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PubMed2005

Stable encapsulation of active enzyme by application of multilayer nanofilm coatings to alginate microspheres.

In an effort to improve the stability for long-term biosensor use, layer-by-layer self-assembly was explored as a potential technique to provide a diffusion barrier to encapsulated glucose oxidase inside alginate microspheres (<5 microm), fabricated using an emulsification technique. The total loss of encapsulated enzyme was reduced to less than 25 and 15% with the application of single PAH/PSS and crosslinked PAH/PAA coatings, respectively, in comparison to at least a 45% loss observed with uncoated and PDDA/PSS-coated microspheres. Furthermore, it was found that enzyme within PAH/PSS- and crosslinked PAH/PAA-coated spheres retained more than 84 and 60% of initial activity, respectively, after three months, whereas uncoated and PDDA/PSS-coated microspheres retained less than 20%.

Alginates↗
PubMed1997

Diffusion of solutes in agarose and alginate gels: 1H and 23Na PFGSE and 23Na TQF NMR studies.

Cells immobilized in gels experience potential metabolic restrictions in the form of reduced diffusion rates of metabolites and ions and their possible selective adsorption on the gel matrix. Diffusion and relaxation characteristics of common solutes in agarose and barium alginate gels were investigated at 37 degrees C by using 1H PFGSE and 23Na TQF NMR spectroscopy. Glucose, glycine, alanine, lactate, sodium ions, and HDO were studied. There were no selective interactions between any of the metabolites and the gel materials but the diffusion coefficients were uniformly reduced. The effects of metabolite diffusion and utilization, in gel beads and threads containing cells, were simulated by using a reaction diffusion model incorporating the measured diffusion coefficients. Metabolism is expected to be very significantly limited by diffusion of solutes to and from the cells that are centrally located within gel threads or spheres of radius approximately 2.0 mm, which is a commonly used size.

Alginates↗
PubMed1990

31P NMR of mammalian cells encapsulated in alginate gels utilizing a new phosphate-free perfusion medium.

A simple technique permitting long term 31P magnetic resonance spectroscopy of cultured mammalian cells is described. Cells are encapsulated in calcium alginate gels and perfused with a new phosphate-fre medium designed for maintenance of high cell viability. Intracellular pH and beta-NTP/Pi ratios, studied in three cell types, remained stable over a 16-20 h NMR monitoring period. The techniques described may be useful for monitoring cell metabolism with a variety of cell types.

Alginates↗
PubMed2000

Differential effects of interleukin-1 on hyaluronan and proteoglycan metabolism in two compartments of the matrix formed by articular chondrocytes maintained in alginate.

Phenotypically stable young adult bovine articular chondrocytes suspended in beads of alginate gel were first cultured for 5 days, using daily changes of medium containing 10% fetal bovine serum and supplements. The cells in the beads were then maintained in culture for a further 3 days in the presence or absence of interleukin-1alpha at 1 ng/ml in the daily change of medium. The exposure to interleukin-1alpha caused the incorporation of (35)S-sulfate into the predominant cartilage proteoglycan, aggrecan, to decrease by approximately 60%. In addition, proteoglycans that had accumulated into the cell-associated matrix during the first 5 days of culture in the absence of interleukin-1alpha moved into the matrix further removed from the cells and from there into the medium. In contrast, the exposure to interleukin-1alpha was found to markedly promote the rate of synthesis of hyaluronan, especially during the first 24 h. Over the 3 days of culture in the presence of interleukin-1alpha, a large proportion of the newly synthesized hyaluronan molecules, as well as those that had previously become residents of the cell-associated matrix, moved out of this compartment and appeared to become permanent residents of the further removed matrix. These results demonstrate that exposure of young adult articular chondrocytes to interleukin-1alpha has profound effects on the metabolism of hyaluronan, a molecule that plays a critical role in the retention of proteoglycan molecules in the matrix. Importantly, the results suggest that exposure of chondrocytes to interleukin-1 in inflamed joints, such as occurs in rheumatoid arthritis, leads to the rapid loss of coordination of the synthesis of aggrecan and hyaluronan, two of the critical constituents of the proteoglycan aggregate. In addition, we present evidence that these interleukin-1-induced effects differentially alter the metabolism of hyaluronan in the metabolically active cell-associated matrix and the metabolically inactive matrix further removed from the chondrocytes.

Aggrecans↗
PubMed1997

Doxyclycline inhibits collagen synthesis by bovine chondrocytes cultured in alginate.

