whereas they maintain their phenotype for long periods of time when they are cultivated in alginate beads. Because cartilage has a low cellularity and is difficult to obtain in large quantities, the number of available cells often becomes a limiting factor in studies of chondrocyte biology. Therefore, we explored the possibility of restoring the differentiated properties of chondrocytes by cultivating them in alginate beads after two multiplication passages in monolayer. This resulted in the reexpression of the two main markers of differentiated chondrocytes: Aggrecan and type II collagen gene expression was strongly reinduced from day 4 after alginate inclusion and paralleled protein expression. However, 2 weeks were necessary for total suppression of type I and III collagen synthesis, indicators of a modulated phenotype. Interleukin-1β, a cytokine that is present in the synovial fluid of rheumatoid arthritis patients, induces many metabolic changes on the chondrocyte biology. Compared with cells in primary culture, the production of nitric oxide and 92-kDa gelatinase in response to interleukin-1β was impaired in cells at passage 2 in monolayer but was fully recovered after their culture in alginate beads for 2 weeks. This suggests that the effects of interleukin-1β on cartilage depend on the differentiation state of chondrocytes. This makes the culture in alginate beads a relevant model for the study of chondrocyte biology in the presence of interleukin-1β and other mediators of cartilage destruction in rheumatoid arthritis and osteoarthrosis. J. Cell. Physiol. 176:303–313, 1998. © 1998 Wiley-Liss, Inc.     

Short-term changes in cell and matrix damage following mechanical injury of articular cartilage explants and modelling of microphysical mediators

The short-term responses of articular cartilage to mechanical injury have important implications for prevention and treatment of degenerative disease. Cell and matrix responses were monitored for 11 days following injurious compression of cartilage in osteochondral explants. Injury was applied as a single ramp compression to 14 MPa peak stress at one of three strain rates: 7 x 10(-1), 7 x 10(-3) or 7 x 10(-5) s(-1). Responses were quantified in terms of the appearance of macroscopic matrix cracks, changes in cell viability, and changes in cartilage wet weights. Loading at the highest strain rate resulted in acute cell death near the superficial zone in association with cracks, followed over the 11 days after compression by a gradual increase in cell death and loss of demarcation between matrix zones containing viable versus nonviable cells. In contrast, loading at the lowest strain rate resulted in more severe, nearly full-depth cell death acutely, but with no apparent worsening over the 11 days following compression. Between days 4 and 11, all mechanically injured explants significantly increased in wet weight, suggesting loss of matrix mechanical integrity independent of compression strain rate. Results demonstrate that short-term responses of cartilage depend upon the biomechanical characteristics of injurious loading, and suggest multiple independent pathways of mechanically-induced cell death and matrix degradation. Modifications to an existing fiber-reinforced poroelastic finite element model were introduced and the model was used for data interpretation and identification of microphysical events involved in cell and matrix injury. The model performed reasonably well at the slower strain rates and exhibited some capacity for anticipating the formation of superficial cracks during injurious loading. However, several improvements appear to be necessary before such a model could reliably be used to draw upon in vitro experimental results for prediction of injurious loading situations in vivo.     

Purification and characterization of small molecular weight myeloperoxidase from human promyelocytic leukemia HL-60 cells

Human myeloperoxidase was purified to homogeneity from human promyelocytic leukemia HL-60 cells. A small molecular weight myeloperoxidase was found in these cells and was separated from three other forms of myeloperoxidase of large molecular weight by carboxymethyl-Sepharose CL-6B column chromatography and Sephacryl S-200 gel filtration. The S20,w values of the molecular weights of the small and large myeloperoxidases were found to be 5.2 and 8.07 S, respectively, by sucrose density gradient centrifugation. From these S20,w values, the molecular weights of the small and large myeloperoxidases were estimated to be 79 000 and 153 000, respectively. On electrophoresis in sodium dodecyl sulfate--polyacrylamide gel, the small and large myeloperoxidases each gave two bands of protein corresponding to molecular weights of 59 300 and 10 150. The small myeloperoxidase could not be distinguished from the large enzymes by the Ouchterlony double immunodiffusion test, but it could be distinguished from them by the microcomplement fixation text. One of the three large molecular weight myeloperoxidases was eluted at a lower concentration of methyl alpha-D-mannoside than the other two on concanavalin A--Sepharose chromatography. This suggested that the heterogeneity of the myeloperoxidases with large molecular weight may be partly due to differences in their sugar moieties.