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Sample records for alginate immobilized cells

  1. Stability of alginate-immobilized algal cells

    SciTech Connect

    Dainty, A.L.; Goulding, K.H.; Robinson, P.K.; Simpkins, I; Trevan, M.D.

    1986-01-01

    Investigations were carried out using immobilized Chlorella cells to determine the diameter, compressibility, tolerance to phosphate chelation, and ability to retain algal cells during incubation of various alginate beads. These physical bead-characteristics were affected by a variety of interactive factors, including multivalent cation type (hardening agent) and cell, cation, and alginate concentration, the latter exhibiting a predominant influence. The susceptibility of alginate beads to phosphate chelation involved a complex interaction of cation type, concentration, and pH of phosphate solution. A scale of response ranging from gel swelling to gel shrinking was observed for a range of conditions. However, stable Ca alginate beads were maintained in incubation media with a pH of 5.5 and a phosphate concentration of 5 micro M. A preliminary investigation into cell leakage from the beads illustrated the importance of maintaining a stable gel structure and limiting cell growth to reduce leakage.

  2. Cellobiose hydrolysis using Pichia etchellsii cells immobilized in calcium alginate

    SciTech Connect

    Jain, D.; Ghose, T.K.

    1984-04-01

    Cellulose degradation rates can be increased by the hydrolysis of cellobiose using immobilized beta-glucosidase. Production of beta-glucosidase in four yeasts was studied and a maximum activity of 1.22 IU/mg cells was obtained in cells of Pichia etchellsii grown on 3% cellobiose. The immobilization of beta-glucosidase containing cells on various solid supports was studied and entrapment in calcium alginate gel beads was found to be the best method. After ten sequential batch uses of the preparation, 96.5% of the initial activity was retained. The pH and temperature optima for free and immobilized cells were pH 6.5 (0.05M Maleate buffer) and 50/sup 0/C however, the enzyme has a better thermal stability at 45/sup 0/C. Beads stored at 4/sup 0/C for six months retain 80% of their activity. Kinetic studies performed on free and immobilized cells show that glucose is a noncompetitive product inhibitor. The immobilized preparation was limited by pore diffusion but exhibited no film-diffusion resistance during packed bed reactor operation. Good plug flow characteristics were observed. A model for reaction with pore diffusion for a noncompetitive type of inhibited system was developed and applied to this system. The reation rate with diffusional limitations was determined by using the model and effectiveness factors were calculated for different particle sizes. The modified rate expression using the effectiveness factor represented batch and packed bed reactor operation satisfactorily. The productivity in the packed bed column fell rapidly with an increase in conversion rate indicating that the operating conditions of the column would have to balance high conversion rates with acceptable productivity. The half-life in the column was affected by temperature, increasing to over seventeen days at 40/sup 0/C and decreasing to less than two days at 50/sup 0/C.

  3. Cellobiose hydrolysis using Pichia etchellsii cells immobilized in calcium alginate

    SciTech Connect

    Jain, D.; Ghose, T.K.

    1984-01-01

    The rate of cellulose degradation, limited by inhibition by cellobiose, can be increased by hydrolysis of cellobiose to glucose using immobilized ..beta..-glucosidase. Production of ..beta..-glucosidase in four yeasts was studied and a maximum activity of 1.22 IU/mg cells was obtained in cells of Pichia etchellsii when grown on 3% cellobiose as the sole carbon source. Immobilization of ..beta..-glucosidase containing cells of Pichia etchellsii on various solid supports was conducted and immobilization by entrapment in calcium alginate gel beads was found to be the most simple and efficient method. The immobilized preparation was found to be limited by pore diffusion but exhibited no film-diffusion resistance during packed bed reactor operation. Good plug flow characteristics were observed in the packed bed column indicated by a low dispersion number of 0.1348. A model for reaction with pore diffusion for a noncompetitive type of inhibited system was developed and applied to the cellobiose hydrolysis system. The rate of reaction with diffusional limitations was determined by using the model and effectiveness factors were calculated for different particle sizes. An effectiveness factor of 0.49 was obtained for a particle diameter of 2.5 mm. The modified rate expression using the effectiveness factor represented batch and packed bed reactor operation satisfactorily. The productivity in the packed bed column was found to fall rapidly with increase in conversion rate indicating that the operating conditions of the column would have to be a compromise between high conversion rates and reasonable productivity. A half-life of over seven days was obtained at the operating temperature of 45/sup 0/C in continuous operation of the packed bed reactor. However, the half-life in the column was found to be greatly affected by temperature, increasing to over seve

  4. Evaluation of the use of Sr2+ in alginate immobilization of cells

    NASA Astrophysics Data System (ADS)

    Widerøe, Hege; Danielsen, Signe

    2001-05-01

    In recent years the method of immobilization of living cells in Ca-alginate beads has gained a wide range of applications. In all cases high chemical stability of the immobilization material and mild conditions for the cells are prerequisites. However, in long-term experiments that may last for several days Ca-alginate may dissolve due to an exchange of Ca2+ with Na+, forming fluid Na-alginate. As well as Ca-alginate, the more chemically stable Sr-alginate and Ba-alginate are materials that have been used for the immobilization of living cells. In this study, the effects of Ca2+, Sr2+ and Ba2+ on growth, viability and intracellular free calcium concentration in a human leukemic T cell line (Jurkat) were investigated. The findings in this study, and the fact that Sr-alginate has a considerably higher chemical stability than Ca-alginate, led to the conclusion that Sr-alginate is a more suitable material for use in the entrapment of living cells in long-term studies.

  5. Evaluation of the use of Sr2+ in alginate immobilization of cells.

    PubMed

    Widerøe, H; Danielsen, S

    2001-05-01

    In recent years the method of immobilization of living cells in Ca-alginate beads has gained a wide range of applications. In all cases high chemical stability of the immobilization material and mild conditions for the cells are prerequisites. However, in long-term experiments that may last for several days Ca-alginate may dissolve due to an exchange of Ca2+ with Na+, forming fluid Na-alginate. As well as Ca-alginate, the more chemically stable Sr-alginate and Ba-alginate are materials that have been used for the immobilization of living cells. In this study, the effects of Ca2+, Sr2+ and Ba2+ on growth, viability and intracellular free calcium concentration in a human leukemic T cell line (Jurkat) were investigated. The findings in this study, and the fact that Sr-alginate has a considerably higher chemical stability than Ca-alginate, led to the conclusion that Sr-alginate is a more suitable material for use in the entrapment of living cells in long-term studies.

  6. Polygalacturonase production by calcium alginate immobilized Enterobacter aerogenes NBO2 cells.

    PubMed

    Darah, I; Nisha, M; Lim, Sheh-Hong

    2015-03-01

    Bacterial cells of Enterobacter aerogenes NBO2 were entrapped in calcium alginate beads in order to enhance polygalacturonase production compared to free cells. The optimized condition of 5 % (w/v) sodium alginate concentration, agitation speed of 250 rpm, and 15 beads of calcium alginate with inoculum size of 4 % (v/v; 5.4 × 10(7) cells/ml) produced 23.48 U/mL of polygalacturonase compared to free cells of 18.54 U/ml. There was about 26.6 % increment in polygalaturonase production. However, in this study, there was 296.6 % of increment in polygalacturonase production after improvement parameters compared to before improvement parameters of calcium alginate bead immobilization cells (5.92 U/ml). This research has indicated that optimized physical parameters of calcium alginate bead immobilization cells have significantly enhanced the production of polygalacturonase.

  7. Immobilization of Bacillus amyloliquefaciens MBL27 cells for enhanced antimicrobial protein production using calcium alginate beads.

    PubMed

    Kumaravel, Vijayalakshmi; Gopal, Suseela Rajakumar

    2010-12-01

    Cell immobilization is one of the common techniques for increasing the overall cell concentration and productivity. Bacillus amyloliquefaciens MBL27 cells were immobilized in calcium alginate beads and it is a promising method for repeated AMP (antimicrobial protein) production. The present study aimed at determining the optimal conditions for immobilization of B. amyloliquefaciens MBL27 cells in calcium alginate beads and the operational stability for enhanced production of the AMP. AMP production with free and immobilized cells was also done. In batch fermentation, maximum AMP production (7300 AU (arbitrary units)/ml against Staphylococcus aureus) was obtained with immobilized cells in shake flasks under optimized parameters such as 3% (w/v) sodium alginate, 136 mM CaCl2 with 350 alginate beads/flask of 2.7-3.0 mm diameter. In repeated cultivation, the highest activity was obtained after the second cycle of use and approx. 94% production was noted up to the fifth cycle. The immobilized cells of B. amyloliquefaciens MBL27 in alginate beads are more efficient for the production of AMP and had good stability. The potential application of AMP as a wound healant and the need for development of economical methods for improved production make whole cell immobilization an excellent alternative method for enhanced AMP production.

  8. Growth and by-product profiles of Kluyveromyces marxianus cells immobilized in foamed alginate.

    PubMed

    Wilkowska, Agnieszka; Kregiel, Dorota; Guneser, Onur; Karagul Yuceer, Yonca

    2015-01-01

    The aim of this research was to study how the yeast cell immobilization technique influences the growth and fermentation profiles of Kluyveromyces marxianus cultivated on apple/chokeberry and apple/cranberry pomaces. Encapsulation of the cells was performed by droplet formation from a foamed alginate solution. The growth and metabolic profiles were evaluated for both free and immobilized cells. Culture media with fruit waste produced good growth of free as well as immobilized yeast cells. The fermentation profiles of K. marxianus were different with each waste material. The most varied aroma profiles were noted for immobilized yeast cultivated on apple/chokeberry pomace. PMID:25277269

  9. Effect of immobilized cells in calcium alginate beads in alcoholic fermentation

    PubMed Central

    2013-01-01

    Saccharomyces cerevisiae cells were immobilized in calcium alginate and chitosan-covered calcium alginate beads and studied in the fermentation of glucose and sucrose for ethanol production. The batch fermentations were carried out in an orbital shaker and assessed by monitoring the concentration of substrate and product with HPLC. Cell immobilization in calcium alginate beads and chitosan-covered calcium alginate beads allowed reuse of the beads in eight sequential fermentation cycles of 10 h each. The final concentration of ethanol using free cells was 40 g L-1 and the yields using glucose and sucrose as carbon sources were 78% and 74.3%, respectively. For immobilized cells in calcium alginate beads, the final ethanol concentration from glucose was 32.9 ± 1.7 g L-1 with a 64.5 ± 3.4% yield, while the final ethanol concentration from sucrose was 33.5 ± 4.6 g L-1 with a 64.5 ± 8.6% yield. For immobilized cells in chitosan-covered calcium alginate beads, the ethanol concentration from glucose was 30.7 ± 1.4 g L-1 with a 61.1 ± 2.8% yield, while the final ethanol concentration from sucrose was 31.8 ± 6.9 g L-1 with a 62.1 ± 12.8% yield. The immobilized cells allowed eight 10 h sequential reuse cycles to be carried out with stable final ethanol concentrations. In addition, there was no need to use antibiotics and no contamination was observed. After the eighth cycle, there was a significant rupture of the beads making them inappropriate for reuse. PMID:23721664

  10. Effect of immobilized cells in calcium alginate beads in alcoholic fermentation.

    PubMed

    Duarte, Juliana C; Rodrigues, J Augusto R; Moran, Paulo J S; Valença, Gustavo P; Nunhez, José R

    2013-05-30

    Saccharomyces cerevisiae cells were immobilized in calcium alginate and chitosan-covered calcium alginate beads and studied in the fermentation of glucose and sucrose for ethanol production. The batch fermentations were carried out in an orbital shaker and assessed by monitoring the concentration of substrate and product with HPLC. Cell immobilization in calcium alginate beads and chitosan-covered calcium alginate beads allowed reuse of the beads in eight sequential fermentation cycles of 10 h each. The final concentration of ethanol using free cells was 40 g L-1 and the yields using glucose and sucrose as carbon sources were 78% and 74.3%, respectively. For immobilized cells in calcium alginate beads, the final ethanol concentration from glucose was 32.9 ± 1.7 g L-1 with a 64.5 ± 3.4% yield, while the final ethanol concentration from sucrose was 33.5 ± 4.6 g L-1 with a 64.5 ± 8.6% yield. For immobilized cells in chitosan-covered calcium alginate beads, the ethanol concentration from glucose was 30.7 ± 1.4 g L-1 with a 61.1 ± 2.8% yield, while the final ethanol concentration from sucrose was 31.8 ± 6.9 g L-1 with a 62.1 ± 12.8% yield. The immobilized cells allowed eight 10 h sequential reuse cycles to be carried out with stable final ethanol concentrations. In addition, there was no need to use antibiotics and no contamination was observed. After the eighth cycle, there was a significant rupture of the beads making them inappropriate for reuse.

  11. Characterization of an encapsulation device for the production of monodisperse alginate beads for cell immobilization.

    PubMed

    Serp, D; Cantana, E; Heinzen, C; Von Stockar, U; Marison, I W

    2000-10-01

    An encapsulation device, designed on the basis of the laminar jet break-up technique, is characterized for cell immobilization with different types of alginate. The principle of operation of the completely sterilizable encapsulator, together with techniques for the continuous production of beads from 250 microm to 1 mm in diameter, with a size distribution below 5%, at a flow rate of 1-15 mL/min, is described. A modification of the device, to incorporate an electrostatic potential between the alginate droplets and an internal electrode, results in enhanced monodispersity with no adverse effects on cell viability. The maximum cell loading capacity of the beads strongly depends on the nozzle diameter as well as the cells used. For the yeast Phaffia rhodozyma, it is possible to generate 700 microm alginate beads with an initial cell concentration of 1 x 10(8) cells/mL of alginate whereas only 1 x 10(6) cells/ml could be entrapped within 400 microm beads. The alginate beads have been characterized with respect to mechanical resistance and size distribution immediately after production and as a function of storage conditions. The beads remain stable in the presence of acetic acid, hydrochloric acid, water, basic water, and sodium ions. The latter stability applies when the ratio of sodium: calcium ions is less than 1/5. Complexing agents such as sodium citrate result in the rapid solubilization of the beads due to calcium removal. The presence of cells does not affect the mechanical resistance of the beads. Finally, the mechanical resistance of alginate beads can be doubled by treatment with 5-10 kDa chitosan, resulting in reduced leaching of cells.

  12. The immobilization of microbial cells, subcellular organelles, and enzymes in calcium alginate gels. Reprinted from Biotechnology and Bioengineering, Vol. XIX, No. 3, Pages 387-397 (1977).

    PubMed

    Kierstan, M; Bucke, C

    2000-03-20

    Saccharomyces cerevisiae cells, Kluyveromyces marxianus cells, inulase, glucose oxidase, chloroplasts, and mitochondria were immobilized in calcium alginate gels. Ethanol production from glucose solutions by an immobilized preparation of S. cerevisiae was demonstrated over a total of twenty-three days, and the half-life of such a preparation was shown to be about ten days. Immobilized K. marxianus, inulase, and glucose oxidase preparations were used to demonstrate the porosity and retraining properties of calcium alginate gels. Calcium alginate-immobilized chloroplasts were shown to perform the Hill reaction. Some experiments with immobilized mitochondria are reported.

  13. Alginate immobilization of Escherichia coli MTCC 1652 whole cells for bioconversion of glycyrrhizinic acid and into 18-beta glycyrrhetinic acid.

    PubMed

    Ahmad, M; Panda, B P

    2013-12-15

    Microbial biotransformation of Glycyrrhizinic acid (GL) into 18-beta Glycyrrhetinic Acid (GA) was achieved using Escherichia coli MTCC 1652 whole cell. The E. coli whole cell was immobilized by entrapment method within calcium alginate beads using cell suspension of equal volume with sodium alginate 8%. The pH of solution, reaction volume and % of GL were optimized during the immobilization procedure and optimum pH 6.5, reaction volume of 4 mL and at 3% GL concentration for 12 h of incubation time showed highest concentration of GA (72.649 microg mL(-1)) with 76% bioconversion of GL to GA. Under optimized condition the immobilized cell produces 58.663 microg per mL of GA in licorice root extract containing 95.118 microg of GL per mL of the extract with 61% conversion at 12 h.

  14. Hydrogen photoproduction by nutrient-deprived Chlamydomonas reinhardtii cells immobilized within thin alginate films under aerobic and anaerobic conditions.

    PubMed

    Kosourov, Sergey N; Seibert, Michael

    2009-01-01

    A new technique for immobilizing H2-photoproducing green algae within a thin (<400 microm) alginate film has been developed. Alginate films with entrapped sulfur/phosphorus-deprived Chlamydomonas reinhardtii, strain cc124, cells demonstrate (a) higher cell density (up to 2,000 microg Chl mL(-1) of matrix), (b) kinetics of H2 photoproduction similar to sulfur-deprived suspension cultures, (c) higher specific rates (up to 12.5 micromol mg(-1) Chl h(-1)) of H2 evolution, (d) light conversion efficiencies to H2 of over 1% and (e) unexpectedly high resistance of the H2-photoproducing system to inactivation by atmospheric O2. The algal cells, entrapped in alginate and then placed in vials containing 21% O2 in the headspace, evolved up to 67% of the H2 gas produced under anaerobic conditions. The results indicate that the lower susceptibility of the immobilized algal H2-producing system to inactivation by O2 depends on two factors: (a) the presence of acetate in the medium, which supports higher rates of respiration and (b) the capability of the alginate polymer itself to effectively separate the entrapped cells from O2 in the liquid and headspace and restrict O2 diffusion into the matrix. The strategy presented for immobilizing algal cells within thin polymeric matrices shows the potential for scale-up and possible future applications. PMID:18823051

  15. Hydrogen Photoproduction by Nutrient-Deprived Chalamydomonas reinhardtii Cells Immobilized Within Thin Alginate Films Under Aerobic and Anaerobic Conditions

    SciTech Connect

    Kosourov, S. N.; Seibert, M.

    2009-01-01

    A new technique for immobilizing H{sub 2}-photoproducing green algae within a thin (<400 {micro}m) alginate film has been developed. Alginate films with entrapped sulfur/phosphorus-deprived Chlamydomonas reinhardtii, strain cc124, cells demonstrate (a) higher cell density (up to 2,000 {micro}g Chl mL{sup -1} of matrix), (b) kinetics of H{sub 2} photoproduction similar to sulfur-deprived suspension cultures, (c) higher specific rates (up to 12.5 {micro}mol mg{sup -1} Chl h{sup -1}) of H{sub 2} evolution, (d) light conversion efficiencies to H{sub 2} of over 1% and (e) unexpectedly high resistance of the H{sub 2}-photoproducing system to inactivation by atmospheric O{sub 2}. The algal cells, entrapped in alginate and then placed in vials containing 21% O{sub 2} in the headspace, evolved up to 67% of the H{sub 2} gas produced under anaerobic conditions. The results indicate that the lower susceptibility of the immobilized algal H{sub 2}-producing system to inactivation by O{sub 2} depends on two factors: (a) the presence of acetate in the medium, which supports higher rates of respiration and (b) the capability of the alginate polymer itself to effectively separate the entrapped cells from O{sub 2} in the liquid and headspace and restrict O{sub 2} diffusion into the matrix. The strategy presented for immobilizing algal cells within thin polymeric matrices shows the potential for scale-up and possible future applications.

  16. In Situ Gelation for Cell Immobilization and Culture in Alginate Foam Scaffolds

    PubMed Central

    Markussen, Christine; Dornish, Michael; Heier-Baardson, Helene; Melvik, Jan Egil; Alsberg, Eben; Christensen, Bjørn E.

    2014-01-01

    Essential cellular functions are often lost under culture in traditional two-dimensional (2D) systems. Therefore, biologically more realistic three-dimensional (3D) cell culture systems are needed that provide mechanical and biochemical cues which may otherwise be unavailable in 2D. For the present study, an alginate-based hydrogel system was used in which cells in an alginate solution were seeded onto dried alginate foams. A uniform distribution of NIH:3T3 and NHIK 3025 cells entrapped within the foam was achieved by in situ gelation induced by calcium ions integrated in the foam. The seeding efficiency of the cells was about 100% for cells added in a seeding solution containing 0.1–1.0% alginate compared with 18% when seeded without alginate. The NHIK 3025 cells were allowed to proliferate and form multi-cellular structures inside the transparent gel that were later vital stained and evaluated by confocal microcopy. Gels were de-gelled at different time points to isolate the multi-cellular structures and to determine the spheroid growth rate. It was also demonstrated that the mechanical properties of the gel could largely be varied through selection of type and concentration of the applied alginate and by immersing the already gelled disks in solutions providing additional gel-forming ions. Cells can efficiently be incorporated into the gel, and single cells and multi-cellular structures that may be formed inside can be retrieved without influencing cell viability or contaminating the sample with enzymes. The data show that the current system may overcome some limitations of current 3D scaffolds such as cell retrieval and in situ cell staining and imaging. PMID:24125496

  17. In situ gelation for cell immobilization and culture in alginate foam scaffolds.

    PubMed

    Andersen, Therese; Markussen, Christine; Dornish, Michael; Heier-Baardson, Helene; Melvik, Jan Egil; Alsberg, Eben; Christensen, Bjørn E

    2014-02-01

    Essential cellular functions are often lost under culture in traditional two-dimensional (2D) systems. Therefore, biologically more realistic three-dimensional (3D) cell culture systems are needed that provide mechanical and biochemical cues which may otherwise be unavailable in 2D. For the present study, an alginate-based hydrogel system was used in which cells in an alginate solution were seeded onto dried alginate foams. A uniform distribution of NIH:3T3 and NHIK 3025 cells entrapped within the foam was achieved by in situ gelation induced by calcium ions integrated in the foam. The seeding efficiency of the cells was about 100% for cells added in a seeding solution containing 0.1-1.0% alginate compared with 18% when seeded without alginate. The NHIK 3025 cells were allowed to proliferate and form multi-cellular structures inside the transparent gel that were later vital stained and evaluated by confocal microscopy. Gels were de-gelled at different time points to isolate the multi-cellular structures and to determine the spheroid growth rate. It was also demonstrated that the mechanical properties of the gel could largely be varied through selection of type and concentration of the applied alginate and by immersing the already gelled disks in solutions providing additional gel-forming ions. Cells can efficiently be incorporated into the gel, and single cells and multi-cellular structures that may be formed inside can be retrieved without influencing cell viability or contaminating the sample with enzymes. The data show that the current system may overcome some limitations of current 3D scaffolds such as cell retrieval and in situ cell staining and imaging.

  18. Modified alginate and chitosan for lactic acid bacteria immobilization.

    PubMed

    Le-Tien, Canh; Millette, Mathieu; Mateescu, Mircea-Alexandru; Lacroix, Monique

    2004-06-01

    Beads with enhanced-stability acid media, which were based on alginate and chitosan functionalized by succinylation (increasing the anionic charges able to retain protons) or by acylation (improving matrix hydrophobicity), were developed for immobilization of bacterial cells. Beads (3 mm diameter) formed by ionotropic gelation with CaCl(2) presented good mechanical characteristics. After 30 min incubation of viable free Lactobacillus rhamnosus cells in simulated gastric fluid (pH 1.5), we noticed that the level of viable bacteria was undetectable. Bacterial immobilization in native-alginate-based beads generated a viable-cell count of 22-26%, whereas, when entrapped in succinylated alginate and chitosan beads, the percentage of viable cells was of 60 and 66%, respectively. Best viability (87%) was found for bacteria immobilized in N -palmitoylaminoethyl alginate, which affords a high protective effect, probably due to long alkyl pendants that improve the beads' hydrophobicity, limiting hydration in the acidic environment.

  19. Simultaneous Alcoholic and Malolactic Fermentations by Saccharomyces cerevisiae and Oenococcus oeni Cells Co-immobilized in Alginate Beads

    PubMed Central

    Bleve, Gianluca; Tufariello, Maria; Vetrano, Cosimo; Mita, Giovanni; Grieco, Francesco

    2016-01-01

    Malolactic fermentation (MLF) usually takes place after the end of alcoholic fermentation (AF). However, the inoculation of lactic acid bacteria together with yeast starter cultures is a promising system to enhance the quality and safety of wine. In recent years, the use of immobilized cell systems has been investigated, with interesting results, for the production of different fermented foods and beverages. In this study we have carried out the simultaneous immobilization of Saccharomyces cerevisiae and Oenococcus oeni in alginate beads and used them in microvinifications tests to produce Negroamaro wine. The process was monitored by chemical and sensorial analyses and dominance of starters and cell leaking from beads were also checked. Co-immobilization of S. cerevisiae and O. oeni allowed to perform an efficient fermentation process, producing low volatile acidity levels and ethanol and glycerol concentrations comparable with those obtained by cell sequential inoculum and co-inoculum of yeast and bacteria cells in free form. More importantly, co-immobilization strategy produced a significant decrease of the time requested to complete AF and MLF. The immobilized cells could be efficiently reused for the wine fermentation at least three times without any apparent loss of cell metabolic activities. This integrated biocatalytic system is able to perform simultaneously AF and MLF, producing wines similar in organoleptic traits in comparison with wines fermented following traditional sequential AF and MLF with free cell starters. The immobilized-cell system, that we here describe for the first time in our knowledge, offers many advantages over conventional free cell fermentations, including: (i) elimination of non-productive cell growth phases; (ii) feasibility of continuous processing; (iii) re-use of the biocatalyst. PMID:27379072

  20. Simultaneous Alcoholic and Malolactic Fermentations by Saccharomyces cerevisiae and Oenococcus oeni Cells Co-immobilized in Alginate Beads.

    PubMed

    Bleve, Gianluca; Tufariello, Maria; Vetrano, Cosimo; Mita, Giovanni; Grieco, Francesco

    2016-01-01

    Malolactic fermentation (MLF) usually takes place after the end of alcoholic fermentation (AF). However, the inoculation of lactic acid bacteria together with yeast starter cultures is a promising system to enhance the quality and safety of wine. In recent years, the use of immobilized cell systems has been investigated, with interesting results, for the production of different fermented foods and beverages. In this study we have carried out the simultaneous immobilization of Saccharomyces cerevisiae and Oenococcus oeni in alginate beads and used them in microvinifications tests to produce Negroamaro wine. The process was monitored by chemical and sensorial analyses and dominance of starters and cell leaking from beads were also checked. Co-immobilization of S. cerevisiae and O. oeni allowed to perform an efficient fermentation process, producing low volatile acidity levels and ethanol and glycerol concentrations comparable with those obtained by cell sequential inoculum and co-inoculum of yeast and bacteria cells in free form. More importantly, co-immobilization strategy produced a significant decrease of the time requested to complete AF and MLF. The immobilized cells could be efficiently reused for the wine fermentation at least three times without any apparent loss of cell metabolic activities. This integrated biocatalytic system is able to perform simultaneously AF and MLF, producing wines similar in organoleptic traits in comparison with wines fermented following traditional sequential AF and MLF with free cell starters. The immobilized-cell system, that we here describe for the first time in our knowledge, offers many advantages over conventional free cell fermentations, including: (i) elimination of non-productive cell growth phases; (ii) feasibility of continuous processing; (iii) re-use of the biocatalyst. PMID:27379072

  1. Effect of concentration and substrate flow rate on isomaltulose production from sucrose by Erwinia sp. cells immobilized in calcium-alginate using packed bed reactor.

    PubMed

    Kawaguti, Haroldo Yukio; Harumi Sato, Hélia

    2010-09-01

    Isomaltulose was obtained from sucrose solution by immobilized cells of Erwinia sp. D12 using a batch and a continuous process. Parameters for sucrose conversion into isomaltulose were evaluated using both experimental design and response surface methodology. Erwinia sp. D12 cells were immobilized in different alginates, and the influence of substrate flow rate and concentration parameters to produce isomaltulose from sucrose were observed. Response surface methodology demonstrated that packed bed columns containing cells immobilized in low-viscosity sodium alginate (250 cP) presented a mean isomaltulose conversion rate of 47%. In a continuous process, both sucrose substrate concentration and substrate flow rate parameters had a significant effect (p < 0.05) and influenced the conversion of sucrose into isomaltulose. Higher conversion rates of sucrose into isomaltulose, from 53-75% were obtained using 75 g of immobilized cells at a substrate flow rate of 0.6 mL/min.

  2. Preparation and characterization of. beta. -D-glucosidase immobilized in calcium alginate

    SciTech Connect

    Woodward, J.; Krasniak, S.R.; Smith, R.D.; Spielberg, F.; Zachry, G.S.

    1982-01-01

    The immobilization of enzymes, organelles, and microbial cells within calcium alginate gel spheres has been reported. Few reports exist, however, on the use of these spheres for enzyme immobilization. The study reported here describes the immobilization of ..beta..-D-glucosidase within calcium alginate gel spheres and addresses the problems of loss of enzyme activity in the spheres via inhibition and leakage.

  3. Enhanced production of alkaline thermostable keratinolytic protease from calcium alginate immobilized cells of thermoalkalophilic Bacillus halodurans JB 99 exhibiting dehairing activity.

    PubMed

    Shrinivas, Dengeti; Kumar, Raghwendra; Naik, G R

    2012-01-01

    The thermoalkalophilic Bacillus halodurans JB 99 cells known for production of novel thermostable alkaline keratinolytic protease were immobilized in calcium alginate matrix. Batch and repeated batch cultivation using calcium alginate immobilized cells were studied for alkaline protease production in submerged fermentation. Immobilized cells with 2.5% alginate and 350 beads/flask of initial cell loading showed enhanced production of alkaline protease by 23.2% (5,275 ± 39.4 U/ml) as compared to free cells (4,280 ± 35.4 U/ml) after 24 h. In the semicontinuous mode of cultivation, immobilized cells under optimized conditions produced an appreciable level of alkaline protease in up to nine cycles and reached a maximal value of 5,975 U/ml after the seventh cycle. The enzyme produced from immobilized cells efficiently degraded chicken feathers in the presence of a reducing agent which can help the poultry industry in the management of keratin-rich waste and obtaining value-added products.

  4. 3D Cell Culture in Alginate Hydrogels.

    PubMed

    Andersen, Therese; Auk-Emblem, Pia; Dornish, Michael

    2015-03-24

    This review compiles information regarding the use of alginate, and in particular alginate hydrogels, in culturing cells in 3D. Knowledge of alginate chemical structure and functionality are shown to be important parameters in design of alginate-based matrices for cell culture. Gel elasticity as well as hydrogel stability can be impacted by the type of alginate used, its concentration, the choice of gelation technique (ionic or covalent), and divalent cation chosen as the gel inducing ion. The use of peptide-coupled alginate can control cell-matrix interactions. Gelation of alginate with concomitant immobilization of cells can take various forms. Droplets or beads have been utilized since the 1980s for immobilizing cells. Newer matrices such as macroporous scaffolds are now entering the 3D cell culture product market. Finally, delayed gelling, injectable, alginate systems show utility in the translation of in vitro cell culture to in vivo tissue engineering applications. Alginate has a history and a future in 3D cell culture. Historically, cells were encapsulated in alginate droplets cross-linked with calcium for the development of artificial organs. Now, several commercial products based on alginate are being used as 3D cell culture systems that also demonstrate the possibility of replacing or regenerating tissue.

  5. 3D Cell Culture in Alginate Hydrogels

    PubMed Central

    Andersen, Therese; Auk-Emblem, Pia; Dornish, Michael

    2015-01-01

    This review compiles information regarding the use of alginate, and in particular alginate hydrogels, in culturing cells in 3D. Knowledge of alginate chemical structure and functionality are shown to be important parameters in design of alginate-based matrices for cell culture. Gel elasticity as well as hydrogel stability can be impacted by the type of alginate used, its concentration, the choice of gelation technique (ionic or covalent), and divalent cation chosen as the gel inducing ion. The use of peptide-coupled alginate can control cell–matrix interactions. Gelation of alginate with concomitant immobilization of cells can take various forms. Droplets or beads have been utilized since the 1980s for immobilizing cells. Newer matrices such as macroporous scaffolds are now entering the 3D cell culture product market. Finally, delayed gelling, injectable, alginate systems show utility in the translation of in vitro cell culture to in vivo tissue engineering applications. Alginate has a history and a future in 3D cell culture. Historically, cells were encapsulated in alginate droplets cross-linked with calcium for the development of artificial organs. Now, several commercial products based on alginate are being used as 3D cell culture systems that also demonstrate the possibility of replacing or regenerating tissue. PMID:27600217

  6. 3D Cell Culture in Alginate Hydrogels

    PubMed Central

    Andersen, Therese; Auk-Emblem, Pia; Dornish, Michael

    2015-01-01

    This review compiles information regarding the use of alginate, and in particular alginate hydrogels, in culturing cells in 3D. Knowledge of alginate chemical structure and functionality are shown to be important parameters in design of alginate-based matrices for cell culture. Gel elasticity as well as hydrogel stability can be impacted by the type of alginate used, its concentration, the choice of gelation technique (ionic or covalent), and divalent cation chosen as the gel inducing ion. The use of peptide-coupled alginate can control cell–matrix interactions. Gelation of alginate with concomitant immobilization of cells can take various forms. Droplets or beads have been utilized since the 1980s for immobilizing cells. Newer matrices such as macroporous scaffolds are now entering the 3D cell culture product market. Finally, delayed gelling, injectable, alginate systems show utility in the translation of in vitro cell culture to in vivo tissue engineering applications. Alginate has a history and a future in 3D cell culture. Historically, cells were encapsulated in alginate droplets cross-linked with calcium for the development of artificial organs. Now, several commercial products based on alginate are being used as 3D cell culture systems that also demonstrate the possibility of replacing or regenerating tissue.

  7. Immobilization of electroporated cells for fabrication of cellular biosensors: physiological effects of the shape of calcium alginate matrices and foetal calf serum.

    PubMed

    Katsanakis, Nikos; Katsivelis, Andreas; Kintzios, Spiridon

    2009-01-01

    In order to investigate the physiological effect of transfected cell immobilization in calcium alginate gels, we immobilized electroporated Vero cells in gels shaped either as spherical beads or as thin membrane layers. In addition, we investigated whether serum addition had a positive effect on cell proliferation and viability in either gel configuration. The gels were stored for four weeks in a medium supplemented or not with 20% (v/v) foetal calf serum. Throughout a culture period of four weeks, cell proliferation and cell viability were assayed by optical microscopy after provision of Trypan Blue. Non-elaborate culture conditions (room temperature, non-CO(2) enriched culture atmosphere) were applied throughout the experimental period in order to evaluate cell viability under less than optimal storage conditions. Immobilization of electroporated cells was associated with an initially reduced cell viability, which was gradually increased. Immobilization was associated with maintenance of cell growth for the duration of the experimental period, whereas electroporated cells essentially died after a week in suspension culture. Considerable proliferation of immobilized cells was observed in spherical alginate beads. In both gel configurations, addition of serum was associated with increased cell proliferation. The results of the present study could contribute to an improvement of the storability of biosensors based on electroporated, genetically or membrane-engineered cells.

  8. Differential effect of the shape of calcium alginate matrices on the physiology of immobilized neuroblastoma N2a and Vero cells: a comparative study.

    PubMed

    Kintzios, S; Yiakoumetis, I; Moschopoulou, G; Mangana, O; Nomikou, K; Simonian, A

    2007-11-30

    In order to investigate the effect of cell immobilization in calcium alginate gels on cell physiology, we immobilized Vero or N2a neuroblastoma cells in gels shaped either as spherical beads or as thin membrane layers. Throughout a culture period of 4 weeks cell viability, RNA and cytoplasmic calcium concentration and glutathione accumulation were assayed by fluorescence microscopy after provision of an appropriate dye. Non-elaborate culture conditions were applied throughout the experimental period in order to evaluate cell viability under less than optimal storage conditions. Vero cell proliferation was observed only in spherical beads, while N2a cell proliferation was observed in both configurations until the third week of culture. Increased [Ca2+]cyt could be associated with cell proliferation only when cells were immobilized in spherical beads, while a considerable decrease in the biosynthesis of reduced glutathione and RNA was observed in cells immobilized in thin membrane layers. The observed effects of the shape of the immobilization matrix may be due to differences in external mass transfer resistance. Therefore, depending on cell type, cell proliferation could have been promoted by either increased (Vero) or decreased (N2a) nutrient and oxygen flow to immobilized cells. The results of the present study could contribute to an improvement of immobilized cell sensor storability.

  9. Immobilization of Erwinia sp. D12 Cells in Alginate-Gelatin Matrix and Conversion of Sucrose into Isomaltulose Using Response Surface Methodology

    PubMed Central

    Kawaguti, Haroldo Yukio; Carvalho, Priscila Hoffmann; Figueira, Joelise Alencar; Sato, Hélia Harumi

    2011-01-01

    Isomaltulose is a noncariogenic reducing disaccharide and also a structural isomer of sucrose and is used by the food industry as a sucrose replacement. It is obtained through enzymatic conversion of microbial sucrose isomerase. An Erwinia sp. D12 strain is capable of converting sucrose into isomaltulose. The experimental design technique was used to study the influence of immobilization parameters on converting sucrose into isomaltulose in a batch process using shaken Erlenmeyer flasks. We assessed the effect of gelatin and transglutaminase addition on increasing the reticulation of granules of Erwinia sp. D12 cells immobilized in alginate. Independent parameters, sodium alginate concentration, cell mass concentration, CaCl2 concentration, gelatin concentration, and transglutaminase concentration had all a significant effect (P < 0.05) on isomaltulose production. Erwinia sp. D12 cells immobilized in 3.0% (w/v) sodium alginate, 47.0% (w/v) cell mass, 0.3 molL−1 CaCl2, 1.7% (w/v) gelatin and 0.15% (w/v) transglutaminase presented sucrose conversion into isomaltulose, of around 50–60% in seven consecutive batches. PMID:21785708

  10. An anomalous behavior of trypsin immobilized in alginate network.

    PubMed

    Ganachaud, Chrystelle; Bernin, Diana; Isaksson, Dan; Holmberg, Krister

    2013-05-01

    Alginate is a biopolymer used in drug formulations and for surgical purposes. In the presence of divalent cations, it forms solid gels, and such gels are of interest for immobilization of cells and enzymes. In this work, we entrapped trypsin in an alginate gel together with a known substrate, N α-benzoyl-L-arginine-4-nitroanilide hydrochloride (L-BAPNA), and in the presence or absence of D-BAPNA, which is known to be a competitive inhibitor. Interactions between alginate and the substrate as well as the enzyme were characterized with transmission electron microscopy, rheology, and nuclear magnetic resonance spectroscopy. The biocatalysis was monitored by spectrophotometry at temperatures ranging from 10 to 42 °C. It was found that at 37 and 42 °C a strong acceleration of the reaction was obtained, whereas at 10 °C and at room temperature, the presence of D-BAPNA leads to a retardation of the reaction rate. The same effect was found when the reaction was performed in a non-cross-linked alginate solution. In alginate-free buffer solution, as well as in a solution of carboxymethylcellulose, a biopolymer that resembles alginate, the normal behavior was obtained; however, with D-BAPNA acting as an inhibitor at all temperatures. A more detailed investigation of the reaction kinetics showed that at higher temperature and in the presence of alginate, the curve of initial reaction rate versus L-BAPNA concentration had a sigmoidal shape, indicating an allosteric behavior. We believe that the anomalous behavior of trypsin in the presence of alginate is due to conformational changes caused by interactions between the positively charged trypsin and the strongly negatively charged alginate.

  11. Removal of uranium and other metals from wastewaters using calcium alginate and bacteria immobilized in calcium alginate

    SciTech Connect

    Gardner, K.R.; Montgomery, J.R.; Adams, D.J.

    1995-12-31

    Calcium alginate beads, containing immobilized bacteria, removed turanium and other metals from mine wastewaters. Batch tests using 2% calcium alginate beads with and without immobilized bacteria showed both were effective at removing uranium. Beads containing live and dead cells removed essentially the same amount of uranium and removed significantly more uranium than beads without bacteria. The bacteria-alginate system was used to treat waters containing 24.5 mg/L uranium and produced effluents containing 1.0 mg/L uranium in 1 hr and <0.1 mg/L uranium in 24 hr. Calcium alginate beads, with and without immobilized bacteria, also removed arsenic, selenium, and other metals from wastewaters. Bacteria investigated for immobilization in the alginate beads include Bacillus and Pseudomonas sp. A spray technique using an air nozzle has been tested to disperse the alginate into mine wastewater eliminating the bead-making process. Ninety-nine percent of the uranium was removed in less than 15 min contact.

  12. Enhanced stability of Bacillus licheniformis L-arabinose isomerase by immobilization with alginate.

    PubMed

    Zhang, Ye-Wang; Prabhu, Ponnandy; Lee, Jung-Kul; Kim, In-Won

    2010-01-01

    Recombinant Escherichia coli whole cells harboring Bacillus licheniformis L-arabinose isomerase (BLAI) were harvested to prepare alginate-immobilized biocatalysts. The operational conditions for immobilization were optimized according to relative activity and the cell leakage of the immobilized cell. The optimal conditions are as follows: alginate concentration, Ca(2+) concentration, cell mass loading, and curing time were 2% (w/v), 0.1 M, 50 g l(-1), and 4 hours, respectively. After immobilization, cross-linking with 0.1% glutaraldehyde significantly reduced cell leakage. The immobilized whole cells harboring BLAI were very stable with 89% residual activity remaining after 33 days of incubation at 50 degrees C and were much more stable than the free enzyme and cells. The results showed that immobilizing whole cells harboring BLAI is suitable for use as a biocatalyst in the production of L-ribulose, largely due to its high stability and low cost.

  13. Calcium alginate bead immobilization of cells containing tyrosine ammonia lyase activity for use in the production of p-hydroxycinnamic acid.

    PubMed

    Trotman, Robert J; Camp, Carl E; Ben-Bassat, Arie; DiCosimo, Robert; Huang, Lixuan; Crum, Grace A; Sariaslani, F Sima; Haynie, Sharon L

    2007-01-01

    An Escherichia coli catalyst with tyrosine ammonia lyase activity (TAL) has been stabilized for repeated use in batch conversions of high tyrosine solids to p-hydroxycinnamic acid (pHCA). The TAL biocatalyst was stabilized by controlling the reaction pH to 9.8 +/- 0.1 and immobilizing the cells within a calcium alginate matrix that was cross-linked with glutaraldehyde and polyethyleneimine (GA/PEI). We found a GA range where the bead-encapsulated TAL was not inactivated, and the resulting cross-linking provided the beads with the mechanical stability necessary for repeated use in consecutive batch reactions with catalyst recycle. The GA/PEI calcium alginate TAL catalyst was used in 41 1-L batch reactions where 50 g L(-1) tyrosine was converted to 39 +/- 4 g L(-1) pHCA in each batch. The practical usefulness and ease of this process was demonstrated by scaling up the TAL bead immobilization and using the immobilized TAL catalyst in four 125-L bioconversion reactions to produce over 12 kg of purified pHCA.

  14. Improved production of isomaltulose by a newly isolated mutant of Serratia sp. cells immobilized in calcium alginate.

    PubMed

    Kim, Yonghwan; Koo, Bong-Seong; Lee, Hyeon-Cheol; Yoon, Youngdae

    2015-03-01

    Isomaltulose, also known as palatinose, is produced by sucrose isomerase and has been highlighted as a sugar substitute due to a number of advantageous properties. For the massive production of isomaltulose, high resistance to sucrose and stability of sucrose isomerase as well as sucrose conversion yields would be critical factors. We describe a series of screening procedures to isolate the mutant strain of Serratia sp. possessing enhanced isomaltulose production with improved stability. The new Serratia sp. isolated from a series of screening procedures allowed us to produce isomaltulose from 60% sucrose solution, with over 90% conversion yield. Moreover, when this strain was immobilized in calcium alginate beads and placed in a medium containing 60% sucrose, it showed over 70% sucrose conversion yields for 30 cycles of repeated-batch reactions. Thus, improved conversion activity and stability of the newly isolated Serratia sp. strain in the present study would be highly valuable for industries related to isomaltulose production.

  15. Immobilization of Candida krusei cells producing phytase in alginate gel beads: an application of the preparation of myo-inositol phosphates.

    PubMed

    Quan, C S; Fan, S D; Ohta, Y

    2003-07-01

    Cells of Candida krusei capable of producing phytase were immobilized in Ca-alginate gel beads and used for the preparation of myo-inositol phosphates. The immobilization yield was increased about 5-fold after the beads were treated for 96 h at pH 4.0, 4 degrees C. The increased yield was retained, even after 1 month, when the cells were kept at this temperature and pH. No shift in the pH optima of phytase of the immobilized cells was observed, compared with that of free cells. However, the optimum temperature for the enzyme of the immobilized cells was 55 degrees C, which was 15 degrees C higher than that of free cells. The degradation characteristics of the phytate in immobilized cells packed in a glass column (i.d. 1.2 cm, length 20 cm) were investigated. The variation in the composition of the products results from a change in the flow rate of phytate solution (5 mM). At a flow rate of 1.30 ml/min, a mixture of myo-inositol-2-monophosphate, myo-inositol-1,2,5-triphosphate and myo-inositol-1,2,5,6-tetrakisphosphate was produced, in which the latter two were physiologically active. Also, it was found by NMR analysis that the enzyme of this strain produced only one isomer of each of the inositol phosphates, with the exception of myo-inositol pentakisphosphate. Therefore, the pure isomers were easily isolated using ion-exchange chromatography.

  16. A Ca-alginate particle co-immobilized with Phanerochaete chrysosporium cells and the combined cross-linked enzyme aggregates from Trametes versicolor.

    PubMed

    Li, Yanchun; Wang, Zhi; Xu, Xudong; Jin, Liqiang

    2015-12-01

    For improving stability of immobilized white-rot fungus to treat various effluents, Phanerochaete chrysosporium cells and the combined cross-link enzyme aggregates (combi-CLEAs) prepared from Trametes versicolor were co-immobilized into the Ca-alginate gel particles in this paper. The activity yields of obtained combi-CLEAs were 42.7% for lignin peroxidases (LiPs), 31.4% for manganese peroxidases (MnPs) and 40.4% for laccase (Lac), respectively. And their specific activities were 30.2U/g as combi-CLEAs-LiPs, 9.5 U/g as combi-CLEAs-MnPs and 28.4 U/g as combi-CLEAs-Lac. Further, the present of the combi-CLEAs in the particles extremely improved their ability to degrade the dyes. Compared to the immobilized Ph. chrysosporium without the combi-CLEAs, the co-immobilized particles enhanced the decolorized rate of Acid Violet 7 (from 45.2% to 93.4%) and Basic Fuchsin (from 12.1% to 67.9%). In addition, the addition of the combi-CLEAs improved the adaptability of the white-rot fungal particles to adverse environmental conditions. PMID:26413897

  17. Optimization of alpha-amylase immobilization in calcium alginate beads.

    PubMed

    Ertan, Figen; Yagar, Hulya; Balkan, Bilal

    2007-01-01

    alpha-Amylase enzyme was produced by Aspergillus sclerotiorum under SSF conditions, and immobilized in calcium alginate beads. Effects of immobilization conditions, such as alginate concentration, CaCl(2) concentration, amount of loading enzyme, bead size, and amount of beads, on enzymatic activity were investigated. Optimum alginate and CaCl(2) concentration were found to be 3% (w/v). Using a loading enzyme concentration of 140 U mL(-1), and bead (diameter 3 mm) amount of 0.5 g, maximum enzyme activity was observed. Beads prepared at optimum immobilization conditions were suitable for up to 7 repeated uses, losing only 35% of their initial activity. Among the various starches tested, the highest enzyme activity (96.2%) was determined in soluble potato starch hydrolysis for 120 min at 40 degrees C.

  18. Immobilization of a Plant Lipase from Pachira aquatica in Alginate and Alginate/PVA Beads.

    PubMed

    Bonine, Bárbara M; Polizelli, Patricia Peres; Bonilla-Rodriguez, Gustavo O

    2014-01-01

    This study reports the immobilization of a new lipase isolated from oleaginous seeds of Pachira aquatica, using beads of calcium alginate (Alg) and poly(vinyl alcohol) (PVA). We evaluated the morphology, number of cycles of reuse, optimum temperature, and temperature stability of both immobilization methods compared to the free enzyme. The immobilized enzymes were more stable than the free enzyme, keeping 60% of the original activity after 4 h at 50°C. The immobilized lipase was reused several times, with activity decreasing to approximately 50% after 5 cycles. Both the free and immobilized enzymes were found to be optimally active between 30 and 40°C.

  19. 3-Chloro-1,2-propanediol biodegradation by Ca-alginate immobilized Pseudomonas putida DSM 437 cells applying different processes: mass transfer effects.

    PubMed

    Konti, Aikaterini; Mamma, Diomi; Hatzinikolaou, Dimitios G; Kekos, Dimitris

    2016-10-01

    3-Chloro-1,2-propanediol (3-CPD) biodegradation by Ca-alginate immobilized Pseudomonas putida cells was performed in batch system, continuous stirred tank reactor (CSTR), and packed-bed reactor (PBR). Batch system exhibited higher biodegradation rates and 3-CPD uptakes compared to CSTR and PBR. The two continuous systems (CSTR and PBR) when compared at 200 mg/L 3-CPD in the inlet exhibited the same removal of 3-CPD at steady state. External mass-transfer limitations are found negligible at all systems examined, since the observable modulus for external mass transfer Ω ≪ 1 and the Biot number Bi > 1. Intra-particle diffusion resistance had a significant effect on 3-CPD biodegradation in all systems studied, but to a different extent. Thiele modulus was in the range of 2.5 in batch system, but it was increased at 11 when increasing cell loading in the beads, thus lowering significantly the respective effectiveness factor. Comparing the systems at the same cell loading in the beads PBR was less affected by internal diffusional limitations compared to CSTR and batch system, and, as a result, exhibited the highest overall effectiveness factor.

  20. 3-Chloro-1,2-propanediol biodegradation by Ca-alginate immobilized Pseudomonas putida DSM 437 cells applying different processes: mass transfer effects.

    PubMed

    Konti, Aikaterini; Mamma, Diomi; Hatzinikolaou, Dimitios G; Kekos, Dimitris

    2016-10-01

    3-Chloro-1,2-propanediol (3-CPD) biodegradation by Ca-alginate immobilized Pseudomonas putida cells was performed in batch system, continuous stirred tank reactor (CSTR), and packed-bed reactor (PBR). Batch system exhibited higher biodegradation rates and 3-CPD uptakes compared to CSTR and PBR. The two continuous systems (CSTR and PBR) when compared at 200 mg/L 3-CPD in the inlet exhibited the same removal of 3-CPD at steady state. External mass-transfer limitations are found negligible at all systems examined, since the observable modulus for external mass transfer Ω ≪ 1 and the Biot number Bi > 1. Intra-particle diffusion resistance had a significant effect on 3-CPD biodegradation in all systems studied, but to a different extent. Thiele modulus was in the range of 2.5 in batch system, but it was increased at 11 when increasing cell loading in the beads, thus lowering significantly the respective effectiveness factor. Comparing the systems at the same cell loading in the beads PBR was less affected by internal diffusional limitations compared to CSTR and batch system, and, as a result, exhibited the highest overall effectiveness factor. PMID:27262716

  1. Immobilization of a Plant Lipase from Pachira aquatica in Alginate and Alginate/PVA Beads

    PubMed Central

    Bonine, Bárbara M.; Polizelli, Patricia Peres; Bonilla-Rodriguez, Gustavo O.

    2014-01-01

    This study reports the immobilization of a new lipase isolated from oleaginous seeds of Pachira aquatica, using beads of calcium alginate (Alg) and poly(vinyl alcohol) (PVA). We evaluated the morphology, number of cycles of reuse, optimum temperature, and temperature stability of both immobilization methods compared to the free enzyme. The immobilized enzymes were more stable than the free enzyme, keeping 60% of the original activity after 4 h at 50°C. The immobilized lipase was reused several times, with activity decreasing to approximately 50% after 5 cycles. Both the free and immobilized enzymes were found to be optimally active between 30 and 40°C. PMID:24818012

  2. Biodegradation of crystal violet using Burkholderia vietnamiensis C09V immobilized on PVA-sodium alginate-kaolin gel beads.