Doxycycline is known for its ability to inhibit matrix metalloproteinases (MMPs), a family of enzymes that play a role in cartilage breakdown in arthritides. Its prophylactic effect in reducing joint degradation in osteoarthritis is mainly attributed to this property. In this study, we show that doxycycline exhibits a profound inhibition of collagen synthesis by bovine articular chondrocytes cultured in alginate. At 25 microM doxycycline, collagen synthesis was decreased by 50%; no effect on cell proliferation (DNA levels) or general protein synthesis (35S-Met and 35S-Cys incorporation) was observed. Messenger RNA levels of type II collagen were also reduced, indicating an effect of doxycycline at the transcriptional level. The concentration of doxycycline needed to downregulate collagen synthesis was > 10-fold lower than that needed to inhibit most of the MMPs. Inasmuch as differentiated chondrocytes in the early stages of osteoarthritis exhibit increased collagen synthesis, the beneficial effect of doxycycline in vivo may involve prevention of changes in chondrocyte phenotype.

Alginates↗
PubMed2001

Regulation of gelatinase-A (MMP-2) production by ovine intervertebral disc nucleus pulposus cells grown in alginate bead culture by Transforming Growth Factor-beta(1)and insulin like growth factor-I.

The aim of this study was to gain information relevant to disc repair processes. Limited degradation of the collagen matrix by matrix metalloproteases (MMPs) may facilitate the loosening of cell-cell and cell-matrix interactions within the injured intervertebral disc (IVD) to favour the penetration of blood vessels and migration of fibroblasts into the defect to promote repair processes. Gelatinase A (MMP-2) has a particularly important role to play in angiogenesis, in the present study we investigated the in vitro regulation of MMP-2 by Transforming Growth Factor-beta 1 (TGF-beta 1) and Insulin-like Growth Factor-1 (beta IGF-I) in cells from the nucleus pulposus (NP) of the ovine IVD. Ovine NP cells were grown in alginate bead cultures in complete medium (10% foetal calf serum) for 7 days, established in serum-free conditions for 24 h, then stimulated with TGF-beta 1 (0.1 or 10 ng/ml) or IGF-I (2 or 50 ng/ml) +/-Concanavalin A (20 microg/ml) for an additional 48 h. Conditioned medium was examined for matrix metalloproteases using gelatin zymography, Tissue Inhibitor of Metalloproteinase 2 (TIMP-2) and Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) were immunolocalised in beads. Pro (72 kDa) and active (59 kDa) MMP-2 were the major gelatinolytic MMPs detected in control cultures, the TGF-beta 1 and IGF-I treatments significantly decreased levels of the active MMP-2, inclusion of Concanavalin A resulted in a complete reversal of this trend with IGF-I, and to a lesser extent with TGF-beta 1. Cell surface levels of TIMP-2 and MT1-MMP were decreased by the TGF-beta 1 treatment while IGF-I only appeared to decrease TIMP-2 expression. The findings of this study provide some insight as to why dense avascular connective tissues such as the intervertebral disc have such a poor healing potential.

Alginates↗
PubMed1997

Human intervertebral disc cells from the annulus: three-dimensional culture in agarose or alginate and responsiveness to TGF-beta1.

Cell culture procedures were developed for use with surgical and normal control specimens of the annulus of the human intervertebral disc. Cells were established in monolayer explant culture and seeded into three-dimensional growth environments of alginate or agarose; under these growth conditions cells assumed a rounded phenotype and formed colonies. A novel method of layering suspensions of cells onto cell well inserts proved technically much easier than the microbead culture method. Immunohistochemistry was utilized to demonstrate in vitro production of the following extracellular matrix components: types I, II, III, and VI collagen, 4-S-chondroitin sulfate, and keratan sulfate. Young and old age- and gender-matched cells grown in the presence of TGF-beta1 showed significant enhancement of proliferation after 4 days of exposure to TGF-beta with a lessened mitogenic response present after 10 days. Molecular studies of proteoglycan gene expression showed that at 4 days young normal cells had increased biglycan, but not decorin, message levels. Decorin expression was unchanged at Day 4 and decreased or shut off by Day 10. Results support the use of three-dimensional culture systems for in vitro evaluation of human disc cell function and expand our understanding of the in vitro behavior of these cells.

Adult↗
PubMed2000

Purification of alpha-amylase isoenzymes from Scytalidium thermophilum on a fluidized bed of alginate beads followed by concanavalin A-agarose column chromatography.

An alpha-amylase has been purified from the thermophilic fungus Scytalidium thermophilum. A ninefold purification was achieved in a single step using fluidized bed chromatography wherein alginate was used as the affinity matrix. There are at least two isoenzymes as shown by concanavalin A (Con A)-agarose column chromatography. The isoenzyme binding to Con A is stable for at least 3 h at 80 degrees C in the presence of calcium ions. The isoenzymes have similar molecular weights of around 45,000 Da as shown by SDS-PAGE analysis. The isoenzymes differ only slightly in their pH optima and temperature optima but the isoenzyme binding to Con A-agarose has slightly higher thermal stability.