    PubMed

    Cheng, Ying; Lin, HongYan; Chen, Zuliang; Megharaj, Mallavarapu; Naidu, Ravi

    2012-09-01

    The strain, Burkholderia vietnamiensis C09V was immobilized on PVA-alginate-kaolin gel beads as a biomaterial to improve the degradation of crystal violet from aqueous solution. The results show that 98.6% (30 mg L(-1)) crystal violet was removed from aqueous solution using immobilized cells on PVA-alginate-kaolin gel beads, while 94.0% crystal violet was removed by free cells after degradation at the pH 5 and 30°C for 30 h. Kinetics studies show that the pseudo-second-order kinetics well described the adsorption of crystal violet on the PVA-alginate-kaolin beads. Biodegradation of crystal violet on immobilized cells was fitted well by first-order reaction kinetics, indicating that CV was adsorbed onto kaolin and followed their degradation by immobilized cells onto the the PVA-alginate-kaolin beads. Characterization with SEM shows that cells attached well to the surface of PVA-alginate-kaolin beads, leading to improved crystal violet transfer from aqueous solution to immobilized cells. In addition, UV-vis show that the absorption peak at 588 nm was reduced by the degraded N-bond linkages, as well as the formation of degrading products were observed by Fourier transform infrared (FTIR). These results suggest that crystal violet was biodegraded to N,N-dimethylaminophenol and Michler's Ketone prior to these intermediates being further degraded. PMID:22789742

  3. Biodegradation of crystal violet using Burkholderia vietnamiensis C09V immobilized on PVA-sodium alginate-kaolin gel beads.

    PubMed

    Cheng, Ying; Lin, HongYan; Chen, Zuliang; Megharaj, Mallavarapu; Naidu, Ravi

    2012-09-01

    The strain, Burkholderia vietnamiensis C09V was immobilized on PVA-alginate-kaolin gel beads as a biomaterial to improve the degradation of crystal violet from aqueous solution. The results show that 98.6% (30 mg L(-1)) crystal violet was removed from aqueous solution using immobilized cells on PVA-alginate-kaolin gel beads, while 94.0% crystal violet was removed by free cells after degradation at the pH 5 and 30°C for 30 h. Kinetics studies show that the pseudo-second-order kinetics well described the adsorption of crystal violet on the PVA-alginate-kaolin beads. Biodegradation of crystal violet on immobilized cells was fitted well by first-order reaction kinetics, indicating that CV was adsorbed onto kaolin and followed their degradation by immobilized cells onto the the PVA-alginate-kaolin beads. Characterization with SEM shows that cells attached well to the surface of PVA-alginate-kaolin beads, leading to improved crystal violet transfer from aqueous solution to immobilized cells. In addition, UV-vis show that the absorption peak at 588 nm was reduced by the degraded N-bond linkages, as well as the formation of degrading products were observed by Fourier transform infrared (FTIR). These results suggest that crystal violet was biodegraded to N,N-dimethylaminophenol and Michler's Ketone prior to these intermediates being further degraded.

  4. Raman probing of molecular interactions of alginate biopolymers with cells

    NASA Astrophysics Data System (ADS)

    Chourpa, Igor; Carpentier, Philippe; Maingault, Philippe; Fetissoff, Franck; Dubois, Pierre

    2000-05-01

    The biological polymers extracted from brown algae, alginates, are novel materials in biotechnology and biomedicine. Their ability to form viscous gels is used to immobilize or encapsulate yeast, enzymes, living cells and drugs. Calcium-alginate fibers are extensively used in wound dressings since exhibit antihaemostatic and healing properties. The problem with alginate-made dressings in surgery is their slow biodegradability: if entrapped within tissues, they can induce a local cellular recruitment with an inflammatory response contemporaneous to the resorption phase. In part, this problem is a consequence of poor solubility of the calcium alginates in water. Although calcium alginate fibers can exchange calcium ions with sodium ions from the wound exudate to create a calcium/sodium alginate fibers, the residual alginates are thought to be not totally degradable in vivo. Rapid and non- destructive characterization of series of the crude alginates and calcium alginate fibers has been performed using Raman spectroscopy with near IR excitation. Study of structural organization of the polymeric chains within calcium alginate fibers have been previously reported as made by confocal Raman multispectral imaging (CRMSI) in visible. Here, the Raman approach has been used to monitor the ion exchange reactions for different types of alginates and their salts in vitro. For in vivo evaluation, histological sections of alginate-treated rat tissue have been analyzed by light microscopy and CRMSI. The in vitro Raman modeling and the histochemical mapping were a necessary precursor for application of the Raman microprobe to follow in a non-invasive way the alginate-cell molecular interactions in rat tissue.

  5. Increased erythromycin production by alginate as a medium ingredient or immobilization support in cultures of Saccharopolyspora erythraea.

    PubMed

    Hamedi, J; Khodagholi, F; Hassani-Nasab, A

    2005-05-01

    Erythromycin production by Saccharopolyspora erythraea immobilized in 2% (w/v) calcium alginate or grown in medium containing 20 g sodium alginate/l inoculated with free cells was almost twice more than that of the control. S. erythraea did not consume alginate, agar, dextran, silicon antifoaming agent or cyclodextrin as a carbon source, although, all of these increased the production of erythromycin. Highest titer of erythromycin (2.3 times more than that of the control) was achieved in medium containing 1 g agar/l.

  6. Gelatin blends with alginate: gels for lipase immobilization and purification.

    PubMed

    Fadnavis, Nitin W; Sheelu, Gurrala; Kumar, Bezavada Mani; Bhalerao, Mahendra U; Deshpande, Ashlesha A

    2003-01-01

    Blends of natural polysaccharide sodium alginate (5%) with gelatin (3%) cross-linked with glutaraldehyde provide beads with excellent compressive strength (8 x 10(4) Pa) and regular structure on treatment with calcium chloride. Lipases from porcine pancreas, Pseudomonas cepacia, and Candida rugosa were immobilized in such a blend with excellent efficiency. The immobilized enzymes were stable and were reused several times without significant loss of enzyme activity both in aqueous and reverse micellar media. The beads were functionalized with succinic anhydride to obtain beads with extra carboxylic acid groups. These functionalized beads were then successfully used for 7.4-fold purification of crude porcine pancreatic lipase in a simple operation of protein binding at pH 5 and release at pH 8.5.

  7. Survival of Bifidobacterium longum immobilized in calcium alginate beads in simulated gastric juices and bile salt solution.

    PubMed

    Lee, K Y; Heo, T R

    2000-02-01

    Bifidobacterium longum KCTC 3128 and HLC 3742 were independently immobilized (entrapped) in calcium alginate beads containing 2, 3, and 4% sodium alginate. When the bifidobacteria entrapped in calcium alginate beads were exposed to simulated gastric juices and a bile salt solution, the death rate of the cells in the beads decreased proportionally with an increase in both the alginate gel concentration and bead size. The initial cell numbers in the beads affected the numbers of survivors after exposure to these solutions; however, the death rates of the viable cells were not affected. Accordingly, a mathematical model was formulated which expressed the influences of several parameters (gel concentration, bead size, and initial cell numbers) on the survival of entrapped bifidobacteria after sequential exposure to simulated gastric juices followed by a bile salt solution. The model proposed in this paper may be useful for estimating the survival of bifidobacteria in beads and establishing optimal entrapment conditions.

  8. Arsenite oxidation in batch reactors with alginate-immobilized ULPAs1 strain.

    PubMed

    Simeonova, Diliana D; Micheva, Kalina; Muller, Daniel A E; Lagarde, Florence; Lett, Marie-Claire; Groudeva, Veneta I; Lièvremont, Didier

    2005-08-20

    Arsenic is one of the major groundwater contaminants worldwide. It was previously demonstrated that the beta-proteobacterium Cenibacterium arsenoxidans has an efficient As[III] oxidation ability. The present study was conducted to evaluate the performance of alginate-immobilized ULPAs1 in the oxidation of As[III] to As[V] in batch reactors. A two-level full factorial experimental design was applied to investigate the influence of main parameters involved in the oxidation process, i.e., pH (7-8), temperature (4 degrees C-25 degrees C), kind of nutrient media (2%-20% sauerkraut brine), and arsenic concentration (10-100 mg/L). One hundred milligram per liter of As[III] was fully oxidized by calcium-alginate immobilized cells in 1 h. It was found that the temperature as well as the kind of nutrient media used were significant parameters at a 95% confidence interval whereas only temperature was a significant parameter at a 99% confidence interval. The immobilization of the As[III] oxidizing strain in alginate beads offers a promising way to implement new treatment processes in the remediation of arsenic contaminated waters.

  9. Optically clear alginate hydrogels for spatially controlled cell entrapment and culture at microfluidic electrode surfaces.

    PubMed

    Betz, Jordan F; Cheng, Yi; Tsao, Chen-Yu; Zargar, Amin; Wu, Hsuan-Chen; Luo, Xiaolong; Payne, Gregory F; Bentley, William E; Rubloff, Gary W

    2013-05-21

    We describe an innovation in the immobilization, culture, and imaging of cells in calcium alginate within microfluidic devices. This technique allows unprecedented optical access to the entirety of the calcium alginate hydrogel, enabling observation of growth and behavior in a chemical and mechanical environment favored by many kinds of cells.

  10. Isomaltulose production from sucrose by Protaminobacter rubrum immobilized in calcium alginate.

    PubMed

    de Oliva-Neto, P; Menão, Paula T P

    2009-09-01

    Different culture conditions for Protaminobacter rubrum and enzymatic reaction parameters were evaluated with the goal of improving isomaltulose production. P. rubrum was grown in a medium with 1% (w/v) cane molasses and 0.5% yeast extract and achieved a maximum cell yield Y(x/s) of 0.295 g of cells/g sucrose and a specific growth rate (mu) of 0.192 h(-1). The immobilization of P. rubrum cells was carried out with calcium alginate, glutaraldehyde and polyethyleneimine. Stabile immobilized cell pellets were obtained and used 24 times in batch processes. Enzymatic conversion was carried out at different sucrose concentrations and in pH 6 medium with 70% (w/v) sucrose at 30 degrees C an isomaltulose yield of 89-94% (w/v) was obtained. The specific activity of the P. rubrum immobilized pellets in calcium alginate at 30 degrees C ranged from 1.6 to 4.0 g isomaltulose g(-1) pellet h(-1), respectively with 70% and 65% sucrose solution, while in lower sucrose concentration had higher specific activities presumably due to substrate inhibition of the isomaltulose synthase in higher sucrose concentrations.

  11. Immobilization of glucose oxidase in alginate-chitosan microcapsules.

    PubMed

    Wang, Xia; Zhu, Ke-Xue; Zhou, Hui-Ming

    2011-01-01

    In order to improve its stability and catalytic rate in flour, the immobilization of glucose oxidase (GOX) was investigated in this work. The enzyme was encapsulated in calcium alginate-chitosan microspheres (CACM) using an emulsification-internal gelation-GOX adsorption-chitosan coating method. The interaction between alginate and chitosan was confirmed by infrared spectroscopy (IR). The resultant CACM in wet state, whose morphology was investigated by scanning electron microscopy (SEM), was spherical with a mean diameter of about 26 μm. The GOX load, encapsulation efficiency and activity of the CACM-GOX were influenced by concentration of chitosan, encapsulation time and encapsulation pH. The highest total enzymatic activity and encapsulation efficiency was achieved when the pH of the adsorption medium was near the isoelectric point (pI) of GOX, approximately pH 4.0. In addition, the molecular weight of chitosan also evidently influenced the encapsulation efficiency. Storage stabilities of GOX samples were investigated continuously over two months and the retained activity of CACM-GOX was 70.4%, markedly higher than the 7.5% of free enzyme. The results reveal the great potential of CACM-GOX as a flour improver.

  12. Enzyme immobilization in novel alginate-chitosan core-shell microcapsules.

    PubMed

    Taqieddin, Ehab; Amiji, Mansoor

    2004-05-01

    Alginate-chitosan core-shell microcapsules were prepared in order to develop a biocompatible matrix for enzyme immobilization, where the protein is retained either in a liquid or solid core and the shell allows permeability control over substrates and products. The permeability coefficients of different molecular weight compounds (vitamin B2, vitamin B12, and myoglobin) were determined through sodium tripolyphosphate (Na-TPP)-crosslinked chitosan membrane. The microcapsule core was formed by crosslinking sodium alginate with either calcium or barium ions. The crosslinked alginate core was uniformly coated with a chitosan layer and crosslinked with Na-TPP. In the case of calcium alginate, the phosphate ions of Na-TPP were able to extract the calcium ions from alginate and liquefy the core. A model enzyme, beta-galactosidase, was immobilized in the alginate core and the catalytic activity was measured with o-nitrophenyl-beta-D-galactopyranoside (ONPG). Change in the activity of free and immobilized enzyme was determined at three different temperatures. Na-TPP crosslinked chitosan membranes were found to be permeable to solutes of up to 17,000Da molecular weight. The enzyme loading efficiency was higher in the barium alginate core (100%) as compared to the calcium alginate core (60%). The rate of ONPG conversion to o-nitrophenol was faster in the case of calcium alginate-chitosan microcapsules as compared to barium alginate-chitosan microcapsules. Barium alginate-chitosan microcapsules, however, did improve the stability of the enzyme at 37 degrees C relative to calcium alginate-chitosan microcapsules or free enzyme. This study illustrates a new method of enzyme immobilization for biotechnology applications using liquid or solid core and shell microcapsule technology.

  13. Affinity covalent immobilization of glucoamylase onto ρ-benzoquinone-activated alginate beads: II. Enzyme immobilization and characterization.

    PubMed

    Eldin, M S Mohy; Seuror, E I; Nasr, M A; Tieama, H A

    2011-05-01

    A novel affinity covalent immobilization technique of glucoamylase enzyme onto ρ-benzoquinone-activated alginate beads was presented and compared with traditional entrapment one. Factors affecting the immobilization process such as enzyme concentration, alginate concentration, calcium chloride concentration, cross-linking time, and temperature were studied. No shift in the optimum temperature and pH of immobilized enzymes was observed. In addition, K (m) values of free and entrapped glucoamylase were found to be almost identical, while the covalently immobilized enzyme shows the lowest affinity for substrate. In accordance, V (m) value of covalently immobilized enzyme was found lowest among free and immobilized counter parts. On the other hand, the retained activity of covalently immobilized glucoamylase has been improved and was found higher than that of entrapped one. Finally, the industrial applicability of covalently immobilized glucoamylase has been investigated through monitoring both shelf and operational stability characters. The covalently immobilized enzyme kept its activity over 36 days of shelf storage and after 30 repeated use runs. Drying the catalytic beads greatly reduced its activity in the beginning but recovered its lost part during use. In general, the newly developed affinity covalent immobilization technique of glucoamylase onto ρ-benzoquinone-activated alginate carrier is simple yet effective and could be used for the immobilization of some other enzymes especially amylases.

  14. [Study on PVA-alginate co-immobilization of Xanthomonas ampelina TS206].

    PubMed

    Chen, Qingsen; Liu, Jian

    2003-08-01

    Ice nucleation activity and anti-leaking capability are both important technical parameters in INA (Ice nucleation active bactetia) bacteria immobilization which can be adopted on freezing concentrate. Both PVA and alginate are good medium for immobilization. They can be used on co-immobilization of ice nucleation-active bacteria (Xanthomonas ampelinaTS206). The results showed that quantity of embedding affects ice nucleation activity greatly. The order of importance to comprehensive scores of technical standard should be concentration of Sodium alginate > Boric acid > PVA > CaCl2. The optimized concentration are PVA 8%, sodium alginate 1%, CaCl2 1.1% and boric acid 5%. A conclusion can be draw that ice nucleation activity increases with the number of beads and shows little pertinency with the time of immobilization, whereas anti-leaking capability can be influenced faintly by the number of beads and the time of immobilization. PMID:16276925

  15. Stabilization of Aspergillus parasiticus cytosine deaminase by immobilization on calcium alginate beads improved enzyme operational stability.

    PubMed

    Zanna, H; Nok, A J; Ibrahim, S; Inuwa, H M

    2013-12-01

    Cytosine deaminase (CD) from Aspergillus parasiticus, which has half-life of 1.10 h at 37°C, was stabilized by immobilization on calcium alginate beads. The immobilized CD had pH and temperature optimum of 5 and 50°C respectively. The immobilized enzyme also stoichiometrically deaminated Cytosine and 5-fluorocytosine (5-FC) with the apparent K(M) values of 0.60 mM and 0.65 mM respectively, displaying activation energy of 10.72 KJ/mol. The immobilization of native CD on calcium alginate beads gave the highest yield of apparent enzymatic activity of 51.60% of the original activity and the enzymatic activity was lost exponentially at 37°C over 12 h with a half-life of 5.80 h. Hence, the operational stability of native CD can be improved by immobilization on calcium alginate beads.

  16. Polydopamine-Mediated Immobilization of Alginate Lyase to Prevent P. aeruginosa Adhesion.

    PubMed

    Alves, Diana; Sileika, Tadas; Messersmith, Phillip B; Pereira, Maria Olívia

    2016-09-01

    Given alginate's contribution to Pseudomonas aeruginosa virulence, it has long been considered a promising target for interventional therapies, which have been performed by using the enzyme alginate lyase. In this work, instead of treating pre-established mucoid biofilms, alginate lyase is immobilized onto a surface as a preventive measure against P. aeruginosa adhesion. A polydopamine dip-coating strategy is employed for functionalization of polycarbonate surfaces. Enzyme immobilization is confirmed by surface characterization. Surfaces functionalized with alginate lyase exhibit anti-adhesive properties, inhibiting the attachment of the mucoid strain. Moreover, surfaces modified with this enzyme also inhibit the adhesion of the tested non-mucoid strain. Unexpectedly, treatment with heat-inactivated enzyme also inhibits the attachment of mucoid and non-mucoid P. aeruginosa strains. These findings suggest that the antibacterial performance of alginate lyase functional coatings is catalysis-independent, highlighting the importance of further studies to better understand its mechanism of action against P. aeruginosa strains. PMID:27198822

  17. Polydopamine-Mediated Immobilization of Alginate Lyase to Prevent P. aeruginosa Adhesion.

    PubMed

    Alves, Diana; Sileika, Tadas; Messersmith, Phillip B; Pereira, Maria Olívia

    2016-09-01

    Given alginate's contribution to Pseudomonas aeruginosa virulence, it has long been considered a promising target for interventional therapies, which have been performed by using the enzyme alginate lyase. In this work, instead of treating pre-established mucoid biofilms, alginate lyase is immobilized onto a surface as a preventive measure against P. aeruginosa adhesion. A polydopamine dip-coating strategy is employed for functionalization of polycarbonate surfaces. Enzyme immobilization is confirmed by surface characterization. Surfaces functionalized with alginate lyase exhibit anti-adhesive properties, inhibiting the attachment of the mucoid strain. Moreover, surfaces modified with this enzyme also inhibit the adhesion of the tested non-mucoid strain. Unexpectedly, treatment with heat-inactivated enzyme also inhibits the attachment of mucoid and non-mucoid P. aeruginosa strains. These findings suggest that the antibacterial performance of alginate lyase functional coatings is catalysis-independent, highlighting the importance of further studies to better understand its mechanism of action against P. aeruginosa strains.

  18. Immobilization of Escherichia coli novablue gamma-glutamyltranspeptidase in Ca-alginate-kappa-carrageenan beads.

    PubMed

    Hung, Chih-Peng; Lo, Huei-Fen; Hsu, Wen-Hwei; Chen, Shih-Chun; Lin, Long-Liu

    2008-08-01

    The recombinant Escherichia coli gamma-glutamyltranspeptidase (EcGGT) was immobilized in Ca-alginate-kappa-carrageenan beads. Effects of alginate concentration, amount of loading enzyme, and bead size on the entrapped activity were investigated. Optimum alginate concentration for EcGGT immobilization was found to be 2% (w/v). Using a loading enzyme concentration of 1.5 mg/g alginate, maximum enzyme activity was observed. With increase in bead size from 1.9 to 3.1 mm, the immobilization efficiency was decreased significantly because of mass transfer resistance. Thermal stability of the free EcGGT was increased as a result of the immobilization. Ca-alginate-kappa-carrageenan-EcGGT beads were suitable for up to six repeated uses, losing only 45% of their initial activity. Upon 30 days of storage the preserved activity of free and immobilized enzyme were found as 4% and 68%, respectively. The synthesis of L: -theanine was performed in 50 mM Tris-HCl buffer (pH 10) containing 25 mM L: -glutamine, 40 mM ethylamine, and 1.5 mg EcGGT/g alginate at 40 degrees C for 12 h, and a conversion rate of 27% was achieved.

  19. Properties and application of immobilized beta-D-glucosidase coentrapped with Zymomonas mobilis in calcium alginate.

    PubMed

    Lee, J M; Woodward, J

    1983-10-01

    The enzyme beta-D-glucosidase has been immobilized on concanavalin A-Sepharose to give a maximum loading of 2050 units/g dry weight of support material. The immobilized beta-D-glucosidase was also entrapped within calcium alginate gel spheres with apparently only 35% retention of activity when assayed with 10mM cellobiose. However, it was discovered that, unlike the immobilized enzyme, the entrapped immobilized enzyme was not subject to substrate inhibition up to 100mM cellobiose, suggesting that a concentration gradient of cellobiose existed between the bulk solution and the interior of the gel sphere. Thus, the activity of the entrapped immobilized enzyme was almost twice as high as that of the immobilized enzyme when assayed with 100mM cellobiose. Concanavalin A-Sepharose-immobilized beta-D-glucosidase and the bacterium Zymomonas mobilis coimmobilized in calcium alginate gel spheres converted cellobiose to ethanol in both batch and continuous-flow fermentation systems.

  20. Improvement of bioethanol productivity of immobilized Saccharomyces bayanus with using sodium alginate-graft-poly(N-vinyl-2-pyrrolidone) matrix.

    PubMed

    İnal, Murat; Yiğitoğlu, Mustafa

    2012-09-01

    In this study, immobilization conditions and bioethanol production characteristics of immobilized Saccharomyces bayanus were investigated into sodium alginate-graft-poly(N-vinyl-2-pyrrolidone; NaAlg-g-PVP) matrix. The matrix that crosslinked with calcium clorid was used for immobilization of S. bayanus. Bioethanol productivity of the NaAlg-g-PVP matrix was found to increase from 4.21 to 4.84 gL(-1) h(-1) when compared with the convential sodium alginate matrix. The production of bioethanol was affected by initial glucose concentration and percentage of immobilized cell beads in fermentation medium. Bioethanol productivity was increased from 3.62 to 4.84 gL(-1) h(-1) while the glucose concentration increasing from 50 to 100 gL(-1). Due to the increase in percentage from 10 to 20 % of immobilized cell beads in the fermentation medium, bioethanol productivity was increased from 4.84 to 8.68 gL(-1) h(-1). The cell immobilized NaAlg-g-PVP beads were protected 92 % of initial activity after six repeated fermentation.

  1. Proteomic analysis of calcium alginate-immobilized Saccharomyces cerevisiae under high-gravity fermentation conditions.

    PubMed

    Pham, Trong Khoa; Wright, Phillip C

    2008-02-01

    Saccharomyces cerevisiae KAY446 cells immobilized in calcium alginate gel, and supplemented with additional amino acids, were successfully used in enhancing ethanol production. This combination succeeded in improving the ethanol yield and reducing the fermentation time. The ethanol yield under these conditions was 0.40 g of ethanol/g of glucose, with a final ethanol concentration of 118 g/L after 72 h. This is compared to yields with immobilized cells alone of 0.35 g of ethanol/g of glucose and freely suspended cells with no amino acid supplementation of 0.30 g of ethanol/g of glucose, under the same VHG conditions. The maximum specific ethanol production rates were 0.98, 0.73, and 0.61 g (g dry weight) (-1) h (-1) for immobilized cells under VHG conditions with and without amino acid supplementation and free cells, respectively. A proteomic analysis showed significant stimulation of many pathways during fermentation under these conditions, including the Ras/cAMP, glycolysis, starch, and sucrose pathways, amino acids biosynthesis, and aminoacyl-tRNA synthetases. The upregulation of ribosomal, heat-shock proteins and proteins involved in cell viability confirmed that protein biosynthesis was accelerated and revealed likely mechanisms for improving cellular viability.

  2. Immobilization of Multi-biocatalysts in Alginate Beads for Cofactor Regeneration and Improved Reusability.

    PubMed

    Gao, Hui; Khera, Eshita; Lee, Jung-Kul; Wen, Fei

    2016-04-22

    We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst immobilization for improved production yield and sustained synthesis. Described herewith is the experimental procedure for the development of such a system consisting of two E. coli strains that express functionally complementary enzymes. Together, these two enzymes can function co-operatively to mediate the regeneration of expensive cofactors for improving the product yield of the bioreaction. In addition, the method of synthesizing an immobilized form of the coupled biocatalytic system by encapsulation of whole cells in calcium alginate beads is reported. As an example, we present the improved biosynthesis of L-xylulose from L-arabinitol by coupling E. coli cells expressing the enzymes L-arabinitol dehydrogenase or NADH oxidase. Under optimal conditions and using an initial concentration of 150 mM L-arabinitol, the maximal L-xylulose yield reached 96%, which is higher than those reported in the literature. The immobilized form of the coupled whole-cell biocatalysts demonstrated good operational stability, maintaining 65% of the yield obtained in the first cycle after 7 cycles of successive re-use, while the free cell system almost completely lost the catalytic activity. Therefore, the methods reported here provides two strategies that could help improve the industrial production of L-xylulose, as well as other value-added compounds requiring the use of cofactors in general.

  3. Characterization of dextransucrase immobilized on calcium alginate beads from Leuconostoc mesenteroides PCSIR-4.

    PubMed

    Ul Qader, Shah Ali; Aman, Afsheen; Syed, Noman; Bano, Saeeda; Azhar, Abid

    2007-06-01

    Immobilization of dextransucrase from Leuconostoc mesenteroides PCSIR-4 on alginate is optimized for application in the production of dextran from sucrose. Dextransucrase was partially purified by ethanol upto 2.5 fold. Properties of dextransucrase were less affected by immobilization on alginate beads from soluble enzyme. Highest activities of both soluble and immobilized dextransucrase found to be at 35 degrees C and optimum pH for activity remain 5.00. Substrate maxima for immobilized enzyme changed from 125 mg/ml to 200 mg/ml. Incubation time for enzyme-substrate reaction for maximum enzyme activity was increased from 15 minutes to 60 minutes in case of immobilized enzyme. Maximum stability of immobilized dextransucrase was achieved at 25 degrees C with respect to time.

  4. Preparation and activity of bubbling-immobilized cellobiase within chitosan-alginate composite.

    PubMed

    Wang, Fang; Su, Rong-Xin; Qi, Wei; Zhang, Ming-Jia; He, Zhi-Min

    2010-01-01

    Cellobiase can hydrolyze cellobiose into glucose; it plays a key role in the process of cellulose hydrolysis by reducing the product inhibition. To reuse the enzyme and improve the economic value of cellulosic ethanol, cellobiase was immobilized using sodium alginate and chitosan as carriers by the bubbling method. The immobilization conditions were optimized as follows: enzyme loading of 100 U cellobiase/g carrier, 30 min immobilization, 3.5 wt% sodium alginate, 0.25 wt% chitosan, and 2 wt% calcium chloride. Compared to free enzyme, the immobilized cellobiase had a decreased apparent K(m) and the maximum activity at a lower pH, indicating its higher acidic and thermal stability. The immobilized cellobiase was further tested in the hydrolysis of cellobiose and various cellulosic substrates (microcrystalline cellulose, filter paper, and ammonia-pretreated corn cobs). Together with cellulases, the immobilized cellobiase converted the cellulosic substrates into glucose with the rate and extent similar to the free enzyme.

  5. Kinetics of fungal extracellular alpha-amylase from Fusarium solani immobilized in calcium alginate beads.

    PubMed

    Kumar, Devendra; Muthukumar, M; Garg, Neelima

    2012-11-01

    Extracellular alpha-amylase mass produced by Fusarium solani using mango kernel as substrate was immobilized in calcium alginate beads through entrapment technique. Maximum enzyme immobilization efficiency was achieved in 2 mm size beads formed by 6.5% (w/v) of sodium alginate in 2% (w/v) calcium chloride. The catalytic properties of the immobilized alpha-amylase were compared with that of free enzyme (soluble). The activity yield of the immobilized enzyme was 81% of the free enzyme. The immobilized enzyme showed optimum activityat pH 4.5-6.0 and temperature 40 degrees C, in contrast to the free enzyme at 5.5 and 30 degrees C, respectively. Thermal stability of the immobilized enzyme was found to be more than the free enzyme over a longer time interval. The immobilized enzyme retained activity upto 20% of optimum even after 180 min. While the free enzyme lost its 80% activity after 60 min and lost total activity down to zero by 120 min. The kinetic constants, viz., K(M) (Michaelis constant), V(max) and activation energy were affected by immobilization. However, the immobilized alpha-amylase in calcium alginate beads supports its long-term storage which has immense industrial applications.

  6. Biosorption of cadmium, lead and copper with calcium alginate xerogels and immobilized Fucus vesiculosus.

    PubMed

    Mata, Y N; Blázquez, M L; Ballester, A; González, F; Muñoz, J A

    2009-04-30

    This paper determines the effect of immobilized brown alga Fucus vesiculosus in the biosorption of heavy metals with alginate xerogels. Immobilization increased the kinetic uptakes and intraparticle diffusion rates of the three metals. The Langmuir maximum biosorption capacity increased twofold for cadmium, 10 times for lead, and decreased by half for copper. According to this model, the affinity of the metals for the biomass was as follows: Cu>Pb>Cd without alga and Pb>Cu>Cd with alga. FITR confirmed that carboxyl groups were the main groups involved in the metal uptake. Calcium in the gels was displaced by heavy metals from solution according to the "egg-box" model. The restructured gel matrix became more uniform and organized as shown by scanning electron microscopy (SEM) characterization. F. vesiculosus immobilized in alginate xerogels constitutes an excellent biosorbent for cadmium, lead and copper, sometimes surpassing the biosorption performance of alginate alone and even the free alga.

  7. Enhanced accumulation of starch and total carbohydrates in alginate-immobilized Chlorella spp. induced by Azospirillum brasilense: II. Heterotrophic conditions.

    PubMed

    Choix, Francisco J; de-Bashan, Luz E; Bashan, Yoav

    2012-10-10

    The effect of the bacterium Azospirillum brasilense jointly immobilized with Chlorella vulgaris or C. sorokiniana in alginate beads on total carbohydrates and starch was studied under dark and heterotrophic conditions for 144 h in synthetic growth medium supplemented with either d-glucose or Na-acetate as carbon sources. In all treatments, enhanced total carbohydrates and starch content per culture and per cell was obtained after 24h; only jointly immobilized C. vulgaris growing on d-glucose significantly increased total carbohydrates and starch content after 96 h. Enhanced accumulation of carbohydrate and starch under jointly immobilized conditions was variable with time of sampling and substrate used. Similar results occurred when the microalgae was immobilized alone. In both microalgae growing on either carbon sources, the bacterium promoted accumulation of carbohydrates and starch; when the microalgae were immobilized alone, they used the carbon sources for cell multiplication. In jointly immobilized conditions with Chlorella spp., affinity to carbon source and volumetric productivity and yield were higher than when Chlorella spp. were immobilized alone; however, the growth rate was higher in microalgae immobilized alone. This study demonstrates that under heterotrophic conditions, A. brasilense promotes the accumulation of carbohydrates in two strains Chlorella spp. under certain time-substrate combinations, producing mainly starch. As such, this bacterium is a biological factor that can change the composition of compounds in microalgae in dark, heterotrophic conditions.

  8. Microfluidic one-step synthesis of alginate microspheres immobilized with antibodies

    PubMed Central

    Chen, Wanyu; Kim, Jong-Hoon; Zhang, Di; Lee, Kyong-Hoon; Cangelosi, G. A.; Soelberg, S. D.; Furlong, C. E.; Chung, Jae-Hyun; Shen, Amy Q.

    2013-01-01

    Micrometre- and submicrometre-size functionalized beads are frequently used to capture targets of interest from a biological sample for biological characterizations and disease diagnosis. The main challenge of the microbead-based assay is in the immobilization of probe molecules onto the microbead surfaces. In this paper, we report a versatile droplet microfluidics method to fabricate alginate microspheres while simultaneously immobilizing anti-Mycobacterium tuberculosis complex IgY and anti-Escherichia coli IgG antibodies primarily on the porous alginate carriers for specific binding and binding affinity tests. The binding affinity of antibodies is directly measured by fluorescence intensity of stained target bacteria on the microspheres. We demonstrate that the functionalized alginate microspheres yield specificity comparable with an enzyme-linked immunosorbent assay. The high surface area-to-volume ratio of the functionalized porous alginate microspheres improves the detection limit. By using the droplet microfluidics, we can easily modify the size and shape of alginate microspheres, and increase the concentration of functionalized alginate microspheres to further enhance binding kinetics and enable multiplexing. PMID:23966617

  9. Microfluidic one-step synthesis of alginate microspheres immobilized with antibodies.

    PubMed

    Chen, Wanyu; Kim, Jong-Hoon; Zhang, Di; Lee, Kyong-Hoon; Cangelosi, G A; Soelberg, S D; Furlong, C E; Chung, Jae-Hyun; Shen, Amy Q

    2013-11-01

    Micrometre- and submicrometre-size functionalized beads are frequently used to capture targets of interest from a biological sample for biological characterizations and disease diagnosis. The main challenge of the microbead-based assay is in the immobilization of probe molecules onto the microbead surfaces. In this paper, we report a versatile droplet microfluidics method to fabricate alginate microspheres while simultaneously immobilizing anti-Mycobacterium tuberculosis complex IgY and anti-Escherichia coli IgG antibodies primarily on the porous alginate carriers for specific binding and binding affinity tests. The binding affinity of antibodies is directly measured by fluorescence intensity of stained target bacteria on the microspheres. We demonstrate that the functionalized alginate microspheres yield specificity comparable with an enzyme-linked immunosorbent assay. The high surface area-to-volume ratio of the functionalized porous alginate microspheres improves the detection limit. By using the droplet microfluidics, we can easily modify the size and shape of alginate microspheres, and increase the concentration of functionalized alginate microspheres to further enhance binding kinetics and enable multiplexing.

  10. Development of a new antibacterial biomaterial by tetracycline immobilization on calcium-alginate beads.

    PubMed

    Ozseker, Emine Erdogan; Akkaya, Alper

    2016-10-20

    In recent years, increasing risk of infection, caused by resistant microorganism to antibiotics, has become the limelight discovery of new and natural antibacterial materials. Heavy metals, such as silver, copper, mercury and titanium, have antibacterial activity. Products, which improved these metals, do not have stable antibacterial property. Therefore, use of these products is restricted. The aim of this study was to immobilize tetracycline to alginate and improve an antibacterial biomaterial. For this purpose, calcium-alginate beads were formed by dropping to calcium-chloride solution and tetracycline was immobilized to beads using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at optimum conditions. After immobilization, actualization of immobilization was investigated by analyzing ATR-FTIR spectrum and SEM images. Also, antibacterial property of obtained product was tested. Improved product demonstrated antibacterial property. It has potential for open wound, surgical drapes, bed and pillow sheath in hospitals and it may also be used for increasing human comfort in daily life. PMID:27474587

  11. Development of a new antibacterial biomaterial by tetracycline immobilization on calcium-alginate beads.

    PubMed

    Ozseker, Emine Erdogan; Akkaya, Alper

    2016-10-20

    In recent years, increasing risk of infection, caused by resistant microorganism to antibiotics, has become the limelight discovery of new and natural antibacterial materials. Heavy metals, such as silver, copper, mercury and titanium, have antibacterial activity. Products, which improved these metals, do not have stable antibacterial property. Therefore, use of these products is restricted. The aim of this study was to immobilize tetracycline to alginate and improve an antibacterial biomaterial. For this purpose, calcium-alginate beads were formed by dropping to calcium-chloride solution and tetracycline was immobilized to beads using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at optimum conditions. After immobilization, actualization of immobilization was investigated by analyzing ATR-FTIR spectrum and SEM images. Also, antibacterial property of obtained product was tested. Improved product demonstrated antibacterial property. It has potential for open wound, surgical drapes, bed and pillow sheath in hospitals and it may also be used for increasing human comfort in daily life.

  12. Effects of Lactobacillus plantarum immobilization in alginate coated with chitosan and gelatin on antibacterial activity.

    PubMed

    Trabelsi, Imen; Ayadi, Dorra; Bejar, Wacim; Bejar, Samir; Chouayekh, Hichem; Ben Salah, Riadh

    2014-03-01

    The present study aimed to investigate and evaluate the efficiency of immobilizing the Lactobacillus plantarum TN9 strain in alginate using chitosan and gelatin as coating materials, in terms of viability and antibacterial activity. The results indicate that maximum concentrations of L. plantarum TN9 strain were produced with 2% sodium alginate, 10(8)UFC/ml, and 1M calcium chloride. The viability and antibacterial activity of the L. plantarum TN9 cultures before and after immobilization in alginate, chitosan-coated alginate, and gelatin-coated alginate, were studied. The findings revealed that the viability of encapsulated L. plantarum could be preserved more than 5.8 log CFU/ml after 35 day of incubation at 4 °C, and no effects were observed when gelatin was used. The antibacterial activity of encapsulated L. plantarum TN9 against Gram-positive and Gram-negative pathogenic bacteria was enhanced in the presence of chitosan coating materials, and no activity was observed in the presence of gelatin. The effects of catalase and proteolytic enzymes on the culture supernatant of L. plantarum TN9 were also investigated, and the results suggested that the antibacterial activity observed was due to the production of organic acids. Taken together, the findings indicated that immobilization in chitosan enhanced the antibacterial activity of L. plantarum TN9 against several pathogenic bacteria. This encapsulated strain could be considered as a potential strong candidate for future application as an additive in the food and animal feed industries.

  13. Preparation and characterization of. beta. -D-glucosidase immobilized in calcium alginate

    SciTech Connect

    Krasniak, S. R.; Smith, R. D.

    1982-01-01

    Enzymatic hydrolysis of biomass to produce glucose may become feasible if an inexpensive method to reuse the enzyme can be found. This study investigated one such method whereby ..beta..-D-glucosidase (E.C. 3.2.1.21) was immobilized in calcium alginate gel spheres, which were shown to catalyze the hydrolysis of cellobiose to glucose. There was a loss of 49% of the enzyme from the alginate slurry during gelation. After gelation, in the stable gel spheres, there was a 37% retention of the enzyme activity that was actually immobilized. The reason for the loss in activity was investigated and may be caused by inhibition of the enzyme within the sphere by the calcium cations and the alginate anions also present. Mass transfer effects were minimal in this system and were not responsible for the activity loss.

  14. Natural impacted freshwaters: in situ use of alginate immobilized algae to the assessment of algal response.

    PubMed

    Corrêa, A X R; Tamanaha, M S; Horita, C O; Radetski, M R; Corrêa, R; Radetski, C M

    2009-05-01

    The objective of this study was to investigate the feasibility of an in situ phytotoxicity test using alginate-immobilized algae for 60 days, in the assessment of water quality in an impacted small peri-urban stream. After laboratory optimization of algae immobilization/de-immobilization processes, the performance of immobilized/de-immobilized algae was compared to the performance of free algae in terms of specific algal growth and sensitivity. This was done by comparing 72 h EC50 values obtained with zinc and the pesticides clomazone and carbofuran. The results showed a similar performance, which allow us to conclude that immobilization for 60 days do not cause any significant alteration in algae physiology. In the field, immobilized algae were exposed at different times (2, 4 and 7 days) to water samples in both disturbed and undisturbed sites. Both laboratory and field experiments indicated that alginate-immobilized algae for 60 days were sufficiently sensitive for use in the in situ assessment of water quality.

  15. Preparation and characterization of. beta. -D-glucosidase immobilized in calcium alginate

    SciTech Connect

    Woodward, J.; Krasniak, S.R.; Smith, R.D.; Spielberg, F.; Zachry, G.S.

    1982-01-01

    This study investigated the immobilization of ..beta..-D-glucosidase (E.C. 3.2.1.21) in calcium alginate gel spheres. The immobilized enzyme catalyzed the hydrolysis of cellobiose to glucose. During preparation of the enzyme-containing spheres, 49% of the initial activity was lost from the alginate slurry. There was a 37% retention of the enzyme activity that was actually immobilized within the spheres. This loss of activity upon immobilization may be caused by inhibition of the enzyme by calcium cations and alginate anions present in the gel. Mass transfer effects were apparently minimal in this system and were not responsible for the activity loss. Leakage of the enzyme from the spheres occurred during storage of the spheres occurred during storage of the spheres at 4/sup 0/C and during their incubation with stirring at 23/sup 0/C. Leakage was severe at pH 5.0 but could be prevented if the enzyme was treated with glutaraldehyde prior to immobilization.

  16. Immobilization of Chlorella sorokiniana GXNN 01 in alginate for removal of N and P from synthetic wastewater.

    PubMed

    Liu, Kai; Li, Jian; Qiao, Hongjin; Lin, Apeng; Wang, Guangce

    2012-06-01

    High costs and issues such as a high cell concentrations in effluents are encountered when utilizing microalgae for wastewater treatment. The present study analyzed nitrogen and phosphate removal under autotrophic, heterotrophic, mixotrophic and micro-aerobic conditions by Chlorella sorokiniana GXNN 01 immobilized in calcium alginate. The immobilized cells grew as well as free-living cells under micro-aerobic conditions and better than free-living cells under the other conditions. The immobilized cells had a higher ammonium removal rate (21.84%, 43.59% and 41.46%) than free living cells (14.35%, 38.57% and 40.59%) under autotrophic, heterotrophic, and micro-aerobic conditions, and higher phosphate removal rate (87.49%, 88.65% and 84.84%) than free living cells (20.21%, 42.27% and 53.52%) under heterotrophic, mixotrophic and micro-aerobic conditions, respectively. The data indicate that immobilized Chlorella sorokiniana GXNN 01 is a suitable species for use in wastewater treatment.

  17. Immobilization of thermoalkalophilic recombinant esterase enzyme by entrapment in silicate coated Ca-alginate beads and its hydrolytic properties.

    PubMed

    Gülay, Seçkin; Şanlı-Mohamed, Gülşah

    2012-04-01

    Thermoalkalophilic esterase enzyme from Balçova (Agamemnon) geothermal site were aimed to be immobilized effectively via a simple and cost-effective protocol in silicate coated Calcium alginate (Ca-alginate) beads by entrapment. The optimal immobilization conditions of enzyme in Ca-alginate beads were investigated and obtained with 2% alginate using 0.5mg/ml enzyme and 0.7 M CaCl(2) solution. In order to prevent enzyme from leaking out of the gel beads, Ca-alginate beads were then coated with silicate. Enzyme loading efficiency and immobilization yield for silicate coated beads was determined as 98.1% and 71.27%, respectively and compared with non-coated ones which were 68.5% and 45.80%, respectively. Surface morphologies, structure and elemental analysis of both silicate coated and non-coated alginate beads were also compared using Fourier Transform Infrared Spectroscopy (FT-IR) and Scanning Electron Microscope (SEM) equipped with Energy-dispersive X-ray spectroscopy (EDX). Moreover, silicate coated alginate beads enhanced reusability of esterase in continuous processes compared to non-coated beads. The hydrolytic properties of free and immobilized enzyme in terms of storage and thermal stability as well as the effects of the temperature and pH were determined. It was observed that operational, thermal and storage stabilities of the esterase were increased with immobilization. PMID:22309712

  18. Immobilization of thermoalkalophilic recombinant esterase enzyme by entrapment in silicate coated Ca-alginate beads and its hydrolytic properties.

    PubMed

    Gülay, Seçkin; Şanlı-Mohamed, Gülşah

    2012-04-01

    Thermoalkalophilic esterase enzyme from Balçova (Agamemnon) geothermal site were aimed to be immobilized effectively via a simple and cost-effective protocol in silicate coated Calcium alginate (Ca-alginate) beads by entrapment. The optimal immobilization conditions of enzyme in Ca-alginate beads were investigated and obtained with 2% alginate using 0.5mg/ml enzyme and 0.7 M CaCl(2) solution. In order to prevent enzyme from leaking out of the gel beads, Ca-alginate beads were then coated with silicate. Enzyme loading efficiency and immobilization yield for silicate coated beads was determined as 98.1% and 71.27%, respectively and compared with non-coated ones which were 68.5% and 45.80%, respectively. Surface morphologies, structure and elemental analysis of both silicate coated and non-coated alginate beads were also compared using Fourier Transform Infrared Spectroscopy (FT-IR) and Scanning Electron Microscope (SEM) equipped with Energy-dispersive X-ray spectroscopy (EDX). Moreover, silicate coated alginate beads enhanced reusability of esterase in continuous processes compared to non-coated beads. The hydrolytic properties of free and immobilized enzyme in terms of storage and thermal stability as well as the effects of the temperature and pH were determined. It was observed that operational, thermal and storage stabilities of the esterase were increased with immobilization.

  19. Mussel-inspired alginate gel promoting the osteogenic differentiation of mesenchymal stem cells and anti-infection.

    PubMed

    Zhang, Shiwen; Xu, Kaige; Darabi, Mohammad Ali; Yuan, Quan; Xing, Malcolm

    2016-12-01

    Alginate hydrogels have been used in cell encapsulation for many years but a prevalent issue with pure alginates is that they are unable to provide enough bioactive properties to interact with mammalian cells. This paper discusses the modification of alginate with mussel-inspired dopamine for cell loading and anti-infection. Mouse bone marrow stem cells were immobilized into alginate and alginate-dopamine beads and fibers. Through live-dead and MTT assay, alginates modified by dopamine promoted cell viability and proliferation. In vitro cell differentiation results showed that such an alginate-dopamine gel can promote the osteogenic differentiation of mesenchymal stem cell after PCR and ALP assays. In addition to that, the adhesive prosperities of dopamine allowed for coating the surface of alginate-dopamine gel with silver nanoparticles, which provided the gel with significant antibacterial characteristics. Overall, these results demonstrate that a dopamine-modified alginate gel can be a great tool for cell encapsulation to promote cell proliferation and can be applied to bone regeneration, especially in contaminated bone defects. PMID:27612740

  20. High-rate ferrous iron oxidation by immobilized Acidithiobacillus ferrooxidans with complex of PVA and sodium alginate.

    PubMed

    Yujian, Wang; Xiaojuan, Yang; Wei, Tu; Hongyu, Li

    2007-02-01

    By four different methods, Acidithiobacillus ferrooxidans cells were immobilized by the complex of PVA and sodium alginate. The beads formed by these different methods were evaluated in terms of relative mechanical strength, biological activity, dilatability, and so on. The results indicate that the technique utilizing the complex of PVA and sodium alginate crosslinked with Ca(NO(3))(2) is more appropriate for the immobilization of A. ferrooxidans than any others. So the PVA-calcium nitrate beads were used in batch and continuous culture. A maximum ferrous iron oxidation rate of 4.6 g/l/h was achieved in batch culture. Long-time performance of packed-bed bioreactor was evaluated systematically over 40 days, depending on the conversion ratio of ferrous iron and the residence time. At a residence time of 2.5 h, 96% of the initial ferrous iron was oxidized. This study shows this new immobilization technique will be a feasible and economical method for A. ferrooxidans.

  1. Biological deterioration of alginate beads containing immobilized microalgae and bacteria during tertiary wastewater treatment.

    PubMed

    Cruz, Ivonne; Bashan, Yoav; Hernàndez-Carmona, Gustavo; de-Bashan, Luz E

    2013-11-01

    Secondary treatment of municipal wastewater affects the mechanical stability of polymer Ca-alginate beads containing the microalgae Chlorella vulgaris that are jointly immobilized with Azospirillum brasilense as treating agents whose presence do not affect bead stability. Nine strains of potential alginate-degrading bacteria were isolated from wastewater and identified, based on their nearly complete 16S rDNA sequence. Still, their population was relatively low. Attempts to enhance the strength of the beads, using different concentrations of alginate and CaCl2 or addition of either of three polymers (polyvinylpyrrolidone, polyvinyl alcohol, carboxymethylcellulose), CaCO3, or SrCl2, failed. Beads lost their mechanical strength after 24 h of incubation but not the integrity of their shape for at least 96 h, a fact that sustained successful tertiary wastewater treatment for 48 h. In small bioreactors, removal of phosphorus was low under sterile conditions but high in unsterile wastewater. Alginate beads did not absorb PO4 (-3) in sterile wastewater, but in natural wastewater, they contained PO4 (-3). Consequently, PO4 (-3) content declined in the wastewater. A supplement of 10 % beads (w/v) was significantly more efficient in removing nutrients than 4 %, especially in a jointly immobilized treatment where >90 % of PO4 (-3) and >50 % ammonium were removed. Tertiary wastewater treatment in 25-L triangular, airlift, autotrophic bioreactors showed, as in small bioreactors, very similar nutrient removal patterns, decline in bead strength phenomena, and increase in total bacteria during the wastewater treatment only in the presence of the immobilized treatment agents. This study demonstrates that partial biological degradation of alginate beads occurred during tertiary wastewater treatment, but the beads survive long enough to permit efficient nutrient removal. PMID:23354446

  2. Reduction of Brochothrix thermosphacta on beef surfaces following immobilization of nisin in calcium alginate gels.

    PubMed

    Cutter, C N; Siragusa, G R

    1996-07-01

    Lean and adipose beef carcass tissues inoculated with Brochothrix thermosphacta (BT) (approx. 4.50 log10 cfu cm-2) were left untreated (U) or treated with 100 micrograms ml-1 nisin (N), calcium alginate (A) or 100 micrograms ml-1 nisin immobilized in a calcium alginate gel (AN). Tissue samples were refrigerated after treatments and bacterial populations and nisin activity were determined at 0, 1, 2 and 7 d. U, A and N treatments of lean and adipose tissues did not suppress bacterial growth ( > 6 log10 cfu cm-2 by day 7) while treatments of lean and adipose tissues with AN suppressed bacteria ( > 2.42 log10 cfu cm-2 by day 7). Bacteriocin titres from both tissues were higher in AN vs N samples after the 7 d incubation. This study demonstrates that immobilization of nisin in a gel may be a more effective delivery system of a bacteriocin to the carcass surface than direct application.

  3. Immobilization of alginate-encapsulated Bacillus thuringiensis var. israelensis containing different multivalent counterions for mosquito control.

    PubMed

    Prabakaran, G; Hoti, S L

    2008-08-01

    Immobilized techniques have been used widely for the controlled release formulation of mosquitoes. Among the microbial formulations, polymeric matrices play an important role in the controlled release of microbial pesticide at rates sufficiently effective to kill mosquitoes in the field. The advantage of these matrices is that they enhance the stability of both spores and toxin against pH, temperature variations, and UV irradiation. The disadvantage of using calcium alginate beads is that they are unstable upon contact with phosphate of potassium or sodium ions rich in the mosquito habitats. To overcome these problems, attempts were made to encapsulate Bacillus thuringiensis var. israelensis within alginate by using different multivalent counterions, namely, calcium chloride, zinc sulfate, copper sulfate, cobalt chloride, and ferric chloride, and the beads formed were tested for its mosquito larvicidal activity. Among all the beads tested, zinc alginate beads resulted in maximum larvicidal activity of 98% (+/-1.40 SE) against Culex quinquefasciatus IIIrd instar larvae and maximum spore count of 3.36 x 10(5) (+/-5291.50 SE) CFU/ml. Zinc alginate beads maintained their structure for up to 48 h when shaken vigorously on a rotary shaker at 180 rpm in the presence of 10 mM potassium phosphate buffer (pH 6.8 +/- 0.1). In conclusion, our results suggest that the use of zinc sulfate as counterions to encapsulate B. thuringiensis var. israelensis within alginate may be a potent mosquito control program in the habitats where more phosphate ions are present.

  4. Removal and recovery of uranium from aqueous solutions by Ca-alginate immobilized Trichoderma harzianum.

    PubMed

    Akhtar, K; Khalid, A M; Akhtar, M W; Ghauri, M A

    2009-10-01

    The ability of Ca-alginate immobilized Trichoderma harzianum has been explored for removal and recovery of uranium from aqueous streams. Ca-alginate as polymeric support was selected after screening different matrices. Immobilization of Trichoderma harzianum to Ca-alginate improved the stability as well as uranium biosorption capacity of biosorbent at 28+/-2 degrees C and 200 rpm. The suitability of packed bed column operations was illustrated by obtaining break through curves at different bed heights, flow rates and inlet uranium concentrations. The adsorption column containing 1.5 g dry weight of immobilized material has purified 8.5L of bacterial leach liquor (58 mg/LU) before break through occurred and the biosorbent became saturated after 25 L of influent. Sorbed uranium was recovered in 200 ml of 0.1N HCl resulting in 98.1-99.3% elution by 0.1N HCl, which regenerated the biosorbent facilitating the sorption-desorption cycles for better economic feasibility without any significant alteration in sorption capacity/elution efficiency.

  5. Removal and recovery of uranium from aqueous solutions by Ca-alginate immobilized Trichoderma harzianum.

    PubMed

    Akhtar, K; Khalid, A M; Akhtar, M W; Ghauri, M A

    2009-10-01

    The ability of Ca-alginate immobilized Trichoderma harzianum has been explored for removal and recovery of uranium from aqueous streams. Ca-alginate as polymeric support was selected after screening different matrices. Immobilization of Trichoderma harzianum to Ca-alginate improved the stability as well as uranium biosorption capacity of biosorbent at 28+/-2 degrees C and 200 rpm. The suitability of packed bed column operations was illustrated by obtaining break through curves at different bed heights, flow rates and inlet uranium concentrations. The adsorption column containing 1.5 g dry weight of immobilized material has purified 8.5L of bacterial leach liquor (58 mg/LU) before break through occurred and the biosorbent became saturated after 25 L of influent. Sorbed uranium was recovered in 200 ml of 0.1N HCl resulting in 98.1-99.3% elution by 0.1N HCl, which regenerated the biosorbent facilitating the sorption-desorption cycles for better economic feasibility without any significant alteration in sorption capacity/elution efficiency. PMID:19467596

  6. Enzymatic cellulose hydrolysis: enzyme reusability and visualization of β-glucosidase immobilized in calcium alginate.

    PubMed

    Tsai, Chien-Tai; Meyer, Anne S

    2014-01-01

    The high cellulase enzyme dosages required for hydrolysis of cellulose is a major cost challenge in lignocellulosic ethanol production. One method to decrease the enzyme dosage and increase biocatalytic productivity is to re-use β-glucosidase (BG) via immobilization. In the present research, glutaraldehyde cross-linked BG was entrapped in calcium alginate gel particles. More than 60% of the enzyme activity could be recovered under optimized conditions, and glutaraldehyde cross-linking decreased leakage of BG from the calcium alginate particles. The immobilized BG aggregates were visualized by confocal laser scanning microscopy (CLSM). The CLSM images, which we believe are the first to be published, corroborate that more BG aggregates were entrapped in the matrix when the enzymes were cross-linked by glutaraldehyde as opposed to when they are not cross-linked. The particles with the immobilized BG were recycled for cellulase catalyzed hydrolysis of Avicel. No significant loss in BG activity was observed for up to 20 rounds of reaction recycle steps of the BG particles of 48 h each, verifying a significant stabilization of the BG by immobilization. Similar high glucose yields were obtained by one round of enzymatic hydrolysis of hydrothermally pretreated barley straw during a 72 h reaction with immobilized BG and free BG. PMID:25429563

  7. Enzymatic cellulose hydrolysis: enzyme reusability and visualization of β-glucosidase immobilized in calcium alginate.

    PubMed

    Tsai, Chien-Tai; Meyer, Anne S

    2014-11-25

    The high cellulase enzyme dosages required for hydrolysis of cellulose is a major cost challenge in lignocellulosic ethanol production. One method to decrease the enzyme dosage and increase biocatalytic productivity is to re-use β-glucosidase (BG) via immobilization. In the present research, glutaraldehyde cross-linked BG was entrapped in calcium alginate gel particles. More than 60% of the enzyme activity could be recovered under optimized conditions, and glutaraldehyde cross-linking decreased leakage of BG from the calcium alginate particles. The immobilized BG aggregates were visualized by confocal laser scanning microscopy (CLSM). The CLSM images, which we believe are the first to be published, corroborate that more BG aggregates were entrapped in the matrix when the enzymes were cross-linked by glutaraldehyde as opposed to when they are not cross-linked. The particles with the immobilized BG were recycled for cellulase catalyzed hydrolysis of Avicel. No significant loss in BG activity was observed for up to 20 rounds of reaction recycle steps of the BG particles of 48 h each, verifying a significant stabilization of the BG by immobilization. Similar high glucose yields were obtained by one round of enzymatic hydrolysis of hydrothermally pretreated barley straw during a 72 h reaction with immobilized BG and free BG.

  8. Continuous bioconversion of starch to ethanol by calcium-alginate immobilized enzymes and yeasts

    SciTech Connect

    McGhee, J.E.; Carr, M.E.; St. Julian, G.

    1984-01-01

    Continuous bioconversion of starch to EtOH by immobilized enzymes and yeasts was studied. Commercial corn starch (10%) was 1st batch-liquefied with bacterial alpha-amylase. In continuous-flow systems, liquefied starch was then converted to glucose with Ca alginate-entrapped fungal glucoamylase, and the resulting glucose was fermented to EtOH by Ca alginate-entrapped active dry yeast. The continuous-flow saccharification-fermentation processes were performed in either 2-stage (sequential) or single-stage (simultaneous) operations. In the single-stage operation, immobilized glucoamylase produced glucose from liquefied starch continuously for 11 days. In the simultaneous saccharification technique using immobilized glucoamylase and yeast mixture in a single-stage column, EtOH production was 69% of theoretical for 5 days. In the 2-stage operation, in which immobilized glucoamylase and yeast were contained in separate columns connected in tandem, EtOH production averaged 97% of theoretical for 5 days. The overall alcoholic production efficiency was significantly greater in the 2-stage system than in the single-stage system.

  9. Degradation of complex carbohydrate: immobilization of pectinase from Bacillus licheniformis KIBGE-IB21 using calcium alginate as a support.

    PubMed

    Rehman, Haneef Ur; Aman, Afsheen; Silipo, Alba; Qader, Shah Ali Ul; Molinaro, Antonio; Ansari, Asma

    2013-08-15

    Pectinases are heterogeneous group of enzymes that catalyse the hydrolysis of pectin substances which is responsible for the turbidity and undesirable cloudiness in fruits juices. In current study, partially purified pectinase from Bacillus licheniformis KIBGE-IB21 was immobilized in calcium alginate beads. The effect of sodium alginate and calcium chloride concentration on immobilization was studied and it was found that the optimal sodium alginate and calcium chloride concentration was 3.0% and 0.2 M, respectively. It was found that immobilization increases the optimal reaction time for pectin degradation from 5 to 10 min and temperature from 45 to 55°C, whereas, the optimal pH remained same with reference to free enzyme. Thermal stability of enzyme increased after immobilization and immobilized pectinase retained more than 80% of its initial activity after 5 days at 30°C as compared with free enzyme which showed only 30% of residual activity. The immobilized enzyme also exhibited good operational stability and 65% of its initial activity was observed during third cycle. In term of pectinase immobilization efficiency and stability, this calcium alginate beads approach seemed to permit good results and can be used to make a bioreactor for various applications in food industries.

  10. Influence of immobilization parameters on growth and lactic acid production by Streptococcus thermophilus and Lactobacillus bulgaricus co-immobilized in calcium alginate gel beads.

    PubMed

    Garbayo, I; Vílchez, C; Vega, J M; Nava-Saucedo, J E; Barbotin, J N

    2004-12-01

    Streptococcus thermophilus and Lactobacillus bulgaricus were co-immobilized in different systems with varying calcium (0.1-1.5 M) and alginate (1-2%, w/v) concentrations. Highest lactic acid production was 35 g l(-1) when both bacteria were in high viscosity beads (1%, w/v alginate) hardened in 0.1 M CaCl2 . The gel bead composition affected size and distribution of entrapped lactic acid bacteria.

  11. Rapid degradation of phenanthrene by using Sphingomonas sp. GY2B immobilized in calcium alginate gel beads.

    PubMed

    Tao, Xue-Qin; Lu, Gui-Ning; Liu, Jie-Ping; Li, Ting; Yang, Li-Ni

    2009-09-01

    The strain Sphingomonas sp. GY2B is a high efficient phenanthrene-degrading strain isolated from crude oil contaminated soils that displays a broad-spectrum degradation ability towards PAHs and related aromatic compounds. This paper reports embedding immobilization of strain GY2B in calcium alginate gel beads and the rapid degradation of phenanthrene by the embedded strains. Results showed that embedded immobilized strains had high degradation percentages both in mineral salts medium (MSM) and 80% artificial seawater (AS) media, and had higher phenanthrene degradation efficiency than the free strains. More than 90% phenanthrene (100 mg x L(-1)) was degraded within 36 h, and the phenanthrene degradation percentages were >99.8% after 72 h for immobilized strains. 80% AS had significant negative effect on the phenanthrene degradation rate (PDR) of strain GY2B during the linear-decreasing stage of incubation and preadsorption of cells onto rice straw could improve the PDR of embedded strain GY2B. The immobilization of strain GY2B possesses a good potential for application in the treatment of industrial wastewater containing phenanthrene and other related aromatic compounds.

  12. Thallium(I) sorption using Prussian blue immobilized in alginate capsules.

    PubMed

    Vincent, Thierry; Taulemesse, Jean-Marie; Dauvergne, Agnès; Chanut, Thomas; Testa, Flaviano; Guibal, Eric

    2014-01-01

    Prussian blue (PB) was immobilized in alginate capsules. The composite sorbent was used for the recovery of Tl(I) ions from slightly acidic solutions: optimum pH being close to 4. The sorption isotherm can be described by the bi-site Langmuir sorption isotherm. This means that the metal ion can be bound through two different sorption sites: one having a strong affinity for Tl(I) (probably PB), the other having a lower affinity (probably the encapsulating material). The kinetics are described by either the pseudo-second order rate equation or the Crank's equation (resistance to intraparticle diffusion). The ionic strength (increased by addition of NaCl, KCl or CaCl₂) slightly decreased sorption capacity. The SEM-EDX analysis of PB-alginate capsules (before and after Tl(I) sorption) shows that the PB is homogeneously distributed in the capsules and that all reactive groups remain available for metal binding.

  13. Preparation of core-shell magnetic polydopamine/alginate biocomposite for Candida rugosa lipase immobilization.

    PubMed

    Hou, Chen; Qi, Zhigang; Zhu, Hao

    2015-04-01

    A flexible, biocompatible and bioadhesive enzyme immobilizing material, which was synthesized based on the covalent assembly of biomimetic polymer and oxidized polysaccharide on magnetic nanoparticles (NPs), has been developed in this feasibility study. In this work, the bio-inspired polymer, polydopamine (PDA), was used to modify the well-monodispersed Fe3O4 NPs (mPDA NPs) with a controllable thickness via a dip-coating process, then the alginate di-aldehyde (ADA) was covalently assembled on the mPDA NPs and employed as a naturally occurring linking agent for Candida rugosa lipase (CRL) immobilization. The resulting support material was characterized by means of the transmission electron microscope (TEM), Fourier transform infrared spectra (FT-IR), X-ray diffraction (XRD), thermogravimetry (TG) analyser, and vibrating sample magnetometer (VSM). It was verified that the prepared mPDA NPs possessed distinct core-shell structure with uniform size and high saturation magnetization. For further application, the mPDA NPs was utilized in CRL immobilizing procedures and demonstrated can facilitate improving the enzyme activities. The optimum amount of lipase was 200 mg g(-1) support, the optimal pH and temperature for the catalyse condition of the immobilized CRL was 7.0 and 40°C, respectively. Moreover, the immobilized CRL kept the high activity at 77% after 12 times of recycling for batch hydrolysis of olive oil emulsion. This magnetic bioadhesive composite with functionalized properties and adhesion strength presents a general strategy for the immobilization of macromolecules.

  14. Biophotolytic H2 production using alginate-immobilized chloroplasts, enzymes and synthetic catalysts

    NASA Astrophysics Data System (ADS)

    Gisby, P. E.; Hall, D. O.

    1980-09-01

    Hydrogen can be produced by illumination of an aqueous mixture of chloroplasts and hydrogenase, in the presence of an electron carrier. This system may have potential for development of a solar converter to produce hydrogen from water if it can be stabilized or constructed as a completely synthetic system. The immobilization of the chloroplasts, or membrane analogues, would make possible a one-stage reactor with all the components in one chamber, or a two-stage reactor if the electron carrier was passed to another chamber to react with an immobilized hydrogen-producing catalyst. However, techniques for immobilizing enzymes tend to yield immobilized chloroplasts that are not very active, and other methods must be used. This paper describes the immobilization of chloroplasts using calcium alginate gels on reinforcing grids of nylon and stainless steel. Chloroplasts thus immobilized are fully active and can be used to produce hydrogen gas. Strengthened films of this sort could provide a good, solid, rigid matrix for a solar converter.

  15. Calcium alginate beads encapsulated PMMA-g-CS nano-particles for α-chymotrypsin immobilization.

    PubMed

    Abd El-Ghaffar, M A; Hashem, M S

    2013-02-15

    Chitosan grafted with polymethyl methacrylate (PMMA-g-CS) was prepared via a free-radicals polymerization technique as a carrier for enzyme immobilization. α-Chymotrypsin (CT), as an enzyme model in this study, was immobilized onto the prepared PMMA-g-CS via covalent bonding. Calcium alginate (CA) beads were developed for encapsulating PMMA-g-CS-CT to produce PMMA-g-CS-CT/CA composite beads. Morphology and size of PMMA-g-CS particles were investigated by TEM and found to be in the nanoscale. The structure and surface morphology of the beads before and after immobilization process were characterized by FT-IR and SEM, respectively. Both the bound CT content and relative activity of immobilized enzyme were measured. A higher retained activity (about 97.7%) obtained for the immobilized CT at pH 9 for 24 h. The results indicated that immobilized CT maintained excellent performance even after 25 reuses and retained 75% from its original activity after 60 days of storage at 25 °C.

  16. Cell immobilization technique for the enhanced production of alpha-galactosidase by Streptomyces griseoloalbus.

    PubMed

    Anisha, G S; Prema, P

    2008-06-01

    Streptomyces griseoloalbus was immobilized in calcium alginate gel and the optimal immobilization parameters (concentrations of sodium alginate and calcium chloride, initial biomass and curing time) for the enhanced production of alpha-galactosidase were determined. The immobilization was most effective with 3% sodium alginate and 0.1M calcium chloride. The optimal initial biomass for immobilization was approximately 2.2g (wet wt.). The alginate-entrapped cells were advantageous because there was a twofold increase in the enzyme yield (55 U/ml) compared to the highest yield obtained with free cells (23.6 U/ml). Moreover, with immobilized cells the maximum yield was reached after 72 h of incubation in batch fermentation under optimal conditions, whereas in the case of free cells the maximum enzyme yield was obtained only after 96 h of incubation. The alginate beads had good stability and also retained 75% ability of enzyme production even after eight cycles of repeated batch fermentation. It is significant that this is the first report on whole-cell immobilization for alpha-galactosidase production.

  17. Polyurethane and alginate immobilized algal biomass for the removal of aqueous toxic metals

    SciTech Connect

    Fry, I.V.; Mehlhorn, R.J.

    1992-12-01

    We describe the development of immobilized, processed algal biomass for use as an adsorptive filter in the removal of toxic metals from waste water. To fabricate an adsorptive filter from precessed biomass several crucial criteria must be met, including: (1) high metal binding capacity, (2) long term stability (both mechanical and chemical), (3) selectivity for metals of concern (with regard to ionic competition), (4) acceptable flow capacity (to handle large volumes in short time frames), (5) stripping/regeneration (to recycle the adsorptive filter and concentrate the toxic metals to manageable volumes). This report documents experiments with processed algal biomass (Spirulina platensis and Spirulina maxima) immobilized in either alginate gel or preformed polyurethane foam. The adsorptive characteristics of these filters were assessed with regard to the criteria listed above.

  18. Degradation of trichloroethylene (TCE) by nanoscale zero-valent iron (nZVI) immobilized in alginate bead.

    PubMed

    Kim, Hojeong; Hong, Hye-Jin; Jung, Juri; Kim, Seong-Hye; Yang, Ji-Won

    2010-04-15

    Nowadays, many researchers have studied the environmental application of the nanoscale zero-valent iron (nZVI) and several field applications for the groundwater remediation have been reported. Still, there are many concerns on the fate and transport of the nZVI and the corresponding risks. To avoid such concerns, it was investigated to immobilize nZVI in a support and then it was applied to degrade trichloroethylene (TCE). The nZVI and palladium-doped nZVI (Fe(0)- and Fe/Pd-alginate) were immobilized in the alginate bead where ferric and barium ions are used as the cross-linking cations of the bead. According to TEM (transmission electron microscopy), the size of the immobilized ZVI was as small as a few nanometers. From the surface analysis of the Fe/Pd-alginate, it is found that the immobilized nZVI has the core-shell structure. The core is composed of single crystal Fe(0), while most of irons on the surface are oxidized to Fe(3+). When 50 g/L of Fe/Pd-alginate (3.7 g Fe/L) was introduced to the aqueous solution, >99.8% of TCE was removed and the release of metal from the support was <3% of the loaded iron. The removal of TCE by Fe/Pd-alginate followed pseudo-first-order kinetics. The observed pseudo-first-order reaction constant (k(obs)) of Fe/Pd-alginate was 6.11 h(-1) and the mass normalized rate constant (k(m)) was 1.6 L h(-1) g(-1). The k(m) is the same order of magnitude with that of iron nanoparticles. In conclusion, it is considered that Fe/Pd-alginate can be used efficiently in the treatment of chlorinated solvent.

  19. Bioacetylation of Seaweed Alginate

    PubMed Central

    Lee, J. W.; Day, D. F.

    1995-01-01

    Seaweed alginate was acetylated by intact, resting cells of Pseudomonas syringae ATCC 19304. Maximum acetylation of this polymer occurred at a pH of 6.0 and a temperature of 25 deg C. Aeration and gluconic acid were required for an optimal reaction. A reactor which contained carbon-immobilized cells was constructed to continuously acetylate alginate. The maximal yield of acetylation was about 90%, and the half-life of this system was 6.5 days. PMID:16534934

  20. Adsorption and desorption of Zn(II) and Cu(II) on Ca- alginate immobilized activated rice bran

    NASA Astrophysics Data System (ADS)

    Suratman, A.; Kamalia, N. Z.; Kusumawati, W. A.

    2016-02-01

    Ca-alginate immobilized activated rice bran has been used for adsorption of Zn(II) and Cu(II) from aqueous solution. The effect of the pH, kinetics model, adsorption isotherm and desorption on the adsorption performance was investigated. Activated rice bran was immobilized by the entrapment in alginate beads. The adsorption strength of Ca-alginate immobilized activated rice bran was compared to Ca-alginate and non-immobilized activated rice bran. The concentrations of adsorbed ions were analyzed using Atomic Absorption Spectrophotometer (AAS). The result showed that pH of 4.0 and the contact time of 120 min are the optimum condition for adsorption of Zn(II) and Cu(II). The adsorption kinetic of Zn(II) and Cu(II) followed the pseudo-second-order model with adsorption rate constant 4.9 x 10-2 and 3.14 g.mg-1.min-1, respectively. The both adsorption processes obeyed Langmuir isotherm with adsorption capacity of 2.03 and 2.42 mg.g-1 of adsorbent, respectively. The strength of Zn adsorption on Ca-alginate immobilized activated rice bran (86.63%) was more effective compared to Ca-alginate beads (60.96%) and activated rice bran (43.85%). The strength of Cu adsorption was 80.00%, 61.50% and 22.10%, respectively. The desorption of Zn(II) and Cu(II) showed that recovery percentage of the adsorption was 76.56% and 57.80% with the condition of using HCl 0.1 M as desorption agent for 1 hour.

  1. Immobilization of coacervate microcapsules in multilayer sodium alginate beads for efficient oral anticancer drug delivery.

    PubMed

    Feng, Chao; Song, Ruixi; Sun, Guohui; Kong, Ming; Bao, Zixian; Li, Yang; Cheng, Xiaojie; Cha, Dongsu; Park, Hyunjin; Chen, Xiguang

    2014-03-10

    We have designed and evaluated coacervate microcapsules-immobilized multilayer sodium alginate beads (CMs-M-ALG-Beads) for oral drug delivery. The CMs-M-ALG-Beads were prepared by immobilization of doxorubicin hydrochloride (DOX) loaded chitosan/carboxymethyl coacervate microcapsules (DOX:CS/CMCS-CMs) in the core and layers of the multilayer sodium alginate beads. The obtained CMs-M-ALG-beads exhibited layer-by-layer structure and rough surface with many nanoscale particles. The swelling characteristic and drug release results indicated that 4-layer CMs-M-ALG-Beads possessed favorable gastric acid tolerance (the swelling rate <5%, the cumulative drug release rate <3.8%). In small intestine, the intact DOX:CS/CMCS-CMs were able to rapidly release from CMs-M-ALG-Beads with the dissolution of ALG matrix. Ex vivo intestinal mucoadhesive and permeation showed that CMs-M-ALG-Beads exhibited continued growth for P(app) values of DOX, which was 1.07-1.15 folds and 1.28-1.38 folds higher than DOX:CS:CMCS-CMs in rat jejunum and ileum, respectively, demonstrating that CMs-M-ALG-Beads were able to enhance the absorption of DOX by controlled releasing DOX:CS/CMCS-CMs and prolonging the contact time between the DOX:CS/CMCS-CMs and small intestinal mucosa.

  2. Removal of mercury (II) from aqueous solution using papain immobilized on alginate bead: optimization of immobilization condition and modeling of removal study.

    PubMed

    Bhattacharyya, Aparupa; Dutta, Susmita; De, Parameswar; Ray, Parthasarathi; Basu, Srabanti

    2010-12-01

    Papain having the characteristics of metal binding ability is immobilized on alginate bead. Design Expert Software (Version 7.1.6) uses Response Surface Methodology (RSM) for statistical designing of operating condition for immobilization of papain on alginate bead considering concentration of papain, concentration of sodium alginate, concentration of calcium chloride and pH as numeric factors and Specific Enzymatic Activity (SEA) of immobilized papain sample as response. Immobilization using 25.96 g/L papain, 20 g/L sodium alginate and 20 g/L calcium chloride at pH 7 gives the desired product as indicated by ANOVA (Analysis of Variance). Three parameters viz., initial concentration of mercury (II), amount of AIP and pH are varied in a systematic manner. Maximum 98.88% removal of mercury (II) has been achieved within 8 min when simulated aqueous solution of mercury (II) with initial concentration of 10mg/L has been contacted with 5 g of AIP at pH 9 and at 35 degrees C in a batch contactor. A mathematical model has been developed and the value of equilibrium constant for binding of mercury (II) with AIP has been found to be 126797.3. PMID:20696575

  3. Removal of mercury (II) from aqueous solution using papain immobilized on alginate bead: optimization of immobilization condition and modeling of removal study.

    PubMed

    Bhattacharyya, Aparupa; Dutta, Susmita; De, Parameswar; Ray, Parthasarathi; Basu, Srabanti

    2010-12-01

    Papain having the characteristics of metal binding ability is immobilized on alginate bead. Design Expert Software (Version 7.1.6) uses Response Surface Methodology (RSM) for statistical designing of operating condition for immobilization of papain on alginate bead considering concentration of papain, concentration of sodium alginate, concentration of calcium chloride and pH as numeric factors and Specific Enzymatic Activity (SEA) of immobilized papain sample as response. Immobilization using 25.96 g/L papain, 20 g/L sodium alginate and 20 g/L calcium chloride at pH 7 gives the desired product as indicated by ANOVA (Analysis of Variance). Three parameters viz., initial concentration of mercury (II), amount of AIP and pH are varied in a systematic manner. Maximum 98.88% removal of mercury (II) has been achieved within 8 min when simulated aqueous solution of mercury (II) with initial concentration of 10mg/L has been contacted with 5 g of AIP at pH 9 and at 35 degrees C in a batch contactor. A mathematical model has been developed and the value of equilibrium constant for binding of mercury (II) with AIP has been found to be 126797.3.

  4. Nanocellulose-alginate hydrogel for cell encapsulation.

    PubMed

    Park, Minsung; Lee, Dajung; Hyun, Jinho

    2015-02-13

    TEMPO-oxidized bacterial cellulose (TOBC)-sodium alginate (SA) composites were prepared to improve the properties of hydrogel for cell encapsulation. TOBC fibers were obtained using a TEMPO/NaBr/NaClO system at pH 10 and room temperature. The fibrillated TOBCs mixed with SA were cross-linked in the presence of Ca(2+) solution to form hydrogel composites. The compression strength and chemical stability of the TOBC/SA composites were increased compared with the SA hydrogel, which indicated that TOBC performed an important function in enhancing the structural, mechanical and chemical stability of the composites. Cells were successfully encapsulated in the TOBC/SA composites, and the viability of cells was investigated. TOBC/SA composites can be a potential candidate for cell encapsulation engineering. PMID:25458293

  5. Preparation of core-shell magnetic polydopamine/alginate biocomposite for Candida rugosa lipase immobilization.

    PubMed

    Hou, Chen; Qi, Zhigang; Zhu, Hao

    2015-04-01

    A flexible, biocompatible and bioadhesive enzyme immobilizing material, which was synthesized based on the covalent assembly of biomimetic polymer and oxidized polysaccharide on magnetic nanoparticles (NPs), has been developed in this feasibility study. In this work, the bio-inspired polymer, polydopamine (PDA), was used to modify the well-monodispersed Fe3O4 NPs (mPDA NPs) with a controllable thickness via a dip-coating process, then the alginate di-aldehyde (ADA) was covalently assembled on the mPDA NPs and employed as a naturally occurring linking agent for Candida rugosa lipase (CRL) immobilization. The resulting support material was characterized by means of the transmission electron microscope (TEM), Fourier transform infrared spectra (FT-IR), X-ray diffraction (XRD), thermogravimetry (TG) analyser, and vibrating sample magnetometer (VSM). It was verified that the prepared mPDA NPs possessed distinct core-shell structure with uniform size and high saturation magnetization. For further application, the mPDA NPs was utilized in CRL immobilizing procedures and demonstrated can facilitate improving the enzyme activities. The optimum amount of lipase was 200 mg g(-1) support, the optimal pH and temperature for the catalyse condition of the immobilized CRL was 7.0 and 40°C, respectively. Moreover, the immobilized CRL kept the high activity at 77% after 12 times of recycling for batch hydrolysis of olive oil emulsion. This magnetic bioadhesive composite with functionalized properties and adhesion strength presents a general strategy for the immobilization of macromolecules. PMID:25784302

  6. Degradation of h-acid by free and immobilized cells of Alcaligenes latus

    PubMed Central

    Usha, M.S.; Sanjay, M.K.; Gaddad, S.M.; Shivannavar, C.T.

    2010-01-01

    Alcaligenes latus, isolated from industrial effluent, was able to grow in mineral salts medium with 50 ppm (0.15 mM) of H-acid as a sole source of carbon. Immobilization of Alcaligenes latus in Ca-alginate and polyurethane foam resulted in cells embedded in the matrices. When free cells and immobilized cells were used for biodegradation studies at concentration ranging from 100 ppm (0.3 mM) to 500 ppm (1.15 mM) degradation rate was enhanced with immobilized cells. Cells immobilized in polyurethane foam showed 100% degradation up to 350 ppm (1.05 mM) and 57% degradation at 500 ppm (1.5 mM). Degradation rate of Ca-alginate immobilized cells was less as compared to that of polyurethane foam immobilized cells. With Ca-alginate immobilized cells 100% degradation was recorded up to 200 ppm (0.6 mM) of H-acid and only 33% degradation was recorded at 500 ppm (1.5 mM) of H-acid. Spectral analysis of the products after H-acid utilization showed that the spent medium did not contain any aromatic compounds indicating H-acid degradation by A. latus. PMID:24031573

  7. High pressure studies on hesperitin production with hesperidinase free and immobilized in calcium alginate beads

    NASA Astrophysics Data System (ADS)

    Furtado, Andreia; Rosário, Pedro M.; Calado, António R. T.; Alfaia, António J. I.; Ribeiro, Maria H. L.

    2012-03-01

    The use of high pressure for the enzymatic synthesis of pharmacologically interesting molecules is a very important tool. Hesperidin and hesperitin exhibit anti-inflammatory, antimicrobial, antioxidant, and anticarcinogenic properties and prevent bone loss. However, hesperidin has a low bioavailability compared with hesperitin, due to the rutinoside moiety attached to the flavonoid. The aim of this work was the enzymatic production of hesperitin from hesperidin (soluble and insoluble) with hesperidinase free and immobilized in Ca-alginate beads, under high pressure conditions. The work was focused on the optimization of enzyme activity, studying the effects: pressure (50-150 MPa), temperature (35-75 °C), concentration of substrate (100-800 mg/L), and immobilization of hesperidinase. An 18-fold increase in hesperidinase residual activity was observed under high pressure conditions of 100 MPa compared to 0.1 MPa. A higher specificity of the hydrolytic reaction under high pressure (100 MPa) with a two-and three-fold increase in the ratio K cat/K M (specificity constant) at 55 °C and 75 °C was observed. A two-fold increase in the maximum activity at 100 MPa was observed with immobilized hesperinase compared to 0.1 MPa. In the second reutilization, almost a four-fold increase was obtained under high pressure conditions in comparison to atmospheric pressure.

  8. Osteogenic behavior of alginate encapsulated bone marrow stromal cells: an in vitro study.

    PubMed

    Abbah, S A; Lu, W W; Chan, D; Cheung, K M C; Liu, W G; Zhao, F; Li, Z Y; Leong, J C Y; Luk, K D K

    2008-05-01

    Sodium alginate is a useful polymer for the encapsulation and immobilization of a variety of cells in tissue engineering because it is biocompatible, biodegradable and easy to process into injectable microbeads. Despite these properties, little is known of the efficacy of calcium cross-linked alginate gel beads as a biodegradable scaffold for osteogenic cell proliferation and differentiation. In this study, we investigated the ability of rabbit derived bone marrow cells (BMCs) to proliferate and differentiate in alginate microbeads and compared them with BMCs cultured in poly-L-lysine (PLL) coated microbeads and on conventional 2D plastic surfaces. Results show that levels of proliferation and differentiation in microbeads and on tissue culture plastics were comparable. Cell proliferation in microbeads however diminished after fortification with a coating layer of PLL. Maximum cell numbers observed were, 3.32 x 10(5) +/- 1.72 x 103; 3.11 x 10(5) +/- 1.52 x 10(3) and 3.28 x 10(5) +/- 1.21 x 10(3 ) for the uncoated, PLL coated and plastic surface groups respectively. Alkaline phosphatase and protein expressions reflected the stage of cell differentiation. We conclude that calcium cross-linked alginate microbeads can act as a scaffold for BMC proliferation and osteogenic differentiation and has potential for use as 3D degradable scaffold.

  9. Immobilization of tomato (Lycopersicon esculentum) pectinmethylesterase in calcium alginate beads and its application in fruit juice clarification.

    PubMed

    Bogra, Pushpa; Kumar, Ashwani; Kuhar, Kalika; Panwar, Surbhi; Singh, Randhir

    2013-11-01

    Clarity of fruit juices is desirable to maintain an aesthetically pleasing quality and international standards. The most commonly used enzymes in juice industries are pectinases. A partially-purified pectinmethylesterase from tomato was entrapped in calcium alginate beads and used for juice clarification. The activity yield was maximum at 1 % (w/v) CaCl2 and 2.5 % (w/v) alginate. The immobilized enzyme retained ~55 % of its initial activity (5.7 × 10(-2) units) after more than ten successive batch reactions. The Km, pH and temperature optima were increased after immobilization. The most effective clarification of fruit juice (%T620 ~60 %) by the immobilized enzyme was at 4 °C with a holding time of 20 min. The viscosity dropped by 56 % and the filterability increased by 260 %. The juice remains clear after 2 months of storage at 4 °C.

  10. Enhanced accumulation of starch and total carbohydrates in alginate-immobilized Chlorella spp. induced by Azospirillum brasilense: I. Autotrophic conditions.

    PubMed

    Choix, Francisco J; de-Bashan, Luz E; Bashan, Yoav

    2012-10-10

    The effect of the microalgae-growth promoting bacterium Azospirillum brasilense on accumulation of total carbohydrates and starch in two species of Chlorella (Chlorella vulgaris and Chlorella sorokiniana), when the bacterium and each microalga were jointly immobilized in alginate beads was studied under autotrophic conditions for 144 h in synthetic medium. The interaction of the bacterium with the microalgae enhanced accumulation of total carbohydrate and starch. Cells of Chlorella accumulated the highest amounts of carbohydrate after incubation for 24h. Yet, this did not coincide with the highest affinity and volumetric productivity measured in these cultures. However, after incubation for 72 h, mainly in jointly immobilized treatments of both microalgae species, the cultures reached their highest total carbohydrate content (mainly as starch) and also the highest affinity and volumetric productivity. These results demonstrate the potential of A. brasilense to affect carbohydrates and starch accumulation in Chlorella spp. when both microorganisms are co-cultured, which can be an important tool for applications of microalgae.

  11. Binary immobilization of tyrosinase by using alginate gel beads and poly(acrylamide-co-acrylic acid) hydrogels.

    PubMed

    Yahşi, Ayşe; Sahin, Ferat; Demirel, Gökhan; Tümtürk, Hayrettin

    2005-09-15

    The use of the immobilized and the stable enzymes has immense potential in the enzymatic analysis of clinical, industrial and environmental samples. However, their widespread uses are limited due to the high cost of their production. In this study, binary immobilization of tyrosinase by using Ca-alginate and poly(acrylamide-co-acrylic acid) [P(AAm-co-AA)] was investigated. Maximum reaction rate (Vmax) and Michaelis-Menten constant (Km) were determined for the free and binary immobilized enzymes. The effects of pH, temperature, storage stability, reuse number and thermal stability on the free and immobilized tyrosinase were also examined. For the free and binary immobilized enzymes on Ca-alginate and P(AAm-co-AA), optimum pH was found to be 7 and 5, respectively. Optimum temperature of the free and immobilized enzymes was observed to be 30 and 35 degrees C, respectively. Reuse number, storage and thermal stability of the free tyrosinase were increased by a result of binary immobilization.

  12. Immobilization of a thermostable alpha-amylase by covalent binding to an alginate matrix increases high temperature usability.

    PubMed

    Tee, Boon L; Kaletunç, Gönül

    2009-01-01

    Thermostable alpha-amylase was covalently bound to calcium alginate matrix to be used for starch hydrolysis at liquefaction temperature of 95 degrees C. 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride (EDAC) was used as crosslinker. EDAC reacts with the carboxylate groups on the calcium alginate matrix and the amine groups of the enzyme. Ethylenediamine tetraacetic acid (EDTA) treatment was applied to increase the number of available carboxylate groups on the calcium alginate matrix for EDAC binding. After the immobilization was completed, the beads were treated with 0.1 M calcium chloride solution to reinstate the bead mechanical strength. Enzyme loading efficiency, activity, and reusability of the immobilized alpha-amylase were investigated. Covalently bound thermostable alpha-amylase to calcium alginate produced a total of 53 g of starch degradation/mg of bound protein after seven consecutive starch hydrolysis cycles of 10 min each at 95 degrees C in a stirred batch reactor. The free and covalently bound alpha-amylase had maximum activity at pH 5.5 and 6.0, respectively. The Michaelis-Menten constant (K(m)) of the immobilized enzyme (0.98 mg/mL) was 2.5 times greater than that of the free enzyme (0.40 mg/mL). The maximum reaction rate (V(max)) of immobilized and free enzyme were determined to be 10.4-mg starch degraded/mL min mg bound protein and 25.7-mg starch degraded/mL min mg protein, respectively. The high cumulative activity and seven successive reuses obtained at liquefaction temperature make the covalently bound thermostable alpha-amylase to calcium alginate matrix, a promising candidate for use in industrial starch hydrolysis process. PMID:19353735

  13. Immobilization of a thermostable alpha-amylase by covalent binding to an alginate matrix increases high temperature usability.

    PubMed

    Tee, Boon L; Kaletunç, Gönül

    2009-01-01

    Thermostable alpha-amylase was covalently bound to calcium alginate matrix to be used for starch hydrolysis at liquefaction temperature of 95 degrees C. 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride (EDAC) was used as crosslinker. EDAC reacts with the carboxylate groups on the calcium alginate matrix and the amine groups of the enzyme. Ethylenediamine tetraacetic acid (EDTA) treatment was applied to increase the number of available carboxylate groups on the calcium alginate matrix for EDAC binding. After the immobilization was completed, the beads were treated with 0.1 M calcium chloride solution to reinstate the bead mechanical strength. Enzyme loading efficiency, activity, and reusability of the immobilized alpha-amylase were investigated. Covalently bound thermostable alpha-amylase to calcium alginate produced a total of 53 g of starch degradation/mg of bound protein after seven consecutive starch hydrolysis cycles of 10 min each at 95 degrees C in a stirred batch reactor. The free and covalently bound alpha-amylase had maximum activity at pH 5.5 and 6.0, respectively. The Michaelis-Menten constant (K(m)) of the immobilized enzyme (0.98 mg/mL) was 2.5 times greater than that of the free enzyme (0.40 mg/mL). The maximum reaction rate (V(max)) of immobilized and free enzyme were determined to be 10.4-mg starch degraded/mL min mg bound protein and 25.7-mg starch degraded/mL min mg protein, respectively. The high cumulative activity and seven successive reuses obtained at liquefaction temperature make the covalently bound thermostable alpha-amylase to calcium alginate matrix, a promising candidate for use in industrial starch hydrolysis process.

  14. Immobilization of cellulase from newly isolated strain Bacillus subtilis TD6 using calcium alginate as a support material.

    PubMed

    Andriani, Dian; Sunwoo, Changshin; Ryu, Hwa-Won; Prasetya, Bambang; Park, Don-Hee

    2012-01-01

    Bacillus subtilis TD6 was isolated from Takifugu rubripes, also known as puffer fish. Cellulase from this strain was partially purified by ammonium sulphate precipitation up to 80% saturation, entrapped in calcium alginate beads, and finally characterized using CMC as the substrate. For optimization, various parameters were observed, including pH maximum, temperature maximum, sodium alginate, and calcium chloride concentration. pH maximum of the enzyme showed no changes before and after immobilization and remained stable at 6.0. The temperature maximum showed a slight increase to 60 °C. Two percent sodium alginate and a 0.15 M calcium chloride solution were the optimum conditions for acquisition of enzyme with greater stability. K (m) and V (max) values for the immobilized enzyme were slightly increased, compared with those of free enzyme, 2.9 mg/ml and 32.1 μmol/min/mL, respectively. As the purpose of immobilization, reusability and storage stability of the enzyme were also observed. Immobilized enzyme retained its activity for a longer period of time and can be reused up to four times. The storage stability of entrapped cellulase at 4 °C was found to be up to 12 days, while at 30 °C, the enzyme lost its activity within 3 days.

  15. Degradation of mix hydrocarbons by immobilized cells of mix culture using a trickle fluidized bed reactor

    SciTech Connect

    Chapatwala, K.D.

    1993-01-01

    The microorganisms, capable of degrading mix hydrocarbons were isolated from the soil samples collected from the hydrocarbon contaminated sites. The mix cultures were immobilized in calcium alginate solution in the form of beads. A trickle fluidized bed air-uplift-type reactor designed to study the degradation of mix hydrocarbons was filled with 0.85% normal saline containing the immobilized cells of mix culture. The immobilized beads were aerated with CO[sub 2]-free air at 200 ml/min. The degradation of different concentrations of hydrocarbons in the presence/absence of commercially available fertilizers by the immobilized cells of mix culture is now in progress.

  16. Dried calcium alginate/magnetite spheres: a new support for chromatographic separations and enzyme immobilization

    SciTech Connect

    Burns, M.A.; Kvesitadze, G.I.; Graves, D.J.

    1985-02-01

    Dried spheres made from an alginate solution containing magnetite particles have excellent potential as a support for enzyme immobilization and chromatographic applications. The beads were found to be much stronger than gels such as polyacrylamide and dextran, indicating that high flow rates and pressures could be used in column separations. The support withstood not only temperatures of up to 120/sup 0/C, but also most pH values and common solvents. While some solutions, such as phosphate buffers, dissolved the spheres, stabilization with Tyzor TE eliminated this problem. The physical properties of the beads include a glasslike density of 2.2 g/mL, excellent sphericity, low porosity, and a narrow size distribution. The magnetite present in the support allows the beads to be used for magnetic separations such as high gradient magnetic filtration. Their high degree of microroughness provides a large exposed surface area for enzyme and ligand binding. Mixed Actinomyces fradiae proteases and Aspergillus niger ..cap alpha..-amylase, two enzymes representative of classes which attack large substrates, were immobilized on the bead's surface with high activity and stability. A cyanuric dye which can be used in chromatographic applications (Cibacron Blue F3GA) was also readily coupled to the surface of this support with good yield.

  17. Combined physical and chemical immobilization of glucose oxidase in alginate microspheres improves stability of encapsulation and activity.

    PubMed

    Zhu, Huiguang; Srivastava, Rohit; Brown, J Quincy; McShane, Michael J

    2005-01-01

    Chemical sensors utilizing immobilized enzymes and proteins are important for monitoring chemical processes and biological systems. In this study, calcium-cross-linked alginate hydrogel microspheres were fabricated as enzyme carriers by an emulsification technique. Glucose oxidase (GOx) was encapsulated in alginate microspheres using three different methods: physical entrapment (emulsion), chemical conjugation (conjugation), and a combination of physical entrapment and chemical conjugation (emulsion-conjugation). Nano-organized coatings were applied on alginate/GOx microspheres using the layer-by-layer self-assembly technique in order to stabilize the hydrogel/enzyme system under biological environment. The encapsulation of GOx and formation of nanofilm coating on alginate microspheres were verified with FTIR spectral analysis, zeta-potential analysis, and confocal laser scanning microscopy. To compare both the immobilization properties of enzyme encapsulation techniques and the influence of nanofilms with uncoated microspheres, the relationship between enzyme loading, release, and effective GOx activity (enzyme activity per unit protein loading) were studied over a period of four weeks. The results produced four key findings: (1) the emulsion-conjugation technique improved the stability of GOx in alginate microspheres compared to the emulsion technique, reducing the GOx leaching from microsphere from 50% to 17%; (2) the polyelectrolyte nanofilm coatings increased the GOx stability over time, but also reduced the effective GOx activity; (3) the effective GOx activity for the emulsion-conjugation technique (about 3.5 x 10(-)(5) AU microg(-)(1) s(-)(1)) was higher than that for other methods, and did not change significantly over four weeks; and (4) the GOx concentration, when compared after one week for microspheres with three bilayers of poly(allylamine hydrochloride)/sodium poly(styrene sulfonate) ({PAH/PSS}) coating, was highest for the emulsion

  18. Degradation potential of protocatechuate 3,4-dioxygenase from crude extract of Stenotrophomonas maltophilia strain KB2 immobilized in calcium alginate hydrogels and on glyoxyl agarose.

    PubMed

    Guzik, Urszula; Hupert-Kocurek, Katarzyna; Krysiak, Marta; Wojcieszyńska, Danuta

    2014-01-01

    Microbial intradiol dioxygenases have been shown to have a great potential for bioremediation; however, their structure is sensitive to various environmental and chemical agents. Immobilization techniques allow for the improvement of enzyme properties. This is the first report on use of glyoxyl agarose and calcium alginate as matrixes for the immobilization of protocatechuate 3,4-dioxygenase. Multipoint attachment of the enzyme to the carrier caused maintenance of its initial activity during the 21 days. Immobilization of dioxygenase in calcium alginate or on glyoxyl agarose resulted in decrease in the optimum temperature by 5 °C and 10 °C, respectively. Entrapment of the enzyme in alginate gel shifted its optimum pH towards high-alkaline pH while immobilization of the enzyme on glyoxyl agarose did not influence pH profile of the enzyme. Protocatechuate 3,4-dioygenase immobilized in calcium alginate showed increased activity towards 2,5-dihydroxybenzoate, caffeic acid, 2,3-dihydroxybenzoate, and 3,5-dihydroxybenzoate. Slightly lower activity of the enzyme was observed after its immobilization on glyoxyl agarose. Entrapment of the enzyme in alginate gel protected it against chelators and aliphatic alcohols while its immobilization on glyoxyl agarose enhanced enzyme resistance to inactivation by metal ions. PMID:24693536

  19. Degradation potential of protocatechuate 3,4-dioxygenase from crude extract of Stenotrophomonas maltophilia strain KB2 immobilized in calcium alginate hydrogels and on glyoxyl agarose.

    PubMed

    Guzik, Urszula; Hupert-Kocurek, Katarzyna; Krysiak, Marta; Wojcieszyńska, Danuta

    2014-01-01

    Microbial intradiol dioxygenases have been shown to have a great potential for bioremediation; however, their structure is sensitive to various environmental and chemical agents. Immobilization techniques allow for the improvement of enzyme properties. This is the first report on use of glyoxyl agarose and calcium alginate as matrixes for the immobilization of protocatechuate 3,4-dioxygenase. Multipoint attachment of the enzyme to the carrier caused maintenance of its initial activity during the 21 days. Immobilization of dioxygenase in calcium alginate or on glyoxyl agarose resulted in decrease in the optimum temperature by 5 °C and 10 °C, respectively. Entrapment of the enzyme in alginate gel shifted its optimum pH towards high-alkaline pH while immobilization of the enzyme on glyoxyl agarose did not influence pH profile of the enzyme. Protocatechuate 3,4-dioygenase immobilized in calcium alginate showed increased activity towards 2,5-dihydroxybenzoate, caffeic acid, 2,3-dihydroxybenzoate, and 3,5-dihydroxybenzoate. Slightly lower activity of the enzyme was observed after its immobilization on glyoxyl agarose. Entrapment of the enzyme in alginate gel protected it against chelators and aliphatic alcohols while its immobilization on glyoxyl agarose enhanced enzyme resistance to inactivation by metal ions.

  20. Oxidized alginate hydrogels as niche environments for corneal epithelial cells

    PubMed Central

    Wright, Bernice; De Bank, Paul A; Luetchford, Kim A; Acosta, Fernando R; Connon, Che J

    2014-01-01

    Chemical and biochemical modification of hydrogels is one strategy to create physiological constructs that maintain cell function. The aim of this study was to apply oxidised alginate hydrogels as a basis for development of a biomimetic niche for limbal epithelial stem cells that may be applied to treating corneal dysfunction. The stem phenotype of bovine limbal epithelial cells (LEC) and the viability of corneal epithelial cells (CEC) were examined in oxidised alginate gels containing collagen IV over a 3-day culture period. Oxidation increased cell viability (P ≤ 0.05) and this improved further with addition of collagen IV (P ≤ 0.01). Oxidised gels presented larger internal pores (diameter: 0.2–0.8 µm) than unmodified gels (pore diameter: 0.05–0.1 µm) and were significantly less stiff (P ≤ 0.001), indicating that an increase in pore size and a decrease in stiffness contributed to improved cell viability. The diffusion of collagen IV from oxidised alginate gels was similar to that of unmodified gels suggesting that oxidation may not affect the retention of extracellular matrix proteins in alginate gels. These data demonstrate that oxidised alginate gels containing corneal extracellular matrix proteins can influence corneal epithelial cell function in a manner that may impact beneficially on corneal wound healing therapy. © 2013 The Authors. Journal of Biomedical Materials Research Part A Published byWiley Periodicals, Inc. Part A: 102A: 3393–3400, 2014. PMID:24142706

  1. Oxidized alginate hydrogels as niche environments for corneal epithelial cells.

    PubMed

    Wright, Bernice; De Bank, Paul A; Luetchford, Kim A; Acosta, Fernando R; Connon, Che J

    2014-10-01

    Chemical and biochemical modification of hydrogels is one strategy to create physiological constructs that maintain cell function. The aim of this study was to apply oxidised alginate hydrogels as a basis for development of a biomimetic niche for limbal epithelial stem cells that may be applied to treating corneal dysfunction. The stem phenotype of bovine limbal epithelial cells (LEC) and the viability of corneal epithelial cells (CEC) were examined in oxidised alginate gels containing collagen IV over a 3-day culture period. Oxidation increased cell viability (P ≤ 0.05) and this improved further with addition of collagen IV (P ≤ 0.01). Oxidised gels presented larger internal pores (diameter: 0.2-0.8 µm) than unmodified gels (pore diameter: 0.05-0.1 µm) and were significantly less stiff (P ≤ 0.001), indicating that an increase in pore size and a decrease in stiffness contributed to improved cell viability. The diffusion of collagen IV from oxidised alginate gels was similar to that of unmodified gels suggesting that oxidation may not affect the retention of extracellular matrix proteins in alginate gels. These data demonstrate that oxidised alginate gels containing corneal extracellular matrix proteins can influence corneal epithelial cell function in a manner that may impact beneficially on corneal wound healing therapy.

  2. Evaluation of sodium alginate for bone marrow cell tissue engineering.

    PubMed

    Wang, L; Shelton, R M; Cooper, P R; Lawson, M; Triffitt, J T; Barralet, J E

    2003-09-01

    Sodium alginate has applications as a material for the encapsulation and immobilisation of a variety of cell types for immunoisolatory and biochemical processing applications. It forms a biodegradable gel when crosslinked with calcium ions and it has been exploited in cartilage tissue engineering since chondrocytes do not dedifferentiate when immobilised in it. Despite its attractive properties of degradability, ease of processing and cell immobilisation, there is little work demonstrating the efficacy of alginate gel as a substrate for cell proliferation, except when RGD is modified. In this study we investigated the ability of rat bone marrow cells to proliferate and differentiate on alginates of differing composition and purity. The mechanical properties of the gels were investigated. It was found that high purity and high G-type alginate retained 27% of its initial strength after 12 days in culture and that comparable levels of proliferation were observed on this material and tissue culture plastic. Depending on composition, calcium crosslinked alginate can act as a substrate for rat marrow cell proliferation and has potential for use as 3D degradable scaffold.

  3. Microalgal immobilization methods.

    PubMed

    Moreno-Garrido, Ignacio

    2013-01-01

    In this review, methods for the most common microalgal immobilization procedures are gathered and described. Passive (due to natural adherence of cells to surfaces) and active immobilization methods should be distinguished. Among active immobilization methods, calcium alginate entrapment is the most widely used method if living cells are intended to be immobilized, due to the chemical, optical, and mechanical characteristics of this substance. Immobilization in synthetic foams, immobilization in agar and carrageenan as well as immobilization in silica-based matrix or filters are also discussed and described. Finally, some considerations on the use of flocculation for microalgae are mentioned.