Adsorption↗
PubMed1990

Storage and growth of neuroblastoma cells immobilized in calcium-alginate beads.

Mouse neuroblastoma cells (N18) were immobilized in calcium-alginate gel beads. Under standard culture conditions (37 degrees C; 5% CO2), cell growth was observed inside the beads. The number of cells increased threefold during 7 days of culture with cell division and differentiation visualized by electron microscopy. Cell properties maintained after short-term storage (2-3 days at 4 degrees C) included: (i) properties of voltage-dependent ionic channels tested by patch-clamp electrophysiological techniques; (ii) expression of cell-adhesion membrane proteins tested by immunohistochemistry (iii) morphological differentiation obtained by depletion of foetal calf serum in culture medium. The advantages of such an immobilization technique as applied to neurone cells are discussed.

Alginates↗
PubMed1992

Controlled mycelial growth for kojic acid production using Ca-alginate-immobilized fungal cells.

Conidia of Aspergillus oryzae were immobilized in Ca-alginate beads and then incubated in a nutrient medium to yield an immobilized biocatalyst producing kojic acid. The immobilized cell cultures produced kojic acid linearly during cultivation. Regardless of the size of the immobilized particles, there existed an optimal nitrogen concentration for the maximum production rate of kojic acid, at which smaller bead sizes resulted in a higher production rate. When the growth of mycelia were confined within the bead surface and segregated from each other by gel material, they produced kojic acid with maximal catalytic activity and exhibited the highest conversion yield of glucose. The extent of mycelial segregation was especially higher in cultures of smaller bead particles, and the depth of mycelial growth was 150 to 250 microns from the gel bead surface in all cultures of different nitrogen concentrations and bead sizes. Therefore, for the maximum expression of catalytic activities of immobilized mycelial cultures, it was found very critical to optimally control the mycelial distribution in gel beads by the culture conditions affecting mycelial growth.

Alginates↗
PubMed1992

Rabbit articular chondrocytes in alginate gel: characterisation of immobilized preparations and potential applications.

Primary cultivated rabbit articular chondrocytes were immobilized in calcium alginate beads. Both free and entrapped cells were allowed to grow under normal conditions. After long-term immobilization, the cells still exhibited metabolic activities, patterns of division, synthesis and secretion of extracellular matrix macromolecules such as type II collagen and proteoglycans. After 38 days, immobilized rabbit articular chondrocytes predominantly expressed type II but not type I collagen. Thus, they maintained their cartilage pheno-type. After bead lysis, harvested cells showed normal growth patterns when resuspended in culture medium. On the basis of these results, long-duration storage and large-scale production of extracellular matrix components are being investigated.

Alginates↗
PubMed1992

Alginate coating of islets of Langerhans: in vitro studies on a new method for microencapsulation for immuno-isolated transplantation.

Immuno-isolated transplantation offers the attractive prospect of being able to transplant xenogeneic islets without immunosuppression. This study introduces a completely new method of coating single islets using a homogeneous alginate membrane approximately 10 microns thick. During glucose challenge (perifusion and static incubation) encapsulated islets show the same pattern and quantity of insulin release as non-encapsulated controls. This encapsulation method markedly reduces the amount of transplanted material by reducing the size of the capsule. It is suggested that encapsulated islets may be transplanted into sites such as the renal capsule or omentum or even by intraportal injection into the liver.

Alginates↗
PubMed1993

Production of monoclonal antibodies against human chorionic gonadotropin by hybridoma cultures in calcium alginate capsules.

A new method of making microcapsules with calcium alginate gel was developed and the cultivation of the encapsulated hybridoma cells producing monoclonal antibodies against human chorionic gonadotropin was investigated. A high cell density of 2.0 x 10(8) cells/cm3 in the capsules led to a high dilution rate of 0.67 per hour and resulted in the high volumetric monoclonal antibody productivity of 652.8 mg/l/day, which was 20-30 times higher than those of traditional continuous suspension cultures. However, long-term continuous culture was not achieved with this capsule system probably because of the limitation in nutrient supply and the accumulation of waste products. Also the analysis of oxygen transfer in this system showed that oxygen supply was not enough to support such a high cell density.

Alginates↗
PubMed1994

Calcium alginate immobilized hybridomas grown using a fluidized-bed perfusion system with a protein-free medium.

Hybridoma SPO1 cells were immobilized in calcium alginate beads and were further grown in a fluidized-bed perfusion system with a protein-free medium. The presence of serum in the steps of entrapment was shown to be helpful for the preservation of cell viability. Each step during immobilization was investigated with respect to the extent of cell damage caused. The immobilization process using small beads caused a lower cell viability initially but allowed a higher rate of cell growth subsequently, compared to those in large beads. In a perfusion system for the continuous production of monoclonal antibodies (MAb), the viable cell density reached 2 x 10(7) cells per ml of beads with a viability of 40%. Compared with the cells in suspension culture, the immobilized SPO1 cells showed higher viable cell based specific rates of substrate uptake (glucose and glutamine) and of MAb production. A significant drop in the formation of lactate after the cell growth entered a steady state suggested a higher activity of the Tricarboxylic Acid Cycle in the cells when the cell density became high.