  4. Production of xanthan gum by free and immobilized cells of Xanthomonas campestris and Xanthomonas pelargonii.

    PubMed

    Niknezhad, Seyyed Vahid; Asadollahi, Mohammad Ali; Zamani, Akram; Biria, Davoud

    2016-01-01

    Production of xanthan gum using immobilized cells of Xanthomonas campestris and Xanthomonas pelargonii grown on glucose or hydrolyzed starch as carbon sources was investigated. Calcium alginate (CA) and calcium alginate-polyvinyl alcohol-boric acid (CA-PVA) beads were used for the immobilization of cells. Xanthan titers of 8.2 and 9.2g/L were obtained for X. campestris cells immobilized in CA-PVA beads using glucose and hydrolyzed starch, respectively, whereas those for X. pelargonii were 8 and 7.9 g/L, respectively. Immobilized cells in CA-PVA beads were successfully employed in three consecutive cycles for xanthan production without any noticeable degradation of the beads whereas the CA beads were broken after the first cycle. The results of this study suggested that immobilized cells are advantageous over the free cells for xanthan production. Also it was shown that the cells immobilized in CA-PVA beads are more efficient than cells immobilized in CA beads for xanthan production. PMID:26526173

  5. Kinetic Characterization and Effect of Immobilized Thermostable β-Glucosidase in Alginate Gel Beads on Sugarcane Juice

    PubMed Central

    Keerti; Gupta, Anuradha; Dubey, Ashutosh; Verma, A. K.

    2014-01-01

    A thermostable β-glucosidase was effectively immobilized on alginate by the method of gel entrapment. After optimization of immobilized conditions, recovered enzyme activity was 60%. Optimum pH, temperature, kinetic parameters, thermal and pH stability, reusability, and storage stability were investigated. The Km and Vmax for immobilized β-glucosidase were estimated to be 5.0 mM and 0.64 U/ml, respectively. When comparing, free and immobilized enzyme, change was observed in optimum pH and temperature from 5.0 to 6.0 and 60°C to 80°C, respectively. Immobilized enzyme showed an increase in pH stability over the studied pH range (3.0–10.0) and stability at temperature up to 80°C. The storage stability and reusability of the immobilized β-glucosidase were improved significantly, with 12.09% activity retention at 30°C after being stored for 25 d and 17.85% residual activity after being repeatedly used for 4 times. The effect of both free and immobilized β-glucosidase enzyme on physicochemical properties of sugarcane juice was also analyzed. PMID:25969764

  6. Immobilization of cells by electrostatic droplet generation: a model system for potential application in medicine

    PubMed Central

    Manojlovic, V; Djonlagic, J; Obradovic, B; Nedovic, V; Bugarski, B

    2006-01-01

    The process of electrostatic extrusion as a method for cell immobilization was investigated that could be used for potential applications in medicine. An attempt was made to assess the effects of cell addition and polymer concentration on the overall entrapment procedure, ie, on each stage of immobilization: polymer-cell suspension rheological characteristics, electrostatic extrusion process, and the process of gelation. The findings should contribute to a better understanding of polymer–cell interactions, which could be crucial in possible medical treatments. Alginate–yeast was used as a model system for carrier-cells. The electrostatic extrusion was considered as a complex two-phase flow system and the effects of cell and alginate concentrations on the resulting microbead size and uniformity were assessed. Under investigated conditions, microbeads 50–600 μm in diameter were produced and the increase in both alginate and cell concentrations resulted in larger microbeads with higher standard deviations in size. We attempted to rationalize the findings by rheological characterization of the cell–alginate suspensions. Rheological characterization revealed non-Newtonian, pseudoplastic behavior of cell-alginate suspensions with higher viscosities at higher alginate concentrations. However, the presence of cells even at high concentrations (5×108 and 1×109 cells/mL) did not significantly affect the rheological properties of Na-alginate solution. Lastly, we investigated the kinetics of alginate gelation with respect to the quantity of Ca2+ ions and cell presence. The gelation kinetics were examined under conditions of limited supply with Ca2+ ions, which can be essential for immobilization of highly sensitive mammalian cells that require minimal exposure to CaCl2 solution. The molar ratio of G units to Ca2+ ions of 3.8:1 provided complete crosslinking, while the increase in alginate concentration resulted in prolonged gelation times but higher strength of the

  7. Physical characteristics of poly(vinyl alcohol) and calcium alginate hydrogels for the immobilization of activated sludge.

    PubMed

    Doria-Serrano, M C; Ruiz-Treviño, F A; Rios-Arciga, C; Hernández-Esparza, M; Santiago, P

    2001-01-01

    Hydrogels based on poly(vinyl alcohol), PVA, and calcium alginate were prepared by a freezing and thawing cycle process and characterized, in terms of the role of the polymer mixture percentage and the number of treatment cycles, on their weight swelling ratio, WSR, gel fraction, and activated sludge entrapment and immobilization. The results show that the morphology of these hydrogels is highly dependent on the PVA-Ca alginate ratio of 5 wt % total polymer content in the initial aqueous solution and that the number of entrapped microorganisms which survive the freezing-thawing procedure is independent of this ratio. For 80/20 PVA-Ca alginate hydrogels, results also show that for up to three freezing and thawing cycles, the WSR, which is in average 24, is not severely affected by the number of the cycles. For the hydrogels with three cycles, the calculated gel fraction for the composite hydrogel is 0.99. Immobilized microorganisms from sedimented activated sludge, constituted by bacteria and fungi, die in high numbers during the freezing and thawing treatment. However, with a proper time of incubation with glucose as carbon source, the population of bacteria is recovered and mainly proliferate inside the hydrogel, attached on top of the fibril network formed by the polymers, while fungi are recovered predominantly on the surface of the spheres.

  8. Effective Immobilization of Agrobacterium sp. IFO 13140 Cells in Loofa Sponge for Curdlan Biosynthesis.

    PubMed

    Martinez, Camila Ortiz; Ruiz, Suelen Pereira; Nogueira, Marcela Tiemi; Bona, Evandro; Portilho, Márcia; Matioli, Graciette

    2015-05-04

    Curdlan production by Agrobacterium sp. IFO13140 immobilized on loofa sponge, alginate and loofa sponge with alginate was investigated. There was no statistically-significant difference in curdlan production when the microorganism was immobilized in different matrices. The loofa sponge was chosen because of its practical application and economy and because it provides a high stability through its continued use. The best conditions for immobilization on loofa sponge were 50 mg of cell, 200 rpm and 72 h of incubation, which provided a curdlan production 1.50-times higher than that obtained by free cells. The higher volumetric productivity was achieved by immobilized cells (0.09 g/L/h) at 150 rpm. The operating stability was evaluated, and until the fourth cycle, immobilized cells retained 87.40% of the production of the first cycle. The immobilized cells remained active after 300 days of storage at 4 °C. The results of this study demonstrate success in immobilizing cells for curdlan biosynthesis, making the process potentially suitable for industrial scale-up. Additional studies may show a possible contribution to the reduction of operating costs.

  9. Collective and individual glycolytic oscillations in yeast cells encapsulated in alginate microparticles

    NASA Astrophysics Data System (ADS)

    Amemiya, Takashi; Obase, Kouhei; Hiramatsu, Naoki; Itoh, Kiminori; Shibata, Kenichi; Takinoue, Masahiro; Yamamoto, Tetsuya; Yamaguchi, Tomohiko

    2015-06-01

    Yeast cells were encapsulated into alginate microparticles of a few hundred micrometers diameter using a centrifuge-based droplet shooting device. We demonstrate the first experimental results of glycolytic oscillations in individual yeast cells immobilized in this way. We investigated both the individual and collective oscillatory behaviors at different cell densities. As the cell density increased, the amplitude of the individual oscillations increased while their period decreased, and the collective oscillations became more synchronized, with an order parameter close to 1 (indicating high synchrony). We also synthesized biphasic-Janus microparticles encapsulating yeast cells of different densities in each hemisphere. The cellular oscillations between the two hemispheres were entrained at both the individual and population levels. Such systems of cells encapsulated into microparticles are useful for investigating how cell-to-cell communication depends on the density and spatial distribution of cells.

  10. Collective and individual glycolytic oscillations in yeast cells encapsulated in alginate microparticles.

    PubMed

    Amemiya, Takashi; Obase, Kouhei; Hiramatsu, Naoki; Itoh, Kiminori; Shibata, Kenichi; Takinoue, Masahiro; Yamamoto, Tetsuya; Yamaguchi, Tomohiko

    2015-06-01

    Yeast cells were encapsulated into alginate microparticles of a few hundred micrometers diameter using a centrifuge-based droplet shooting device. We demonstrate the first experimental results of glycolytic oscillations in individual yeast cells immobilized in this way. We investigated both the individual and collective oscillatory behaviors at different cell densities. As the cell density increased, the amplitude of the individual oscillations increased while their period decreased, and the collective oscillations became more synchronized, with an order parameter close to 1 (indicating high synchrony). We also synthesized biphasic-Janus microparticles encapsulating yeast cells of different densities in each hemisphere. The cellular oscillations between the two hemispheres were entrained at both the individual and population levels. Such systems of cells encapsulated into microparticles are useful for investigating how cell-to-cell communication depends on the density and spatial distribution of cells. PMID:26117131

  11. Single-step laser-based fabrication and patterning of cell-encapsulated alginate microbeads

    PubMed Central

    Kingsley, DM; Dias, AD; Chrisey, DB; Corr, DT

    2014-01-01

    Alginate can be used to encapsulate mammalian cells and for the slow release of small molecules. Packaging alginate as microbead structures allows customizable delivery for tissue engineering, drug release, or contrast agents for imaging. However, state-of-the-art microbead fabrication has limited range in achievable bead sizes, and poor control over bead placement, which may be desired to localize cellular signaling or delivery. Herein, we present a novel, laser-based method for single-step fabrication and precise planar placement of alginate microbeads. Our results show that bead size is controllable within 8%, and fabricated microbeads can remain immobilized within 2% of their target placement. Demonstration of this technique using human breast cancer cells shows that cells encapsulated within these microbeads survive at a rate of 89.6%, decreasing to 84.3% after five days in culture. Infusing rhodamine dye into microbeads prior to fluorescent microscopy shows their 3D spheroidal geometry and the ability to sequester small molecules. Microbead fabrication and patterning is compatible with conventional cellular transfer and patterning by laser direct-write, allowing location-based cellular studies. While this method can also be used to fabricate microbeads en masse for collection, the greatest value to tissue engineering and drug delivery studies and applications lies in the pattern registry of printed microbeads. PMID:24192221

  12. Study of the potential of the air lift bioreactor for xylitol production in fed-batch cultures by Debaryomyces hansenii immobilized in alginate beads.

    PubMed

    Pérez-Bibbins, Belinda; de Souza Oliveira, Ricardo Pinheiro; Torrado, Ana; Aguilar-Uscanga, María Guadalupe; Domínguez, José Manuel

    2014-01-01

    Cell immobilization has shown to be especially adequate for xylitol production. This work studies the suitability of the air lift bioreactor for xylitol production by Debaryomyces hansenii immobilized in Ca-alginate operating in fed-batch cultures to avoid substrate inhibition. The results showed that the air lift bioreactor is an adequate system since the minimum air flow required for fluidization was even lower than that leading to the microaerobic conditions that trigger xylitol accumulation by this yeast, also maintaining the integrity of the alginate beads and the viability of the immobilized cells until 3 months of reuses. Maximum productivities and yields of 0.43 g/l/h and 0.71 g/g were achieved with a xylose concentration of 60 g/l after each feeding. The xylose feeding rate, the air flow, and the biomass concentration at the beginning of the fed-batch operation have shown to be critical parameters for achieving high productivities and yields. Although a maximum xylitol production of 139 g/l was obtained, product inhibition was evidenced in batch experiments, which allowed estimating at 200 and 275 g/l the IC50 for xylitol productivity and yield, respectively. The remarkable production of glycerol in the absence of glucose was noticeable, which could not only be attributed to the osmoregulatory function of this polyol in conditions of high osmotic pressure caused by high xylitol concentrations but also to the role of the glycerol synthesis pathway in the regeneration of NAD(+) in conditions of suboptimal microaeration caused by insufficient aeration or high oxygen demand when high biomass concentrations were achieved.

  13. Industrial applications of immobilized cells

    SciTech Connect

    Linko, P.; Linko, Y.Y.

    1984-01-01

    Although the application of the natural attraction of many microorganisms to surfaces has been applied in vinegar production since the early 1980s, and has long been utilized in waste water purification, the development of microbial cell immobilization techniques for special applications dates back only to the early 1960s. The immobilization may involve whole cells, cell fragments, or lysed cells. Whole cells may retain their metabolic activity with their complex multienzyme systems and cofactor regeneration mechanisms intact, or they may be killed in the process with only a few desired enzymes remaining active in the final biocatalyst. Cells may also be coimmobilized with an enzyme to carry out special reactions. Although relatively few industrial scale applications exist today, some are of very large scale. Current applications vary from relatively small scale steroid conversions to amino acid production and high fructose syrup manufacture. A vast number of potential applications are already known, and one of the most interesting applications may be in continuous fermentation such as ethanol production by immobilized living microorganisms. 373 references.

  14. Enhancement of chitosanase production by cell immobilization of Gongronella sp. JG.

    PubMed

    Zhang, Pingping; Zhou, Wei; Wang, Peng; Wang, Li; Tang, Mingli

    2013-01-01

    Chitosanase production of Gongronella sp. JG cells immobilized in calcium alginate gel and polyurethane foam was compared with that of the free cells, there was a 60% increase in the enzyme yield (2429 U/L) compared to the highest yield obtained from free cells (1513 U/L). The optimal immobilization parameters (concentrations of sodium alginate, calcium chloride, bead inoculums, bead diameter, etc) for the enhanced production of chitosanase were determined as: sodium alginate 2% (w/v), 0.1 M calcium chloride, inoculum 10 mL beads to 100 mL production media and 2.7 mm bead diameter. Maximum chitosanase production was achieved with initial pH of 5.5 and temperature of 30 °C. The alginate beads had well stability, retained 85% ability of enzyme production even after 7 cycles of repeated batch fermentation. These results showed the immobilization technique was a feasible and economical method for chitosansase production by Gongronella sp. JG.

  15. Decolorization of textile effluent by bitter gourd peroxidase immobilized on concanavalin A layered calcium alginate-starch beads.

    PubMed

    Matto, Mahreen; Husain, Qayyum

    2009-05-30

    Bitter gourd peroxidase immobilized on the surface of concanavalin A layered calcium alginate-starch beads was used for the successful and effective decolorization of textile industrial effluent. Effluent was recalcitrant to the action of bitter gourd peroxidase; however, in the presence of some redox mediators, it was successfully decolorized. Effluent decolorization was maximum (70%) in the presence of 1.0mM 1-hydroxybenzotriazole within 1h of incubation. However, immobilized bitter gourd peroxidase showed maximum decolorization at pH 5.0 and 40 degrees C. Immobilized bitter gourd peroxidase decolorized more than 90% effluent after 3h of incubation in a batch process. The two-reactor system, one reactor containing immobilized peroxidase and the other had activated silica, was quite effective in the decolorization of textile effluent. The system was capable of decolorizing 40% effluent even after 2 months of continuous operation. The absorption spectra of the untreated and treated effluent exhibited a marked difference in absorbance at various wavelengths. Immobilized peroxidase/1-hydroxybenzotriazole system could be employed for the treatment of a large volume of effluent in a continuous reactor.

  16. Alginate gel microwell arrays using electrodeposition for three-dimensional cell culture.

    PubMed

    Ozawa, Fumisato; Ino, Kosuke; Arai, Toshiharu; Ramón-Azcón, Javier; Takahashi, Yasufumi; Shiku, Hitoshi; Matsue, Tomokazu

    2013-08-01

    In this study, we developed a novel method for fabricating microwell arrays constructed from alginate gels, and the alginate gel microwells were used for three-dimensional (3D) cell culture. The alginate gel microwells were fabricated on a patterned ITO electrode using alginate gel electrodeposition. Embryonic stem (ES) cells or hepatocellular carcinoma cells (HepG2) were cultured in the alginate gel microwells containing 3T3 cells. During the culture, embryoid bodies (EBs) or HepG2 spheroids were successfully fabricated in the alginate gel microwells. The oxygen consumption of the EBs indicated that they were successfully cultured. Liver-specific gene expressions of the HepG2 spheroids apparently increased by performing 3D co-culture in the microwell arrays with 3T3 cells. These results show that the alginate gel microwells are a useful 3D culture system.

  17. Calcium Alginate Gels as Stem Cell Matrix – Making Paracrine Stem Cell Activity Available for Enhanced Healing after Surgery

    PubMed Central

    Schmitt, Andreas; Rödel, Philipp; Anamur, Cihad; Seeliger, Claudine; Imhoff, Andreas B.; Herbst, Elmar; Vogt, Stephan; van Griensven, Martijn; Winter, Gerhard; Engert, Julia

    2015-01-01

    Regeneration after surgery can be improved by the administration of anabolic growth factors. However, to locally maintain these factors at the site of regeneration is problematic. The aim of this study was to develop a matrix system containing human mesenchymal stem cells (MSCs) which can be applied to the surgical site and allows the secretion of endogenous healing factors from the cells. Calcium alginate gels were prepared by a combination of internal and external gelation. The gelling behaviour, mechanical stability, surface adhesive properties and injectability of the gels were investigated. The permeability of the gels for growth factors was analysed using bovine serum albumin and lysozyme as model proteins. Human MSCs were isolated, cultivated and seeded into the alginate gels. Cell viability was determined by AlamarBlue assay and fluorescence microscopy. The release of human VEGF and bFGF from the cells was determined using an enzyme-linked immunoassay. Gels with sufficient mechanical properties were prepared which remained injectable through a syringe and solidified in a sufficient time frame after application. Surface adhesion was improved by the addition of polyethylene glycol 300,000 and hyaluronic acid. Humans MSCs remained viable for the duration of 6 weeks within the gels. Human VEGF and bFGF was found in quantifiable concentrations in cell culture supernatants of gels loaded with MSCs and incubated for a period of 6 weeks. This work shows that calcium alginate gels can function as immobilization matrices for human MSCs. PMID:25793885

  18. Calcium alginate gels as stem cell matrix-making paracrine stem cell activity available for enhanced healing after surgery.

    PubMed

    Schmitt, Andreas; Rödel, Philipp; Anamur, Cihad; Seeliger, Claudine; Imhoff, Andreas B; Herbst, Elmar; Vogt, Stephan; van Griensven, Martijn; Winter, Gerhard; Engert, Julia

    2015-01-01

    Regeneration after surgery can be improved by the administration of anabolic growth factors. However, to locally maintain these factors at the site of regeneration is problematic. The aim of this study was to develop a matrix system containing human mesenchymal stem cells (MSCs) which can be applied to the surgical site and allows the secretion of endogenous healing factors from the cells. Calcium alginate gels were prepared by a combination of internal and external gelation. The gelling behaviour, mechanical stability, surface adhesive properties and injectability of the gels were investigated. The permeability of the gels for growth factors was analysed using bovine serum albumin and lysozyme as model proteins. Human MSCs were isolated, cultivated and seeded into the alginate gels. Cell viability was determined by AlamarBlue assay and fluorescence microscopy. The release of human VEGF and bFGF from the cells was determined using an enzyme-linked immunoassay. Gels with sufficient mechanical properties were prepared which remained injectable through a syringe and solidified in a sufficient time frame after application. Surface adhesion was improved by the addition of polyethylene glycol 300,000 and hyaluronic acid. Humans MSCs remained viable for the duration of 6 weeks within the gels. Human VEGF and bFGF was found in quantifiable concentrations in cell culture supernatants of gels loaded with MSCs and incubated for a period of 6 weeks. This work shows that calcium alginate gels can function as immobilization matrices for human MSCs.

  19. Biotransformation of L-tyrosine to Dopamine by a Calcium Alginate Immobilized Mutant Strain of Aspergillus oryzae.

    PubMed

    Ali, Sikander; Nawaz, Wajeeha

    2016-08-01

    The present research work is concerned with the biotransformation of L-tyrosine to dopamine (DA) by calcium alginate entrapped conidiospores of a mutant strain of Aspergillus oryzae. Different strains of A. oryzae were isolated from soil. Out of 13 isolated strains, isolate-2 (I-2) was found to be a better DA producer. The wild-type I-2 was chemically improved by treating it with different concentrations of ethyl methyl sulfonate (EMS). Among seven mutant variants, EMS-6 exhibiting maximal DA activity of 43 μg/ml was selected. The strain was further exposed with L-cysteine HCl to make it resistant against diversion and environmental stress. The conidiospores of selected mutant variant A. oryzae EMS-6 strain were entrapped in calcium alginate beads. Different parameters for immobilization were investigated. The activity was further improved from 44 to 62 μg/ml under optimized conditions (1.5 % sodium alginate, 2 ml inoculum, and 2 mm bead size). The best resistant mutant variable exhibited over threefold increase in DA activity (62 μg/ml) than did wild-type I-2 (21 μg/ml) in the reaction mixture. From the results presented in the study, it was observed that high titers of DA activity in vitro could effectively be achieved by the EMS-induced mutagenesis of filamentous fungus culture used.

  20. Development of highly sensitive amperometric biosensor for glucose using carbon nanosphere/sodium alginate composite matrix for enzyme immobilization.

    PubMed

    Han, En; Li, Xia; Cai, Jian-Rong; Cui, Hai-Ying; Zhang, Xing-Ai

    2014-01-01

    In this study, we developed a highly sensitive amperometric biosensor for glucose detection based on glucose oxidase immobilized in a novel carbon nanosphere (CNS)/sodium alginate (SA) composite matrix. This hybrid material combined the advantages of CNS and natural biopolymer SA. This composite film was characterized by scanning electron microscope, electrochemical impedance spectroscopy and UV-vis, which indicated that the hybrid material was suitable for immobilization of glucose oxidase. Various experimental conditions were investigated that influenced the performance of the biosensor, such as pH, applied potential and temperature. Under the optimum conditions, the biosensor showed excellent performance for glucose over a wide linear concentration range from 1.0 × 10(-6) to 4.6 × 10(-3) M with a detection limit of 0.5 μM based on a signal-to-noise ratio of 3. Furthermore, the biosensor exhibited excellent long-term stability and satisfactory reproducibility. PMID:25213818

  1. Evaluation of various parameters of calcium-alginate immobilization method for enhanced alkaline protease production by Bacillus licheniformis NCIM-2042 using statistical methods.

    PubMed

    Potumarthi, Ravichandra; Subhakar, Ch; Pavani, A; Jetty, Annapurna

    2008-04-01

    Calcium-alginate immobilization method for the production of alkaline protease by Bacillus licheniformis NCIM-2042 was optimized statistically. Four variables, such as sodium-alginate concentration, calcium chloride concentration, inoculum size and agitation speed were optimized by 2(4) full factorial central composite design and subsequent analysis and model validation by a second-order regression equation. Eleven carbon, 11 organic nitrogen and seven inorganic nitrogen sources were screened by two-level Plackett-Burman design for maximum alkaline protease production by using optimized immobilized conditions. The levels of four variables, such as Na-alginate 2.78%; CaCl(2), 2.15%; inoculum size, 8.10% and agitation, 139 rpm were found to be optimum for maximal production of protease. Glucose, soybean meal and ammonium sulfate were resulted in maximum protease production at 644 U/ml, 720 U/ml, and 806 U/ml when screened for carbon, organic nitrogen and inorganic nitrogen sources, respectively, using optimized immobilization conditions. Repeated fed batch mode of operation, using optimized immobilized conditions, resulted in continuous operation for 12 cycles without disintegration of beads. Cross-sectional scanning electron microscope images have shown the growth pattern of B. licheniformis in Ca-alginate immobilized beads.

  2. Biodegradation of tetrahydrofuran by Pseudomonas oleovorans DT4 immobilized in calcium alginate beads impregnated with activated carbon fiber: mass transfer effect and continuous treatment.

    PubMed

    Chen, Dong-Zhi; Fang, Jun-Yi; Shao, Qian; Ye, Jie-Xu; Ouyang, Du-Juan; Chen, Jian-Meng

    2013-07-01

    A novel entrapment matrix, calcium alginate (CA) coupled with activated carbon fiber (ACF), was prepared to immobilize Pseudomonas oleovorans DT4 for degrading tetrahydrofuran (THF). The addition of 1.5% ACF increased the adsorption capacity of the immobilized bead, thus resulting in an enhanced average removal rate of 30.3mg/(Lh). The synergism between adsorption and biodegradation was observed in the hybrid CA-ACF beads instead of in the system comprising CA beads and freely suspended ACF. The effective diffusion coefficient of the CA-ACF bead was not significantly affected by bead size, but the bead's value of 1.14×10(-6)cm(2)/s (for the bead diameter of 0.4 cm) was larger than that of the CA bead by almost one order of magnitude based on the intraparticle diffusion-reaction kinetics analysis. Continuous treatment of the THF-containing wastewater was succeeded by CA-ACF immobilized cells in a packed-bed reactor for 54 d with a >90% removal efficiency. PMID:23644074

  3. Biodegradation of tetrahydrofuran by Pseudomonas oleovorans DT4 immobilized in calcium alginate beads impregnated with activated carbon fiber: mass transfer effect and continuous treatment.

    PubMed

    Chen, Dong-Zhi; Fang, Jun-Yi; Shao, Qian; Ye, Jie-Xu; Ouyang, Du-Juan; Chen, Jian-Meng

    2013-07-01

    A novel entrapment matrix, calcium alginate (CA) coupled with activated carbon fiber (ACF), was prepared to immobilize Pseudomonas oleovorans DT4 for degrading tetrahydrofuran (THF). The addition of 1.5% ACF increased the adsorption capacity of the immobilized bead, thus resulting in an enhanced average removal rate of 30.3mg/(Lh). The synergism between adsorption and biodegradation was observed in the hybrid CA-ACF beads instead of in the system comprising CA beads and freely suspended ACF. The effective diffusion coefficient of the CA-ACF bead was not significantly affected by bead size, but the bead's value of 1.14×10(-6)cm(2)/s (for the bead diameter of 0.4 cm) was larger than that of the CA bead by almost one order of magnitude based on the intraparticle diffusion-reaction kinetics analysis. Continuous treatment of the THF-containing wastewater was succeeded by CA-ACF immobilized cells in a packed-bed reactor for 54 d with a >90% removal efficiency.

  4. Engineered yeast whole-cell biocatalyst for direct degradation of alginate from macroalgae and production of non-commercialized useful monosaccharide from alginate.

    PubMed

    Takagi, Toshiyuki; Yokoi, Takahiro; Shibata, Toshiyuki; Morisaka, Hironobu; Kuroda, Kouichi; Ueda, Mitsuyoshi

    2016-02-01

    Alginate is a major component of brown macroalgae. In macroalgae, an endolytic alginate lyase first degrades alginate into oligosaccharides. These oligosaccharides are further broken down into monosaccharides by an exolytic alginate lyase. In this study, genes encoding various alginate lyases derived from alginate-assimilating marine bacterium Saccharophagus degradans were isolated, and their enzymes were displayed using the yeast cell surface display system. Alg7A-, Alg7D-, and Alg18J-displaying yeasts showed endolytic alginate lyase activity. On the other hand, Alg7K-displaying yeast showed exolytic alginate lyase activity. Alg7A, Alg7D, Alg7K, and Alg18J, when displayed on yeast cell surface, demonstrated both polyguluronate lyase and polymannuronate lyase activities. Additionally, polyguluronic acid could be much easily degraded by Alg7A, Alg7K, and Alg7D than polymannuronic acid. In contrast, polymannuronic acid could be much easily degraded by Alg18J than polyguluronic acid. We further constructed yeasts co-displaying endolytic and exolytic alginate lyases. Degradation efficiency by the co-displaying yeasts were significantly higher than single alginate lyase-displaying yeasts. Alg7A/Alg7K co-displaying yeast had maximum alginate degrading activity, with production of 1.98 g/L of reducing sugars in a 60-min reaction. This system developed, along with our findings, will contribute to the efficient utilization and production of useful and non-commercialized monosaccharides from alginate by Saccharomyces cerevisiae. PMID:26490549

  5. Production, partial characterization, and immobilization in alginate beads of an alkaline protease from a new thermophilic fungus Myceliophthora sp.

    PubMed

    Zanphorlin, Letícia Maria; Facchini, Fernanda Dell Antonio; Vasconcelos, Filipe; Bonugli-Santos, Rafaella Costa; Rodrigues, André; Sette, Lara Durães; Gomes, Eleni; Bonilla-Rodriguez, Gustavo Orlando

    2010-06-01

    Thermophilic fungi produce thermostable enzymes which have a number of applications, mainly in biotechnological processes. In this work, we describe the characterization of a protease produced in solidstate (SSF) and submerged (SmF) fermentations by a newly isolated thermophilic fungus identified as a putative new species in the genus Myceliophthora. Enzyme-production rate was evaluated for both fermentation processes, and in SSF, using a medium composed of a mixture of wheat bran and casein, the proteolytic output was 4.5-fold larger than that obtained in SmF. Additionally, the peak of proteolytic activity was obtained after 3 days for SSF whereas for SmF it was after 4 days. The crude enzyme obtained by both SSF and SmF displayed similar optimum temperature at 50 degrees C, but the optimum pH shifted from 7 (SmF) to 9(SSF). The alkaline protease produced through solid-state fermentation (SSF), was immobilized on beads of calcium alginate, allowing comparative analyses of free and immobilized proteases to be carried out. It was observed that both optimum temperature and thermal stability of the immobilized enzyme were higher than for the free enzyme. Moreover, the immobilized enzyme showed considerable stability for up to 7 reuses.

  6. Poly(vinyl alcohol) and alginate cross-linked matrix with immobilized Prussian blue and ion exchange resin for cesium removal from waters.

    PubMed

    Lai, Yu-Chen; Chang, Yin-Ru; Chen, Man-Li; Lo, Yu-Kuo; Lai, Juin-Yih; Lee, Duu-Jong

    2016-08-01

    Cesium (Cs) removal from contaminated water bodies is an emerging issue after the disaster at the Fukushima Daiichi Nuclear Power Plant. The Prussian blue (PB) is an effective Cs adsorbent but will release hexacyanoferrate fragments from the adsorbent matrix during adsorption. Alginate is an affordable biopolymer for PB particles immobilization. This study synthesized poly(vinyl alcohol) (PVA) and alginate cross-linked matrix for immobilization of PB nano-sized particles and a surface-modified styrene-ethyl styrene divinyl benzene resin and tested their swelling stability and Cs adsorption performance in fresh water and in seawater. The PVA-alginate granules have high structural stability in both fresh water and seawater, with the Cs adsorption capability higher for the former than the latter. The adopted resin effectively remove released PB fragments from the tested granules. The transport and reaction parameters for the granules and for the sand filter bed were estimated. PMID:27132227

  7. An electrohydrodynamic bioprinter for alginate hydrogels containing living cells.

    PubMed

    Gasperini, Luca; Maniglio, Devid; Motta, Antonella; Migliaresi, Claudio

    2015-02-01

    In this work we present a bioprinting technique that exploits the electrohydrodynamic process to obtain a jet of liquid alginate beads containing cells. A printer is used to microfabricate hydrogels block by block following a bottom-up approach. Alginate beads constitute the building blocks of the microfabricated structures. The beads are placed at predefined position on a target substrate made of calcium-enriched gelatin, where they crosslink upon contact without the need of further postprocessing. The printed sample can be easily removed from the substrate at physiological temperature. Three-dimensional printing is accomplished by the deposition of multiple layers of hydrogel. We have investigated the parameters influencing the process, the compatibility of the printing procedure with cells, and their survival after printing.

  8. Enzymatic production of atranorin: a component of the oak moss absolute by immobilized lichen cells.

    PubMed

    Vicente, C; Fontaniella, B; Millanes, A M; Sebastián, B; Legaz, M E

    2003-04-01

    Cells of the lichen, Evernia prunastri, immobilized in calcium alginate were able to produce the depside atranorin from acetate. The synthesis of the depside was enhanced by molecular oxygen and NADH. This enhancement suggested the participation of an oxidase and an alcohol dehydrogenase to produce an aldehyde-substituted phenolic acid, hematommic acid, as the most probable precursor of atranorin. The participation of both enzymes was confirmed by loading immobilized cells with sodium azide, an inhibitor of several metallo-oxidases, and pyrazole, an inhibitor of alcohol dehydrogenase, which impeded atranorin production and accumulated beta-methyl orsellinate (after azide loading) or its alcohol derivative (after pirazole treatment). PMID:18494879

  9. Combined of ultrasound irradiation with high hydrostatic pressure (US/HHP) as a new method to improve immobilization of dextranase onto alginate gel.

    PubMed

    Bashari, Mohanad; Abbas, Shabbar; Xu, Xueming; Jin, Zhengyu

    2014-07-01

    In this research work, dextranase was immobilized onto calcium alginate beads by the combination of ultrasonic irradiation and high hydrostatic pressure (US/HHP) treatments. Effects of US/HHP treatments on loading efficiency and immobilization yield of dextranase enzyme onto calcium alginate beads were investigated. Furthermore, the activities of immobilized enzymes prepared with and without US/HHP treatments and that prepared with ultrasonic irradiation (US) and high hydrostatic pressure (HHP), as a function of pH, temperature, recyclability and enzyme kinetic parameters, were compared with that for free enzyme. The maximum loading efficiency and the immobilization yield were observed when the immobilized dextranase was prepared with US (40 W at 25 kHz for 15 min) combined with HHP (400 MPa for 15 min), under which the loading efficiency and the immobilization yield increased by 88.92% and 80.86%, respectively, compared to immobilized enzymes prepared without US/HHP treatment. On the other hand, immobilized enzyme prepared with US/HHP treatment showed Vmax, KM, catalytic and specificity constants values higher than that for the immobilized enzyme prepared with HHP treatment, indicated that, this new US/HHP method improved the catalytic kinetics activity of immobilized dextranase at all the reaction conditions studied. Compared to immobilized enzyme prepared either with US or HHP, the immobilized enzymes prepared with US/HHP method exhibited a higher: pH optimum, optimal reaction temperature, thermal stability and recyclability, and lower activation energy, which, illustrating the effectiveness of the US/HHP method. These results indicated that, the combination of US and HHP treatments could be an effective method for improving the immobilization of enzymes in polymers.

  10. Biodegradation of cypermethrin by immobilized cells of Micrococcus sp. strain CPN 1.

    PubMed

    Tallur, Preeti N; Mulla, Sikandar I; Megadi, Veena B; Talwar, Manjunatha P; Ninnekar, Harichandra Z

    2015-01-01

    Pyrethroid pesticide cypermethrin is a environmental pollutant because of its widespread use, toxicity and persistence. Biodegradation of such chemicals by microorganisms may provide an cost-effective method for their detoxification. We have investigated the degradation of cypermethrin by immobilized cells of Micrococcus sp. strain CPN 1 in various matrices such as, polyurethane foam (PUF), polyacrylamide, sodium alginate and agar. The optimum temperature and pH for the degradation of cypermethrin by immobilized cells of Micrococcus sp. were found to be 30 °C and 7.0, respectively. The rate of degradation of 10 and 20 mM of cypermethrin by freely suspended cells were compared with that of immobilized cells in batches and semi-continuous with shaken cultures. PUF-immobilized cells showed higher degradation of cypermethrin (10 mM and 20 mM) than freely suspended cells and cells immobilized in other matrices. The PUF-immobilized cells of Micrococcus sp. strain CPN 1 were retain their degradation capacity. Thus, they can be reused for more than 32 cycles, without losing their degradation capacity. Hence, the PUF-immobilized cells of Micrococcus sp. could potentially be used in the bioremediation of cypermethrin contaminated water.

  11. Biodegradation of cypermethrin by immobilized cells of Micrococcus sp. strain CPN 1.

    PubMed

    Tallur, Preeti N; Mulla, Sikandar I; Megadi, Veena B; Talwar, Manjunatha P; Ninnekar, Harichandra Z

    2015-01-01

    Pyrethroid pesticide cypermethrin is a environmental pollutant because of its widespread use, toxicity and persistence. Biodegradation of such chemicals by microorganisms may provide an cost-effective method for their detoxification. We have investigated the degradation of cypermethrin by immobilized cells of Micrococcus sp. strain CPN 1 in various matrices such as, polyurethane foam (PUF), polyacrylamide, sodium alginate and agar. The optimum temperature and pH for the degradation of cypermethrin by immobilized cells of Micrococcus sp. were found to be 30 °C and 7.0, respectively. The rate of degradation of 10 and 20 mM of cypermethrin by freely suspended cells were compared with that of immobilized cells in batches and semi-continuous with shaken cultures. PUF-immobilized cells showed higher degradation of cypermethrin (10 mM and 20 mM) than freely suspended cells and cells immobilized in other matrices. The PUF-immobilized cells of Micrococcus sp. strain CPN 1 were retain their degradation capacity. Thus, they can be reused for more than 32 cycles, without losing their degradation capacity. Hence, the PUF-immobilized cells of Micrococcus sp. could potentially be used in the bioremediation of cypermethrin contaminated water. PMID:26413046

  12. Biodegradation of cypermethrin by immobilized cells of Micrococcus sp. strain CPN 1

    PubMed Central

    Tallur, Preeti N.; Mulla, Sikandar I.; Megadi, Veena B.; Talwar, Manjunatha P.; Ninnekar, Harichandra Z.

    2015-01-01

    Pyrethroid pesticide cypermethrin is a environmental pollutant because of its widespread use, toxicity and persistence. Biodegradation of such chemicals by microorganisms may provide an cost-effective method for their detoxification. We have investigated the degradation of cypermethrin by immobilized cells of Micrococcus sp. strain CPN 1 in various matrices such as, polyurethane foam (PUF), polyacrylamide, sodium alginate and agar. The optimum temperature and pH for the degradation of cypermethrin by immobilized cells of Micrococcus sp. were found to be 30 °C and 7.0, respectively. The rate of degradation of 10 and 20 mM of cypermethrin by freely suspended cells were compared with that of immobilized cells in batches and semi-continuous with shaken cultures. PUF-immobilized cells showed higher degradation of cypermethrin (10 mM and 20 mM) than freely suspended cells and cells immobilized in other matrices. The PUF-immobilized cells of Micrococcus sp. strain CPN 1 were retain their degradation capacity. Thus, they can be reused for more than 32 cycles, without losing their degradation capacity. Hence, the PUF-immobilized cells of Micrococcus sp. could potentially be used in the bioremediation of cypermethrin contaminated water. PMID:26413046

  13. Transplantation of bovine adrenocortical cells encapsulated in alginate

    PubMed Central

    Balyura, Mariya; Gelfgat, Evgeny; Ehrhart-Bornstein, Monika; Ludwig, Barbara; Gendler, Zohar; Barkai, Uriel; Zimerman, Baruch; Rotem, Avi; Block, Norman L.; Schally, Andrew V.; Bornstein, Stefan R.

    2015-01-01

    Current treatment options for adrenal insufficiency are limited to corticosteroid replacement therapies. However, hormone therapy does not replicate circadian rhythms and has unpleasant side effects especially due to the failure to restore normal function of the hypothalamic–pituitary–adrenal (HPA) axis. Adrenal cell transplantation and the restoration of HPA axis function would be a feasible and useful therapeutic strategy for patients with adrenal insufficiency. We created a bioartificial adrenal with 3D cell culture conditions by encapsulation of bovine adrenocortical cells (BACs) in alginate (enBACs). We found that, compared with BACs in monolayer culture, encapsulation in alginate significantly increased the life span of BACs. Encapsulation also improved significantly both the capacity of adrenal cells for stable, long-term basal hormone release as well as the response to pituitary adrenocorticotropic hormone (ACTH) and hypothalamic luteinizing hormone-releasing hormone (LHRH) agonist, [D-Trp6]LHRH. The enBACs were transplanted into adrenalectomized, immunodeficient, and immunocompetent rats. Animals received enBACs intraperitoneally, under the kidney capsule (free cells or cells encapsulated in alginate slabs) or s.c. enclosed in oxygenating and immunoisolating βAir devices. Graft function was confirmed by the presence of cortisol in the plasma of rats. Both types of grafted encapsulated cells, explanted after 21–25 d, preserved their morphology and functional response to ACTH stimulation. In conclusion, transplantation of a bioartificial adrenal with xenogeneic cells may be a treatment option for patients with adrenocortical insufficiency and other stress-related disorders. Furthermore, this model provides a microenvironment that ensures 3D cell–cell interactions as a unique tool to investigate new insights into cell biology, differentiation, tissue organization, and homeostasis. PMID:25675525

  14. Synthesis of superparamagnetic carboxymethyl chitosan/sodium alginate nanosphere and its application for immobilizing α-amylase.

    PubMed

    Jiang, Jianfang; Chen, Yongzheng; Wang, Wei; Cui, Baodong; Wan, Nanwei

    2016-10-20

    In order to improve catalytic activity, increase recycling times, reduce use cost for enzyme, superparamagnetic carboxymethyl chitosan/sodium alginate nanosphere (SM/CMC/SA) has been synthesized via an improved hydrothermal method, molecular self-assembly technology, electrostatic interaction and amide linkage. Its mean diameter was 65nm, zeta potential was -36.9mV and BET was 53.8m(2)/g. α-Amylase was selected as a simulation object to manufacture an immobilized enzyme (SM/CMC/CA/α-Amy), and its catalytic activity, release behavior, reusability and stability were researched. Immobilization increased 4.67 times to catalytic activity, slowed down release rate and improved reusable performance. SM/CMC/CA/α-Amy showed higher activity over a wider pH range, especially in strong acidic and alkaline environments. The thermal stability and storage stability were improved remarkably too. All these results indicated that SM/CMC/SA was an ideal carrier for immobilizing enzyme. PMID:27474605

  15. Use of Ca-alginate as a novel support for TiO2 immobilization in methylene blue decolorisation.

    PubMed

    Albarelli, Juliana Q; Santos, Diego T; Murphy, Sharon; Oelgemöller, Michael

    2009-01-01

    This study provides a preliminary contribution to the development of an industrial process for the UV/TiO(2) water treatment by introducing a novel support for TiO(2) immobilization. For the following study, Methylene Blue (MB) was chosen as the model dye to evaluate this novel immobilization system. The results showed that TiO(2) immobilized in a Ca-alginate bead retained its photoactivity during all of the experiments and the TiO(2)-gel beads presented good stability in water for maintaining its shape after several uses. When a proportion of 10% (v/v) of these beads was used, the configuration system demonstrated an improved mass transfer and consequently enhanced degradation efficiency. Experiments were also performed using 'recycled' beads. The results showed an increase in the degradation efficiency when the beads were reused, with an eventual 'self-destructive' effect. These studies showed great promise regarding the recyclable reagents with a reduction in waste at no greater cost or reduction in efficiency. Therefore, the potential of TiO(2)-gel beads as a simple and environmentally friendly catalyst for continuous use was developed.

  16. Enzymatic detection of mercuric ions in ground-water from vegetable wastes by immobilizing pumpkin (Cucumis melo) urease in calcium alginate beads.

    PubMed

    Prakash, Om; Talat, Mahe; Hasan, Syed Hadi; Pandey, Rajesh K

    2008-07-01

    Present report describes a quick and simple test based on enzyme inhibition for the detection of mercury in aqueous medium by urease immobilized in alginate beads. Urease was extracted from the discarded seeds of pumpkin (Cucumis melo) and was purified to apparent homogeneity (5.2-fold) by heat treatment at 48+/-0.1 degrees C and gel filtration through Sephadex G-200. The homogeneous enzyme preparation (Sp activity 353 U/mg protein, A(280)/A(260)=1.12) was immobilized in 3.5% alginate leading to 86% immobilization. Effect of mercuric ion on the activity of soluble as well as immobilized enzyme was investigated. Hg(2+) exhibited a concentration-dependent inhibition both in the presence and absence of the substrate. The alginate immobilized enzyme showed less inhibition. There was no leaching of the enzyme over a period of 15 days at 4 degrees C. The inhibition was non-competitive and the K(i) was found to be 1.26x10(-1)microM. Time-dependent interaction of urease with Hg(2+) exhibited a biphasic inhibition behavior in which approximately half of the initial activity was lost rapidly (within 10 min) and reminder in a slow phase. Binding of Hg(2+) with the enzyme was largely irreversible, as the activity could not be restored by dialysis. The significance of the observations is discussed.

  17. Ethanol production from nonsterilized carob pod extract by free and immobilized Saccharomyces cerevisiae cells using fed-batch culture

    SciTech Connect

    Roukas, T. . Dept. of Food Science and Technology)

    1994-02-05

    The production of ethanol from carob pod extract by free and immobilized Saccharomyces cerevisiae cells in batch and fed-batch culture was investigated. Fed-batch culture proved to be a better fermentation system for the production of ethanol than batch culture. In fed-batch culture, both free and immobilized S. cerevisiae cells gave the same maximum concentration of final ethanol at an initial sugar concentration of 300 g/L and F = 167 mL/h. The maximum ethanol productivity was obtained with both free and immobilized cells at a substrate concentration of 300 g/L and F = 334 mL/h. In repeated fed-batch culture, immobilized S. cerevisiae cells gave a higher overall ethanol concentration compared with the free cells. The immobilized S. cerevisiae cells in Ca-alginate beads retained their ability to produce ethanol for 10 days.

  18. pH-controlled construction of chitosan/alginate multilayer film: characterization and application for antibody immobilization.

    PubMed

    Yuan, Weiyong; Dong, Hua; Li, Chang Ming; Cui, Xiaoqiang; Yu, Ling; Lu, Zhisong; Zhou, Qin

    2007-12-18

    In this work, a chitosan/alginate multilayer film was constructed via layer-by-layer self-assembly and studied by in situ surface plasmon resonance monitoring and contact angle measurements. The results demonstrate that the surface composition of the self-assembled multilayer film can be simply tailored through pH control during the assembly process. The biological property of the assembled film was further characterized via antigen-antibody interactions, showing that the loading capacity of the antibody on the multilayered film and the binding activity of the antigen to the immobilized antibody could be also well-tuned by pH control. This work can provide more scientific insight in the interaction between protein and polymer matrix and render a novel simple approach to build high-performance biointerfaces through pH control for potential applications of highly sensitive immunosensors. PMID:18004897

  19. Enhanced U(VI) bioreduction by alginate-immobilized uranium-reducing bacteria in the presence of carbon nanotubes and anthraquinone-2,6-disulfonate.

    PubMed

    Wang, Weida; Feng, Yali; Tang, Xinhua; Li, Haoran; Du, Zhuwei; Yi, Aifei; Zhang, Xu

    2015-05-01

    Uranium-reducing bacteria were immobilized with sodium alginate, anthraquinone-2,6-disulfonate (AQDS), and carbon nanotubes (CNTs). The effects of different AQDS-CNTs contents, U(IV) concentrations, and metal ions on U(IV) reduction by immobilized beads were examined. Over 97.5% U(VI) (20 mg/L) was removed in 8 hr when the beads were added to 0.7% AQDS-CNTs, which was higher than that without AQDS-CNTs. This result may be attributed to the enhanced electron transfer by AQDS and CNTs. The reduction of U(VI) occurred at initial U(VI) concentrations of 10 to 100 mg/L and increased with increasing AQDS-CNT content from 0.1% to 1%. The presence of Fe(III), Cu(II) and Mn(II) slightly increased U(VI) reduction, whereas Cr(VI), Ni(II), Pb(II), and Zn(II) significantly inhibited U(VI) reduction. After eight successive incubation-washing cycles or 8 hr of retention time (HRT) for 48 hr of continuous operation, the removal efficiency of uranium was above 90% and 92%, respectively. The results indicate that the AQDS-CNT/AL/cell beads are suitable for the treatment of uranium-containing wastewaters.

  20. Surface modified alginate microcapsules for 3D cell culture

    NASA Astrophysics Data System (ADS)

    Chen, Yi-Wen; Kuo, Chiung Wen; Chueh, Di-Yen; Chen, Peilin

    2016-06-01

    Culture as three dimensional cell aggregates or spheroids can offer an ideal platform for tissue engineering applications and for pharmaceutical screening. Such 3D culture models, however, may suffer from the problems such as immune response and ineffective and cumbersome culture. This paper describes a simple method for producing microcapsules with alginate cores and a thin shell of poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) to encapsulate mouse induced pluripotent stem (miPS) cells, generating a non-fouling surface as an effective immunoisolation barrier. We demonstrated the trapping of the alginate microcapsules in a microwell array for the continuous observation and culture of a large number of encapsulated miPS cells in parallel. miPS cells cultured in the microcapsules survived well and proliferated to form a single cell aggregate. Droplet formation of monodisperse microcapsules with controlled size combined with flow cytometry provided an efficient way to quantitatively analyze the growth of encapsulated cells in a high-throughput manner. The simple and cost-effective coating technique employed to produce the core-shell microcapsules could be used in the emerging field of cell therapy. The microwell array would provide a convenient, user friendly and high-throughput platform for long-term cell culture and monitoring.

  1. Production of endothelial cell-enclosing alginate-based hydrogel fibers with a cell adhesive surface through simultaneous cross-linking by horseradish peroxidase-catalyzed reaction in a hydrodynamic spinning process.

    PubMed

    Liu, Yang; Sakai, Shinji; Taya, Masahito

    2012-09-01

    We developed an alginate-based hydrogel fiber enabling to enclose endothelial cells, degradable on-demand by alginate lyase, and having a cell adhesive surface. The hydrogel fiber was obtained by extruding an aqueous solution of 4% (w/v) alginate derivative possessing phenolic hydroxyl moieties (Alg-Ph) and horseradish peroxidase (HRP) into a flow of aqueous solution containing 0.3 mM H(2)O(2) and gelatin derivative possessing Ph moieties (Gelatin-Ph). In the process, cross-linking of Alg-Ph resulting in a hydrogel fiber and immobilization of Gelatin-Ph on the surface of the hydrogel fiber were simultaneously accomplished by an HRP-catalyzed cross-linking reaction between Ph moieties. The diameter of the hydrogel fiber and the quantity of immobilized Gelatin-Ph on the fiber were controllable by changing the flow rates of the solutions and the concentration of HRP in the Alg-Ph-containing solution, respectively. The viability of the human endothelial cells enclosed in the hydrogel fibers obtained by 10 s of flowing in the H(2)O(2)-containing solution was 87.1%. In addition, the cells harvested from the hydrogel fibers through degradation using alginate lyase grew on tissue culture dishes in the same fashion as the cells seeded by a conventional subculture protocol. Human smooth muscle cells adhered, grew and achieved confluence on the surface of the hydrogel fibers. By degrading the hydrogel fibers using alginate lyase, a tubular cell construct was successfully obtained.

  2. Survival of different cell lines in alginate-agarose microcapsules.

    PubMed

    Orive, G; Hernández, R M; Gascón, A R; Igartua, M; Pedraz, J L

    2003-01-01

    Cell microencapsulation has emerged as a promising therapeutic strategy to treat a wide range of diseases. The optimisation of this technology depends on several critical issues such as the careful selection of the cell line, the controlled manufacture of microcapsules and the suitable adaptation of the construct design to the selected cell line. In this work, we studied the behavior of hybridoma cells once enclosed in solid and liquefied core alginate-agarose beads. Results show that hybridoma cells presented a better growing pattern and improved their viability and antibody production within liquefied beads. However, when these beads were evaluated with a compression resistance study, they were found to be mechanically more fragile than solid ones. To address this problem, we entrapped non-autologous cells (BHK fibroblast and C2C12 myoblast) in solid alginate-agarose beads and observed that they showed an improved growing profile and prolonged their viability up to 70 days in comparison to the 15 days seen for the hybridoma cells.

  3. Surface characteristics determining the cell compatibility of ionically cross-linked alginate gels.

    PubMed

    Machida-Sano, Ikuko; Hirakawa, Makoto; Matsumoto, Hiroki; Kamada, Mitsuki; Ogawa, Sakito; Satoh, Nao; Namiki, Hideo

    2014-04-01

    In this study we investigated differences in the characteristics determining the suitability of five types of ion (Fe(3+), Al(3+), Ca(2+), Ba(2+) and Sr(2+))-cross-linked alginate films as culture substrates for cells. Human dermal fibroblasts were cultured on each alginate film to examine the cell affinity of the alginates. Since cell behavior on the surface of a material is dependent on the proteins adsorbed to it, we investigated the protein adsorption ability and surface features (wettability, morphology and charge) related to the protein adsorption abilities of alginate films. We observed that ferric, aluminum and barium ion-cross-linked alginate films supported better cell growth and adsorbed higher amounts of serum proteins than other types. Surface wettability analysis demonstrated that ferric and aluminum ion-cross-linked alginates had moderate hydrophilic surfaces, while other types showed highly hydrophilic surfaces. The roughness was exhibited only on barium ion-cross-linked alginate surface. Surface charge measurements revealed that alginate films had negatively charged surfaces, and showed little difference among the five types of gel. These results indicate that the critical factors of ionically cross-linked alginate films determining the protein adsorption ability required for their cell compatibility may be surface wettability and morphology. PMID:24496019

  4. General Approach to the Immobilization of Glycoenzyme Chains Inside Calcium Alginate Beads for Bioassay.

    PubMed

    Mallardi, Antonia; Angarano, Valeria; Magliulo, Maria; Torsi, Luisa; Palazzo, Gerardo

    2015-11-17

    A general method to obtain the efficient entrapment of mixtures of glycoenzymes in calcium alginate hydrogel is proposed in this paper. As a proof of principle, three glycoenzymes acting in series (trehalase, glucose oxidase, and horseradish peroxidase) have been coimmobilized in calcium alginate beads. The release of the enzymes from the hydrogel mesh (leakage) is avoided by exploiting the enzyme's aggregation induced by the concanavalin A. The aggregation process has been monitored by dynamic light scattering technique, while both enzyme encapsulation efficiency and leakage have been quantified spectrophotometrically. Obtained data show an encapsulation efficiency above 95% and a negligible leakage from the beads when enzyme aggregates are larger than 300 nm. Operational stability of "as prepared" beads has been largely improved by a coating of alternated shells of polycation poly(diallyldimethylammonium chloride) and of alginate. As a test for the effectiveness of the overall procedure, analytical bioassays exploiting the enzyme-containing beads have been developed for the optical determination of glucose and trehalose, and limit of detection values of 0.2 and of 40 μM, respectively, have been obtained.

  5. Biodegradation of pesticide profenofos by the free and immobilized cells of Pseudoxanthomonas suwonensis strain HNM.

    PubMed

    Talwar, Manjunatha P; Ninnekar, Harichandra Z

    2015-09-01

    Profenofos is an organophosphate pesticide used extensively in agriculture to control pests. A bacterium capable of degrading profenofos was isolated from pesticide-contaminated soil samples and identified as Pseudoxanthomonas suwonensis strain HNM based on its morphological and biochemical characteristics and phylogenetic analysis of 16S rRNA gene sequences. 4-Bromo-2-chlorophenol was identified as a metabolite of profenofos degradation by HPLC and GC-MS analysis. The organism degraded profenofos by hydrolysis to yield 4-bromo-2-chlorophenol which was further utilized as carbon source for growth. The organism utilized various organophosphate pesticides such as temephos, quinalphos, and chloropyrifos as carbon sources. The optimum conditions for degradation of profenofos by P. suwonensis strain HMN were found to be at pH 7 and 30 °C. We have investigated the rate of degradation of profenofos by the free and immobilized cells of P. suwonensis strain HNM in various matrices such as sodium alginate (SA), sodium alginate-polyvinyl alcohol (SA-PVA), and SA-bentonite clay. The rate of degradation of 3 and 6 mM profenofos by the freely suspended cells were compared with that by immobilized cells in batches and semi-continuous with shaken cultures. The SA-bentonite clay-immobilized cells showed higher rate of degradation of 3 and 6 mM profenofos then freely suspended cells and cells immobilized in SA and SA-PVA. The SA-bentonite clay-immobilized cells of P. suwonensis strain HNM could be reused for more than 32 cycles without losing their degradation capacity. Thus, the immobilized cells are more efficient than freely suspended cells for the degradation of organophosphate pesticide contaminated water.

  6. Biodegradation of pesticide profenofos by the free and immobilized cells of Pseudoxanthomonas suwonensis strain HNM.

    PubMed

    Talwar, Manjunatha P; Ninnekar, Harichandra Z

    2015-09-01

    Profenofos is an organophosphate pesticide used extensively in agriculture to control pests. A bacterium capable of degrading profenofos was isolated from pesticide-contaminated soil samples and identified as Pseudoxanthomonas suwonensis strain HNM based on its morphological and biochemical characteristics and phylogenetic analysis of 16S rRNA gene sequences. 4-Bromo-2-chlorophenol was identified as a metabolite of profenofos degradation by HPLC and GC-MS analysis. The organism degraded profenofos by hydrolysis to yield 4-bromo-2-chlorophenol which was further utilized as carbon source for growth. The organism utilized various organophosphate pesticides such as temephos, quinalphos, and chloropyrifos as carbon sources. The optimum conditions for degradation of profenofos by P. suwonensis strain HMN were found to be at pH 7 and 30 °C. We have investigated the rate of degradation of profenofos by the free and immobilized cells of P. suwonensis strain HNM in various matrices such as sodium alginate (SA), sodium alginate-polyvinyl alcohol (SA-PVA), and SA-bentonite clay. The rate of degradation of 3 and 6 mM profenofos by the freely suspended cells were compared with that by immobilized cells in batches and semi-continuous with shaken cultures. The SA-bentonite clay-immobilized cells showed higher rate of degradation of 3 and 6 mM profenofos then freely suspended cells and cells immobilized in SA and SA-PVA. The SA-bentonite clay-immobilized cells of P. suwonensis strain HNM could be reused for more than 32 cycles without losing their degradation capacity. Thus, the immobilized cells are more efficient than freely suspended cells for the degradation of organophosphate pesticide contaminated water. PMID:25832924

  7. Co-immobilization of Pseudomonas stutzeri YHA-13 and Alcaligenes sp. ZGED-12 with polyvinyl alcohol-alginate for removal of nitrogen and phosphorus from synthetic wastewater.

    PubMed

    Han, Yonghe; Zhang, Wenxian; Lu, Wenxian; Zhou, Zhihua; Zhuang, Zhigang; Li, Min

    2014-01-01

    Nitrogen (N) and phosphorus (P) are the two main factors causing water eutrophication. Immobilized micro-organisms have been widely studied in N and P removal. However, the effects of various immobilizing conditions on the removal efficiency of N and P using immobilized micro-organism beads (IMOBs) remain unclear. Polyvinyl alcohol (PVA) and alginate, as the two frequently immobilizing-used matrixes, were used for co-immobilizing Pseudomonas stutzeri YHA-13 and Alcaligenes sp. ZGED-12. PVA, alginate and CaCl₂contents, immobilization time and different wet biomass ratios of P. stutzeri to Alcaligenes sp. were conducted to elucidate their roles in and influences on the removal efficiency of N and P from synthetic wastewater. The application potential of IMOBs was estimated as well. Results showed that IMOBs prepared by cross-link of 4% PVA and 2-3% alginate with 5% CaCl₂and saturated boric acid solution for 10-15 min are the best ones in removal of N and P. Though IMOBs containing P. stutzeri and/or Alcaligenes sp. were capable of removal of the two nutrients, the highest removal efficiency was observed when the wet biomass ratio of P. stutzeri to Alcaligenes sp. was adjusted to 2:2. In addition, the IMOBs were of good ability to remove chemical oxygen demand (COD), NO(3)(-), NO(2)(-), NH(4)(+)- N, total nitrogen (TN) and total phosphorus (TP) from artificial wastewater. Of which, micro-organisms immobilized in matrixes were mainly responsible for NO(3)(-) and TP removal. Therefore, P. stutzeri YHA-13 and Alcaligenes sp. ZGED-12 are reliable bioresources to remove N and P from wastewater.

  8. Preparation of aminated chitosan/alginate scaffold containing halloysite nanotubes with improved cell attachment.

    PubMed

    Amir Afshar, Hamideh; Ghaee, Azadeh

    2016-10-20

    The chemical nature of biomaterials play important role in cell attachment, proliferation and migration in tissue engineering. Chitosan and alginate are biodegradable and biocompatible polymers used as scaffolds for various medical and clinical applications. Amine groups of chitosan scaffolds play an important role in cell attachment and water adsorption but also associate with alginate carboxyl groups via electrostatic interactions and hydrogen bonding, consequently the activity of amine groups in the scaffold decreases. In this study, chitosan/alginate/halloysite nanotube (HNTs) composite scaffolds were prepared using a freeze-drying method. Amine treatment on the scaffold occurred through chemical methods, which in turn caused the hydroxyl groups to be replaced with carboxyl groups in chitosan and alginate, after which a reaction between ethylenediamine, 1-ethyl-3,(3-dimethylaminopropyl) carbodiimide (EDC) and scaffold triggered the amine groups to connect to the carboxyl groups of chitosan and alginate. The chemical structure, morphology and mechanical properties of the composite scaffolds were investigated by FTIR, CHNS, SEM/EDS and compression tests. The electrostatic attraction and hydrogen bonding between chitosan, alginate and halloysite was confirmed by FTIR spectroscopy. Chitosan/alginate/halloysite scaffolds exhibit significant enhancement in compressive strength compared with chitosan/alginate scaffolds. CHNS and EDS perfectly illustrate that amine groups were effectively introduced in the aminated scaffold. The growth and cell attachment of L929 cells as well as the cytotoxicity of the scaffolds were investigated by SEM and Alamar Blue (AB). The results indicated that the aminated chitosan/alginate/halloysite scaffold has better cell growth and cell adherence in comparison to that of chitosan/alginate/halloysite samples. Aminated chitosan/alginate/halloysite composite scaffolds exhibit great potential for applications in tissue engineering, ideally in

  9. Preparation of aminated chitosan/alginate scaffold containing halloysite nanotubes with improved cell attachment.

    PubMed

    Amir Afshar, Hamideh; Ghaee, Azadeh

    2016-10-20

    The chemical nature of biomaterials play important role in cell attachment, proliferation and migration in tissue engineering. Chitosan and alginate are biodegradable and biocompatible polymers used as scaffolds for various medical and clinical applications. Amine groups of chitosan scaffolds play an important role in cell attachment and water adsorption but also associate with alginate carboxyl groups via electrostatic interactions and hydrogen bonding, consequently the activity of amine groups in the scaffold decreases. In this study, chitosan/alginate/halloysite nanotube (HNTs) composite scaffolds were prepared using a freeze-drying method. Amine treatment on the scaffold occurred through chemical methods, which in turn caused the hydroxyl groups to be replaced with carboxyl groups in chitosan and alginate, after which a reaction between ethylenediamine, 1-ethyl-3,(3-dimethylaminopropyl) carbodiimide (EDC) and scaffold triggered the amine groups to connect to the carboxyl groups of chitosan and alginate. The chemical structure, morphology and mechanical properties of the composite scaffolds were investigated by FTIR, CHNS, SEM/EDS and compression tests. The electrostatic attraction and hydrogen bonding between chitosan, alginate and halloysite was confirmed by FTIR spectroscopy. Chitosan/alginate/halloysite scaffolds exhibit significant enhancement in compressive strength compared with chitosan/alginate scaffolds. CHNS and EDS perfectly illustrate that amine groups were effectively introduced in the aminated scaffold. The growth and cell attachment of L929 cells as well as the cytotoxicity of the scaffolds were investigated by SEM and Alamar Blue (AB). The results indicated that the aminated chitosan/alginate/halloysite scaffold has better cell growth and cell adherence in comparison to that of chitosan/alginate/halloysite samples. Aminated chitosan/alginate/halloysite composite scaffolds exhibit great potential for applications in tissue engineering, ideally in

  10. Metabolic Responses of Bacterial Cells to Immobilization.

    PubMed

    Żur, Joanna; Wojcieszyńska, Danuta; Guzik, Urszula

    2016-01-01

    In recent years immobilized cells have commonly been used for various biotechnological applications, e.g., antibiotic production, soil bioremediation, biodegradation and biotransformation of xenobiotics in wastewater treatment plants. Although the literature data on the physiological changes and behaviour of cells in the immobilized state remain fragmentary, it is well documented that in natural settings microorganisms are mainly found in association with surfaces, which results in biofilm formation. Biofilms are characterized by genetic and physiological heterogeneity and the occurrence of altered microenvironments within the matrix. Microbial cells in communities display a variety of metabolic differences as compared to their free-living counterparts. Immobilization of bacteria can occur either as a natural phenomenon or as an artificial process. The majority of changes observed in immobilized cells result from protection provided by the supports. Knowledge about the main physiological responses occurring in immobilized cells may contribute to improving the efficiency of immobilization techniques. This paper reviews the main metabolic changes exhibited by immobilized bacterial cells, including growth rate, biodegradation capabilities, biocatalytic efficiency and plasmid stability. PMID:27455220

  11. Mechanically reinforced cell-laden scaffolds formed using alginate-based bioink printed onto the surface of a PCL/alginate mesh structure for regeneration of hard tissue.

    PubMed

    Kim, Yong Bok; Lee, Hyeongjin; Yang, Gi-Hoon; Choi, Chang Hyun; Lee, DaeWeon; Hwang, Heon; Jung, Won-Kyo; Yoon, Hyeon; Kim, Geun Hyung

    2016-01-01

    Cell-printing technology has provided a new paradigm for biofabrication, with potential to overcome several shortcomings of conventional scaffold-based tissue regeneration strategies via controlled delivery of various cell types in well-defined target regions. Here we describe a cell-printing method to obtain mechanically reinforced multi-layered cell-embedded scaffolds, formed of micron-scale poly(ε-caprolactone) (PCL)/alginate struts coated with alginate-based bioink. To compare the physical and cellular activities, we used a scaffold composed of pure alginate (without cells) coated PCL/alginate struts as a control. We systematically varied the ratio of alginate cross-linking agent, and determined the optimal cell-coating conditions to form the PCL/alginate struts. Following fabrication of the cell (MG63)-laden PCL/alginate scaffold, the bioactivity was evaluated in vitro. The laden cells exhibited a substantially more developed cytoskeleton compared with those on a control scaffold consisting of the same material composition. Based on these results, the printed cells exhibited a significantly more homogenous distribution within the scaffold compared with the control. Cell proliferation was determined via MTT assays at 1, 3, 7, and 14 days of culture, and the proliferation of the cell-printed scaffold was substantially in excess (∼2.4-fold) of that on the control. Furthermore, the osteogenic activity such as ALP was measured, and the cell-laden scaffold exhibited significantly greater activity (∼3.2-fold) compared with the control scaffold.

  12. Chemically crosslinked alginate porous microcarriers modified with bioactive molecule for expansion of human hepatocellular carcinoma cells.

    PubMed

    Li, Chunge; Zhao, Shuang; Zhao, Yanyan; Qian, Yufeng; Li, Junjie; Yin, Yuji

    2014-11-01

    Microcarrier is an essential matrix for the large-scale culture of anchorage-dependent cells. In this study, chemical cross-linked alginate porous microcarriers (AMC) were prepared using microemulsion and freeze-drying technology. Moreover, chitosan was coated on the surface of microcarriers (AMC-CS) via electrostatic interactions to improve the mechanical strength. The size of AMC can be modulated through adjusting the concentration of alginate, amount of dispersant and stirring rate. The surface chemical characteristics and morphology of AMC-CS were evaluated by Fourier transformed infrared, X-ray photoelectron spectroscopy, and scanning electron microscope. Fibronectin (Fn) or heparin/basic fibroblast growth factor (bFGF) was then immobilized on the surface of microcarriers via layer-by-layer technology to improve the cytocompatibility. Our data suggested that the size of AMC can be accurately modulated from 90 μm to 900 μm with a narrow size distribution. Micropore structures of AMC-CS were relatively disordered and the pore size ranged between 20 μm and 100 μm. Using AMC after modified with Fn or bFGF as the cell expansion microcarriers, we showed that the proliferation rates of HepG2 cells increased significantly, reaching to more than 30-fold of cell expansion after 10 days of culture, with minor cellular damage caused by the microcarriers. Moreover, the AMC microcarriers modified with Fn or bFGF can increase albumin secretion of HepG2. We suggest that our new modified AMC-based microcarriers will be an attractive candidate for the large-scale cell culture of therapeutic cells.

  13. Digital microfluidic three-dimensional cell culture and chemical screening platform using alginate hydrogels

    PubMed Central

    2015-01-01

    Electro wetting-on-dielectric (EWOD) digital microfluidics (DMF) can be used to develop improved chemical screening platforms using 3-dimensional (3D) cell culture. Alginate hydrogels are one common method by which a 3D cell culture environment is created. This paper presents a study of alginate gelation on EWOD DMF and investigates designs to obtain uniform alginate hydrogels that can be repeatedly addressed by any desired liquids. A design which allows for gels to be retained in place during liquid delivery and removal without using any physical barriers or hydrophilic patterning of substrates is presented. A proof of concept screening platform is demonstrated by examining the effects of different concentrations of a test chemical on 3D cells in alginate hydrogels. In addition, the temporal effects of the various chemical concentrations on different hydrogel posts are demonstrated, thereby establishing the benefits of an EWOD DMF 3D cell culture and chemical screening platform using alginate hydrogels. PMID:25945142

  14. Digital microfluidic three-dimensional cell culture and chemical screening platform using alginate hydrogels.

    PubMed

    George, Subin M; Moon, Hyejin

    2015-03-01

    Electro wetting-on-dielectric (EWOD) digital microfluidics (DMF) can be used to develop improved chemical screening platforms using 3-dimensional (3D) cell culture. Alginate hydrogels are one common method by which a 3D cell culture environment is created. This paper presents a study of alginate gelation on EWOD DMF and investigates designs to obtain uniform alginate hydrogels that can be repeatedly addressed by any desired liquids. A design which allows for gels to be retained in place during liquid delivery and removal without using any physical barriers or hydrophilic patterning of substrates is presented. A proof of concept screening platform is demonstrated by examining the effects of different concentrations of a test chemical on 3D cells in alginate hydrogels. In addition, the temporal effects of the various chemical concentrations on different hydrogel posts are demonstrated, thereby establishing the benefits of an EWOD DMF 3D cell culture and chemical screening platform using alginate hydrogels. PMID:25945142

  15. Alginate Encapsulation Parameters Influence the Differentiation of Microencapsulated Embryonic Stem Cell Aggregates

    PubMed Central

    Wilson, Jenna L.; Najia, Mohamad Ali; Saeed, Rabbia; McDevitt, Todd C.

    2014-01-01

    Pluripotent embryonic stem cells (ESCs) have tremendous potential as tools for regenerative medicine and drug discovery, yet the lack of processes to manufacture viable and homogenous cell populations of sufficient numbers limits the clinical translation of current and future cell therapies. Microencapsulation of ESCs within microbeads can shield cells from hydrodynamic shear forces found in bioreactor environments while allowing for sufficient diffusion of nutrients and oxygen through the encapsulation material. Despite initial studies examining alginate microbeads as a platform for stem cell expansion and directed differentiation, the impact of alginate encapsulation parameters on stem cell phenotype has not been thoroughly investigated. Therefore, the objective of this study was to systematically examine the effects of varying alginate compositions on microencapsulated ESC expansion and phenotype. Pre-formed aggregates of murine ESCs were encapsulated in alginate microbeads composed of a high or low ratio of guluronic to mannuronic acid residues (High G and High M, respectively), with and without a poly-l-lysine (PLL) coating, thereby providing four distinct alginate bead compositions for analysis. Encapsulation in all alginate compositions was found to delay differentiation, with encapsulation within High G alginate yielding the least differentiated cell population. The addition of a PLL coating to the High G alginate prevented cell escape from beads for up to 14 days. Furthermore, encapsulation within High M alginate promoted differentiation toward a primitive endoderm phenotype. Taken together, the findings of this study suggest that distinct ESC expansion capacities and differentiation trajectories emerge depending on the alginate composition employed, indicating that encapsulation material physical properties can be used to control stem cell fate. PMID:24166004

  16. Biomimetic Mineralization of the Alginate/Gelatin/Calcium Oxalate Matrix for Immobilization of Pectinase: Influence of Matrix on the Pectinolytic Activity.

    PubMed

    Bustamante-Vargas, Cindy Elena; de Oliveira, Débora; Valduga, Eunice; Venquiaruto, Luciana Dornelles; Paroul, Natalia; Backes, Geciane Toniazzo; Dallago, Rogério Marcos

    2016-07-01

    Pectinases catalyze the degradation of pectic substances and are used in several processes, mainly in food and textile industries. In this study, a biomimetic matrix of alginate/gelatin/calcium oxalate (AGOCa) was synthesized for the in situ immobilization via encapsulation of crude pectinase from Aspergillus niger ATCC 9642, obtaining an immobilization efficiency of about 61.7 %. To determine the performance of AGOCa matrix, this was compared to control matrices of alginate/calcium oxalate (AOxal) and alginate/water (ACa). By the evaluation of pH and temperature effects on the enzyme activity, it was observed an increase on pectinolytic activity for both three tested matrices with an increase on pH and temperature. The kinetic parameters for pectinase immobilized in the three matrices were determined using citric pectin as substrate. Values of K m of 0.003, 0.0013, and 0.0022 g mL(-1) and V max of 3.85, 4.32, and 3.17 μmol min(-1) g(-1) for AGOCa, AOxal, and ACa matrices were obtained, respectively. After 33 days of storage, the pectinase immobilized in the three different matrices kept its initial activity, but that immobilized in AGOCa presented high stability to the storage with a relative activity of about 160 %. The enzyme immobilized in AGOCa, AOxal, and ACa could be used in 10, 8, and 7 cycles, respectively, keeping 40 % of its initial activity.

  17. Degradation of mix hydrocarbons by immobilized cells of mix culture using a trickle fluidized bed reactor. Annual progress report, June 1992--May 1993

    SciTech Connect

    Chapatwala, K.D.

    1993-06-01

    The microorganisms, capable of degrading mix hydrocarbons were isolated from the soil samples collected from the hydrocarbon contaminated sites. The mix cultures were immobilized in calcium alginate solution in the form of beads. A trickle fluidized bed air-uplift-type reactor designed to study the degradation of mix hydrocarbons was filled with 0.85% normal saline containing the immobilized cells of mix culture. The immobilized beads were aerated with CO{sub 2}-free air at 200 ml/min. The degradation of different concentrations of hydrocarbons in the presence/absence of commercially available fertilizers by the immobilized cells of mix culture is now in progress.

  18. Immobilization of whole cells using polymeric coatings

    SciTech Connect

    Lawton, C.W.; Klei, H.E.; Sunstrom, D.V.; Voronka, P.J.; Scott, C.D.

    1986-01-01

    A cell immobilization procedure was developed using latex coatings on solid particles. The method's widespread applicability has been demonstrated by successfully immobilizing Saccharomyces cerevisiae (ethanol production), Bacillus subtilis (tryptophan production). Penicillium chrysogenum (penicillin G production), and Escherichia coli (aspartic acid production). In contrast to other immobilization methods, this procedure produces a pellicular particle that is porous, allowing rapid substrate and gas transfer, has a hard core to avoid compression in large beds, and is dense to allow use in fluidized beds. The immobilization procedure was optimized with S. cerevisiae. Kinetic constants obtained were used to calculate effectiveness factors to show that there was minimal intraparticle diffusion resistance. Reactors utilizing the optimized particles were run for 300 hours to evaluate immobilized particle half-life which was 250 hours.

  19. Immobilization of Lactobacillus rhamnosus in mesoporous silica-based material: An efficiency continuous cell-recycle fermentation system for lactic acid production.

    PubMed

    Zhao, Zijian; Xie, Xiaona; Wang, Zhi; Tao, Yanchun; Niu, Xuedun; Huang, Xuri; Liu, Li; Li, Zhengqiang

    2016-06-01

    Lactic acid bacteria immobilization methods have been widely used for lactic acid production. Until now, the most common immobilization matrix used is calcium alginate. However, Ca-alginate gel disintegrated during lactic acid fermentation. To overcome this deficiency, we developed an immobilization method in which Lactobacillus rhamnosus cells were successfully encapsulated into an ordered mesoporous silica-based material under mild conditions with a high immobilization efficiency of 78.77% by using elemental analysis. We also optimized the cultivation conditions of the immobilized L. rhamnosus and obtained a high glucose conversion yield of 92.4%. Furthermore, L. rhamnosus encapsulated in mesoporous silica-based material exhibited operational stability during repeated fermentation processes and no decrease in lactic acid production up to 8 repeated batches.

  20. Use of immobilized cells of Zymomonas mobilis in a novel fluidized bioreactor to produce ethanol

    SciTech Connect

    Margaritis, A.; Wallace, J.B.

    1982-01-01

    A novel 2-L fluidized bioreactor was designed and built and its performance characteristics were compared with those of an ordinary stirred-tank bioreactor. Calcium alginate beads containing immobilized cells of Zymomonas mobilis were used in both types of bioreactors to produce ethanol from glucose. The Ca-alginate beads were exposed to low shear rates inside the fluidized bioreactor and as a result the cell leakage from the beads was found to be about 100 times less than the cell leakage obtained in the mechanically stirred bioreactor. For the operating conditions studied, the fluidized bioreactor system gave at least 64% higher maximum rate of ethanol production and the power consumption per unit volume was found to be about four times less than that obtained with the mechanically stirred bioreactor. 9 figures, 1 table.

  1. Surface cell immobilization within perfluoroalkoxy microchannels

    NASA Astrophysics Data System (ADS)

    Stojkovič, Gorazd; Krivec, Matic; Vesel, Alenka; Marinšek, Marjan; Žnidaršič-Plazl, Polona

    2014-11-01

    Perfluoroalkoxy (PFA) is one of the most promising materials for the fabrication of cheap, solvent resistant and reusable microfluidic chips, which have been recently recognized as effective tools for biocatalytic process development. The application of biocatalysts significantly depends on efficient immobilization of enzymes or cells within the reactor enabling long-term biocatalyst use. Functionalization of PFA microchannels by 3-aminopropyltriethoxysilane (ATPES) and glutaraldehyde was used for rapid preparation of microbioreactors with surface-immobilized cells. X-ray photoelectron spectroscopy and scanning electron microscopy were used to accurately monitor individual treatment steps and to select conditions for cell immobilization. The optimized protocol for Saccharomyces cerevisiae immobilization on PFA microchannel walls comprised ethanol surface pretreatment, 4 h contacting with 10% APTES aqueous solution, 10 min treatment with 1% glutaraldehyde and 20 min contacting with cells in deionized water. The same protocol enabled also immobilization of Escherichia coli, Pseudomonas putida and Bacillus subtilis cells on PFA surface in high densities. Furthermore, the developed procedure has been proved to be very efficient also for surface immobilization of tested cells on other materials that are used for microreactor fabrication, including glass, polystyrene, poly (methyl methacrylate), polycarbonate, and two olefin-based polymers, namely Zeonor® and Topas®.

  2. Complete sucrose hydrolysis by heat-killed recombinant Pichia pastoris cells entrapped in calcium alginate

    PubMed Central

    2014-01-01

    Background An ideal immobilized biocatalyst for the industrial-scale production of invert sugar should stably operate at elevated temperatures (60-70°C) and high sucrose concentrations (above 60%, w/v). Commercial invertase from the yeast Saccharomyces cerevisiae is thermolabile and suffers from substrate inhibition. Thermotoga maritima β-fructosidase (BfrA) is the most thermoactive and thermostable sucrose-hydrolysing enzyme so far identified and allows complete inversion of the substrate in highly concentrated solutions. Results In this study, heat-killed Pichia pastoris cells bearing N-glycosylated BfrA in the periplasmic space were entrapped in calcium alginate beads. The immobilized recombinant yeast showed maximal sucrose hydrolysis at pH 5–7 and 90°C. BfrA was 65% active at 60°C and had no activity loss after incubation without the substrate at this temperature for 15 h. Complete inversion of cane sugar (2.04 M) at 60°C was achieved in batchwise and continuous operation with respective productivities of 4.37 and 0.88 gram of substrate hydrolysed per gram of dry beads per hour. The half-life values of the biocatalyst were 14 and 20 days when operated at 60°C in the stirred tank and the fixed-bed column, respectively. The reaction with non-viable cells prevented the occurrence of sucrose fermentation and the formation of by-products. Six-month storage of the biocatalyst in 1.46 M sucrose (pH 5.5) at 4°C caused no reduction of the invertase activity. Conclusions The features of the novel thermostable biocatalyst developed in this study are more attractive than those of immobilized S. cerevisiae cells for application in the enzymatic manufacture of inverted sugar syrup in batch and fixed-bed reactors. PMID:24943124

  3. Antibacterial Performance of Alginic Acid Coating on Polyethylene Film

    PubMed Central

    Karbassi, Elika; Asadinezhad, Ahmad; Lehocký, Marian; Humpolíček, Petr; Vesel, Alenka; Novák, Igor; Sáha, Petr

    2014-01-01

    Alginic acid coated polyethylene films were examined in terms of surface properties and bacteriostatic performance against two most representative bacterial strains, that is, Escherichia coli and Staphylococcus aureus. Microwave plasma treatment followed by brush formation in vapor state from three distinguished precursors (allylalcohol, allylamine, hydroxyethyl methacrylate) was carried out to deposit alginic acid on the substrate. Surface analyses via various techniques established that alginic acid was immobilized onto the surface where grafting (brush) chemistry influenced the amount of alginic acid coated. Moreover, alginic acid was found to be capable of bacterial growth inhibition which itself was significantly affected by the brush type. The polyanionic character of alginic acid as a carbohydrate polymer was assumed to play the pivotal role in antibacterial activity. The cell wall composition of two bacterial strains along with the substrates physicochemical properties accounted for different levels of bacteriostatic performance. PMID:25196604

  4. Antibacterial performance of alginic acid coating on polyethylene film.

    PubMed

    Karbassi, Elika; Asadinezhad, Ahmad; Lehocký, Marian; Humpolíček, Petr; Vesel, Alenka; Novák, Igor; Sáha, Petr

    2014-01-01

    Alginic acid coated polyethylene films were examined in terms of surface properties and bacteriostatic performance against two most representative bacterial strains, that is, Escherichia coli and Staphylococcus aureus. Microwave plasma treatment followed by brush formation in vapor state from three distinguished precursors (allylalcohol, allylamine, hydroxyethyl methacrylate) was carried out to deposit alginic acid on the substrate. Surface analyses via various techniques established that alginic acid was immobilized onto the surface where grafting (brush) chemistry influenced the amount of alginic acid coated. Moreover, alginic acid was found to be capable of bacterial growth inhibition which itself was significantly affected by the brush type. The polyanionic character of alginic acid as a carbohydrate polymer was assumed to play the pivotal role in antibacterial activity. The cell wall composition of two bacterial strains along with the substrates physicochemical properties accounted for different levels of bacteriostatic performance. PMID:25196604

  5. Glucosyltransferase production by Klebsiella sp. K18 and conversion of sucrose to palatinose using immobilized cells

    PubMed Central

    Orsi, Daniela C.; Kawaguti, Haroldo Y.; Sato, Hélia H.

    2009-01-01

    The strain Klebsiella sp. K18 produces the enzyme glucosyltransferase and catalyses the conversion of sucrose to palatinose, an alternative sugar that presents low cariogenicity. Response Surface Methodology was successfully employed to determine the optimal concentration of culture medium components. Maximum glucosyltransferase production (21.78 U mL-1) was achieved using the optimized medium composed by sugar cane molasses (80 g L-1), bacteriological peptone (7 g L-1) and yeast extract (20 g L-1), after 8 hours of fermentation at 28°C. The conversion of sucrose to palatinose was studied utilizing immobilized cells in calcium alginate. The effects of the alginate concentration (2-4%), cell mass concentration (20-40%) and substrate concentration (25-45%) were evaluated and the yield of palatinose was approximately 62.5%. PMID:24031319

  6. Batch and continuous flow studies of adsorptive removal of Cr(VI) by adapted bacterial consortia immobilized in alginate beads.

    PubMed

    Samuel, Jastin; Pulimi, Mrudula; Paul, Madona L; Maurya, Arun; Chandrasekaran, Natarajan; Mukherjee, Amitava

    2013-01-01

    The adsorptive removal of Cr(VI) by alginate beads containing Cr(VI)-adapted Acinetobacter junii, Escherichia coli and Bacillus subtilis in batch and continuous packed bed column reactors was investigated. Under optimized conditions (pH 3.0; contact time, 180 min; 30 °C; initial Cr(VI) concentration of 100 mg/L), 65.86 mg/g adsorption capacity was recorded in the batch study. When an adsorbent dosage of 1g/L, a flow rate of 5 mL/min, a bed height of 20 cm, an initial Cr(VI) concentration of 300 mg/L was employed, a capacity of 657 mg/g was noted for the continuous column assay. The batch sorption data followed the Langmuir isotherm and pseudo second order kinetics. Five sorption/desorption cycles yielded 100%, 99.63%, 95.31%, 80.7% and 74.22% regeneration, respectively. Cr(VI) adsorption studies using spiked ground water, freshwater and domestic wastewater in a packed bed reactor demonstrated Cr(VI) removals of 64.8%, 55.08%, 56.86% respectively. Cr(VI) sorption on immobilized bacteria was confirmed with Fourier-transform infrared and Energy dispersive X-ray spectroscopy. PMID:23201524

  7. Ethanol fermentation in an immobilized cell reactor using Saccharomyces cerevisiae.

    PubMed

    Najafpour, Ghasem; Younesi, Habibollah; Syahidah Ku Ismail, Ku

    2004-05-01

    Fermentation of sugar by Saccharomyces cerevisiae, for production of ethanol in an immobilized cell reactor (ICR) was successfully carried out to improve the performance of the fermentation process. The fermentation set-up was comprised of a column packed with beads of immobilized cells. The immobilization of S. cerevisiae was simply performed by the enriched cells cultured media harvested at exponential growth phase. The fixed cell loaded ICR was carried out at initial stage of operation and the cell was entrapped by calcium alginate. The production of ethanol was steady after 24 h of operation. The concentration of ethanol was affected by the media flow rates and residence time distribution from 2 to 7 h. In addition, batch fermentation was carried out with 50 g/l glucose concentration. Subsequently, the ethanol productions and the reactor productivities of batch fermentation and immobilized cells were compared. In batch fermentation, sugar consumption and ethanol production obtained were 99.6% and 12.5% v/v after 27 h while in the ICR, 88.2% and 16.7% v/v were obtained with 6 h retention time. Nearly 5% ethanol production was achieved with high glucose concentration (150 g/l) at 6 h retention time. A yield of 38% was obtained with 150 g/l glucose. The yield was improved approximately 27% on ICR and a 24 h fermentation time was reduced to 7 h. The cell growth rate was based on the Monod rate equation. The kinetic constants (K(s) and mu(m)) of batch fermentation were 2.3 g/l and 0.35 g/lh, respectively. The maximum yield of biomass on substrate (Y(X-S)) and the maximum yield of product on substrate (Y(P-S)) in batch fermentations were 50.8% and 31.2% respectively. Productivity of the ICR were 1.3, 2.3, and 2.8 g/lh for 25, 35, 50 g/l of glucose concentration, respectively. The productivity of ethanol in batch fermentation with 50 g/l glucose was calculated as 0.29 g/lh. Maximum production of ethanol in ICR when compared to batch reactor has shown to increase

  8. Continuous ethanol production from Jerusalem artichoke tubers. II. Use of immobilized cells of Kluyveromyces marxianus

    SciTech Connect

    Margaritis, A.; Bajpai, P.

    1982-07-01

    Kluyveromyces marxianus UCD (FST) 55-82 cells were immobilized in Na alginate beads and used in a packed-bed bioreactor system for the continuous production of ethanol from the extract of Jerusalem artichoke tubers. Volumetric ethanol productivities of 104 and 80 g ethanol/L/h were obtained at 80 and 92% sugar utilization, respectively. The maximum volumetric ethanol productivity of the immobilized cell bioreactor system was found to be 15 times higher than that of an ordinary continuous-stirred-tank (CST) bioreactor using free cells of Kluyveromyces marxianus. The immobilized cell bioreactor system was operated continuously at a constant dilution rate of 0.66/h for 12 days resulting in only an 8% loss of the original immobilized cell activity, which corresponds to an estimated half-life of ca. 72 days. The maximum specific ethanol productivity and maximum specific sugar uptake rate of the immobilized cells were found to be 0.55 g ethanol/g biomass/h and 1.21 g sugars/g biomass/h, respectively. (Refs. 27).

  9. The secretome of alginate-encapsulated limbal epithelial stem cells modulates corneal epithelial cell proliferation.

    PubMed

    Wright, Bernice; Hopkinson, Andrew; Leyland, Martin; Connon, Che J

    2013-01-01

    Limbal epithelial stem cells may ameliorate limbal stem cell deficiency through secretion of therapeutic proteins, delivered to the cornea in a controlled manner using hydrogels. In the present study the secretome of alginate-encapsulated limbal epithelial stem cells is investigated. Conditioned medium was generated from limbal epithelial stem cells encapsulated in 1.2% (w/v) calcium alginate gels. Conditioned medium proteins separated by 1-D gel electrophoresis were visualized by silver staining. Proteins of interest including secreted protein acidic and rich in cysteine, profilin-1, and galectin-1 were identified by immunoblotting. The effect of conditioned medium (from alginate-encapsulated limbal epithelial stem cells) on corneal epithelial cell proliferation was quantified and shown to significantly inhibit (P≤0.05) their growth. As secreted protein acidic and rich in cysteine was previously reported to attenuate proliferation of epithelial cells, this protein may be responsible, at least in part, for inhibition of corneal epithelial cell proliferation. We conclude that limbal epithelial stem cells encapsulated in alginate gels may regulate corneal epithelialisation through secretion of inhibitory proteins.

  10. Behavior of encapsulated MG-63 cells in RGD and gelatine-modified alginate hydrogels.

    PubMed

    Grigore, Alexandra; Sarker, Bapi; Fabry, Ben; Boccaccini, Aldo R; Detsch, Rainer

    2014-08-01

    Achieving cell spreading and proliferation inside hydrogels that are compatible with microencapsulation technology represents a major challenge for tissue engineering scaffolding and for the development of three-dimensional cell culture models. In this study, microcapsules of 650-900 μm in diameter were fabricated from oxidized alginate covalently cross-linked with gelatine (AlGel). Schiff's base bond formed in AlGel, detected by Fourier transform infrared spectroscopy, which confirmed the cross-linking of oxidized alginate with gelatine. Biological properties of alginate based hydrogels were studied by comparing the viability and morphology of MG-63 osteosarcoma cells encapsulated in gelatine and RGD-modified alginate. We hypothesized that the presence of gelatine and RGD will support cell adhesion and spreading inside the microcapsules and finally, also vascular endothelial growth factor (VEGF) secretion. After 4 days of incubation, cells formed extensive cortical protrusions and after 2 weeks they proliferated, migrated, and formed cellular networks through the AlGel material. In contrast, cells encapsulated in pure alginate and in RGD-modified alginate formed spherical aggregates with limited cell mobility and VEGF secretion. Metabolic activity was doubled after 5 days of incubation, making AlGel a promising material for cell encapsulation.

  11. Three-dimensional bioprinting of complex cell laden alginate hydrogel structures.

    PubMed

    Tabriz, Atabak Ghanizadeh; Hermida, Miguel A; Leslie, Nicholas R; Shu, Wenmiao

    2015-01-01

    Different bioprinting techniques have been used to produce cell-laden alginate hydrogel structures, however these approaches have been limited to 2D or simple three-dimension (3D) structures. In this study, a new extrusion based bioprinting technique was developed to produce more complex alginate hydrogel structures. This was achieved by dividing the alginate hydrogel cross-linking process into three stages: primary calcium ion cross-linking for printability of the gel, secondary calcium cross-linking for rigidity of the alginate hydrogel immediately after printing and tertiary barium ion cross-linking for long-term stability of the alginate hydrogel in culture medium. Simple 3D structures including tubes were first printed to ensure the feasibility of the bioprinting technique and then complex 3D structures such as branched vascular structures were successfully printed. The static stiffness of the alginate hydrogel after printing was 20.18 ± 1.62 KPa which was rigid enough to sustain the integrity of the complex 3D alginate hydrogel structure during the printing. The addition of 60 mM barium chloride was found to significantly extend the stability of the cross-linked alginate hydrogel from 3 d to beyond 11 d without compromising the cellular viability. The results based on cell bioprinting suggested that viability of U87-MG cells was 93 ± 0.9% immediately after bioprinting and cell viability maintained above 88% ± 4.3% in the alginate hydrogel over the period of 11 d. PMID:26689257

  12. Three-dimensional bioprinting of complex cell laden alginate hydrogel structures.

    PubMed

    Tabriz, Atabak Ghanizadeh; Hermida, Miguel A; Leslie, Nicholas R; Shu, Wenmiao

    2015-12-21

    Different bioprinting techniques have been used to produce cell-laden alginate hydrogel structures, however these approaches have been limited to 2D or simple three-dimension (3D) structures. In this study, a new extrusion based bioprinting technique was developed to produce more complex alginate hydrogel structures. This was achieved by dividing the alginate hydrogel cross-linking process into three stages: primary calcium ion cross-linking for printability of the gel, secondary calcium cross-linking for rigidity of the alginate hydrogel immediately after printing and tertiary barium ion cross-linking for long-term stability of the alginate hydrogel in culture medium. Simple 3D structures including tubes were first printed to ensure the feasibility of the bioprinting technique and then complex 3D structures such as branched vascular structures were successfully printed. The static stiffness of the alginate hydrogel after printing was 20.18 ± 1.62 KPa which was rigid enough to sustain the integrity of the complex 3D alginate hydrogel structure during the printing. The addition of 60 mM barium chloride was found to significantly extend the stability of the cross-linked alginate hydrogel from 3 d to beyond 11 d without compromising the cellular viability. The results based on cell bioprinting suggested that viability of U87-MG cells was 93 ± 0.9% immediately after bioprinting and cell viability maintained above 88% ± 4.3% in the alginate hydrogel over the period of 11 d.

  13. Calcium alginate matrix increases the stability and recycling capability of immobilized endo-β-1,4-xylanase from Geobacillus stearothermophilus KIBGE-IB29.

    PubMed

    Bibi, Zainab; Qader, Shah Ali Ul; Aman, Afsheen

    2015-07-01

    Exploration of microbial pool from extremely diversified ecosystem is significantly important for various industrial applications. Bacterial communities from extreme habitats including volcanic vents, hot springs, and industrial sectors are eagerly explored for the isolation of thermophiles. Geobacillus stearothermophilus KIBGE-IB29, isolated from blast furnace site of a steel processing industry, is capable of producing thermostable endo-β-1,4-xylanase. In the current study, this enzyme was immobilized within calcium alginate beads using entrapment technique. Amalgamation of sodium alginate (40.0 gL(-1)) and calcium chloride (0.4 M) was used for the formation of immobilized beads. It was observed that temperature (50 °C) and pH (7.0) optima of immobilized enzyme remained same, but enzyme-substrate reaction time increased from 5.0 to 30.0 min as compared to free enzyme. Diffusion limit of high molecular weight xylan (corncob) caused a decline in V max of immobilized enzyme from 4773 to 203.7 U min(-1), whereas K m value increased from 0.5074 to 0.5722 mg ml(-1) with reference to free enzyme. Immobilized endo-β-1,4-xylanase showed its stability even at high temperatures as compared to free enzyme and retained 18 and 9 % residual activity at 70 and 80 °C, respectively. Immobilized enzyme also exhibited sufficient recycling efficiency up to five reaction cycles which indicated that this enzyme can be a plausible candidate in paper and pulp industry. PMID:26001519

  14. Enhancing isomaltulose production by recombinant Escherichia coli producing sucrose isomerase: culture medium optimization containing agricultural wastes and cell immobilization.

    PubMed

    Li, Sha; Xu, Hong; Yu, Jianguang; Wang, Yanyuan; Feng, Xiaohai; Ouyang, Pingkai

    2013-10-01

    Isomaltulose is a structural isomer of sucrose commercially used in food industries. In this work, recombinant Escherichia coli producing sucrose isomerase (SIase) was used to convert sucrose into isomaltulose. To develop an economical industrial medium, untreated cane molasses (10.63 g l⁻¹), yeast extract (25.93 g l⁻¹), and corn steep liquor (10.45 g l⁻¹) were used as main culture compositions for SIase production. The relatively high SIase activity (14.50 ± 0.11 U mg DCW⁻¹) was obtained by the recombinant cells. To the best of our knowledge, this is the first investigation on SIase production by engineered E. coli using untreated cane molasses. The recombinant E. coli cells expressing the SIase gene were immobilized in calcium alginate gel in order to improve the efficiency of recycling. The immobilization was most effective with 2 % (w/v) sodium alginate and 3 % (w/v) calcium chloride. The optimal initial biomass for immobilization was 20 % (w/v, wet wt.), with a hardening time of 8 h for cell immobilization. The immobilized E. coli cells exhibited good stability for 30 batches with the productivity of 0.45 g isomaltulose g pellet⁻¹ h⁻¹. A continuous isomaltulose formation process using a column reactor remained stable for 40 days with 83 ± 2 % isomaltulose yield, which would be beneficial for economical production of isomaltulose. PMID:23300051

  15. Enhancing isomaltulose production by recombinant Escherichia coli producing sucrose isomerase: culture medium optimization containing agricultural wastes and cell immobilization.

    PubMed

    Li, Sha; Xu, Hong; Yu, Jianguang; Wang, Yanyuan; Feng, Xiaohai; Ouyang, Pingkai

    2013-10-01

    Isomaltulose is a structural isomer of sucrose commercially used in food industries. In this work, recombinant Escherichia coli producing sucrose isomerase (SIase) was used to convert sucrose into isomaltulose. To develop an economical industrial medium, untreated cane molasses (10.63 g l⁻¹), yeast extract (25.93 g l⁻¹), and corn steep liquor (10.45 g l⁻¹) were used as main culture compositions for SIase production. The relatively high SIase activity (14.50 ± 0.11 U mg DCW⁻¹) was obtained by the recombinant cells. To the best of our knowledge, this is the first investigation on SIase production by engineered E. coli using untreated cane molasses. The recombinant E. coli cells expressing the SIase gene were immobilized in calcium alginate gel in order to improve the efficiency of recycling. The immobilization was most effective with 2 % (w/v) sodium alginate and 3 % (w/v) calcium chloride. The optimal initial biomass for immobilization was 20 % (w/v, wet wt.), with a hardening time of 8 h for cell immobilization. The immobilized E. coli cells exhibited good stability for 30 batches with the productivity of 0.45 g isomaltulose g pellet⁻¹ h⁻¹. A continuous isomaltulose formation process using a column reactor remained stable for 40 days with 83 ± 2 % isomaltulose yield, which would be beneficial for economical production of isomaltulose.

  16. Enhanced dibenzothiophene biodesulfurization by immobilized cells of Brevibacterium lutescens in n-octane-water biphasic system.

    PubMed

    Dai, Yong; Shao, Rong; Qi, Gang; Ding, Bin-Bin

    2014-11-01

    In this study, it was the first report that the Brevibacterium lutescens CCZU12-1 was employed as a sulfur removing bacteria. Using dibenzothiophene (DBT) as the sole sulfur source, B. lutescens could selectively degrade DBT into 2-hydroxybiphenyl (2-HBP) via the "4S" pathway. In the basal salt medium (BSM) supplemented with 0.25 mM DBT and 0.5 g/L Tween-80, high desulfurization rate (100 %) was obtained by growth cells after 60 h. Furthermore, the n-octane-water (10:90, v/v) biphasic system was built for the biodesulfurization by resting cells. Moreover, a combination of magnetic nano Fe3O4 particles with calcium alginate immobilization was used for enhancing biodesulfurization. In this n-octane-water biphasic system, immobilized B. lutescens cells could be reused for not less than four times. Therefore, B. lutescens CCZU12-1 shows high potential in the biodesulfurization.

  17. Bioluminescence tracking of alginate micro-encapsulated cell transplants.

    PubMed

    Tiernan, Aubrey R; Sambanis, Athanassios

    2014-07-22

    Cell-based therapies to treat loss-of-function hormonal disorders such as diabetes and Parkinson's disease are routinely coupled with encapsulation strategies, but an understanding of when and why grafts fail in vivo is lacking. Consequently, investigators cannot clearly define the key factors that influence graft success. Although bioluminescence is a popular method to track the survival of free cells transplanted in preclinical models, little is known of the ability to use bioluminescence for real-time tracking of microencapsulated cells. Furthermore, the impact that dynamic imaging distances may have, due to freely-floating microcapsules in vivo, on cell survival monitoring is unknown. This work addresses these questions by applying bioluminescence to a pancreatic substitute based on microencapsulated cells. Recombinant insulin-secreting cells were transduced with a luciferase lentivirus and microencapsulated in Ba(2+) crosslinked alginate for in vitro and in vivo studies. In vitro quantitative bioluminescence monitoring was possible and viable microencapsulated cells were followed in real time under both normoxic and anoxic conditions. Although in vivo dispersion of freely-floating microcapsules in the peritoneal cavity limited the analysis to a qualitative bioluminescence evaluation, signals consistently four orders of magnitude above background were clear indicators of temporal cell survival. Strong agreement between in vivo and in vitro cell proliferation over time was discovered by making direct bioluminescence comparisons between explanted microcapsules and parallel in vitro cultures. Broader application of this bioluminescence approach to retrievable transplants, in supplement to currently used end-point physiological tests, could improve understanding and accelerate development of cell-based therapies for critical clinical applications. Copyright © 2014 John Wiley & Sons, Ltd.

  18. Efficient immobilization of mushroom tyrosinase utilizing whole cells from Agaricus bisporus and its application for degradation of bisphenol A.

    PubMed

    Kampmann, Markus; Boll, Stefan; Kossuch, Jan; Bielecki, Julia; Uhl, Stefan; Kleiner, Beatrice; Wichmann, Rolf

    2014-06-15

    A simple and efficient procedure for preparation and immobilization of tyrosinase enzyme was developed utilizing whole cells from the edible mushroom Agaricus bisporus, without the need for enzyme purification. Tyrosinase activity in the cell preparation remained constant during storage at 21 °C for at least six months. The cells were entrapped in chitosan and alginate matrix capsules and characterized with respect to their resulting tyrosinase activity. A modification of the alginate with colloidal silica enhanced the activity due to retention of both cells and tyrosinase from fractured cells, which otherwise leached from matrix capsules. The observed activity was similar to the activity that was obtained with immobilized isolated tyrosinase in the same material. Mushroom cells in water were susceptible to rapid inactivation, whereas the immobilized cells maintained 73% of their initial activity after 30 days of storage in water. Application in repeated batch experiments resulted in almost 100% conversion of endocrine disrupting bisphenol A (BPA) for 11 days, under stirring conditions, and 50-60% conversion after 20 days, without stirring under continuous usage. The results represent the longest yet reported application of immobilized tyrosinase for degradation of BPA in environmental water samples.

  19. Brown adipogenesis of mouse embryonic stem cells in alginate microstrands

    NASA Astrophysics Data System (ADS)

    Unser, Andrea Mannarino

    The ability of brown adipocytes (fat cells) to dissipate energy as heat shows great promise for the treatment of obesity and other metabolic disorders. Employing pluripotent stem cells, with an emphasis on directed differentiation, may overcome many issues currently associated with primary fat cell cultures. However, brown adipocytes are difficult to transplant in vivo due to the instability of fat, in terms of necrosis and neovascularization, once injected. Thus, 3D cell culture systems that have the potential to mimic adipogenic microenvironments are needed, not only to advance brown fat implantation, but also to better understand the role of brown adipocytes in treating obesity. To address this need, we created 3D "Brown-Fat-in-Microstrands" by microfluidic synthesis of alginate hydrogel microstrands that encapsulated cells and directly induced cell differentiation into brown adipocytes, using mouse embryonic stem cells (ESCs) as a model of pluripotent stem cells and brown preadipocytes as a positive control. The effect of hydrogel formation parameters on brown adipogenesis was studied, leading to the establishment of "Brown-Fat-in-Microstrands". Brown adipocyte differentiation within microstrands was confirmed by lipid droplet accumulation, immunocytochemistry and qPCR analysis of gene expression of brown adipocyte marker uncoupling protein 1 (UCP1) in addition to adipocyte marker expression. Compared to a 2D approach, 3D differentiated "Brown-Fat-in-Microstrands" exhibited higher level of brown adipocyte marker expression. The functional analysis of "Brown-Fat-in-Microstrands" was attempted by measuring the mitochondrial activity of ESC-differentiated brown adipocytes in 3D using Seahorse XF24 3 Extracellular Flux Analyzer. The ability to create "Brown-Fat-in-Microstrands" from pluripotent stem cells opens up a new arena to understanding brown adipogenesis and its implications in obesity and metabolic disorders.