Alginates↗
PubMed1992

Application of polyuronides for removing heavy metals from vegetable oils. III. Application of alginic acid, pectic and pectinic acids for demetalization of hydrogenated sunflower oil.

Laboratory experiments have been carried out for the removal of heavy metals from hydrogenated vegetable oils using hydrated polyuronides (degree of swelling from 4 to 12.8 ml/g) such as alginic acid, pectic and pectinic acids. The effect of the type of polyuronide, degree of esterification and oil treatment on the degree of demetalization has been studied. It has been shown that with increase in the degree of esterification of the polyuronide the efficiency of demetalization decreases. The second and third treatment of the hydrogenated oil with pectinic acid resulted in a high degree of heavy metal removal. The possibility of efficient demetalization of hydrogenated oils by treatment with water solutions of pectinic acids has also been demonstrated. The degree of metal ion removal increases with decreasing concentration of pectinic acids in the water solution.

Adsorption↗
PubMed1987

Double-blind controlled study on the efficacy of sodium alginate (Gaviscon) in reducing gastroesophageal reflux assessed by 24 h continuous pH monitoring in infants and children.

We studied the effects of an alginate compound (Gaviscon) on the frequency and the duration of gastroesophageal reflux (GOR) episodes in children. Twenty infants and children with characteristic symptoms of GOR were divided at random into two groups which were given either Gaviscon (ten patients, mean age: 21 months) or a placebo (ten patients, mean age: 35 months). A continuous pH probe monitoring of the lower oesophageal third was performed in all the patients before and after 8 days of treatment. Before the trial, sensitive pH monitoring variables of acid reflux (Euler-Byrne index, percentage of total reflux time per 24 h, mean duration and percentage of reflux time during sleep, total number of reflux episodes per 24 h and number of reflux episodes per 2 h post-cibal periods) were abnormal in all the patients tested. The oesophagram revealed a GOR in 13 of the 20 patients; none of the children who underwent an endoscopy had evidence of oesophagitis. Episodes of regurgitation reported by the parents decreased during Gaviscon therapy while no clinical improvement was noticed in the placebo group. No adverse effects were observed. After 8 days of treatment with Gaviscon, results of all the pH monitoring variables were significantly (P less than 0.05) reduced between -35% and -61% of the initial values recorded. In the placebo treated group, the mean values remained little changes (-9.5 to +8.2% of initial values). These data suggest that Gaviscon may prove useful in the medical management of GOR in infants and children.

Alginates↗
PubMed1989

Metabolic characteristics of bacterial cells entrapped in beaded calcium alginate and/or pectate gels.

A mixture of heterotrophic bacteria and collection strains of Escherichia coli and Pseudomonas fluorescens were immobilized in calcium alginate or pectate gels. Comparison of respiratory activity, substrate uptake and biosynthetic capacity of immobilized cells showed that both types of carriers permit a prolonged preservation of metabolic activity but the transfer of substances through the gel is faster in the pectate. Morphological changes include some intracellular structures, partial shrinkage of the plasma membrane of immobilized cells, and transformation of a rod-like cell shape to an oval one.

Alginates↗
PubMed2006

Effect of additives on the viability of bifidobacteria loaded in alginate poly-l-lysine microparticles during the freeze-drying process.

Bifidobacteria-loaded alginate poly-l-lysine microparticles (bap microparticles) were prepared using an air atomization method and then freeze-dried. The viability of the bap microparticles was investigated as a function of the amount of the bifidobacteria cultures, and the addition of a yeast extract, cryoprotectants, antioxidants and neutralizer. The size of the bap microparticles with and without the bifidobacteria was 84.8 +/- 28.5 microm (mean +/- standard deviation) and 113.1 +/- 38.5 microm, respectively. The surface morphology was slightly ellipsoid and wrinkled regardless of the incorporating bifidobacteria. The viability gradually decreased with increasing freeze-drying time. Free-flowing powdered bap microparticles were obtained at least 12 h after freeze-drying the wetted slurry of bap microparticles. However, the particles tended to aggregate when either lactose or ascorbic acid was added. The addition of a yeast extract, cryoprotectants (glycerol and lactose), antioxidants (NaHSO3 and ascorbic acid) and neutralizer (Mg3(PO4)2) resulted in a significantly higher viability of the bifidobacteria in the bap microparticles after freeze-drying (0.34-1.84 log) compared with the culture alone.

Alginates↗