  20. Biodegradation of Methyl Orange by alginate-immobilized Aeromonas sp. in a packed bed reactor: external mass transfer modeling.

    PubMed

    Kathiravan, Mathur Nadarajan; Praveen, Siluvai Antony; Gim, Geun Ho; Han, Gui Hawn; Kim, Si Wouk

    2014-11-01

    Azo dyes are recalcitrant and xenobiotic nature makes these compounds a challenging task for continuous biodegradation up to satisfactorily levels in large-scale. In the present report, the biodegradation efficiency of alginate immobilized indigenous Aeromonas sp. MNK1 on Methyl Orange (MO) in a packed bed reactor was explored. The experimental results were used to determine the external mass transfer model. Complete MO degradation and COD removal were observed at 0.20 cm bead size and 120 ml/h flow rate at 300 mg/l of initial dye concentration. The degradation of MO decreased with increasing bead sizes and flow rates, which may be attributed to the decrease in surface of the beads and higher flux of MO, respectively. The experimental rate constants (k ps) for various beads sizes and flow rates were calculated and compared with theoretically obtained rate constants using external film diffusion models. From the experimental data, the external mass transfer effect was correlated with a model J D = K Re (-(1 - n)). The model was tested with K value (5.7) and the Colburn factor correlation model for 0.20, 0.40 and 0.60 bead sizes were J D = 5.7 Re (-0.15), J D = 5.7 Re (-0.36) and J D = 5.7 Re (-0.48), respectively. Based on the results, the Colburn factor correlation models were found to predict the experimental data accurately. The proposed model was constructive to design and direct industrial applications in packed bed reactors within acceptable limits.

  1. Mechanically reinforced cell-laden scaffolds formed using alginate-based bioink printed onto the surface of a PCL/alginate mesh structure for regeneration of hard tissue.

    PubMed

    Kim, Yong Bok; Lee, Hyeongjin; Yang, Gi-Hoon; Choi, Chang Hyun; Lee, DaeWeon; Hwang, Heon; Jung, Won-Kyo; Yoon, Hyeon; Kim, Geun Hyung

    2016-01-01

    Cell-printing technology has provided a new paradigm for biofabrication, with potential to overcome several shortcomings of conventional scaffold-based tissue regeneration strategies via controlled delivery of various cell types in well-defined target regions. Here we describe a cell-printing method to obtain mechanically reinforced multi-layered cell-embedded scaffolds, formed of micron-scale poly(ε-caprolactone) (PCL)/alginate struts coated with alginate-based bioink. To compare the physical and cellular activities, we used a scaffold composed of pure alginate (without cells) coated PCL/alginate struts as a control. We systematically varied the ratio of alginate cross-linking agent, and determined the optimal cell-coating conditions to form the PCL/alginate struts. Following fabrication of the cell (MG63)-laden PCL/alginate scaffold, the bioactivity was evaluated in vitro. The laden cells exhibited a substantially more developed cytoskeleton compared with those on a control scaffold consisting of the same material composition. Based on these results, the printed cells exhibited a significantly more homogenous distribution within the scaffold compared with the control. Cell proliferation was determined via MTT assays at 1, 3, 7, and 14 days of culture, and the proliferation of the cell-printed scaffold was substantially in excess (∼2.4-fold) of that on the control. Furthermore, the osteogenic activity such as ALP was measured, and the cell-laden scaffold exhibited significantly greater activity (∼3.2-fold) compared with the control scaffold. PMID:26409783

  2. Simultaneous wastewater treatment, electricity generation and biomass production by an immobilized photosynthetic algal microbial fuel cell.

    PubMed

    He, Huanhuan; Zhou, Minghua; Yang, Jie; Hu, Youshuang; Zhao, Yingying

    2014-05-01

    A photosynthetic algal microbial fuel cell (PAMFC) was constructed by the introduction of immobilized microalgae (Chlorella vulgaris) into the cathode chamber of microbial fuel cells to fulfill electricity generation, biomass production and wastewater treatment. The immobilization conditions, including the concentration of immobilized matrix, initial inoculation concentration and cross-linking time, were investigated both for the growth of C. vulgaris and power generation. It performed the best at 5 % sodium alginate and 2 % calcium chloride as immobilization matrix, initial inoculation concentration of 10(6) cell/mL and cross-linking time of 4 h. Our findings indicated that C. vulgaris immobilization was an effective and promising approach to improve the performance of PAMFC, and after optimization the power density and Coulombic efficiency improved by 258 and 88.4 %, respectively. Important parameters such as temperature and light intensity were optimized on the performance. PAMFC could achieve a COD removal efficiency of 92.1 %, and simultaneously the maximum power density reached 2,572.8 mW/m(3) and the Coulombic efficiency was 14.1 %, under the light intensity of 5,000 lux and temperature at 25 °C.

  3. Cell(MC3T3-E1)-printed poly(ϵ-caprolactone)/alginate hybrid scaffolds for tissue regeneration.

    PubMed

    Lee, Hyeongjin; Ahn, SeungHyun; Bonassar, Lawrence J; Kim, GeunHyung

    2013-01-25

    A new cell-printed scaffold consisting of poly(ϵ-caprolactone) (PCL) and cell-embedded alginate struts is designed. The PCL and alginate struts are stacked in an interdigitated pattern in successive layers to acquire a three-dimensional (3D) shape. The hybrid scaffold exhibits a two-phase structure consisting of cell (MC3T3-E1)-laden alginate struts able to support biological activity and PCL struts able to provide controllable mechanical support of the cell-laden alginate struts. The hybrid scaffolds exhibit an impressive increase in tensile modulus and maximum strength compared to pure alginate scaffolds. Laden cells are homogeneously distributed throughout the alginate struts and the entire scaffold, resulting in cell viability of approximately 84%.

  4. Statistical optimization of alpha-amylase production by Streptomyces erumpens MTCC 7317 cells in calcium alginate beads using response surface methodology.

    PubMed

    Kar, Shaktimay; Ray, Ramesh C

    2008-01-01

    Alpha-amylase has a wide range of applications in starch industries, i.e. baking, brewing, distillery, etc. The alpha-amylase production from Streptomyces erumpens MTCC 7317 immobilized cells was compared with that of free cells. The immobilized cells of S. erumpens in calcium alginate beads were more effective for production of alpha-amylase (12.2% more yield) than free cells. Response surface methodology (RSM) was used to evaluate the effect of main variables, i.e. incubation period, pH and temperature on enzyme production with immobilized cells. A full factorial Central Composite Design (CCD) was applied to study these main factors that affected alpha-amylase production. The experimental results showed that the optimum incubation period, pH and temperature were 36 h, 6.0 and 50 degrees C, respectively for immobilized cells. Repeated batch fermentation of immobilized cells in shake flasks carried out in starch-beef extract medium showed that S. erumpens cells were physiologically active on the support even after four cycles of fermentation.

  5. Encapsulating Non-Human Primate Multipotent Stromal Cells in Alginate via High Voltage for Cell-Based Therapies and Cryopreservation

    PubMed Central

    Gryshkov, Oleksandr; Pogozhykh, Denys; Hofmann, Nicola; Pogozhykh, Olena; Mueller, Thomas; Glasmacher, Birgit

    2014-01-01

    Alginate cell-based therapy requires further development focused on clinical application. To assess engraftment, risk of mutations and therapeutic benefit studies should be performed in an appropriate non-human primate model, such as the common marmoset (Callithrix jacchus). In this work we encapsulated amnion derived multipotent stromal cells (MSCs) from Callithrix jacchus in defined size alginate beads using a high voltage technique. Our results indicate that i) alginate-cell mixing procedure and cell concentration do not affect the diameter of alginate beads, ii) encapsulation of high cell numbers (up to 10×106 cells/ml) can be performed in alginate beads utilizing high voltage and iii) high voltage (15–30 kV) does not alter the viability, proliferation and differentiation capacity of MSCs post-encapsulation compared with alginate encapsulated cells produced by the traditional air-flow method. The consistent results were obtained over the period of 7 days of encapsulated MSCs culture and after cryopreservation utilizing a slow cooling procedure (1 K/min). The results of this work show that high voltage encapsulation can further be maximized to develop cell-based therapies with alginate beads in a non-human primate model towards human application. PMID:25259731

  6. Design and performance of a sericin-alginate interpenetrating network hydrogel for cell and drug delivery.

    PubMed

    Zhang, Yeshun; Liu, Jia; Huang, Lei; Wang, Zheng; Wang, Lin

    2015-01-01

    Although alginate hydrogels have been extensively studied for tissue engineering applications, their utilization is limited by poor mechanical strength, rapid drug release, and a lack of cell adhesive ability. Aiming to improve these properties, we employ the interpenetrating hydrogel design rationale. Using alginate and sericin (a natural protein with many unique properties and a major component of silkworm silk), we develop an interpenetrating polymer network (IPN) hydrogel comprising interwoven sericin and alginate double networks. By adjusting the sericin-to-alginate ratios, IPNs' mechanical strength can be adjusted to meet stiffness requirements for various tissue repairs. The IPNs with high sericin content show increased stability during degradation, avoiding pure alginate's early collapse. These IPNs have high swelling ratios, benefiting various applications such as drug delivery. The IPNs sustain controlled drug release with the adjustable rates. Furthermore, these IPNs are adhesive to cells, supporting cell proliferation, long-term survival and migration. Notably, the IPNs inherit sericin's photoluminescent property, enabling bioimaging in vivo. Together, our study indicates that the sericin-alginate IPN hydrogels may serve as a versatile platform for delivering cells and drugs, and suggests that sericin may be a building block broadly applicable for generating IPN networks with other biomaterials for diverse tissue engineering applications. PMID:26205586

  7. Cell growth on immobilized cell growth factor. 8. Protein-free cell culture on insulin-immobilized microcarriers.

    PubMed

    Ito, Y; Uno, T; Liu, S Q; Imanishi, Y

    1992-12-01

    In order to develop a new protein-free cell culture system, microcarriers immobilized with insulin were synthesized. For the synthesis, glass and polyacrylamide beads were treated for the introduction of amino groups on the surface, and insulin was immobilized on the surface by using several method. Anchorage-dependent cells. mouse fibroblast cells STO and fibroic sarcoma cells HSDM(1)C(1), and the anchorage-independent cells, mouse hybridoma cells SJK132-20 and RDP 45/20 were cultivated on the microcarriers immobilized with insulin. The insulin-immobilized microcarriers did not have any effect on the proliferation of the anchorage independent cells but promoted the growth of anchorage-dependent cells remarkably. The activity of immobilized insulin was larger than that of free or adsorbed insulin. The repeated use of the insulin-immobilized microcarrier was possible, and the promotion activity in the the repeated use was greater than that in the use.

  8. Invertase in immobilized cells of Papaver somniferum L.

    PubMed

    Stano, J; Nemec, P; Bezáková, L; Kovács, P; Kákoniova, D; Neubert, K; Lisková, D

    1997-03-01

    Papaver somniferum L., (opium poppy) cells were after permeabilization in Tween 80 immobilized by glutaraldehyde without any carrier. Cells immobilized by cross-linking performed the hydrolysis of sucrose. The immobilized cells were characterized by high invertase activity and appropriate physico-mechanical properties.

  9. Ethanol tolerance of immobilized brewers' yeast cells.

    PubMed

    Norton, S; Watson, K; D'Amore, T

    1995-04-01

    A method based on the survival of yeast cells subjected to an ethanol or heat shock was utilized to compare the stress resistance of free and carrageenan-immobilized yeast cells. Results demonstrated a significant increase of yeast survival against ethanol for immobilized cells as compared to free cells, while no marked difference in heat resistance was observed. When entrapped cells were released by mechanical disruption of the gel beads and submitted to the same ethanol stress, they exhibited a lower survival rate than entrapped cells, but a similar or slightly higher survival rate than free cells. The incidence of ethanol- or heat-induced respiratory-deficient mutants of entrapped cells was equivalent to that of control or non-stressed cells (1.3 +/- 0.5%) whereas ethanol- and heat-shocked free and released cells exhibited between 4.4% and 10.9% average incidence of respiration-deficient mutants. It was concluded that the carrageenan gel matrix provided a protection against ethanol, and that entrapped cells returned to normal physiological behaviour as soon as they were released. The cell growth rate was a significant factor in the resistance of yeast to high ethanol concentrations. The optimum conditions to obtain reliable and reproducible results involved the use of slow-growing cells after exhaustion of the sugar substrate.

  10. Optimization of ethanol production from carob pod extract using immobilized Saccharomyces cerevisiae cells in a stirred tank bioreactor.

    PubMed

    Ercan, Yatmaz; Irfan, Turhan; Mustafa, Karhan

    2013-05-01

    In this study, optimization of ethanol production from carob pod extract was carried out by immobilized Saccharomyces cerevisiae. Results showed that Ca-alginate concentration and the amount of immobilized cells had significant effects on yield. Optimum conditions for ethanol fermentation were determined to be 2% Ca-alginate concentration, 150 rpm agitation rate, 5% yeast cells entrapped in beads and pH 5.5. After validation experiments; ethanol concentration, yield, production rate and sugar utilization rate were respectively 40.10 g/L, 46.32%, 3.19 g/L/h and 90.66%; and the fermentation time was decreased to 24 h. In addition, the immobilized cells were shown to be reusable for five cycles, though a decrease in yield was observed. Finally, carob pod extract was used for ethanol fermentation by controlled and uncontrolled pH without any enrichment, and the results suggest that carob extract can be utilized effectively by immobilized-cell fermentation without the use of enrichments to facilitate yeast growth.

  11. Methylene blue adsorption on graphene oxide/calcium alginate composites.

    PubMed

    Li, Yanhui; Du, Qiuju; Liu, Tonghao; Sun, Jiankun; Wang, Yonghao; Wu, Shaoling; Wang, Zonghua; Xia, Yanzhi; Xia, Linhua

    2013-06-01

    Graphene oxide has been used as an adsorbent in wastewater treatment. However, the dispersibility in aqueous solution and the biotoxicity to human cells of graphene oxide limits its practical application in environmental protection. In this research, a novel environmental friendly adsorbent, calcium alginate immobilized graphene oxide composites was prepared. The effects of pH, contact time, temperature and dosage on the adsorption properties of methylene blue onto calcium alginate immobilized graphene oxide composites were investigated. The equilibrium adsorption data were described by the Langmuir and Freundlich isotherms. The maximum adsorption capacity obtained from Langmuir isotherm equation was 181.81 mg/g. The pseudo-first order, pseudo-second order, and intraparticle diffusion equation were used to evaluate the kinetic data. Thermodynamic analysis of equilibriums indicated that the adsorption reaction of methylene blue onto calcium alginate immobilized graphene oxide composites was exothermic and spontaneous in nature.

  12. Alginate hydrogel protects encapsulated hepatic HuH-7 cells against hepatitis C virus and other viral infections.

    PubMed

    Tran, Nhu-Mai; Dufresne, Murielle; Helle, François; Hoffmann, Thomas Walter; François, Catherine; Brochot, Etienne; Paullier, Patrick; Legallais, Cécile; Duverlie, Gilles; Castelain, Sandrine

    2014-01-01

    Cell microencapsulation in alginate hydrogel has shown interesting applications in regenerative medicine and the biomedical field through implantation of encapsulated tissue or for bioartificial organ development. Although alginate solution is known to have low antiviral activity, the same property regarding alginate gel has not yet been studied. The aim of this work is to investigate the potential protective effect of alginate encapsulation against hepatitis C virus (HCV) infection for a hepatic cell line (HuH-7) normally permissive to the virus. Our results showed that alginate hydrogel protects HuH-7 cells against HCV when the supernatant was loaded with HCV. In addition, alginate hydrogel blocked HCV particle release out of the beads when the HuH-7 cells were previously infected and encapsulated. There was evidence of interaction between the molecules of alginate hydrogel and HCV, which was dose- and incubation time-dependent. The protective efficiency of alginate hydrogel towards HCV infection was confirmed against a variety of viruses, whether or not they were enveloped. This promising interaction between an alginate matrix and viruses, whose chemical mechanisms are discussed, is of great interest for further medical therapeutic applications based on tissue engineering.

  13. Alginate Hydrogel Protects Encapsulated Hepatic HuH-7 Cells against Hepatitis C Virus and Other Viral Infections

    PubMed Central

    Tran, Nhu-Mai; Dufresne, Murielle; Helle, François; Hoffmann, Thomas Walter; François, Catherine; Brochot, Etienne; Paullier, Patrick; Legallais, Cécile; Duverlie, Gilles; Castelain, Sandrine

    2014-01-01

    Cell microencapsulation in alginate hydrogel has shown interesting applications in regenerative medicine and the biomedical field through implantation of encapsulated tissue or for bioartificial organ development. Although alginate solution is known to have low antiviral activity, the same property regarding alginate gel has not yet been studied. The aim of this work is to investigate the potential protective effect of alginate encapsulation against hepatitis C virus (HCV) infection for a hepatic cell line (HuH-7) normally permissive to the virus. Our results showed that alginate hydrogel protects HuH-7 cells against HCV when the supernatant was loaded with HCV. In addition, alginate hydrogel blocked HCV particle release out of the beads when the HuH-7 cells were previously infected and encapsulated. There was evidence of interaction between the molecules of alginate hydrogel and HCV, which was dose- and incubation time-dependent. The protective efficiency of alginate hydrogel towards HCV infection was confirmed against a variety of viruses, whether or not they were enveloped. This promising interaction between an alginate matrix and viruses, whose chemical mechanisms are discussed, is of great interest for further medical therapeutic applications based on tissue engineering. PMID:25310111

  14. Design and performance of a sericin-alginate interpenetrating network hydrogel for cell and drug delivery

    NASA Astrophysics Data System (ADS)

    Zhang, Yeshun; Liu, Jia; Huang, Lei; Wang, Zheng; Wang, Lin

    2015-07-01

    Although alginate hydrogels have been extensively studied for tissue engineering applications, their utilization is limited by poor mechanical strength, rapid drug release, and a lack of cell adhesive ability. Aiming to improve these properties, we employ the interpenetrating hydrogel design rationale. Using alginate and sericin (a natural protein with many unique properties and a major component of silkworm silk), we develop an interpenetrating polymer network (IPN) hydrogel comprising interwoven sericin and alginate double networks. By adjusting the sericin-to-alginate ratios, IPNs’ mechanical strength can be adjusted to meet stiffness requirements for various tissue repairs. The IPNs with high sericin content show increased stability during degradation, avoiding pure alginate’s early collapse. These IPNs have high swelling ratios, benefiting various applications such as drug delivery. The IPNs sustain controlled drug release with the adjustable rates. Furthermore, these IPNs are adhesive to cells, supporting cell proliferation, long-term survival and migration. Notably, the IPNs inherit sericin’s photoluminescent property, enabling bioimaging in vivo. Together, our study indicates that the sericin-alginate IPN hydrogels may serve as a versatile platform for delivering cells and drugs, and suggests that sericin may be a building block broadly applicable for generating IPN networks with other biomaterials for diverse tissue engineering applications.

  15. Enzymatically cross-linked alginic-hyaluronic acid composite hydrogels as cell delivery vehicles.

    PubMed

    Ganesh, Nitya; Hanna, Craig; Nair, Shantikumar V; Nair, Lakshmi S

    2013-04-01

    An injectable composite gel was developed from alginic and hyaluronic acid. The enzymatically cross-linked injectable gels were prepared via the oxidative coupling of tyramine modified sodium algiante and sodium hyaluronate in the presence of horse radish peroxidase (HRP) and hydrogen peroxide (H2O2). The composite gels were prepared by mixing equal parts of the two tyraminated polymer solutions in 10U HRP and treating with 1.0% H2O2. The properties of the alginate gels were significantly affected by the addition of hyaluronic acid. The percentage water absorption and storage modulus of the composite gels were found to be lower than the alginate gels. The alginate and composite gels showed lower protein release compared to hyaluronate gels in the absence of hyaluronidase. Even hyaluronate gels showed only approximately 10% protein release after 14 days incubation in phosphate buffer solution. ATDC-5 cells encapsulated in the injectable gels showed high cell viability. The composite gels showed the presence of enlarged spherical cells with significantly higher metabolic activity compared to cells in hyaluronic and alginic acid gels. The results suggest the potential of the composite approach to develop covalently cross-linked hydrogels with tuneable physical, mechanical, and biological properties. PMID:23357799

  16. Enzymatically Cross-linked Alginic-Hyaluronic acid Composite Hydrogels As Cell Delivery Vehicles

    PubMed Central

    Ganesh, Nitya; Hanna, Craig; Nair, Shantikumar V.; Nair, Lakshmi S.

    2013-01-01

    An injectable composite gel was developed from alginic and hyaluronic acid. The ezymatically cross-linked injectable gels were prepared via the oxidative coupling of tyramine modified sodium algiante and sodium hyaluronate in the presence of horse radish peroxidase (HRP) and hydrogen peroxide (H2O2). The composite gels were prepared by mixing equal parts of the two tryaminated polymer solutions in 10U HRP and treating with 1.0% H2O2. The properties of the alginate gels were significanly affected by the addition of hyaluronic acid. The percentage water absorption and storage modulus of the composite gels were found to be lower than the alginate gels. The alginate and composite gels showed lower protein release compared to hyaluronate gels in the absence of hyaluronidase. Even hyaluronate gels showed only approximately 10% protein release after 14 days incubation in phosphate buffer solution. ATDC-5 cells encapsulated in the injectable gels showed high cell viability. The composite gels showed the presence of enlarged spherical cells with significantly higher metabolic activity compared to cells in hyaluronic and alginic acid gels. The results suggest the potential of the composite approach to develop covalently cross-linked hydrogels with tuneable physical, mechanical, and biological properties. PMID:23357799

  17. Design and performance of a sericin-alginate interpenetrating network hydrogel for cell and drug delivery

    PubMed Central

    Zhang, Yeshun; Liu, Jia; Huang, Lei; Wang, Zheng; Wang, Lin

    2015-01-01

    Although alginate hydrogels have been extensively studied for tissue engineering applications, their utilization is limited by poor mechanical strength, rapid drug release, and a lack of cell adhesive ability. Aiming to improve these properties, we employ the interpenetrating hydrogel design rationale. Using alginate and sericin (a natural protein with many unique properties and a major component of silkworm silk), we develop an interpenetrating polymer network (IPN) hydrogel comprising interwoven sericin and alginate double networks. By adjusting the sericin-to-alginate ratios, IPNs’ mechanical strength can be adjusted to meet stiffness requirements for various tissue repairs. The IPNs with high sericin content show increased stability during degradation, avoiding pure alginate’s early collapse. These IPNs have high swelling ratios, benefiting various applications such as drug delivery. The IPNs sustain controlled drug release with the adjustable rates. Furthermore, these IPNs are adhesive to cells, supporting cell proliferation, long-term survival and migration. Notably, the IPNs inherit sericin’s photoluminescent property, enabling bioimaging in vivo. Together, our study indicates that the sericin-alginate IPN hydrogels may serve as a versatile platform for delivering cells and drugs, and suggests that sericin may be a building block broadly applicable for generating IPN networks with other biomaterials for diverse tissue engineering applications. PMID:26205586

  18. Transplantation of Neural Stem Cells Cultured in Alginate Scaffold for Spinal Cord Injury in Rats

    PubMed Central

    Sharafkhah, Ali; Koohi-Hosseinabadi, Omid; Semsar-Kazerooni, Maryam

    2016-01-01

    Study Design This study investigated the effects of transplantation of alginate encapsulated neural stem cells (NSCs) on spinal cord injury in Sprague-Dawley male rats. The neurological functions were assessed for 6 weeks after transplantation along with a histological study and measurement of caspase-3 levels. Purpose The aim of this study was to discover whether NSCs cultured in alginate transplantation improve recovery from spinal cord injury. Overview of Literature Spinal cord injury is one of the leading causes of disability and it has no effective treatment. Spinal cord injury can also cause sensory impairment. With an impetus on using stem cells therapy in various central nervous system settings, there is an interest in using stem cells for addressing spinal cord injury. Neural stem cell is one type of stem cells that is able to differentiate to all three neural lineages and it shows promise in spinal injury treatment. Furthermore, a number of studies have shown that culturing NSCs in three-dimensional (3D) scaffolds like alginate could enhance neural differentiation. Methods The NSCs were isolated from 14-day-old rat embryos. The isolated NSCs were cultured in growth media containing basic fibroblast growth factor and endothelial growth factor. The cells were characterized by differentiating to three neural lineages and they were cultured in an alginate scaffold. After 7 days the cells were encapsulated and transplanted in a rat model of spinal cord injury. Results Our data showed that culturing in an alginate 3D scaffold and transplantation of the NSCs could improve neurological outcome in a rat model of spinal cord injury. The inflammation scores and lesion sizes and also the activity of caspase-3 (for apoptosis evaluation) were less in encapsulated neural stem cell transplantation cases. Conclusions Transplantation of NSCs that were cultured in an alginate scaffold led to a better clinical and histological outcome for recovery from spinal cord injury in

  19. Influence of mechanical properties of alginate-based substrates on the performance of Schwann cells in culture.

    PubMed

    Ning, Liqun; Xu, Yitong; Chen, Xiongbiao; Schreyer, David J

    2016-06-01

    In tissue engineering, artificial tissue scaffolds containing living cells have been studied for tissue repair and regeneration. Notably, the performance of these encapsulated-in-scaffolds cells in terms of cell viability, proliferation, and expression of function during and after the scaffold fabrication process, has not been well documented because of the influence of mechanical, chemical, and physical properties of the scaffold substrate materials. This paper presents our study on the influence of mechanical properties of alginate-based substrates on the performance of Schwann cells, which are the major glial cells of peripheral nervous system. Given the fact that alginate polysaccharide hydrogel has poor cell adhesion properties, in this study, we examined several types of cell-adhesion supplements and found that alginate covalently modified with RGD peptide provided improved cell proliferation and adhesion. We prepared alginate-based substrates for cell culture using varying alginate concentrations for altering their mechanical properties, which were confirmed by compression testing. Then, we examined the viability, proliferation, morphology, and expression of the extracellular matrix protein laminin of Schwann cells that were seeded on the surface of alginate-based substrates (or 2D culture) or encapsulated within alginate-based substrates (3D cultures), and correlated the examined cell performance to the alginate concentration (or mechanical properties) of hydrogel substrates. Our findings suggest that covalent attachment of RGD peptide can improve the success of Schwann cell encapsulation within alginate-based scaffolds, and provide guidance for regulating the mechanical properties of alginate-based scaffolds containing Schwann cells for applications in peripheral nervous system regeneration and repair. PMID:27012482

  20. Influence of mechanical properties of alginate-based substrates on the performance of Schwann cells in culture.

    PubMed

    Ning, Liqun; Xu, Yitong; Chen, Xiongbiao; Schreyer, David J

    2016-06-01

    In tissue engineering, artificial tissue scaffolds containing living cells have been studied for tissue repair and regeneration. Notably, the performance of these encapsulated-in-scaffolds cells in terms of cell viability, proliferation, and expression of function during and after the scaffold fabrication process, has not been well documented because of the influence of mechanical, chemical, and physical properties of the scaffold substrate materials. This paper presents our study on the influence of mechanical properties of alginate-based substrates on the performance of Schwann cells, which are the major glial cells of peripheral nervous system. Given the fact that alginate polysaccharide hydrogel has poor cell adhesion properties, in this study, we examined several types of cell-adhesion supplements and found that alginate covalently modified with RGD peptide provided improved cell proliferation and adhesion. We prepared alginate-based substrates for cell culture using varying alginate concentrations for altering their mechanical properties, which were confirmed by compression testing. Then, we examined the viability, proliferation, morphology, and expression of the extracellular matrix protein laminin of Schwann cells that were seeded on the surface of alginate-based substrates (or 2D culture) or encapsulated within alginate-based substrates (3D cultures), and correlated the examined cell performance to the alginate concentration (or mechanical properties) of hydrogel substrates. Our findings suggest that covalent attachment of RGD peptide can improve the success of Schwann cell encapsulation within alginate-based scaffolds, and provide guidance for regulating the mechanical properties of alginate-based scaffolds containing Schwann cells for applications in peripheral nervous system regeneration and repair.

  1. Alginate Microfiber System for Expansion and Direct Differentiation of Human Embryonic Stem Cells.

    PubMed

    Leong, Meng Fatt; Lu, Hong Fang; Lim, Tze Chiun; Narayanan, Karthikeyan; Gao, Shujun; Wang, Luna Yue; Toh, Rebecca P K; Funke, Henrik; Abdul Samad, Muhammad Hamizan; Wan, Andrew C A; Ying, Jackie Y

    2016-09-01

    Pluripotent human embryonic stem cells (hESCs) are a potential renewable cell source for regenerative medicine and drug testing. To obtain adequate cell numbers for these applications, there is a need to develop scalable cell culture platforms to propagate hESCs. In this study, we encapsulated hESCs in calcium alginate microfibers as single cells, for expansion and differentiation under chemically defined conditions. hESCs were suspended in 1% (w/v) alginate solution at high cell density (>10(7) cells/mL) and extruded at 5 m/min into a low calcium concentration bath (10 mM) for gelation. Mild citrate buffer (2.5 mM), which did not affect hESCs viability, was used to release the cells from the calcium alginate hydrogel. Encapsulation as single cells was critical, as this allowed the hESCs to grow in the form of relatively small and uniform aggregates. This alginate microfiber system allowed for expansion of an hESC line, HUES7, for up to five passages while maintaining pluripotency. Immunohistochemistry, polymerase chain reaction, and other analyses showed that passage 5 (P5) HUES7 cells expressed proteins and genes characteristic of pluripotent stem cells, possessed normal karyotype, and were able to form representative tissues of the three embryonic germ layers in vitro and in vivo. Encapsulated HUES7 cells at P5 could also be induced to directly differentiate into liver-like cells. Collectively, our experiments show that the alginate microfiber system can be used as a three-dimensional cell culture platform for long-term expansion and differentiation of hESCs under defined conditions. PMID:27484909

  2. The role of alginate in Pseudomonas aeruginosa EPS adherence, viscoelastic properties and cell attachment.

    PubMed

    Orgad, Oded; Oren, Yoram; Walker, Sharon L; Herzberg, Moshe

    2011-08-01

    Among various functions, extracellular polymeric substances (EPS) provide microbial biofilms with mechanical stability and affect initial cell attachment, the first stage in the biofilm formation process. The role of alginate, an abundant polysaccharide in Pseudomonas aeruginosa biofilms, in the viscoelastic properties and adhesion kinetics of EPS was analyzed using a quartz crystal microbalance with dissipation (QCM-D) monitoring technology. EPS was extracted from two P. aeruginosa biofilms, a wild type strain, PAO1, and a mucoid strain, PAOmucA22 that over-expresses alginate production. The higher alginate content in the EPS originating from the mucoid biofilms was clearly shown to increase both the rate and the extent of attachment of the EPS, as well as the layer's thickness. Also, the presence of calcium and elevated ionic strength increased the thickness of the EPS layer. Dynamic light scattering (DLS) showed that the presence of calcium and elevated ionic strength induced intermolecular attractive interactions in the mucoid EPS molecules. For the wild type EPS, in the presence of calcium, an elevated shift in the distribution of the diffusion coefficients was observed with DLS due to a more compacted conformation of the EPS molecules. Moreover, the alginate over-expression effect on EPS adherence was compared to the effect of alginate over-expression on P. aeruginosa cell attachment. In a parallel plate flow cell, under similar hydraulic and aquatic conditions as those applied for the EPS adsorption tests in the QCM-D flow cell, reduced adherence of the mucoid strain was clearly observed compared to the wild type isogenic bacteria. The results suggest that alginate contributes to steric hindrance and shielding of cell surface features and adhesins that are known to promote cell attachment.

  3. Hemicellulosic Ethanol Production by Immobilized Wild Brazilian Yeast Scheffersomyces shehatae UFMG-HM 52.2: Effects of Cell Concentration and Stirring Rate.

    PubMed

    Antunes, F A F; Santos, J C; Chandel, A K; Milessi, T S S; Peres, G F D; da Silva, S S

    2016-02-01

    The use of sugarcane bagasse hemicellulosic hydrolysates presents an interesting alternative to second generation (2G) ethanol production. Techniques to enhance the fermentation process, e.g., the use of immobilized cells, is one of the key factors for efficient production. Here, the effect of two important parameters (cell concentration in immobilized system and stirring rate) on the 2G ethanol production using the wild Brazilian yeast S. shehatae UFMG-HM 52.2 immobilized in calcium alginate matrix are presented. A 2(2) full factorial design of experiments was carried out to evaluate the effect of cell concentrations in sodium alginate solution for immobilized bead production (3.0, 6.0, and 9.0 g/L) and stirring rate (150, 200, and 250 rpm) for 2G ethanol production. Statistical analysis showed that the use of both variables at low levels enhanced ethanol yield (YP/S). Under these process conditions, YP/S of 0.31 g/g and ethanol productivity (Qp) of 0.12 g/L h were achieved. Results showed the potential of this immobilized yeast in 2G ethanol production from C5 sugars and demonstrate the importance of adequate cell concentration in immobilized systems, a finding that stands to increase bioprocesses yields and productivity.

  4. Insights in Behavior of Variably Formulated Alginate-Based Microcapsules for Cell Transplantation

    PubMed Central

    Montanucci, Pia; Terenzi, Silvia; Pennoni, Ilaria; Basta, Giuseppe; Calafiore, Riccardo

    2015-01-01

    Alginate-based microencapsulation of live cells may offer the opportunity to treat chronic and degenerative disorders. So far, a thorough assessment of physical-chemical behavior of alginate-based microbeads remains cloudy. A disputed issue is which divalent cation to choose for a high performing alginate gelling process. Having selected, in our system, high mannuronic (M) enriched alginates, we studied different gelling cations and their combinations to determine their eventual influence on physical-chemical properties of the final microcapsules preparation, in vitro and in vivo. We have shown that used of ultrapure alginate allows for high biocompatibility of the formed microcapsules, regardless of gelation agents, while use of different gelling cations is associated with corresponding variable effects on the capsules' basic architecture, as originally reported in this work. However, only the final application which the capsules are destined to will ultimately guide the selection of the ideal, specific gelling divalent cations, since in principle there are no capsules that are better than others. PMID:26078974

  5. Adsorption of ochratoxin A from grape juice by yeast cells immobilised in calcium alginate beads.

    PubMed

    Farbo, Maria Grazia; Urgeghe, Pietro Paolo; Fiori, Stefano; Marceddu, Salvatore; Jaoua, Samir; Migheli, Quirico

    2016-01-18

    Grape juice can be easily contaminated with ochratoxin A (OTA), one of the known mycotoxins with the greatest public health significance. Among the different approaches to decontaminate juice from this mycotoxin, microbiological methods proved efficient, inexpensive and safe, particularly the use of yeast or yeast products. To ascertain whether immobilisation of the yeast biomass would lead to successful decontamination, alginate beads encapsulating Candida intermedia yeast cells were used in our experiments to evaluate their OTA-biosorption efficacy. Magnetic calcium alginate beads were also prepared by adding magnetite in the formulation to allow fast removal from the aqueous solution with a magnet. Calcium alginate beads were added to commercial grape juice spiked with 20 μg/kg OTA and after 48 h of incubation a significant reduction (>80%), of the total OTA content was achieved, while in the subsequent phases (72-120 h) OTA was slowly released into the grape juice by alginate beads. Biosorption properties of alginate-yeast beads were tested in a prototype bioreactor consisting in a glass chromatography column packed with beads, where juice amended with OTA was slowly flowed downstream. The adoption of an interconnected scaled-up bioreactor as an efficient and safe tool to remove traces of OTA from liquid matrices is discussed.

  6. Release of angiogenic growth factors from cells encapsulated in alginate beads with bioactive glass.

    PubMed

    Keshaw, Hussila; Forbes, Alastair; Day, Richard M

    2005-07-01

    Attempts to stimulate therapeutic angiogenesis using gene therapy or delivery of recombinant growth factors, such as vascular endothelial growth factor (VEGF), have failed to demonstrate unequivocal efficacy in human trials. Bioactive glass stimulates fibroblasts to secrete significantly increased amounts of angiogenic growth factors and therefore has a number of potential applications in therapeutic angiogenesis. The aim of this study was to assess whether it is possible to encapsulate specific quantities of bioactive glass and fibroblasts into alginate beads, which will secrete growth factors capable of stimulating angiogenesis. Human fibroblasts (CCD-18Co) were encapsulated in alginate beads with specific quantities of 45S5 bioactive glass and incubated in culture medium (0-17 days). The conditioned medium was collected and assayed for VEGF or used to assess its ability to stimulate angiogenesis by measuring the proliferation of human dermal microvascular endothelial cells. At 17 days the beads were lysed and the amount of VEGF retained by the beads measured. Fibroblasts encapsulated in alginate beads containing 0.01% and 0.1% (w/v) 45S5 bioactive glass particles secreted increased quantities of VEGF compared with cells encapsulated with 0% or 1% (w/v) 45S5 bioactive glass particles. Lysed alginate beads containing 0.01% and 0.1% (w/v) 45S5 bioactive glass contained significantly more VEGF (p<0.01) compared with beads containing no glass particles. Endothelial cell proliferation was significantly increased (p<0.01) by conditioned medium collected from alginate beads containing 0.1% (w/v) 45S5 bioactive glass particles. The results of this study demonstrate that bioactive glass and fibroblasts can be successfully incorporated into alginate beads for use in delivering angiogenic growth factors. With further optimization, this technique offers a novel delivery device for stimulating therapeutic angiogenesis. PMID:15664644

  7. Chitosan and alginate types of bio-membrane in fuel cell application: An overview

    NASA Astrophysics Data System (ADS)

    Shaari, N.; Kamarudin, S. K.

    2015-09-01

    The major problems of polymer electrolyte membrane fuel cell technology that need to be highlighted are fuel crossovers (e.g., methanol or hydrogen leaking across fuel cell membranes), CO poisoning, low durability, and high cost. Chitosan and alginate-based biopolymer membranes have recently been used to solve these problems with promising results. Current research in biopolymer membrane materials and systems has focused on the following: 1) the development of novel and efficient biopolymer materials; and 2) increasing the processing capacity of membrane operations. Consequently, chitosan and alginate-based biopolymers seek to enhance fuel cell performance by improving proton conductivity, membrane durability, and reducing fuel crossover and electro-osmotic drag. There are four groups of chitosan-based membranes (categorized according to their reaction and preparation): self-cross-linked and salt-complexed chitosans, chitosan-based polymer blends, chitosan/inorganic filler composites, and chitosan/polymer composites. There are only three alginate-based membranes that have been synthesized for fuel cell application. This work aims to review the state-of-the-art in the growth of chitosan and alginate-based biopolymer membranes for fuel cell applications.

  8. Functional cell-laden alginate scaffolds consisting of core/shell struts for tissue regeneration.

    PubMed

    Ahn, SeungHyun; Lee, HyeongJin; Kim, GeunHyung

    2013-10-15

    We report an innovative cell-dispensing process using a three-axis robot system coupled with a micro-core/shell nozzle and an aerosol cross-linking process to achieve controlled mechanical properties and high cell viability of porous cell-laden alginate scaffolds. The scaffolds were fabricated into layer-by-layer struts, which were used to design the pore structure. The struts consisted of a core/shell region; a low weight fraction of alginate and cells (MC3T3-E1) was injected in the shell region to efficiently exchange nutrients and metabolic wastes, while a high weight fraction of alginate without cells was deposited in the core region to improve the mechanical properties of the cell-laden scaffold. After 10 days of cell culture, the cell viability (95%) in the shell region improved significantly compared to 70% for the cells homogeneously distributed in the struts, and the mechanical properties were enhanced from 1.4 to 15.7 kPa. Stained nuclei and F-actin images showed that the laden cells proliferated well on the functional hydrogel scaffold after 20 days of cell culture, indicating that the cells concentrated in the shell region of the struts survived and increased their metabolic functions during several incubation periods compared to the standard cell-laden scaffold. This innovative cell-dispensing technique represents a promising fabrication tool for obtaining bottom-up scaffolds for various tissue regenerations.

  9. Wound Dressing Model of Human Umbilical Cord Mesenchymal Stem Cells-Alginates Complex Promotes Skin Wound Healing by Paracrine Signaling

    PubMed Central

    Yang, Huachao; Tang, Zhenrui; Long, Gang; Huang, Wen

    2016-01-01

    Purpose. To probe growth characteristics of human umbilical cord mesenchymal stem cells (hUCMSCs) cultured with alginate gel scaffolds, and to explore feasibility of wound dressing model of hUCMSCs-alginates compound. Methods. hUCMSCs were isolated, cultured, and identified in vitro. Then cells were cultivated in 100 mM calcium alginate gel, and the capacity of proliferation and migration and the expression of vascular endothelial growth factors (VEGF) were investigated regularly. Wound dressing model of hUCMSCs-alginate gel mix was transplanted into Balb/c mice skin defects. Wound healing rate and immunohistochemistry were examined. Results. hUCMSCs grew well but with little migration ability in the alginate gel. Compared with control group, a significantly larger cell number and more VEGF expression were shown in the gel group after culturing for 3–6 days (P < 0.05). In addition, a faster skin wound healing rate with more neovascularization was observed in the hUCMSCs-alginate gel group than in control groups at 15th day after surgery (P < 0.05). Conclusion. hUCMSCs can proliferate well and express massive VEGF in calcium alginate gel porous scaffolds. Wound dressing model of hUCMSCs-alginate gel mix can promote wound healing through paracrine signaling. PMID:26880953

  10. Aroma formation by immobilized yeast cells in fermentation processes.

    PubMed

    Nedović, V; Gibson, B; Mantzouridou, T F; Bugarski, B; Djordjević, V; Kalušević, A; Paraskevopoulou, A; Sandell, M; Šmogrovičová, D; Yilmaztekin, M

    2015-01-01

    Immobilized cell technology has shown a significant promotional effect on the fermentation of alcoholic beverages such as beer, wine and cider. However, genetic, morphological and physiological alterations occurring in immobilized yeast cells impact on aroma formation during fermentation processes. The focus of this review is exploitation of existing knowledge on the biochemistry and the biological role of flavour production in yeast for the biotechnological production of aroma compounds of industrial importance, by means of immobilized yeast. Various types of carrier materials and immobilization methods proposed for application in beer, wine, fruit wine, cider and mead production are presented. Engineering aspects with special emphasis on immobilized cell bioreactor design, operation and scale-up potential are also discussed. Ultimately, examples of products with improved quality properties within the alcoholic beverages are addressed, together with identification and description of the future perspectives and scope for cell immobilization in fermentation processes.

  11. Immobilization of Bacillus acidocaldarius whole-cell rhodanese in polysaccharide and insolubilized gelatin gels

    SciTech Connect

    De Riso, L.; Alteriis, E. de; Parascandola, P. |; La Cara, F.; Sada, A.

    1996-04-01

    The presence of rhodanese activity has been investigated in two strains of thermophilic eubacteria and two strains of extremophiles. Bacillus acidocaldarius, a thermoacidophilic eubacterium, showed the highest levels of enzyme activity. Whole cells, previously subjected to one cycle of freeze-thawing, were immobilized by entrapment in the polysaccharide matrices Ca-alginate, {kappa}-carrageenan and chitosan, and in an insolubilized gelatin gel. The results obtained with the different immobilizates in terms of activity yield, possibility of regeneration and operative stability were evaluated with the aim of setting up a continuous system. This was achieved with a system consisting of B. acidocaldarius cells entrapped in an insolubilized gelatin matrix. The latter, in the form of a thin membrane, was employed in a custom-conceived reactor operating as a plug flow reactor. 21 refs., 3 figs., 2 tabs.

  12. Hydrodynamic deposition: a novel method of cell immobilization.

    PubMed

    Salter, G J; Kell, D B; Ash, L A; Adams, J M; Brown, A J; James, R

    1990-06-01

    A novel method of cell immobilization is described. The cell support consists of ceramic microspheres of approximately 50-75 microns diameter. The spheres are hollow, having a wall thickness of 10-15 microns and one entrance (ca. 20 microns diameter). The walls are porous with a mean pore size of approximately 90 nm. When a cell suspension (of S. cerevisiae) is passed through a column of such particles, cells are immobilized. Conditions are devised such that the overwhelming majority of cells are held in the central cavity of the support and not between the particles. Provided turbulence is avoided, the distribution of cells along the column length in the steady state is rather homogeneous. The facts that (a) essentially all particles, regardless of orientation, entrap cells, and (b) nonporous particles also entrap cells with high efficiency, indicate that filtration effects are irrelevant and that heretofore unrecognized hydrodynamic forces are alone responsible for the cell immobilization. Cells can be immobilized to high biomass densities, while the hydrodynamic properties of columns containing such immobilized cells are excellent. We describe an on-line electronic method for the real-time measurement of immobilized cellular biomass. Cell growth (so recorded) and metabolism continue to occur in such particles at high rates. Using the glycolytic production of ethanol by S. cerevisiae as a model reaction, volumetric productivities as great as any published are obtained. Thus the "lobster-pot effect" or "hydrodynamic deposition" represents a novel, promising, and generally applicable method of cell immobilization.

  13. Effect of immobile isolated enzymes from rumen liquid by using alginate matrices on the bay leaf extraction

    NASA Astrophysics Data System (ADS)

    Paramita, Vita; Yulianto, Mohammad Endy; Yohana, Eflita; Arifan, Fahmi; Hanifah, Amjad, Muhammad Taqiyuddin

    2015-12-01

    This research aims to develop the enzymatically of bay leaves phytochemical extraction process. The novelty and the main innovations of this research is the development of extraction process by using enzymatic extractor and isolate the enzymes from rumen liquid to shift the equilibrium phase, increase the extraction rate and increase the extraction yield. The activity of rumen liquid enzyme was represented by the activity of cellulase and protease. The analyze of total flavonoid content was performed by using UV-Vis Spectrofometry. The activity of immobilized enzyme of cellulase (0.08±0.00 U/ml) was lower than the un-immobilized one (0.23±0.00 U/ml). However, there was no difference activity of the immobilized (0.75±0.00 U/ml) and un-immobilized (0.76±0.01 U/ml) of protease. The model of mass transfer of un-immobilized enzyme can be fitted on the experimental data, however the model of mass transfer of immobilized enzyme did not match with the experimental data. The mass transfer coefficient of enzymatic extraction flavonoids bay leaf without immobilization was 0.17167 s-1 which greater than the reported value of obtained KLa from extraction by using electric heating.

  14. Encapsulation of fibroblasts causes accelerated alginate hydrogel degradation.

    PubMed

    Hunt, N C; Smith, A M; Gbureck, U; Shelton, R M; Grover, L M

    2010-09-01

    Calcium-alginate hydrogel has been widely studied as a material for cell encapsulation for tissue engineering. At present, the effect that cells have on the degradation of alginate hydrogel is largely unknown. We have shown that fibroblasts encapsulated at a density of 7.5 x 10(5) cells ml(-1) in both 2% and 5% w/v alginate remain viable for at least 60 days. Rheological analysis was used to study how the mechanical properties exhibited by alginate hydrogel changed during 28 days in vitro culture. Alginate degradation was shown to occur throughout the study but was greatest within the first 7 days of culture for all samples, which correlated with a sharp release of calcium ions from the construct. Fibroblasts were shown to increase the rate of degradation during the first 7 days when compared with acellular samples in both 2% and 5% w/v gels, but after 28 days both acellular and cell-encapsulating samples retained disc-shaped morphologies and gel-like spectra. The results demonstrate that although at an early stage cells influence the mechanical properties of encapsulating alginate, over a longer period of culture, the hydrogels retain sufficient mechanical integrity to exhibit gel-like properties. This allows sustained immobilization of the cells at the desired location in vivo where they can produce extracellular matrix and growth factors to expedite the healing process.

  15. 3D Porous Calcium-Alginate Scaffolds Cell Culture System Improved Human Osteoblast Cell Clusters for Cell Therapy

    PubMed Central

    Chen, Ching-Yun; Ke, Cherng-Jyh; Yen, Ko-Chung; Hsieh, Hui-Chen; Sun, Jui-Sheng; Lin, Feng-Huei

    2015-01-01

    Age-related orthopedic disorders and bone defects have become a critical public health issue, and cell-based therapy is potentially a novel solution for issues surrounding bone tissue engineering and regenerative medicine. Long-term cultures of primary bone cells exhibit phenotypic and functional degeneration; therefore, culturing cells or tissues suitable for clinical use remain a challenge. A platform consisting of human osteoblasts (hOBs), calcium-alginate (Ca-Alginate) scaffolds, and a self-made bioreactor system was established for autologous transplantation of human osteoblast cell clusters. The Ca-Alginate scaffold facilitated the growth and differentiation of human bone cell clusters, and the functionally-closed process bioreactor system supplied the soluble nutrients and osteogenic signals required to maintain the cell viability. This system preserved the proliferative ability of cells and cell viability and up-regulated bone-related gene expression and biological apatite crystals formation. The bone-like tissue generated could be extracted by removal of calcium ions via ethylenediaminetetraacetic acid (EDTA) chelation, and exhibited a size suitable for injection. The described strategy could be used in therapeutic application and opens new avenues for surgical interventions to correct skeletal defects. PMID:25825603

  16. A tunable silk-alginate hydrogel scaffold for stem cell culture and transplantation.

    PubMed

    Ziv, Keren; Nuhn, Harald; Ben-Haim, Yael; Sasportas, Laura S; Kempen, Paul J; Niedringhaus, Thomas P; Hrynyk, Michael; Sinclair, Robert; Barron, Annelise E; Gambhir, Sanjiv S

    2014-04-01

    One of the major challenges in regenerative medicine is the ability to recreate the stem cell niche, which is defined by its signaling molecules, the creation of cytokine gradients, and the modulation of matrix stiffness. A wide range of scaffolds has been developed in order to recapitulate the stem cell niche, among them hydrogels. This paper reports the development of a new silk-alginate based hydrogel with a focus on stem cell culture. This biocomposite allows to fine tune its elasticity during cell culture, addressing the importance of mechanotransduction during stem cell differentiation. The silk-alginate scaffold promotes adherence of mouse embryonic stem cells and cell survival upon transplantation. In addition, it has tunable stiffness as function of the silk-alginate ratio and the concentration of crosslinker--a characteristic that is very hard to accomplish in current hydrogels. The hydrogel and the presented results represents key steps on the way of creating artificial stem cell niche, opening up new paths in regenerative medicine. PMID:24484675

  17. Development of a cell culture surface conversion technique using alginate thin film for evaluating effect upon cellular differentiation

    NASA Astrophysics Data System (ADS)

    Nakashima, Y.; Tsusu, K.; Minami, K.; Nakanishi, Y.

    2014-06-01

    Here, we sought to develop a cell culture surface conversion technique that would not damage living cells. An alginate thin film, formed on a glass plate by spin coating of sodium alginate solution and dipping into calcium chloride solution, was used to inhibit adhesion of cells. The film could be removed by ethylenediaminetetraacetate (EDTA) at any time during cell culture, permitting observation of cellular responses to conversion of the culture surface in real time. Additionally, we demonstrated the validity of the alginate thin film coating method and the performance of the film. The thickness of the alginate thin film was controlled by varying the rotation speed during spin coating. Moreover, the alginate thin film completely inhibited the adhesion of cultured cells to the culture surface, irrespective of the thickness of the film. When the alginate thin film was removed from the culture surface by EDTA, the cultured cells adhered to the culture surface, and their morphology changed. Finally, we achieved effective differentiation of C2C12 myoblasts into myotube cells by cell culture on the convertible culture surface, demonstrating the utility of our novel technique.

  18. Development of a cell culture surface conversion technique using alginate thin film for evaluating effect upon cellular differentiation

    SciTech Connect

    Nakashima, Y.; Tsusu, K.; Minami, K.; Nakanishi, Y.

    2014-06-15

    Here, we sought to develop a cell culture surface conversion technique that would not damage living cells. An alginate thin film, formed on a glass plate by spin coating of sodium alginate solution and dipping into calcium chloride solution, was used to inhibit adhesion of cells. The film could be removed by ethylenediaminetetraacetate (EDTA) at any time during cell culture, permitting observation of cellular responses to conversion of the culture surface in real time. Additionally, we demonstrated the validity of the alginate thin film coating method and the performance of the film. The thickness of the alginate thin film was controlled by varying the rotation speed during spin coating. Moreover, the alginate thin film completely inhibited the adhesion of cultured cells to the culture surface, irrespective of the thickness of the film. When the alginate thin film was removed from the culture surface by EDTA, the cultured cells adhered to the culture surface, and their morphology changed. Finally, we achieved effective differentiation of C2C12 myoblasts into myotube cells by cell culture on the convertible culture surface, demonstrating the utility of our novel technique.

  19. Immobilization of the Methanogenic bacterium methanosarcina barkeri

    SciTech Connect

    Scherer, P.; Kluge, M.; Klein, J.; Sahm, H.

    1981-05-01

    Whole cells of the methanogen Methanosarcina barkeri were immobilized in an alginate network which was crosslinked with Ca/sup 2+/ calcium ions. The rates of methanol conversion to methane of entrapped cells were found to be in the same range as the corresponding rates of free cells. Furthermore, immobilized cells were active for a longer period than free cells. The particle size of the spherical alginate beads and thus diffusion has no obvious influence on the turnover of methanol. The half-value period for methanol conversion activity determined in a buffer medium was approximately 4 days at 37/degree/C for entrapped cells. The high rates of methanol degradation indicated that the immobilization technique preserved the cellular functions of this methanogenic bacterium. 24 refs.

  20. Cesium accumulation of Rhodococcus erythropolis CS98 strain immobilized in hydrogel matrices.

    PubMed

    Takei, Takayuki; Yamasaki, Mika; Yoshida, Masahiro

    2014-04-01

    Agarose gels were superior to calcium-alginate gels for immobilizing Rhodococcus erythropolis CS98 strain to remove cesium from water. Suitable incubation time of the immobilized cells in cesium solutions, cell number in the gels and volume ratio of the cesium solution to the gels for efficient cesium removal were identified.

  1. Importance of solute partitioning in biphasic oxidation of benzyl alcohol by free and immobilized whole cells of Pichia pastoris

    SciTech Connect

    Kawakami, Koei; Nakahara, Takehiko . Dept. of Chemical Engineering)

    1994-04-25

    Using free and immobilized whole cells of Pichia pastoris, the biocatalytic oxidation of benzyl alcohol was investigated in different two-phase systems. This reaction was strongly influenced by both the substrate and product inhibitions, and the production rate of benzaldehyde in the aqueous system became maximum at the initial substrate concentration of ca. 29 g/L with the aldehyde formation less than 4 to 5 g/L even after a longer reaction period. The reaction rates in the two-liquid phase systems were predominantly determined by the partitioning behaviors of the substrate and the product between the two phases rather than by enzyme deactivation by the organic solvents. In the two-liquid phase systems, consequently, the organic solvent acted as a reservoir to reduce these inhibitory effects, and it was essential to select the organic solvent providing the optimal partitioning of the substrate into the aqueous phase as well as the preferential extraction of the product into the organic phase. The whole cells immobilized in a mixed matrix composed of silicone polymer and Ca alginate gel worked well in the xylene and decane media, providing comparable activities with the free cells. The production rate of aldehyde was also influenced by the solute partitioning into the hydrophilic alginate phase where the cells existed.

  2. Advances in ethanol production using immobilized cell systems

    SciTech Connect

    Margaritis, A.; Merchant, F.J.A.

    1984-01-01

    The application of immobilized cell systems for the production of ethanol has resulted in substantial improvements in the efficiency of the process when compared to the traditional free cell system. In this review, the various methods of cell immobilization employed in ethanol production systems have been described in detail. Their salient features, performance characteristics, advantages and limitations have been critically assessed. More recently, these immobilized cell systems have also been employed for the production of ethanol from non-conventional feedstocks such as Jerusalem artichoke extracts, cheese whey, cellulose, cellobiose and xylose. Ethanol production by immobilized yeast and bacterial cells has been attempted in various bioreactor types. Although most of these studies have been carried out using laboratory scale prototype bioreactors, it appears that only fluidized bed, horizontally packed bed bioreactors and tower fermenters may find application on scale-up. Several studies have indicated that upon immobilization, yeast cells performing ethanol fermentation exhibit more favourable physiological and metabolic properties. This, in addition to substantial improvements in ethanol productivities by immobilized cell systems, is indicative of the fact that future developments in the production of ethanol and alcoholic beverages will be directed towards the use of immobilized cell systems. 291 references.

  3. Hydrogen production by immobilized whole cells of Clostridium butyricum

    NASA Astrophysics Data System (ADS)

    Suzuki, S.; Karube, I.

    Immobilized microbial cells were used in a batch system in an attempt to achieve continuous hydrogen production from glucose and waste waters. Clostridium butyricum IFO 3847 was immobilized in polyacrylamide gel and continuously produced hydrogen from glucose. The hydrogen producing bacteria were then immobilized in 2% agar gel and showed continuous hydrogen production from an alcohol factory's waste waters. The hydrogen production rate became constant above BOD 1500 ppm when performed with a batch system. The immobilized whole cells continuously produced hydrogen over a 20 day period, producing about 6 ml/min/kg wet gels. Hydrogen production by bacteria immobilized in acetylcellulose filters was six times higher than that by cells entrapped in agar gels.

  4. Utilizing Core–Shell Fibrous Collagen-Alginate Hydrogel Cell Delivery System for Bone Tissue Engineering

    PubMed Central

    Perez, Roman A.; Kim, Meeju; Kim, Tae-Hyun; Kim, Joong-Hyun; Lee, Jae Ho; Park, Jeong-Hui; Knowles, Jonathan C.

    2014-01-01

    Three-dimensional matrices that encapsulate and deliver stem cells with defect-tuned formulations are promising for bone tissue engineering. In this study, we designed a novel stem cell delivery system composed of collagen and alginate as the core and shell, respectively. Mesenchymal stem cells (MSCs) were loaded into the collagen solution and then deposited directly into a fibrous structure while simultaneously sheathing with alginate using a newly designed core–shell nozzle. Alginate encapsulation was achieved by the crosslinking within an adjusted calcium-containing solution that effectively preserved the continuous fibrous structure of the inner cell-collagen part. The constructed hydrogel carriers showed a continuous fiber with a diameter of ∼700–1000 μm for the core and 200–500 μm for the shell area, which was largely dependent on the alginate concentration (2%–5%) as well as the injection rate (20–80 mL/h). The water uptake capacity of the core–shell carriers was as high as 98%, which could act as a pore channel to supply nutrients and oxygen to the cells. Degradation of the scaffolds showed a weight loss of ∼22% at 7 days and ∼43% at 14 days, suggesting a possible role as a degradable tissue-engineered construct. The MSCs encapsulated within the collagen core showed excellent viability, exhibiting significant cellular proliferation up to 21 days with levels comparable to those observed in the pure collagen gel matrix used as a control. A live/dead cell assay also confirmed similar percentages of live cells within the core–shell carrier compared to those in the pure collagen gel, suggesting the carrier was cell compatible and was effective for maintaining a cell population. Cells allowed to differentiate under osteogenic conditions expressed high levels of bone-related genes, including osteocalcin, bone sialoprotein, and osteopontin. Further, when the core–shell fibrous carriers were implanted in a rat calvarium defect, the bone

  5. Continuous fermentation: improvement of cell immobilization by zeta potential measurement

    SciTech Connect

    Michaux, M.; Paquot, M.; Baijot, B.; Thonart, P.

    1982-01-01

    The influence of various agents such as inulin, proteins, and cationic polymers on the zeta potential of yeast cells has been studied. With various agents, it appears feasible to give a positive charge to the cell and a negative one to the carrier. Under these conditions, the yield of immobilization (mg cells/g support) is significantly improved. This is the case with various agents such as proteins and cationic polymers. For example, the yeild of cell immobilization (Saccharomyces) on sawdust varied from 94.2 to 145.8 mg/g of support with addition of gelatin (0.05%). The results demonstrate the influence of zeta potential on cell immobilization by adsorption. These immobilized cells are advantageously used in continuous production of ethanol. 3 figures, 8 tables.

  6. Nitrogen fixation by immobilized NIF derepressed Klebsiella pneumoniae cells

    SciTech Connect

    Venkatasubramanian, K.; Toda, Y.

    1980-01-01

    In vitro production of ammonia through biological means poses a number of challenges. The organisms should be able to accumulate considerable concentrations of ammonia in the medium. Secondly, nonphotosynthetic organisms must be supplied with high-energy substrates to carry out the fixation reaction. Thirdly, the organisms must be kept in a viable state to produce ammonia over long periods of time. In this article, preliminary results on the production of ammonia by a mutant strain of Klebsiella pneumoniae in continuous reactor systems are discussed. Continuous production of ammonia becomes feasible through the immobilization of the whole microbial cells and then through the use of the resulting catalyst system in a flow-through reactor. The rationale for immobilizing microbial cells and the advantages of such an approach over traditional fermentation processes are briefly described as they relate to the microbial production of ammonia. The microbial cells can be immobilized in such a way that their viability is still maintained in the immobilized state. This, in turn, obviates addition of cofactors, which is often an expensive step associated with immobilized multi-enzyme systems. Reconstituted bovine-hide collagen as the carrier matrix for fixing the cells was the carrier of choice for our work on immobilized Klebsiella cells. Polyacrylamide gels were examined as an alternate carrier matrix but results from this were found to be inferior to those collagen immobilized cell system.

  7. Hydroxyapatite-doped alginate beads as scaffolds for the osteoblastic differentiation of mesenchymal stem cells.

    PubMed

    Wang, Martha O; Bracaglia, Laura; Thompson, Joshua A; Fisher, John P

    2016-09-01

    This work investigates the role of an osteoblastic matrix component, hydroxyapatite (HA), in modular alginate scaffolds to support osteoblastic differentiation of human mesenchymal stem cells for the purpose of tissue engineered bone constructs. This system is first evaluated in a tubular perfusion bioreactor, which has been shown to improve osteoblastic differentiation over static culture conditions. HMSCs in alginate scaffolds that contain HA show increased osteoblastic gene expression compared to cells in pure alginate scaffolds, as well as significantly more matrix production and mineralization. The differentiated hMSCs and cell-laid matrix are ultimately evaluated in an in vivo site specific model. Implantation of these scaffolds with preformed matrix into the rat femoral condyle defects results in abundant bone growth and significant incorporation of the scaffold into the surrounding tissue. The developed mineralized matrix, induced in part by the HA component in the scaffold, could lead to increased tissue development in critically sized defects, and should be included in future implant strategies. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2325-2333, 2016.

  8. Alginate Encapsulation of Human Embryonic Stem Cells to Enhance Directed Differentiation to Pancreatic Islet-Like Cells

    PubMed Central

    Richardson, Thomas; Kumta, Prashant N.

    2014-01-01

    The pluripotent property of human embryonic stem cells (hESCs) makes them attractive for treatment of degenerative diseases such as diabetes. We have developed a stage-wise directed differentiation protocol to produce alginate-encapsulated islet-like cells derived from hESCs, which can be directly implanted for diabetes therapy. The advantage of alginate encapsulation lies in its capability to immunoisolate, along with the added possibility of scalable culture. We have evaluated the possibility of encapsulating hESCs at different stages of differentiation. Encapsulation of predifferentiated cells resulted in insufficient cellular yield and differentiation. On the other hand, encapsulation of undifferentiated hESCs followed by differentiation induction upon encapsulation resulted in the highest viability and differentiation. More striking was that alginate encapsulation resulted in a much stronger differentiation compared to parallel two-dimensional cultures, resulting in 20-fold increase in c-peptide protein synthesis. To elucidate the mechanism contributing to encapsulation-mediated enhancement in hESC maturation, investigation of the signaling pathways revealed interesting insight. While the phospho-protein levels of all the tested signaling molecules were lower under encapsulation, the ratio of pSMAD/pAKT was significantly higher, indicating a more efficient signal transduction under encapsulation. These results clearly demonstrate that alginate encapsulation of hESCs and differentiation to islet-cell types provides a potentially translatable treatment option for type 1 diabetes. PMID:24881778

  9. Microfabrication of a tunable collagen/alginate-chitosan hydrogel membrane for controlling cell-cell interactions.

    PubMed

    Song, Yizhe; Zhang, Demeng; Lv, Yan; Guo, Xin; Lou, Ruyun; Wang, Shujun; Wang, Xiuli; Yu, Weiting; Ma, Xiaojun

    2016-11-20

    Indirect cell contact co-culture system is increasingly becoming more attractable owing to their advantages of easy cell separation and desirable outcomes for cell-cell interactions. However, how to precisely control the spatial position of cells within multicellular co-cultures is still experimentally challenging due to the incapability of the conventional methods in vitro. In the present study, a tunable collagen/alginate-chitosan (Col/Alg-Chi) membrane was established, which was capable of controlling intercellular distance between the neighboring cells at a level of micrometer resolution. It was showed that intercellular distance between the hepatocytes and the fibroblasts exerted significant influence on hepatic function in vitro. In particular, maintenance of the functionality of primary hepatocytes requires direct contact between the hepatocytes and their supportive stromal cells, and their effective contact distance is within 30μm. This technical platform would potentially enable investigations of dynamic cell-cell interaction in a multitude of applications including organogenesis, development or even neoplastic transformation. PMID:27561537

  10. Tris-sucrose buffer system: a new specially designed medium for extracellular invertase production by immobilized cells of isolated yeast Cryptococcus laurentii MT-61.

    PubMed

    Aydogan, Mehmet Nuri; Taskin, Mesut; Canli, Ozden; Arslan, Nazli Pinar; Ortucu, Serkan

    2014-01-01

    The aims of the present study were to isolate new yeasts with high extracellular (exo) invertase activity and to investigate the usability of buffer systems as invertase production media by immobilized yeast cells. Among 70 yeast isolates, Cryptococcus laurentii MT-61 had the highest exo-invertase activity. Immobilization of yeast cells was performed using sodium alginate. Higher exo-invertase activity for immobilized cells was achieved in tris-sucrose buffer system (TSBS) compared to sodium acetate buffer system and potassium phosphate buffer system. TSBS was prepared by dissolving 30 g of sucrose in 1 L of tris buffer solution. The optimum pH, temperature, and incubation time for invertase production with immobilized cells were determined as 8.0, 35 °C and 36 h in TSBS, respectively. Under optimized conditions, maximum exo-invertase activity was found to be 28.4 U/mL in sterile and nonsterile TSBS. Immobilized cells could be reused in 14 and 12 successive cycles in sterile and nonsterile TSBS without any loss in the maximum invertase activity, respectively. This is the first report which showed that immobilized microbial cells could be used as a biocatalyst for exo-invertase production in buffer system. As an additional contribution, a new yeast strain with high invertase activity was isolated.

  11. Immobilization of Pichia pastoris cells containing alcohol oxidase activity

    PubMed Central

    Maleknia, S; Ahmadi, H; Norouzian, D

    2011-01-01

    Background and Objectives The attempts were made to describe the development of a whole cell immobilization of P. pastoris by entrapping the cells in polyacrylamide gel beads. The alcohol oxidase activity of the whole cell Pichia pastoris was evaluated in comparison with yeast biomass production. Materials and Methods Methylotrophic yeast P. pastoris was obtained from Collection of Standard Microorganisms, Department of Bacterial Vaccines, Pasteur Institute of Iran (CSMPI). Stock culture was maintained on YPD agar plates. Alcohol oxidase was strongly induced by addition of 0.5% methanol as the carbon source. The cells were harvested by centrifugation then permeabilized. Finally the cells were immobilized in polyacrylamide gel beads. The activity of alcohol oxidase was determined by method of Tane et al. Results At the end of the logarithmic phase of cell culture, the alcohol oxidase activity of the whole cell P. Pastoris reached the highest level. In comparison, the alcohol oxidase activity was measured in an immobilized P. pastoris when entrapped in polyacrylamide gel beads. The alcohol oxidase activity of cells was induced by addition of 0.5% methanol as the carbon source. The cells were permeabilized by cetyltrimethylammonium bromide (CTAB) and immobilized. CTAB was also found to increase the gel permeability. Alcohol oxidase activity of immobilized cells was then quantitated by ABTS/POD spectrophotometric method at OD 420. There was a 14% increase in alcohol oxidase activity in immobilized cells as compared with free cells. By addition of 2-butanol as a substrate, the relative activity of alcohol oxidase was significantly higher as compared with other substrates added to the reaction media. Conclusion Immobilization of cells could eliminate lengthy and expensive procedures of enzyme separation and purification, protect and stabilize enzyme activity, and perform easy separation of the enzyme from the reaction media. PMID:22530090

  12. Psychrotrophic Streptomyces spp. cells immobilisation in alginate microspheres produced by emulsification-internal gelation.

    PubMed

    Cotârleţ, Mihaela; Dima, Stefan; Bahrim, Gabriela

    2014-01-01

    The objective of the investigations was the optimisation of the parameters for cold-adapted Streptomyces MIUG 4 Alga strain cells immobilisation using emulsification-internal gelation technique in calcium alginate microspheres and testing their ability to produce cold-active β-amylase. By Box-Behnken design and response surface methodology, the effects of independent variables were established, which included sodium alginate concentration (A), sodium alginate:living cell ratio (B) and the Span 80 concentration (C) upon microspheres formation and their functionality. Mean diameter of formed microspheres with immobilised biomass and cold-active β-amylase production were chosen as dependent variables in order to increase the yield of starch hydrolysis. Diameters of microspheres <25.5 μm provided large yield of cold-active β-amylase comparing with microspheres with bigger diameter. A 1.5-fold increase in the substrate hydrolysis yield was achieved using the immobilised biocatalyst compared with the crude enzyme extract, after 96 h of substrate bioconversion.

  13. 3 dimensional cell cultures: a comparison between manually and automatically produced alginate beads.

    PubMed

    Lehmann, R; Gallert, C; Roddelkopf, T; Junginger, S; Wree, A; Thurow, K

    2016-08-01

    Cancer diseases are a common problem of the population caused by age and increased harmful environmental influences. Herein, new therapeutic strategies and compound screenings are necessary. The regular 2D cultivation has to be replaced by three dimensional cell culturing (3D) for better simulation of in vivo conditions. The 3D cultivation with alginate matrix is an appropriate method for encapsulate cells to form cancer constructs. The automated manufacturing of alginate beads might be an ultimate method for large-scaled manufacturing constructs similar to cancer tissue. The aim of this study was the integration of full automated systems for the production, cultivation and screening of 3D cell cultures. We compared the automated methods with the regular manual processes. Furthermore, we investigated the influence of antibiotics on these 3D cell culture systems. The alginate beads were formed by automated and manual procedures. The automated steps were processes by the Biomek(®) Cell Workstation (celisca, Rostock, Germany). The proliferation and toxicity were manually and automatically evaluated at day 14 and 35 of cultivation. The results visualized an accumulation and expansion of cell aggregates over the period of incubation. However, the proliferation and toxicity were faintly and partly significantly decreased on day 35 compared to day 14. The comparison of the manual and automated methods displayed similar results. We conclude that the manual production process could be replaced by the automation. Using automation, 3D cell cultures can be produced in industrial scale and improve the drug development and screening to treat serious illnesses like cancer.

  14. Factors influencing alginate gel biocompatibility.

    PubMed

    Tam, Susan K; Dusseault, Julie; Bilodeau, Stéphanie; Langlois, Geneviève; Hallé, Jean-Pierre; Yahia, L'Hocine

    2011-07-01

    Alginate remains the most popular polymer used for cell encapsulation, yet its biocompatibility is inconsistent. Two commercially available alginates were compared, one with 71% guluronate (HiG), and the other with 44% (IntG). Both alginates were purified, and their purities were verified. After 2 days in the peritoneal cavity of C57BL/6J mice, barium (Ba)-gel and calcium (Ca)-gel beads of IntG alginate were clean, while host cells were adhered to beads of HiG alginate. IntG gel beads, however, showed fragmentation in vivo while HiG gel beads stayed firm. The physicochemical properties of the sodium alginates and their gels were thoroughly characterized. The intrinsic viscosity of IntG alginate was 2.5-fold higher than that of HiG alginate, suggesting a greater molecular mass. X-ray photoelectron spectroscopy indicated that both alginates were similar in elemental composition, including low levels of counterions in all gels. The wettabilities of the alginates and gels were also identical, as measured by contact angles of water on dry films. Ba-gel beads of HiG alginate resisted swelling and degradation when immersed in water, much more than the other gel beads. These results suggest that the main factors contributing to the biocompatibility of gels of purified alginate are the mannuronate/guluronate content and/or intrinsic viscosity.

  15. Flicking technique for microencapsulation of cells in calcium alginate leading to the microtissue formation.

    PubMed

    Wong, Soon Chuan; Soon, Chin Fhong; Leong, Wai Yean; Tee, Kian Sek

    2016-01-01

    Microbeads have wide applications in biomedical engineering field that include drug delivery, encapsulation of biomolecules, tissue padding and tissue regeneration. In this paper, we report a simple, yet efficient, flicking technique to produce microcapsules of calcium alginate at a narrow distribution of size. The system consists of an infusion pump and a customised flicker that taps the syringe needle for dispersing microcapsules of sodium alginate that polymerised in the calcium chloride solution. The flow rate of the syringe pump and the velocity of the flicker were studied to achieve a well controlled and tunable size distribution of microbeads ranging from 200 to 400 μm. At a flow rate of 4 μl/min and flicking rate of 80 rpm, a narrow size distribution of microbeads were produced. Via this technique, HaCaT cells were encapsulated in calcium alginate microbeads that grown into microtissues with a size ranging from 100 to 300 μm after two weeks of culture. These microtissues could be potentially useful for pharmacological application. PMID:26878098

  16. Layer-shaped alginate hydrogels enhance the biological performance of human adipose-derived stem cells

    PubMed Central

    2012-01-01

    Background The reconstruction of adipose tissue defects is often challenged by the complications that may occur following plastic and reconstructive surgery, including donor-site morbidity, implant migration and foreign body reaction. To overcome these problems, adipose tissue engineering (ATE) using stem cell-based regeneration strategies has been widely explored in the last years. Mounting evidence has shown that adipose-derived stem cells (ADSCs) represent a promising cell source for ATE. In the context of a small number of reports concerning adipose tissue regeneration using three-dimensional (3-D) systems, the present study was designed to evaluate the biological performance of a novel alginate matrix that incorporates human ADSCs (hADSCs). Results Culture-expanded cells isolated from the stromal vascular fraction (SVF), corresponding to the third passage which showed the expression of mesenchymal stem cell (MSC) markers, were used in the 3-D culture systems. The latter represented a calcium alginate hydrogel, obtained by the diffusion of calcium gluconate (CGH matrix), and shaped as discoid-thin layer. For comparative purposes, a similar hADSC-laden alginate hydrogel cross-linked with calcium chloride was considered as reference hydrogel (RH matrix). Both hydrogels showed a porous structure under scanning electron microscopy (SEM) and the hADSCs embedded displayed normal spherical morphologies, some of them showing signs of mitosis. More than 85% of the entrapped cells survived throughout the incubation period of 7 days. The percentage of viable cells was significantly higher within CGH matrix at 2 days post-seeding, and approximately similar within both hydrogels after 7 days of culture. Moreover, both alginate-based hydrogels stimulated cell proliferation. The number of hADSC within hydrogels has increased during the incubation period of 7 days and was higher in the case of CGH matrix. Cells grown under adipogenic conditions for 21 days showed

  17. A tunable silk-alginate hydrogel scaffold for stem cell culture and transplantation

    PubMed Central

    Ziv, Keren; Nuhn, Harald; Ben-Haim, Yael; Sasportas, Laura S.; Kempen, Paul J.; Niedringhaus, Thomas P.; Hrynyk, Michael; Sinclair, Robert; Barron, Annelise E.; Gambhir, Sanjiv S.

    2014-01-01

    One of the major challenges in regenerative medicine is the ability to recreate the stem cell niche, which is defined by its signaling molecules, the creation of cytokine gradients, and the modulation of matrix stiffness. A wide range of scaffolds has been developed in order to recapitulate the stem cell niche, among them hydrogels. This paper reports the development of a new silk-alginate based hydrogel with a focus on stem cell culture. This biocomposite allows to fine tune its elasticity during cell culture, addressing the importance of mechanotransduction during stem cell differentiation. The silk-alginate scaffold promotes adherence of mouse embryonic stem cells and cell survival upon transplantation. In addition, it has tunable stiffness as function of the silk-alginte ratio and the concentration of crosslinker - a characteristic that is very hard to accomplish in current hydrogels. The hydrogel and the presented results represents key steps on the way of creating artificial stem cell niche, opening up new paths in regenerative medicine. PMID:24484675

  18. Evaluation of an alginate-gelatine crosslinked hydrogel for bioplotting.

    PubMed

    Zehnder, Tobias; Sarker, Bapi; Boccaccini, Aldo R; Detsch, Rainer

    2015-04-08

    Using additive manufacturing to create hydrogel scaffolds which incorporate homogeneously distributed, immobilized cells in the context of biofabrication approaches represents an emerging and expanding field in tissue engineering. Applying hydrogels for additive manufacturing must consider the material processing properties as well as their influence on the immobilized cells. In this work alginate-dialdehyde (ADA), a partially oxidized alginate, was used as a basic material to improve the physico-chemical properties of the hydrogel for cell immobilization. At first, the processing ability of the gel using a bioplotter and the compatibility of the process with MG-63 osteoblast like cells were investigated. The metabolic and mitochondrial activities increased at the beginning of the incubation period and they balanced at a relatively high level after 14-28 days of incubation. During this incubation period the release of vascular endothelial growth factor-A also increased. After 28 days of incubation the cell morphology showed a spreading morphology and cells were seen to move out of the scaffold struts covering the whole scaffold structure. The reproducible processing capability of alginate-gelatine (ADA-GEL) and the compatibility with MG-63 cells were proven, thus the ADA-GEL material is highlighted as a promising matrix for applications in biofabrication.

  19. Invert sugar formation with Saccharomyces cerevisiae cells encapsulated in magnetically responsive alginate microparticles

    NASA Astrophysics Data System (ADS)

    Safarik, Ivo; Sabatkova, Zdenka; Safarikova, Mirka

    2009-05-01

    Invert sugar (an equimolar mixture of glucose and fructose prepared by sucrose hydrolysis) is a very important food component. We have prepared magnetically responsive alginate microbeads containing entrapped Saccharomyces cerevisiae cells and magnetite microparticles which can be easily separated in an appropriate magnetic separator. The microbeads (typical diameter between 50 and 100 μm) were prepared using the water-in-oil emulsification process. The prepared microbeads containing yeast cells with invertase activity enabled efficient sucrose conversion. The biocatalyst was quite stable; the same catalytic activity was observed after one month storage at 4 °C and the microbeads could be used at least six times.

  20. Crystal structure of bacterial cell-surface alginate-binding protein with an M75 peptidase motif

    SciTech Connect

    Maruyama, Yukie; Ochiai, Akihito; Mikami, Bunzo; Hashimoto, Wataru; Murata, Kousaku

    2011-02-18

    Research highlights: {yields} Bacterial alginate-binding Algp7 is similar to component EfeO of Fe{sup 2+} transporter. {yields} We determined the crystal structure of Algp7 with a metal-binding motif. {yields} Algp7 consists of two helical bundles formed through duplication of a single bundle. {yields} A deep cleft involved in alginate binding locates around the metal-binding site. {yields} Algp7 may function as a Fe{sup 2+}-chelated alginate-binding protein. -- Abstract: A gram-negative Sphingomonas sp. A1 directly incorporates alginate polysaccharide into the cytoplasm via the cell-surface pit and ABC transporter. A cell-surface alginate-binding protein, Algp7, functions as a concentrator of the polysaccharide in the pit. Based on the primary structure and genetic organization in the bacterial genome, Algp7 was found to be homologous to an M75 peptidase motif-containing EfeO, a component of a ferrous ion transporter. Despite the presence of an M75 peptidase motif with high similarity, the Algp7 protein purified from recombinant Escherichia coli cells was inert on insulin B chain and N-benzoyl-Phe-Val-Arg-p-nitroanilide, both of which are substrates for a typical M75 peptidase, imelysin, from Pseudomonas aeruginosa. The X-ray crystallographic structure of Algp7 was determined at 2.10 A resolution by single-wavelength anomalous diffraction. Although a metal-binding motif, HxxE, conserved in zinc ion-dependent M75 peptidases is also found in Algp7, the crystal structure of Algp7 contains no metal even at the motif. The protein consists of two structurally similar up-and-down helical bundles as the basic scaffold. A deep cleft between the bundles is sufficiently large to accommodate macromolecules such as alginate polysaccharide. This is the first structural report on a bacterial cell-surface alginate-binding protein with an M75 peptidase motif.

  1. Physiological and Morphological Modifications in Immobilized Gibberella fujikuroi Mycelia

    PubMed Central

    Saucedo, José Edmundo Nava; Barbotin, Jean-Noël; Thomas, Daniel

    1989-01-01

    Constraints created by immobilization conditions modified the physiological behavior and morphological characteristics of Gibberella fujikuroi mycelia in comparison with their development in free-cell conditions. G. fujikuroi mycelia were immobilized in different support matrices (polyurethane, carrageenan, and alginate) and showed a variety of reactions in response to the different microenvironmental factors encountered during and after immobilization. The best support with respect to gibberellic acid yield and biocatalyst stability was found to be an alginate with a high degree of polymerization. The most visible effects of immobilization included changes in growth development, morphological appearance, metabolite production, mycelial pigmentation, mycelial viability under starvation conditions, and induction of resting forms when previously immobilized mycelia were subcultured. Images PMID:16348017

  2. Silver nanoparticles impregnated alginate-chitosan-blended nanocarrier induces apoptosis in human glioblastoma cells.

    PubMed

    Sharma, Shilpa; Chockalingam, S; Sanpui, Pallab; Chattopadhyay, Arun; Ghosh, Siddhartha Sankar

    2014-01-01

    Herein, a green method for the development of a novel biodegradable silver nanoparticles (NPs) impregnated alginate-chitosan-blended nanocarrier (Ag NPs-Alg-Chi NC) is reported. The synthesis of Ag NPs-Alg-Chi NC is based on the polyelectrolyte complex formation between alginate and chitosan. The composite NC is characterized by ultraviolet-visible spectroscopy, transmission electron microscopy, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy, and X-ray diffraction. The Ag NPs in the NC are found to elicit anticell proliferative effect on refractory U87MG (human glioblastoma) cells at IC50 of 2.4 μg mL(-1) for Ag NPs. The cell cycle analysis shows extensive DNA damage. Elevation in reactive oxygen species level indicates induction of oxidative stress in treated cells. Mitochondrial dysfunction in cell death is evident from the depolarization of mitochondrial membrane potential (ΔΨm ). Fluorescence and SEM images of the treated cells reveal nuclear and morphological changes characteristic of apoptosis, which is further confirmed by TUNEL assay. The induction of apoptosis at low concentration of Ag NPs present in Ag NPs-Alg-Chi NC in comparison with free Ag NPs makes it a promising tool for cancer therapy.

  3. Fluid and cell behaviors along a 3D printed alginate/gelatin/fibrin channel.

    PubMed

    Xu, Yufan; Wang, Xiaohong

    2015-08-01

    Three-dimensional (3D) cell manipulation is available with the integration of microfluidic technology and rapid prototyping techniques. High-Fidelity (Hi-Fi) constructs hold enormous therapeutic potential for organ manufacturing and regenerative medicine. In the present paper we introduced a quasi-three-dimensional (Q3D) model with parallel biocompatible alginate/gelatin/fibrin hurdles. The behaviors of fluids and cells along the microfluidic channels with various widths were studied. Cells inside the newly designed microfluidic channels attached and grew well. Morphological changes of adipose-derived stem cells (ADSCs) in both two-dimensional (2D) and 3D milieu were found on the printed constructs. Endothelialization occurred with the co-cultures of ADSCs and hepatocytes. This study provides insights into the interactions among fluids, cells and biomaterials, the behaviors of fluids and cells along the microfluidic channels, and the applications of Q3D techniques.

  4. Microenvironment of alginate-based microcapsules for cell culture and tissue engineering.

    PubMed

    Huang, Xiaobo; Zhang, Xiangyu; Wang, Xiaoguang; Wang, Chan; Tang, Bin

    2012-07-01

    As a type of 3D model, the technology of microencapsulation holds significant promise for tissue engineering and cell therapy due to its unique performance. The microenvironmental factors within microcapsules play an important role in influencing the behaviors of encapsulated cells. The aim of this review article is to give an overview on the construction of the microenvironmental factors, which include 3D space, physicochemical properties of alginate matrix, cell spheroids, nutritional status, and so on. Furthermore, we clarified the effect of microenvironmental factors on the behaviors of encapsulated cells and the methods about improving the microenvironment of microcapsules. This review will help to understand the interaction of the microenvironment and the encapsulated cells and lay a solid foundation for microcapsule-based cell therapy and tissue engineering.

  5. Direct electrochemistry and electrocatalysis of myoglobin immobilized on Fe2O3 nanoparticle-sodium alginate-ionic liquid composite-modified electrode.

    PubMed

    Zhan, Tianrong; Xi, Mengying; Wang, Yan; Sun, Wei; Hou, Wanguo

    2010-06-01

    A biocomposite material composed of sodium alginate (SA), Fe(2)O(3) nanoparticles, and ionic liquid 1-decyl-3-methylimidazolium bromide ([DMIM]Br) was fabricated and used for the immobilization of myoglobin (Mb) on the surface of a carbon ionic liquid electrode (CILE). The CILE was fabricated by mixing graphite powder with ionic liquid N-butylpyridinium hexafluorophosphate (BPPF(6)) together. UV-Vis absorption and FTIR spectroscopic results indicated that Mb retained its native structure in the composite material. A pair of well-defined redox peaks appeared on the cyclic voltammogram in pH 7.0 phosphate buffer solution (PBS) with the formal peak potential (E(0')) at -0.256 V (versus SCE), which was the typical electrochemical behavior of Mb heme Fe(III)/Fe(II) redox couples. The Mb-modified electrode showed good electrocatalytic activity to the reduction of trichloroacetic acid (TCA) and NaNO(2) with wide linear range, good sensitivity, and reproducibility. The calibration range for TCA detection was between 0.6 and 12.0 mmol L(-1) with the linear regression equation as Iss (μA)=42.44C (mmol L(-1))+50.57 and a detection limit of 0.4 mmol L(-1) (3σ). The Mb-modified electrode also applied to NaNO(2) determination in the concentration range from 4.0 to 100.0 mmol L(-1) with a detection limit of 1.3 mmol L(-1) (3σ). So the proposed electrode has potential applications as third-generation biosensors.

  6. Adsorption of As(III), As(V) and Cu(II) on zirconium oxide immobilized alginate beads in aqueous phase.

    PubMed

    Kwon, Oh-Hun; Kim, Jong-Oh; Cho, Dong-Wan; Kumar, Rahul; Baek, Seung Han; Kurade, Mayur B; Jeon, Byong-Hun

    2016-10-01

    A composite adsorbent to remove arsenite [As(III)], arsenate [As(V)], and copper [Cu(II)] from aqueous phase was synthesized by immobilizing zirconium oxide on alginate beads (ZOAB). The composition (wt%) of ZOAB (Zr-34.0; O-32.7; C-21.3; Ca-1.0) was confirmed by energy dispersive X-ray (EDX) analysis. Sorption studies were conducted on single and binary sorbate systems, and the effects of contact time, initial adsorbate concentration, and pH on the adsorption performance of ZOAB (pHPZC = 4.3) were monitored. The sorption process for As(III)/As(V) and Cu(II) reached an equilibrium state within 240 h and 24 h, respectively, with maximum sorption capacities of 32.3, 28.5, and 69.9 mg g(-1), respectively. The addition of Cu(II) was favorable for As(V) sorption in contrast to As(III). In the presence of 48.6 mg L(-1) Cu(II), the sorption capacity of As(V) increased from 1.5 to 3.8 mg g(-1) after 240 h. The sorption data for As(III)/As(V) and Cu(II) conformed the Freundlich and Langmuir isotherm models, respectively. The adsorption of As(III), As(V), and Cu(II) followed pseudo second order kinetics. The effect of arsenic species on Cu(II) sorption was insignificant. The results of present study demonstrated that the synthesized sorbent could be useful for the simultaneous removal of both anionic and cationic contaminants from wastewaters.

  7. Adsorption of As(III), As(V) and Cu(II) on zirconium oxide immobilized alginate beads in aqueous phase.

    PubMed

    Kwon, Oh-Hun; Kim, Jong-Oh; Cho, Dong-Wan; Kumar, Rahul; Baek, Seung Han; Kurade, Mayur B; Jeon, Byong-Hun

    2016-10-01

    A composite adsorbent to remove arsenite [As(III)], arsenate [As(V)], and copper [Cu(II)] from aqueous phase was synthesized by immobilizing zirconium oxide on alginate beads (ZOAB). The composition (wt%) of ZOAB (Zr-34.0; O-32.7; C-21.3; Ca-1.0) was confirmed by energy dispersive X-ray (EDX) analysis. Sorption studies were conducted on single and binary sorbate systems, and the effects of contact time, initial adsorbate concentration, and pH on the adsorption performance of ZOAB (pHPZC = 4.3) were monitored. The sorption process for As(III)/As(V) and Cu(II) reached an equilibrium state within 240 h and 24 h, respectively, with maximum sorption capacities of 32.3, 28.5, and 69.9 mg g(-1), respectively. The addition of Cu(II) was favorable for As(V) sorption in contrast to As(III). In the presence of 48.6 mg L(-1) Cu(II), the sorption capacity of As(V) increased from 1.5 to 3.8 mg g(-1) after 240 h. The sorption data for As(III)/As(V) and Cu(II) conformed the Freundlich and Langmuir isotherm models, respectively. The adsorption of As(III), As(V), and Cu(II) followed pseudo second order kinetics. The effect of arsenic species on Cu(II) sorption was insignificant. The results of present study demonstrated that the synthesized sorbent could be useful for the simultaneous removal of both anionic and cationic contaminants from wastewaters. PMID:27372261

  8. Alginate Inhibits Iron Absorption from Ferrous Gluconate in a Randomized Controlled Trial and Reduces Iron Uptake into Caco-2 Cells

    PubMed Central

    Wawer, Anna A.; Harvey, Linda J.; Dainty, Jack R.; Perez-Moral, Natalia; Sharp, Paul; Fairweather-Tait, Susan J.

    2014-01-01

    Previous in vitro results indicated that alginate beads might be a useful vehicle for food iron fortification. A human study was undertaken to test the hypothesis that alginate enhances iron absorption. A randomised, single blinded, cross-over trial was carried out in which iron absorption was measured from serum iron appearance after a test meal. Overnight-fasted volunteers (n = 15) were given a test meal of 200 g cola-flavoured jelly plus 21 mg iron as ferrous gluconate, either in alginate beads mixed into the jelly or in a capsule. Iron absorption was lower from the alginate beads than from ferrous gluconate (8.5% and 12.6% respectively, p = 0.003). Sub-group B (n = 9) consumed the test meals together with 600 mg calcium to determine whether alginate modified the inhibitory effect of calcium. Calcium reduced iron absorption from ferrous gluconate by 51%, from 11.5% to 5.6% (p = 0.014), and from alginate beads by 37%, from 8.3% to 5.2% (p = 0.009). In vitro studies using Caco-2 cells were designed to explore the reasons for the difference between the previous in vitro findings and the human study; confirmed the inhibitory effect of alginate. Beads similar to those used in the human study were subjected to simulated gastrointestinal digestion, with and without cola jelly, and the digestate applied to Caco-2 cells. Both alginate and cola jelly significantly reduced iron uptake into the cells, by 34% (p = 0.009) and 35% (p = 0.003) respectively. The combination of cola jelly and calcium produced a very low ferritin response, 16.5% (p<0.001) of that observed with ferrous gluconate alone. The results of these studies demonstrate that alginate beads are not a useful delivery system for soluble salts of iron for the purpose of food fortification. Trial Registration ClinicalTrials.gov NCT01528644 PMID:25391138

  9. Covalent immobilization of p-selectin enhances cell rolling.

    PubMed

    Hong, Seungpyo; Lee, Dooyoung; Zhang, Huanan; Zhang, Jennifer Q; Resvick, Jennifer N; Khademhosseini, Ali; King, Michael R; Langer, Robert; Karp, Jeffrey M

    2007-11-20

    Cell rolling is an important physiological and pathological process that is used to recruit specific cells in the bloodstream to a target tissue. This process may be exploited for biomedical applications to capture and separate specific cell types. One of the most commonly studied proteins that regulate cell rolling is P-selectin. By coating surfaces with this protein, biofunctional surfaces that induce cell rolling can be prepared. Although most immobilization methods have relied on physisorption, chemical immobilization has obvious advantages, including longer functional stability and better control over ligand density and orientation. Here we describe chemical methods to immobilize P-selectin covalently on glass substrates. The chemistry was categorized on the basis of the functional groups on modified glass substrates: amine, aldehyde, and epoxy. The prepared surfaces were first tested in a flow chamber by flowing microspheres functionalized with a cell surface carbohydrate (sialyl Lewis(x)) that binds to P-selectin. Adhesion bonds between P-selectin and sialyl Lewis(x) dissociate readily under shear forces, leading to cell rolling. P-selectin immobilized on the epoxy glass surfaces exhibited enhanced long-term stability of the function and better homogeneity as compared to that for surfaces prepared by other methods and physisorbed controls. The microsphere rolling results were confirmed in vitro with isolated human neutrophils. This work is essential for the future development of devices for isolating specific cell types based on cell rolling, which may be useful for hematologic cancers and certain metastatic cancer cells that are responsive to immobilized selectins.

  10. Cell immobilization in kappa-carrageenan for ethanol production.

    PubMed

    Luong, J H

    1985-12-01

    A combination of extended Monod kinetics and the diffusional equation was used for evaluating the effectiveness factor of entrapped immobilized cells. Based on the kinetics of Zymomonas mobilis reported in the literature, the numerical results have revealed that the problem of mass transfer diffusional restrictions can be neglected by using small beads (1 mm in diameter) with a corresponding cell loading up to 276 g/L gel. On the basis of the numerical results obtained, the application of immobilized cells for continuous ethanol production was investigated. The kappa-carrageenan method was utilized to entrap Z. mobilis CP4, a potential ethanol producer. A two stage fermentation process has also been developed for ethanol production by the Z. mobilis carrageenan-bound cells. About 90 g/L ethanol was produced by immobilized cells at a total residence time of 1.56 h. The ethanol yield was estimated to be 93% of theoretical. The results obtained in this study also indicated that the control of optimum pH in an immobilized cell column is necessary to enhance the rate of ethanol production. PMID:18553626

  11. Alginate cell encapsulation: new advances in reproduction and cartilage regenerative medicine

    PubMed Central

    Ghidoni, Ilaria; Chlapanidas, Theodora; Bucco, Massimo; Crovato, Francesca; Marazzi, Mario; Vigo, Daniele; Torre, Maria Luisa

    2008-01-01

    Cell encapsulation, a strategy whereby a pool of live cells is entrapped within a semipermeable membrane, represents an evolving branch of biotechnology and regenerative medicine. For example, over the last 20 years, male and female gametes and embryos have been encapsulated with or without somatic cells for different purposes, such as in vitro gametogenesis, embryo culture, cell preservation and semen controlled release. Beside that, cell encapsulation technology in alginate, which is a natural biodegradable polymer that mimics the extracellular matrix and supports both cell functions and metabolism, has been developed with the aim of obtaining three-dimensional (3D) cultures. In this context, adipose-derived stromal vascular fraction (SVF) has attracted more and more attention because of its enormous potential in tissue regeneration. In fact, the SVF represents a rich source of mesenchymal cells (ADSCs), potentially able to differentiate into adipocytes, chondrocytes, osteoblasts, myocytes, cardiomyocytes, hepatocytes, and neuronal, epithelial and endothelial cells. These cells are ideal candidates for use in regenerative medicine, tissue engineering, including gene therapy and cell replacement cancer therapies. As long as technological resources are available for large-scale cell encapsulation intended for advanced therapies (gene therapy, somatic cell therapy and tissue engineering), the state-of-the-art in this field is reviewed in terms of scientific literature. PMID:19002776

  12. Alginate bead fabrication and encapsulation of living cells under centrifugally induced artificial gravity conditions.

    PubMed

    Haeberle, Stefan; Naegele, Lars; Burger, Robert; von Stetten, Felix; Zengerle, Roland; Ducrée, Jens

    2008-06-01

    This study presents a novel method for the direct, centrifugally induced fabrication of small, Ca2+-hardened alginate beads at polymer-tube micronozzles. The bead diameter can arbitrarily be adjusted between 180-800 microm by the nozzle geometry and spinning frequencies between 5-28 Hz. The size distribution of the main peak features a CV of 7-16%, only. Up to 600 beads per second and channel are issued from the micronozzle through an air gap towards the curing agent contained in a standard lab tube ('Eppi'). Several tubes can be mounted on a 'flying bucket' rotor where they align horizontally under rotation and return to a vertical position as soon as the rotor is at rest. The centrifugally induced, ultra-high artificial gravity conditions (up to 180 g) even allow the micro-encapsulation of alginate solutions displaying viscosities up to 50 Pa s, i.e. approximately 50,000 times the viscosity of water! With this low cost technology for microencapsulation, HN25 and PC12 cells have successfully been encapsulated while maintaining vitality.

  13. Oral delivery of probiotic expressing M cell homing peptide conjugated BmpB vaccine encapsulated into alginate/chitosan/alginate microcapsules.

    PubMed

    Jiang, Tao; Singh, Bijay; Maharjan, Sushila; Li, Hui-Shan; Kang, Sang-Kee; Bok, Jin-Duck; Cho, Chong-Su; Choi, Yun-Jaie

    2014-11-01

    Oral administration of live probiotics as antigen delivery vectors is a promising approach in vaccine development. However, the low survival of probiotics in the gastrointestinal tract limits this approach. Therefore, the aim of this study was the encapsulation of probiotic expressing vaccine into alginate/chitosan/alginate (ACA) microcapsules (MCs) for efficient oral vaccine delivery. Here, recombinant Lactobacillus plantarum 25 (LP25) expressing M cell homing peptide fused BmpB protein was used as a model probiotic. The viability of LP25 in ACA MCs was more than 65% in simulated gastric fluid (SGF, pH 2.0) and 75% in simulated small intestinal fluid (SIF, pH 7.2) up to 2h. Encapsulated LP25 was completely released from ACA MCs in SIF within 12h. When stored at room temperature (RT) or 4°C, the viability of LP25 in ACA MCs was higher than free LP25. Interestingly, the viability of LP25 in ACA MCs at 4°C for 5weeks was above 58%, whereas viability of free LP25 stored at RT up to 5weeks was zero. After 4weeks from the first immunization, LP25-M-BmpB-loaded ACA MCs induced a stronger BmpB-specific IgG and IgA production in mice. Collectively, these findings suggest that encapsulation of probiotic by ACA MCs is a promising delivery system for oral administration of probiotic expressing vaccine.

  14. Proliferation and osteogenic differentiation of human bone marrow stromal cells on alginate-gelatine-hydroxyapatite scaffolds with anisotropic pore structure.

    PubMed

    Bernhardt, A; Despang, F; Lode, A; Demmler, A; Hanke, T; Gelinsky, M

    2009-01-01

    Porous mineralized scaffolds are required for various applications in bone engineering. In particular, tube-like pores with controlled orientation inside the scaffold may support homogeneous cell seeding as well as sufficient nutrient supply and may facilitate blood vessel ingrowth. Scaffolds with parallely orientated tube-like pores were generated by diffusion-controlled ionotropic gelation of alginate. Incorporation of hydroxyapatite (HA) during the gelation process yielded stable scaffolds with an average pore diameter of approximately 90 microm. To evaluate the potential use of alginate-gelatine-HA scaffolds for bone tissue engineering, in vitro tests with human bone marrow stromal cells (hBMSCs) were carried out. We analysed biocompatibility and cell penetration into the capillary pores by microscopic methods. hBMSCs were also cultivated on alginate-gelatine-HA scaffolds for 3 weeks in the presence and absence of osteogenic supplements. We studied proliferation and osteogenic differentiation in terms of total lactate dehydrogenase (LDH) activity, DNA content and alkaline phosphatase (ALP) activity and found a 10-14-fold increase of cell number after 2 weeks of cultivation, as well as an increase of specific ALP activity for osteogenic-induced hBMSCs. Furthermore, the expression of bone-related genes [ALP, bone sialoprotein II (BSPII)] was analysed. We found an increase of ALP as well as BSPII expression for osteogenic-induced hBMSCs on alginate-gelatin-HA scaffolds.

  15. Dental mesenchymal stem cells encapsulated in an alginate hydrogel co-delivery microencapsulation system for cartilage regeneration.

    PubMed

    Moshaverinia, Alireza; Xu, Xingtian; Chen, Chider; Akiyama, Kentaro; Snead, Malcolm L; Shi, Songtao

    2013-12-01

    Dental-derived mesenchymal stem cells (MSCs) are promising candidates for cartilage regeneration, with a high capacity for chondrogenic differentiation. This property helps make dental MSCs an advantageous therapeutic option compared to current treatment modalities. The MSC delivery vehicle is the principal determinant for the success of MSC-mediated cartilage regeneration therapies. The objectives of this study were to: (1) develop a novel co-delivery system based on TGF-β1 loaded RGD-coupled alginate microspheres encapsulating periodontal ligament stem cells (PDLSCs) or gingival mesenchymal stem cells (GMSCs); and (2) investigate dental MSC viability and chondrogenic differentiation in alginate microspheres. The results revealed the sustained release of TGF-β1 from the alginate microspheres. After 4 weeks of chondrogenic differentiation in vitro, PDLSCs and GMSCs as well as human bone marrow mesenchymal stem cells (hBMMSCs) (as positive control) revealed chondrogenic gene expression markers (Col II and Sox-9) via qPCR, as well as matrix positively stained by Toluidine Blue and Safranin-O. In animal studies, ectopic cartilage tissue regeneration was observed inside and around the transplanted microspheres, confirmed by histochemical and immunofluorescent staining. Interestingly, PDLSCs showed more chondrogenesis than GMSCs and hBMMSCs (p<0.05). Taken together, these results suggest that RGD-modified alginate microencapsulating dental MSCs make a promising candidate for cartilage regeneration. Our results highlight the vital role played by the microenvironment, as well as value of presenting inductive signals for viability and differentiation of MSCs. PMID:23891740

  16. Siderophore production by using free and immobilized cells of two pseudomonads cultivated in a medium enriched with Fe and/or toxic metals (Cr, Hg, Pb).

    PubMed

    Braud, Armelle; Jézéquel, Karine; Léger, Marie-Anne; Lebeau, Thierry

    2006-08-20

    Pseudomonads are serious candidates for siderophore production applied to toxic metal (TM) solubilization. The bioaugmentation of contaminated soils by these TM-solubilizing bacteria combined with phytoextraction is an emerging clean-up technology. Unfortunately, siderophore synthesis may be drastically reduced by soluble iron in soils and bacteria can suffer from TM toxicity. In this study, we compared siderophore production by Pseudomonas aeruginosa and Pseudomonas fluorescens by using free and immobilized cells in Ca-alginate beads incubated in a medium containing Fe and/or TM (mixture of Cr, Hg, and Pb in concentrations which represented the soluble fraction of a contaminated agricultural soil). Free cell growth was stimulated by Fe, whatever the microorganism, the inoculum size and the presence or not of TM might have been. P. aeruginosa was less sensitive to TM than P. fluorescens. By comparison with free cells, immobilization with the high inoculum size showed less sensitivity to TM most probably because of lower metal diffusion in beads. Indeed, a maximum of 99.1% of Cr, 57.4% of Hg, and 99.6% of Pb were adsorbed onto beads. The addition of iron in the culture medium reduced significantly siderophore production of free cells while it led only to a low decrease with their immobilized counterparts, in particular with P. aeruginosa. In culture medium enriched with Fe and/or TM, siderophore-specific production of immobilized cells was higher than for free cells. PMID:16586510

  17. Bioglass/alginate composite hydrogel beads as cell carriers for bone regeneration.

    PubMed

    Zeng, Qiongyu; Han, Yan; Li, Haiyan; Chang, Jiang

    2014-01-01

    Cell carrier is a useful biomedical tool to deliver particular kind of cells to a specific site for cell therapy or tissue regeneration. In the current study, 45S5 bioglass (BG) was introduced into alginate (ALG) to generate BG/ALG composite hydrogel beads as cell carriers. The ions releasing behavior, dimensional stability and in vitro bioactivity of the beads were investigated. Results showed that the BG/ALG beads revealed similar calcium ion releasing behavior as compared with ALG beads. In addition, silicon ion releasing was detected in BG/ALG beads. BG/ALG and ALG beads shared similar dimensional stability, and BG/ALG beads could induce apatite deposition on their surface after being soaked in stimulated body fluid. Then, the effects of ion extracts from hydrogel beads on cell behavior were investigated. Results confirmed that extracts of BG/ALG beads could simulate proliferation and osteogenic differentiation of mesenchymal stem cells as well as angiogenesis of endothelial cells. Furthermore, MC3T3-E1 cells were successfully encapsulated in hydrogel beads. BG/ALG beads enhanced the cell proliferation and stimulated osteogenic differentiation of the encapsulated MC3T3-E1 cells as compared with ALG beads. Therefore, BG/ALG composite hydrogel beads loaded with bone forming cells may be useful tools for bone regeneration and tissue engineering applications.

  18. Neuralization of mouse embryonic stem cells in alginate hydrogels under retinoic acid and SAG treatment.

    PubMed

    Delivopoulos, Evangelos; Shakesheff, Kevin M; Peto, Heather

    2015-08-01

    This paper examines the differentiation of a mouse embryonic stem cell line (CGR8) into neurons, under retinoic acid (RA) and smoothened agonist (SAG) treatment. When stem cells underwent through an embryoid body (EB) formation stage, dissociation and seeding on glass coverslips, immunofluorescent labelling for neuronal markers (Nestin, b-Tubulin III, MAP2) revealed the presence of both immature neural progenitors and mature neurons. Undifferentiated CGR8 were also encapsulated in tubular, alginate-gelatin hydrogels and incubated in differentiation media containing retinoic acid (RA) and smoothened agonist (SAG). Cryo-sections of the hydrogel tubes were positive for Nestin, Pax6 and b-Tubulin III, verifying the presence of neurons and neural progenitors. Provided neural induction can be more precisely directed in the tubular hydrogels, these scaffolds will become a powerful model of neural tube development in embryos and will highlight potential strategies for spinal cord regeneration. PMID:26737053

  19. Ca-alginate hydrogel mechanical transformations--the influence on yeast cell growth dynamics.

    PubMed

    Pajić-Lijaković, Ivana; Plavsić, Milenko; Bugarski, Branko; Nedović, Viktor

    2007-05-01

    A mathematical model was formulated to describe yeast cell growth within the Ca-alginate microbead during air-lift bioreactor cultivation. Model development was based on experimentally obtained data for the intra-bead cell concentration profile, after reached the equilibrium state, as well as, total yeast cell concentration per microbed and microbead volume as function of time. Relatively uniform cell concentration in the carrier matrix indicated that no internal nutrient diffusion limitations, but microenvironmental restriction, affected dominantly the dynamics of cell growth. Also interesting phenomenon of very different rates of cell number growth during cultivation is observed. After some critical time, the growth rate of cell colonies decreased drastically, but than suddenly increased again under all other experimental condition been the same. It is interpreted as disintegration of gel network and opening new free space for growth of cell clusters. These complex phenomena are modeled using the thermodynamical, free energy formalism. The particular form of free energy functional is proposed to describe various kinds of interactions, which affected the dynamics of cell growth and cause pseudo-phase transition of hydrogel. The good agreement of experimentally obtained data and model predictions are obtained. In that way the model provides both, the quantitative tools for further technological optimization of the process and deeper insight into dynamics of cell growth mechanism.

  20. Enhancing cell migration in shape-memory alginate-collagen composite scaffolds: In vitro and ex vivo assessment for intervertebral disc repair.

    PubMed

    Guillaume, Olivier; Naqvi, Syeda Masooma; Lennon, Kerri; Buckley, Conor Timothy

    2015-04-01

    Lower lumbar disc disorders pose a significant problem in an aging society with substantial socioeconomic consequences. Both inner tissue (nucleus pulposus) and outer tissue (annulus fibrosus) of the intervertebral disc are affected by such debilitating disorders and can lead to disc herniation and lower back pain. In this study, we developed an alginate-collagen composite porous scaffold with shape-memory properties to fill defects occurring in annulus fibrosus tissue of degenerated intervertebral discs, which has the potential to be administered using minimal invasive surgery. In the first part of this work, we assessed how collagen incorporation on preformed alginate scaffolds influences the physical properties of the final composite scaffold. We also evaluated the ability of annulus fibrosus cells to attach, migrate, and proliferate on the composite alginate-collagen scaffolds compared to control scaffolds (alginate only). In vitro experiments, performed in intervertebral disc-like microenvironmental conditions (low glucose and low oxygen concentrations), revealed that for alginate only scaffolds, annulus fibrosus cells agglomerated in clusters with limited infiltration and migration capacity. In comparison, for alginate-collagen scaffolds, annulus fibrosus cells readily attached and colonized constructs, while preserving their typical fibroblastic-like cell morphology with spreading behavior and intense cytoskeleton expression. In a second part of this study, we investigated the effects of alginate-collagen scaffold when seeded with bone marrow derived mesenchymal stem cells. In vitro, we observed that alginate-collagen porous scaffolds supported cell proliferation and extracellular matrix deposition (collagen type I), with secretion amplified by the local release of transforming growth factor-β3. In addition, when cultured in ex vivo organ defect model, alginate-collagen scaffolds maintained viability of transplanted mesenchymal stem cells for up to 5

  1. Growth and immobilization of tripterygium wilfordii cultured cells.

    PubMed

    Pépin, M F; Chavarie, C; Archambault, J

    1991-12-20

    The plant Tripterygium wilfordii produces di- and triterpenes of interest for male contraception and treatment of arthritis and skin disorders. Cell line TRP4a obtained form this plant in 1981 was reported to produce these valuable compounds at yields ( approximately 0.04% of the biomass dry weight) higher than found in the plant (0.001%). In order to improve this production, studies were carried out to determine the feasibility of eliminating the troublesome component of coconut milk originally used to culture this cell line. A defined formulation suitable for growth ad maintenance has been developed. This medium consisted of Gamborg's PRL4 or B5 medium supplemented with 2 mg L(-1) 2,4-dichlorophenoxyacetic acid and 20 g L(-1) sucrose. Furthermore, monitoring of carbohydrate uptake revealed that T. wilfordii cells, contrary to many plant cell species, did not hydrolyze sucrose extra-cellularly before uptake. Replacement of this disaccharide by glucose or fructose increased specific growth rate from 0.15 to 0.25 day(-1). As tripdiolide is reported to be present in broth extract in significant amounts, plant cell immobilization technology offers a promising alternative to suspension cultures, especially in view to on line harvesting of the product. Surface immobilized T. wilfordii cell cultures were successfully carried out in 2-L bioreactors. Their biomass production and carbohydrate uptake were comparable to those observed for shake flask grown suspension cultures. Higher nitrate and ammonium uptake were found in immobilized cultures. PMID:18600729

  2. The study of MCF-7 cell aggregates encapsulated in alginate microbeads under the modeled weightlessness

    NASA Astrophysics Data System (ADS)

    Li, Yu; Zheng, Hongxia; Tian, Weiming; Yu, Lei; Zhang, Yao; Han, Fengtong

    Assessing the risks associated with exposure to the microgravity encountered in space is essential for the success of long-term space exploration. For understanding of the effects of microgravity, either traditional 2-dimensional (2D) cell cultures of adherent cell populations or animal models were typically used. The 2D in vitro systems do not allow assessment of the dynamic effects of intercellular interactions within tissues, whereas potentially confounding factors tend to be overlooked in animal models. Therefore novel cell culture model representative of the cellular interactions and with extracellular matrix present in tissues needs to be used. Toward this overall goal, 3D cell aggregates of breast cancer cell line MCF-7 was constructed by encapsu-lating in alginate microbeads. With this model we studied the simulated microgravity effects on MCF-7 by incubating the microbeads in NASA rotary cell culture system with a rate of 15rpm.We found MCF-7 showed higher proliferation rate than that of control group character-ized by up regulation of Ki-67. Further study showed that the effect was achieved by activating the MAPK intercellular signaling pathway. The result is inconsistent with previous conclusion obtained with 2D cell culture model which indicated that 3D cell culture model maybe better for elucidating the microgravity effects.

  3. New biosensor for detection of copper ions in water based on immobilized genetically modified yeast cells.

    PubMed

    Vopálenská, Irena; Váchová, Libuše; Palková, Zdena

    2015-10-15

    Contamination of water by heavy metals represents a potential risk for both aquatic and terrestrial organisms, including humans. Heavy metals in water resources can come from various industrial activities, and drinking water can be ex-post contaminated by heavy metals such as Cu(2+) from house fittings (e.g., water reservoirs) and pipes. Here, we present a new copper biosensor capable of detecting copper ions at concentrations of 1-100 μM. This biosensor is based on cells of a specifically modified Saccharomyces cerevisiae strain immobilized in alginate beads. Depending on the concentration of copper, the biosensor beads change color from white, when copper is present in concentrations below the detection limit, to pink or red based on the increase in copper concentration. The biosensor was successfully tested in the determination of copper concentrations in real samples of water contaminated with copper ions. In contrast to analytical methods or other biosensors based on fluorescent proteins, the newly designed biosensor does not require specific equipment and allows the quick detection of copper in many parallel samples.

  4. New biosensor for detection of copper ions in water based on immobilized genetically modified yeast cells.

    PubMed

    Vopálenská, Irena; Váchová, Libuše; Palková, Zdena

    2015-10-15

    Contamination of water by heavy metals represents a potential risk for both aquatic and terrestrial organisms, including humans. Heavy metals in water resources can come from various industrial activities, and drinking water can be ex-post contaminated by heavy metals such as Cu(2+) from house fittings (e.g., water reservoirs) and pipes. Here, we present a new copper biosensor capable of detecting copper ions at concentrations of 1-100 μM. This biosensor is based on cells of a specifically modified Saccharomyces cerevisiae strain immobilized in alginate beads. Depending on the concentration of copper, the biosensor beads change color from white, when copper is present in concentrations below the detection limit, to pink or red based on the increase in copper concentration. The biosensor was successfully tested in the determination of copper concentrations in real samples of water contaminated with copper ions. In contrast to analytical methods or other biosensors based on fluorescent proteins, the newly designed biosensor does not require specific equipment and allows the quick detection of copper in many parallel samples. PMID:25982723

  5. Effect of Alginate Concentration on Alginate-TiO2 Hydrogel for Lead Ion Removal

    NASA Astrophysics Data System (ADS)

    Teoh, W. T.; Saito, N.; Sato, K.

    2011-03-01

    Alginate-TiO2 hydrogel was investigated for lead ion (Pb(II)) removal. By immobilizing TiO2 powder onto an alginate biopolymer, it is possible to utilize the ion exchange properties of the alginate and the photoreducibility of TiO2 to recover Pb(II). However, these photocatalytic activities degrade the alginate biopolymer in addition to removing Pb(II). This study examines photolytic degradation of alginate-TiO2 hydrogels prepared with alginate concentrations of 1, 1.5, 2, and 2.5%w/v; the same amount (0.4%w/v) of TiO2 was added to each alginate solution. The alginate-TiO2 hydrogels were formed by dripping the alginate-TiO2 suspension into a 0.2 M calcium chloride solution. The samples were washed and dried and then photoirradiated. The samples with alginate concentrations of 1 and 1.5%w/v were depolymerized, whereas the surface morphology of the sample that prepared from the 2%w/v alginate solution remained unchanged. The samples prepared from 1.5, 2, and 2.5%w/v alginate solutions had Pb(II) uptakes of 24.0, 39.8, and 39.7 mg/g, respectively.

  6. Biodegradable photo-crosslinked alginate nanofibre scaffolds with tuneable physical properties, cell adhesivity and growth factor release.

    PubMed

    Jeong, Sung In; Jeon, Oju; Krebs, Melissa D; Hill, Michael C; Alsberg, Eben

    2012-01-01

    Nanofibrous scaffolds are of interest in tissue engineering due to their high surface area to volume ratio, interconnected pores, and architectural similarity to the native extracellular matrix. Our laboratory recently developed a biodegradable, photo-crosslinkable alginate biopolymer. Here, we show the capacity of the material to be electrospun into a nanofibrous matrix, and the ability to enhance cell adhesion and proliferation on these matrices by covalent modification with cell adhesion peptides. Additionally, the potential of covalently incorporating heparin into the hydrogels during the photopolymerisation process to sustain the release of a heparin binding growth factor via affinity interactions was demonstrated. Electrospun photo-crosslinkable alginate nanofibrous scaffolds endowed with cell adhesion ligands and controlled delivery of growth factors may allow for improved regulation of cell behaviour for regenerative medicine. PMID:23070945

  7. BIODEGRADABLE PHOTO-CROSSLINKED ALGINATE NANOFIBRE SCAFFOLDS WITH TUNEABLE PHYSICAL PROPERTIES, CELL ADHESIVITY AND GROWTH FACTOR RELEASE

    PubMed Central

    Jeong, Sung In; Jeon, Oju; Krebs, Melissa D.; Hill, Michael C.; Alsberg, Eben

    2012-01-01

    Nanofibrous scaffolds are of interest in tissue engineering due to their high surface area to volume ratio, interconnected pores, and architectural similarity to the native extracellular matrix. Our laboratory recently developed a biodegradable, photo-crosslinkable alginate biopolymer. Here, we show the capacity of the material to be electrospun into a nanofibrous matrix, and the ability to enhance cell adhesion and proliferation on these matrices by covalent modification with cell adhesion peptides. Additionally, the potential of covalently incorporating heparin into the hydrogels during the photopolymerisation process to sustain the release of a heparin binding growth factor via affinity interactions was demonstrated. Electrospun photo-crosslinkable alginate nanofibrous scaffolds endowed with cell adhesion ligands and controlled delivery of growth factors may allow for improved regulation of cell behaviour for regenerative medicine. PMID:23070945

  8. Continuous glutamate production using an immobilized whole-cell system

    SciTech Connect

    Kim, H.S.; Ryu, D.D.Y.

    1982-10-01

    For the purpose of saving the energy and raw materials required in a glutamate fermentation, an immobilized whole-cell system was prepared and its performance in a continuous reactor system was evaluated. Corynebacterium glutamicum (a mutant strain of ATCC 13058) whole cell was immobilized in k-carrageenan matrix and the gel structure was strengthened by treatment with a hardening agent. The effective diffusivities of carrageenan gel for glucose and oxygen were formed to decrease significantly with an increase in carrageenan concentration, while the gel strength showed an increasing trend. Based on the physical and chemical properties of carrageenan gel, the immobilized method was improved and the operation of the continuous reactor system was partially optimized. In an air-stirred fermentor, the continuous production of glutamate was carried out. The effect of the dilution rate of glutamate production and operation stability was investigated. The performance of the continuous wbole-cell reactor system was evaluated by measuring glutamate productivity for a period of 30 days; it was found to be far superior to the performance of convention batch reactor systems using free cells.

  9. Simulation and analysis of immobilized-cell fermentors

    SciTech Connect

    Heyman, E.C.; Nguyen, X.N.

    1982-06-01

    Immobilized cell bioreactors are important for developing high-productivity fermentation processes. A computer simulation program has been devised to aid in the design of three basic fermentor types including batch, tubular, and continuous stirred tank and the analysis of important process variables. Parametric studies on the sensitivity of fermentor residence time for the glucose to ethanol yeast fermentation system with changes kinetic constants, film/pore diffusion limitations, gel support characteristics and recycle of product flow are discussed.

  10. Conformal coating of mammalian cells immobilized onto magnetically driven beads.

    PubMed

    Khademhosseini, Ali; May, Michael H; Sefton, Michael V

    2005-01-01

    A novel cell bead system, comprising a magnetic core, a spherical annulus of agarose-immobilized cells, all conformally coated within a synthetic polymer, is proposed as a means of immunoisolating mammalian cells in a system that provides a balance between low total implant volume, retrievability, and diffusion limitations. A successful immunoisolation system could be used to transplant cells without eliciting an inappropriate host response. Chinese hamster ovary (CHO) cells were immobilized at the periphery of large (approximately 2 mm) agarose beads containing inert magnetic cores (< or = 1 mm) and coated in a hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA) copolymer by interfacial precipitation. The beads were coated in liquid gradients containing polyethylene glycol 200 (PEG) or bromooctane. Although many cells were adversely affected by the coating process, the cells that did survive (30-50% of those loaded into the beads) remained viable for a period of at least 2 weeks. This viability was much higher than achieved previously because of a number of factors, such as the aqueous agarose, the hydrophobic bromooctane intermediate layer, and faster coating times that minimize the exposure of the cells to organic solvents. Also, a mathematical model was used to describe oxygen transport within the annular agarose beads. These results provide evidence that the proposed geometry and the fabrication approach may be useful for a variety of applications that involve cell encapsulation.

  11. Immobilization of microbial cell and yeast cell and its application to biomass conversion using radiation techniques

    NASA Astrophysics Data System (ADS)

    Kaetsu, Isao; Kumakura, Minoru; Fujimura, Takashi; Kasai, Noboru; Tamada, Masao

    The recent results of immobilization of cellulase-producing cells and ethanol-fermentation yeast by radiation were reported. The enzyme of cellulase produced by immobilized cells was used for saccharification of lignocellulosic wastes and immobilized yeast cells were used for fermentation reaction from glucose to ethanol. The wastes such as chaff and bagasse were treated by γ-ray or electron-beam irradiation in the presence of alkali and subsequent mechanical crushing, to form a fine powder less than 50 μm in diameter. On the other hand, Trichoderma reesei as a cellulase-producing microbial cell was immobilized on a fibrous carrier having a specific porous structure and cultured to produce cellulase. The enzymatic saccharification of the pretreated waste was carried out using the produced cellulase. The enhanced fermentation process to produce ethanol from glucose with the immobilized yeast by radiation was also studied. The ethanol productivity of immobilized growing yeast cells thus obtained was thirteen times that of free yeast cells in a 1:1 volume of liquid medium to immobilized yeast cells.

  12. Cephalosporin C production by immobilized Cephalosporium acremonium cells in a repeated batch tower bioreactor.

    PubMed

    Cruz, Antonio J G; Pan, Tai; Giordano, Roberto C; Araujo, Maria Lucia G C; Hokka, Carlos O

    2004-01-01

    The industrial production of antibiotics with filamentous fungi is usually carried out in conventional aerated and agitated tank fermentors. Highly viscous non-Newtonian broths are produced and a compromise must be found between convenient shear stress and adequate oxygen transfer. In this work, cephalosporin C production by bioparticles of immobilized cells of Cephalosporium acremonium ATCC 48272 was studied in a repeated batch tower bioreactor as an alternative to the conventional process. Also, gas-liquid oxygen transfer volumetric coefficients, k(L)a, were determined at various air flow-rates and alumina contents in the bioparticle. The bioparticles were composed of calcium alginate (2.0% w/w), alumina ( < 44 micra), cells, and water. A model describing the cell growth, cephalosporin C production, oxygen, glucose, and sucrose consumption was proposed. To describe the radial variation of oxygen concentration within the pellet, the reaction-diffusion model forecasting a dead core bioparticle was adopted. The k(L)a measurements with gel beads prepared with 0.0, 1.0, 1.5, and 2.0% alumina showed that a higher k(L)a value is attained with 1.5 and 2.0%. An expression relating this coefficient to particle density, liquid density, and air velocity was obtained and further utilized in the simulation of the proposed model. Batch, followed by repeated batch experiments, were accomplished by draining the spent medium, washing with saline solution, and pouring fresh medium into the bioreactor. Results showed that glucose is consumed very quickly, within 24 h, followed by sucrose consumption and cephalosporin C production. Higher productivities were attained during the second batch, as cell concentration was already high, resulting in rapid glucose consumption and an early derepression of cephalosporin C synthesizing enzymes. The model incorporated this improvement predicting higher cephalosporin C productivity.

  13. Influence of hydrophobic modification in alginate-based hydrogels for biomedical applications

    NASA Astrophysics Data System (ADS)

    Choudhary, Soumitra

    Alginate has been exploited commercially for decades in foods, textiles, paper, pharmaceutical industries, and also as a detoxifier for removing heavy metals. Alginate is also popular in cell encapsulation because of its relatively mild gelation protocol and simple chemistry with which biological active entities can be immobilized. Surface modification of alginate gels has been explored to induce desired cell interactions with the gel matrix. These modifications alter the bulk properties, which strongly determine on how cells feel and response to the three-dimensional microenvironment. However, there is a need to develop strategies to engineer functionalities into bulk alginate hydrogels that not only preserve their inherent qualities but are also less toxic. In this thesis, our main focus was to optimize the mechanical properties of alginate-based hydrogels, and by doing so control the performance of the biomaterials. In the first scheme, we used alginate and hydrophobically modified ethyl hydroxy ethyl cellulose as components in interpenetrating polymer network (IPN) gels. The second network was used to control gelation time and rheological properties. We believe these experiments also may provide insight into the mechanical and structural properties of more complex biopolymer gels and naturally-occurring IPNs. Next, we worked on incorporating a hydrophobic moiety directly into the alginate chain, resulting in materials for extended release of hydrophobic drugs. We successfully synthesized hydrophobically modified alginate (HMA) by attaching octylamine groups onto the alginate backbone by standard carbodiimide based amide coupling reaction. Solubility of several model hydrophobic drugs in dilute HMA solutions was found to be increased by more than an order of magnitude. HMA hydrogels, prepared by crosslinking the alginate chains with calcium ions, were found to exhibit excellent mechanical properties (modulus ˜100 kPa) with release extended upto 5 days. Ability

  14. An understanding of potential and limitations of alginate/PLL microcapsules as a cell retention system for perfusion cultures.

    PubMed

    Demont, Aurelie; Cole, Harriet; Marison, Ian W

    2016-02-01

    Microcapsules for high cell density culture of mammalian cells have found an increasing interest, however, the poor stability of the microcapsules and the lack of characterisation methods led to few quantitative results. Alginate-poly-L-lysine (PLL) microcapsules have been studied in detail in order to form a basis for comparison of capsules made from different polymers. Since the microcapsules can be easily retained in the bioreactor without the need for a cell separation device, high cell densities were achieved with a maximum of 4 × 10(7) cell/ml(microcapsules), corresponding to a colonisation of 5% of the internal capsule volume. Measurement of microcapsule integrity and mechanical resistance showed that alginate-PLL microcapsules are not suitable for perfusion cultures since they are very sensitive to media composition, mainly the presence of non-gelling ions that have a higher affinity for alginate than PLL and Ca(2+), leading to the leakage of PLL and Ca(2+), and to microcapsule rupture.

  15. Human hepatoma cell lines on gas foaming templated alginate scaffolds for in vitro drug-drug interaction and metabolism studies.

    PubMed

    Stampella, A; Rizzitelli, G; Donati, F; Mazzarino, M; de la Torre, X; Botrè, F; Giardi, M F; Dentini, M; Barbetta, A; Massimi, M

    2015-12-25

    Liver in vitro systems that allow reliable prediction of major human in vivo metabolic pathways have a significant impact in drug screening and drug metabolism research. In the present study, a novel porous scaffold composed of alginate was prepared by employing a gas-in-liquid foaming approach. Galactose residues were introduced on scaffold surfaces to promote cell adhesion and to enhance liver specific functions of the entrapped HepG2/C3A cells. Hepatoma cells in the gal-alginate scaffold showed higher levels of liver specific products (albumin and urea) and were more responsive to specific inducers (e.g. dexamethasone) and inhibitors (e.g. ketoconazole) of the CYP3A4 system than in conventional monolayer culture. HepG2/C3A cells were also more efficient in terms of rapid elimination of testosterone, used as a model substance, at rates comparable to those of in vivo excretion. In addition, an improvement in metabolism of testosterone, in terms of phase II metabolite formation, was also observed when the more differentiated HepaRG cells were used. Together the data suggest that hepatocyte/gas templated alginate-systems provide an innovative high throughput platform for in vitro drug metabolism and drug-drug interaction studies, with broad fields of application, and might provide a valid tool for minimizing animal use in preclinical testing of human relevance.

  16. Design and Fabrication of a Biodegradable, Covalently Crosslinked Shape-Memory Alginate Scaffold for Cell and Growth Factor Delivery

    PubMed Central

    Wang, Lin; Shansky, Janet; Borselli, Cristina; Mooney, David

    2012-01-01

    The successful use of transplanted cells and/or growth factors for tissue repair is limited by a significant cell loss and/or rapid growth factor diffusion soon after implantation. Highly porous alginate scaffolds formed with covalent crosslinking have been used to improve cell survival and growth factor release kinetics, but require open-wound surgical procedures for insertion and have not previously been designed to readily degrade in vivo. In this study, a biodegradable, partially crosslinked alginate scaffold with shape-memory properties was fabricated for minimally invasive surgical applications. A mixture of high and low molecular weight partially oxidized alginate modified with RGD peptides was covalently crosslinked using carbodiimide chemistry. The scaffold was compressible 11-fold and returned to its original shape when rehydrated. Scaffold degradation properties in vitro indicated ∼85% mass loss by 28 days. The greater than 90% porous scaffolds released the recombinant growth factor insulin-like growth factor-1 over several days in vitro and allowed skeletal muscle cell survival, proliferation, and migration from the scaffold over a 28-day period. The compressible scaffold thus has the potential to be delivered by a minimally invasive technique, and when rehydrated in vivo with cells and/or growth factors, could serve as a temporary delivery vehicle for tissue repair. PMID:22646518

  17. Preparation and characterization of immobilized [A336][MTBA] in PVA-alginate gel beads as novel solid-phase extractants for an efficient recovery of Hg (II) from aqueous solutions.

    PubMed

    Zhang, Yun; Kogelnig, Daniel; Morgenbesser, Cornelia; Stojanovic, Anja; Jirsa, Franz; Lichtscheidl-Schultz, Irene; Krachler, Regina; Li, Yanfeng; Keppler, Bernhard K

    2011-11-30

    The coarse PVA-alginate matrix gel beads entrapping the micro-droplets of the ionic liquid tricaprylylmethylammonium 2-(methylthio) benzoate ([A336][MTBA]) as novel solid-phase extractants were prepared for the removal of mercury (II) from aqueous media. The ionic liquid [A336][MTBA] immobilized PVA-alginate beads (PVA/IL) have been characterized by FTIR, SEM and TGA. The influence of the uptake conditions was investigated including aqueous pH, PVA/IL dosage, the content of [A336][MTBA] and initial Hg (II) concentration; maximum Hg (II) ion adsorption capacity obtained was 49.89 (± 0.11)mgg(-1) at pH 5.8 with adsorptive removal of approximately 99.98%. The selectivity of the PVA/IL beads towards Hg (II), Pb (II) and Cu (II) ions tested was Hg>Pb>Cu. The rate kinetic study was found to follow second-order and the applicability of Langmuir, Freundlich and Tempkin adsorption isotherm model were tested as well. The results of the study showed that PVA/IL beads could be efficiently used as novel extractants for the removal of divalent mercury from aqueous solutions under comparatively easy operation conditions.

  18. Degradation of mix hydrocarbons by immobilized cells of mix culture using a trickle fluidized bed reactor. Final report: June 1992--June 1994

    SciTech Connect

    Chapatwala, K.D.

    1994-12-01

    The microorganisms capable of degrading mix hydrocarbons were isolated from the soil samples collected from the hydrocarbon contaminated sites. The mix cultures were identified as Pseudomonas acidovorans, Flavobacterium indoltheticum and Phyllobacterium rubiaceum. The bacterial cells of mix cultures were immobilized in calcium-alginate solution in the form of beads. A trickle fluidized bed air-uplift-type reactor designed to study the degradation of mix hydrocarbons was filled with 0.85% normal saline containing the immobilized cells of mix culture. The immobilized beads were aerated with different amounts of CO{sub 2}-free air. The normal saline saturated with BTXs was circulated in the bioreactors at the rate of 2--4 ml/min. The biodegradation of BTXs by the immobilized beads of mix culture was monitored by determining the concentrations of the BTXs and the metabolites formed during their degradation in the samples at regular intervals using GC. The peaks obtained through the degradation of BTXs were not identified and quantified in this study.

  19. Interaction between extracellular lipase LipA and the polysaccharide alginate of Pseudomonas aeruginosa

    PubMed Central

    2013-01-01

    Background As an opportunistic human pathogen Pseudomonas aeruginosa is able to cause acute and chronic infections. The biofilm mode of life significantly contributes to the growth and persistence of P. aeruginosa during an infection process and mediates the pathogenicity of the bacterium. Within a biofilm mucoid strains of P. aeruginosa simultaneously produce and secrete several hydrolytic enzymes and the extracellular polysaccharide alginate. The focus of the current study was the interaction between extracellular lipase LipA and alginate, which may be physiologically relevant in biofilms of mucoid P. aeruginosa. Results Fluorescence microscopy of mucoid P. aeruginosa biofilms were performed using fluorogenic lipase substrates. It showed a localization of the extracellular enzyme near the cells. A microtiter plate-based binding assay revealed that the polyanion alginate is able to bind LipA. A molecular modeling approach showed that this binding is structurally based on electrostatic interactions between negatively charged residues of alginate and positively charged amino acids of the protein localized opposite of the catalytic centre. Moreover, we showed that the presence of alginate protected the lipase activity by protection from heat inactivation and from degradation by the endogenous, extracellular protease elastase LasB. This effect was influenced by the chemical properties of the alginate molecules and was enhanced by the presence of O-acetyl groups in the alginate chain. Conclusion We demonstrate that the extracellular lipase LipA from P. aeruginosa interacts with the polysaccharide alginate in the self-produced extracellular biofilm matrix of P. aeruginosa via electrostatic interactions suggesting a role of this interaction for enzyme immobilization and accumulation within biofilms. This represents a physiological advantage for the cells. Especially in the biofilm lifestyle, the enzyme is retained near the cell surface, with the catalytic centre exposed

  20. Ultra-structural changes and expression of chondrogenic and hypertrophic genes during chondrogenic differentiation of mesenchymal stromal cells in alginate beads

    PubMed Central

    Dashtdar, Havva; Selvaratnam, Lakshmi; Balaji Raghavendran, Hanumantharao; Suhaeb, Abdulrazzaq Mahmod; Ahmad, Tunku Sara

    2016-01-01

    Chondrogenic differentiation of mesenchymal stromal cells (MSCs) in the form of pellet culture and encapsulation in alginate beads has been widely used as conventional model for in vitro chondrogenesis. However, comparative characterization between differentiation, hypertrophic markers, cell adhesion molecule and ultrastructural changes during alginate and pellet culture has not been described. Hence, the present study was conducted comparing MSCs cultured in pellet and alginate beads with monolayer culture. qPCR was performed to assess the expression of chondrogenic, hypertrophic, and cell adhesion molecule genes, whereas transmission electron microscopy (TEM) was used to assess the ultrastructural changes. In addition, immunocytochemistry for Collagen type II and aggrecan and glycosaminoglycan (GAG) analysis were performed. Our results indicate that pellet and alginate bead cultures were necessary for chondrogenic differentiation of MSC. It also indicates that cultures using alginate bead demonstrated significantly higher (p < 0.05) chondrogenic but lower hypertrophic (p < 0.05) gene expressions as compared with pellet cultures. N-cadherin and N-CAM1 expression were up-regulated in second and third weeks of culture and were comparable between the alginate bead and pellet culture groups, respectively. TEM images demonstrated ultrastructural changes resembling cell death in pellet cultures. Our results indicate that using alginate beads, MSCs express higher chondrogenic but lower hypertrophic gene expression. Enhanced production of extracellular matrix and cell adhesion molecules was also observed in this group. These findings suggest that alginate bead culture may serve as a superior chondrogenic model, whereas pellet culture is more appropriate as a hypertrophic model of chondrogenesis. PMID:26966647

  1. Effect of temperature and pH on ethanol production by free and immobilized cells of Kluyveromyces marxianus grown on Jerusalem artichoke extract

    SciTech Connect

    Bajpai, P.; Margaritis, A.

    1987-01-01

    The effect of temperature and pH on the kinetics of ethanol production by free and calcium alginate immobilized cells of Kluyveromyces marxianus grown on Jerusalem artichoke extract was investigated. With the free cells, the ethanol and biomass yields were relatively constant over the temperature range 25-35 degrees C, but dropped sharply beyond 35 degrees C. Other kinetic parameters, specific growth rate, specific ethanol production rate, and specific total sugar uptake rate were maximum at 35 degrees C. However, with the immobilized cells, ethanol yield remained almost constant in the temperatue range 25-45 degrees C, and the specific ethanol production rate and specific total sugar uptake rate attained their maximum values at 40 degrees C. For the pH range between 3 and 7, the free-cell optimum for growth and product formation was found to be circa pH 5. At this pH, the specific growth rate was 0.35/h and specific ethanol production rate was 2.83 g/g/h. At values higher or lower than pH 5, a sharp decrease in specific ethanol production rate as well as specific growth rate was observed. In comparison, the immobilized cells showed a broad optimum pH profile. The best ethanol production rates were observed between pH 4 and 6. (Refs. 22).

  2. Cell Alignment Driven by Mechanically Induced Collagen Fiber Alignment in Collagen/Alginate Coatings

    PubMed Central

    Chaubaroux, Christophe; Perrin-Schmitt, Fabienne; Senger, Bernard; Vidal, Loïc; Voegel, Jean-Claude; Schaaf, Pierre; Haikel, Youssef; Boulmedais, Fouzia; Lavalle, Philippe

    2015-01-01

    For many years it has been a major challenge to regenerate damaged tissues using synthetic or natural materials. To favor the healing processes after tendon, cornea, muscle, or brain injuries, aligned collagen-based architectures are of utmost interest. In this study, we define a novel aligned coating based on a collagen/alginate (COL/ALG) multilayer film. The coating exhibiting a nanofibrillar structure is cross-linked with genipin for stability in physiological conditions. By stretching COL/ALG-coated polydimethylsiloxane substrates, we developed a versatile method to align the collagen fibrils of the polymeric coating. Assays on cell morphology and alignment were performed to investigate the properties of these films. Microscopic assessments revealed that cells align with the stretched collagen fibrils of the coating. The degree of alignment is tuned by the stretching rate (i.e., the strain) of the COL/ALG-coated elastic substrate. Such coatings are of great interest for strategies that require aligned nanofibrillar biological material as a substrate for tissue engineering. PMID:25658028

  3. Enhanced antiproliferative activity of carboplatin-loaded chitosan-alginate nanoparticles in a retinoblastoma cell line.

    PubMed

    Parveen, Suphiya; Mitra, Moutushy; Krishnakumar, S; Sahoo, Sanjeeb K

    2010-08-01

    In the present study the potential of carboplatin-loaded chitosan-alginate nanoparticles (CANPs) for the treatment of retinoblastoma was investigated. The carboplatin-loaded CANPs were approximately 300 nm in size, exhibited a high zeta potential of approximately 36 mV and drug encapsulation of approximately 20 wt.%. The CANPs were further characterized by Fourier transform infrared spectroscopy, differential scanning calorimetry and transmission electron microscopy. In vitro release studies revealed fast release of approximately 25% of the drug during the first 24h, followed by sustained release. CANPs demonstrated greater and sustained antiproliferative activity of the drug in a dose- and time-dependent manner (carboplatin IC(50)=0.56 microg ml(-1), carboplatin-loaded CANPs IC(50)=0.004 microg ml(-1)), as well as an enhanced apoptotic effect as compared with the drug in solution in a retinoblastoma cell line (Y79). The higher cytotoxic effect of CANPs may be due to their greater cellular uptake as compared with native carboplatin. It was also demonstrated that clathrin-mediated endocytosis plays a key role in the internalization of CANPs in the Y79 cell line. In conclusion, biodegradable chitosan nanoparticles could be used as an effective ocular drug delivery system for sustained intracellular delivery of carboplatin for the treatment of retinoblastoma. PMID:20149903

  4. Effect of sodium-alginate and laminaran on Salmonella Typhimurium infection in human enterocyte-like HT-29-Luc cells and BALB/c mice.

    PubMed

    Kuda, Takashi; Kosaka, Misa; Hirano, Shino; Kawahara, Miho; Sato, Masahiro; Kaneshima, Tai; Nishizawa, Makoto; Takahashi, Hajime; Kimura, Bon

    2015-07-10

    Brown algal polysaccharides such as alginate, polymers of uronic acids, and laminaran, beta-1,3 and 1,6-glucan, can be fermented by human intestinal microbiota. To evaluate the effects of these polysaccharides on infections caused by food poisoning pathogens, we investigated the adhesion and invasion of pathogens (Salmonella Typhimurium, Listeria monocytogenes and Vibrio parahaemolyticus) in human enterocyte-like HT-29-Luc cells and in infections caused in BALB/c mice. Both sodium Na-alginate and laminaran (0.1% each) inhibited the adhesion of the pathogens to HT-29-Luc cells by approximately 70-90%. The invasion of S. Typhimurium was also inhibited by approximately 70 and 80% by Na-alginate and laminaran, respectively. We observed that incubation with Na-alginate for 18 h increased the transepithelial electrical resistance of HT-29-Luc monolayer cells. Four days after inoculation with 7 log CFU/mouse of S. Typhimurium, the faecal pathogen count in mice that were not fed polysaccharides (control mice) was about 6.5 log CFU/g while the count in mice that were fed Na-alginate had decreased to 5.0 log CFU/g. The liver pathogen count, which was 4.1 log CFU/g in the control mice, was also decreased in mice that were fed Na-alginate. In contrast, the mice that were fed laminaran exhibited a more severe infection than that exhibited by control mice.

  5. Encapsulated human hepatocellular carcinoma cells by alginate gel beads as an in vitro metastasis model

    SciTech Connect

    Xu, Xiao-xi; Liu, Chang; Liu, Yang; Li, Nan; Guo, Xin; Wang, Shu-jun; Sun, Guang-wei; Wang, Wei; Ma, Xiao-jun

    2013-08-15

    Hepatocellular carcinoma (HCC) is the most common primary liver cancer and often forms metastases, which are the most important prognostic factors. For further elucidation of the mechanism underlying the progression and metastasis of HCC, a culture system mimicking the in vivo tumor microenvironment is needed. In this study, we investigated the metastatic ability of HCC cells cultured within alginate gel (ALG) beads. In the culture system, HCC cells formed spheroids by proliferation and maintained in nuclear abnormalities. The gene and protein expression of metastasis-related molecules was increased in ALG beads, compared with the traditional adhesion culture. Furthermore, several gene expression levels in ALG bead culture system were even closer to liver cancer tissues. More importantly, in vitro invasion assay showed that the invasion cells derived from ALG beads was 7.8-fold higher than adhesion cells. Our results indicated that the in vitro three-dimensional (3D) model based on ALG beads increased metastatic ability compared with adhesion culture, even partly mimicked the in vivo tumor tissues. Moreover, due to the controllable preparation conditions, steady characteristics and production at large-scale, the 3D ALG bead model would become an important tool used in the high-throughput screening of anti-metastasis drugs and the metastatic mechanism research. -- Highlights: •We established a 3D metastasis model mimicking the metastatic ability in vivo. •The invasion ability of cells derived from our model was increased significantly. •The model is easy to reproduce, convenient to handle, and amenable for large-scale.

  6. Microfabricated glass devices for rapid single cell immobilization in mouse zygote microinjection.

    PubMed

    Liu, Xinyu; Sun, Yu

    2009-12-01

    This paper presents the design and microfabrication of a vacuum-based cell holding device for single-cell immobilization and the use of the device in mouse zygote microinjection. The device contains many through-holes, constructed via two-sided glass wet etching and polydimethylsiloxane (PDMS)-glass bonding. Experimental results of mouse zygote immobilization and microinjection demonstrate that the device is effective for rapid cell immobilization and does not produce negative effect on embryonic development.

  7. Geometrical design of immobilized cell modula units for ethanol fermentation

    SciTech Connect

    Bourassa, J.F.; LeDuy, A.

    1987-06-01

    Formula are developed for calculating the performance characteristics (surface-to-total-volume ratio, surface-to-packing-volume ratio, and void volume fraction) of four different types of immobilized cell modular units (ICMUs) for ethanol fermentation: plate-type, spiral-type, beehive-type and bead-type ICMUs. Examples are used to illustrate how the formulas are useful for investigating the effects of characteristic dimensions of packing geometry, as well as the effect of scale on the performance characteristics of the ICMUs. (Refs. 4).

  8. Geometrical design of immobilized cell modular units for ethanol fermentation.

    PubMed

    Bourassa, J F; Leduy, A

    1987-06-01

    Formula are developed for calculating the performance characteristics (surface-to-total-volume ratio, surface-to-packing-volume ratio, and void volume fraction) of four different types of immobilized cell modular units (ICMUs) for ethanol fermentation: plate-type, spiral-type, beehive-type and bead-type ICMUs. Examples are used to illustrate how the formulas are useful for investigating the effects of characteristic dimensions of packing geometry, as well as the effect of scale on the performance characteristics of the ICMUs. PMID:18576567

  9. Drying of micro-encapsulated lactic acid bacteria — Effects of trehalose and immobilization on cell survival and release properties

    NASA Astrophysics Data System (ADS)

    Li, Xiaoyan; Chen, Xiguang

    2009-03-01

    Lactic acid bacteria (LAB) were encapsulated with alginate, gelatin and trehalose additives by the extrusion method and dried at 4 °C. The microcapsules were generally spherical and had a wrinkled surface with a size of 1.7 mm ± 0.2 mm. Trehalose as a carbohydrate source in the culture medium could reduce acid production and performed no function in the positive proliferation of LAB. Using trehalose as a carbohydrate source and protective medium simultaneously had a benefit in the protection of LAB cells during the storage at 4 °C. The density of live LAB cells could be 107 CFU g-1 after 8 weeks of storage. Cells of LAB could be continuously released from the capsules from the acidic (pH 1.2) to neutral conditions (pH 6.8). The release amounts and proliferation speeds of LAB cells in neutral medium were much larger and faster than those in acidic conditions. Additionally, immobilization of LAB could improve the survival of cells when they were exposed to acidic medium (pH 1.2) with a survival rate of 76 %.

  10. A simple method for encapsulating single cells in alginate microspheres allows for direct PCR and whole genome amplification.

    PubMed

    Bigdeli, Saharnaz; Dettloff, Roger O; Frank, Curtis W; Davis, Ronald W; Crosby, Laurel D

    2015-01-01

    Microdroplets are an effective platform for segregating individual cells and amplifying DNA. However, a key challenge is to recover the contents of individual droplets for downstream analysis. This paper offers a method for embedding cells in alginate microspheres and performing multiple serial operations on the isolated cells. Rhodobacter sphaeroides cells were diluted in alginate polymer and sprayed into microdroplets using a fingertip aerosol sprayer. The encapsulated cells were lysed and subjected either to conventional PCR, or whole genome amplification using either multiple displacement amplification (MDA) or a two-step PCR protocol. Microscopic examination after PCR showed that the lumen of the occupied microspheres contained fluorescently stained DNA product, but multiple displacement amplification with phi29 produced only a small number of polymerase colonies. The 2-step WGA protocol was successful in generating fluorescent material, and quantitative PCR from DNA extracted from aliquots of microspheres suggested that the copy number inside the microspheres was amplified up to 3 orders of magnitude. Microspheres containing fluorescent material were sorted by a dilution series and screened with a fluorescent plate reader to identify single microspheres. The DNA was extracted from individual isolates, re-amplified with full-length sequencing adapters, and then a single isolate was sequenced using the Illumina MiSeq platform. After filtering the reads, the only sequences that collectively matched a genome in the NCBI nucleotide database belonged to R. sphaeroides. This demonstrated that sequencing-ready DNA could be generated from the contents of a single microsphere without culturing. However, the 2-step WGA strategy showed limitations in terms of low genome coverage and an uneven frequency distribution of reads across the genome. This paper offers a simple method for embedding cells in alginate microspheres and performing PCR on isolated cells in common bulk

  11. A simple method for encapsulating single cells in alginate microspheres allows for direct PCR and whole genome amplification.

    PubMed

    Bigdeli, Saharnaz; Dettloff, Roger O; Frank, Curtis W; Davis, Ronald W; Crosby, Laurel D

    2015-01-01

    Microdroplets are an effective platform for segregating individual cells and amplifying DNA. However, a key challenge is to recover the contents of individual droplets for downstream analysis. This paper offers a method for embedding cells in alginate microspheres and performing multiple serial operations on the isolated cells. Rhodobacter sphaeroides cells were diluted in alginate polymer and sprayed into microdroplets using a fingertip aerosol sprayer. The encapsulated cells were lysed and subjected either to conventional PCR, or whole genome amplification using either multiple displacement amplification (MDA) or a two-step PCR protocol. Microscopic examination after PCR showed that the lumen of the occupied microspheres contained fluorescently stained DNA product, but multiple displacement amplification with phi29 produced only a small number of polymerase colonies. The 2-step WGA protocol was successful in generating fluorescent material, and quantitative PCR from DNA extracted from aliquots of microspheres suggested that the copy number inside the microspheres was amplified up to 3 orders of magnitude. Microspheres containing fluorescent material were sorted by a dilution series and screened with a fluorescent plate reader to identify single microspheres. The DNA was extracted from individual isolates, re-amplified with full-length sequencing adapters, and then a single isolate was sequenced using the Illumina MiSeq platform. After filtering the reads, the only sequences that collectively matched a genome in the NCBI nucleotide database belonged to R. sphaeroides. This demonstrated that sequencing-ready DNA could be generated from the contents of a single microsphere without culturing. However, the 2-step WGA strategy showed limitations in terms of low genome coverage and an uneven frequency distribution of reads across the genome. This paper offers a simple method for embedding cells in alginate microspheres and performing PCR on isolated cells in common bulk

  12. A Simple Method for Encapsulating Single Cells in Alginate Microspheres Allows for Direct PCR and Whole Genome Amplification

    PubMed Central

    Bigdeli, Saharnaz; Dettloff, Roger O.; Frank, Curtis W.; Davis, Ronald W.; Crosby, Laurel D.

    2015-01-01

    Microdroplets are an effective platform for segregating individual cells and amplifying DNA. However, a key challenge is to recover the contents of individual droplets for downstream analysis. This paper offers a method for embedding cells in alginate microspheres and performing multiple serial operations on the isolated cells. Rhodobacter sphaeroides cells were diluted in alginate polymer and sprayed into microdroplets using a fingertip aerosol sprayer. The encapsulated cells were lysed and subjected either to conventional PCR, or whole genome amplification using either multiple displacement amplification (MDA) or a two-step PCR protocol. Microscopic examination after PCR showed that the lumen of the occupied microspheres contained fluorescently stained DNA product, but multiple displacement amplification with phi29 produced only a small number of polymerase colonies. The 2-step WGA protocol was successful in generating fluorescent material, and quantitative PCR from DNA extracted from aliquots of microspheres suggested that the copy number inside the microspheres was amplified up to 3 orders of magnitude. Microspheres containing fluorescent material were sorted by a dilution series and screened with a fluorescent plate reader to identify single microspheres. The DNA was extracted from individual isolates, re-amplified with full-length sequencing adapters, and then a single isolate was sequenced using the Illumina MiSeq platform. After filtering the reads, the only sequences that collectively matched a genome in the NCBI nucleotide database belonged to R. sphaeroides. This demonstrated that sequencing-ready DNA could be generated from the contents of a single microsphere without culturing. However, the 2-step WGA strategy showed limitations in terms of low genome coverage and an uneven frequency distribution of reads across the genome. This paper offers a simple method for embedding cells in alginate microspheres and performing PCR on isolated cells in common bulk

  13. Metal organic frameworks for enzyme immobilization in biofuel cells

    NASA Astrophysics Data System (ADS)

    Bodell, JaDee

    Interest in biofuel cells has been rapidly expanding as an ever-growing segment of the population gains access to electronic devices. The largest areas of growth for new populations using electronic devices are often in communities without electrical infrastructure. This lack of infrastructure in remote environments is one of the key driving factors behind the development of biofuel cells. Biofuel cells employ biological catalysts such as enzymes to catalyze oxidation and reduction reactions of select fuels to generate power. There are several benefits to using enzymes to catalyze reactions as compared to traditional fuel cells which use metal catalysts. First, enzymes are able to catalyze reactions at or near room temperature, whereas traditional metal catalysts are only efficient at very high temperatures. Second, biofuel cells can operate under mild pH conditions which is important for the eventual design of safe, commercially viable devices. Also, biofuel cells allow for implantable and flexible technologies. Finally, enzymes exhibit high selectivity and can be combined to fully oxidize or reduce the fuel which can generate several electrons from a single molecule of fuel, increasing the overall device efficiency. One of the main challenges which persist in biofuel cells is the instability of enzymes over time which tend to denature after hours or days. For a viable commercial biofuel cell to be produced, the stability of enzymes must be extended to months or years. Enzymes have been shown to have improved stability after being immobilized. The focus of this research was to find a metal organic framework (MOF) structure which could successfully immobilize enzymes while still allowing for electron transport to occur between the catalytic center of the enzyme and the electrode surface within a biofuel cell for power generation. Four MOF structures were successfully synthesized and were subsequently tested to determine the MOF's ability to immobilize the following

  14. The promotion of in vitro vessel-like organization of endothelial cells in magnetically responsive alginate scaffolds

    PubMed Central

    Sapir, Yulia; Cohen, Smadar; Friedman, Gary; Polyak, Boris

    2012-01-01

    One of the major challenges in engineering thick, complex tissues such as cardiac muscle, is the need to pre-vascularize the engineered tissue in vitro to enable its efficient integration with host tissue upon implantation. Herein, we explored new magnetic alginate composite scaffolds to provide means of physical stimulation to cells. Magnetite-impregnated alginate scaffolds seeded with aortic endothelial cells stimulated during the first 7 days out of a total 14 day experimental course showed significantly elevated metabolic activity during the stimulation period. Expression of proliferating cell nuclear antigen (PCNA) indicated that magnetically stimulated cells had a lower proliferation index as compared to the non-stimulated cells. This suggests that the elevated metabolic activity could instead be related to cell migration and re-organization. Immunostaining and confocal microscopy analyses supported this observation showing that on day 14 in magnetically stimulated scaffolds without supplementation of any growth factors, cellular vessel-like (loop) structures, known as indicators of vasculogenesis and angiogenesis were formed as compared to cell sheets or aggregates observed in the non-stimulated (control) scaffolds. This work is the first step in our understanding of how to accurately control cellular organization to form tissue engineered constructs, which together with additional molecular signals could lead to a creation of an efficient pre-vascularized tissue construct with potential applicability for transplantation. PMID:22417620

  15. How to build an adapted and bioactive cell microenvironment? A chemical interaction study of the structure of Ca-alginate matrices and their repercussion on confined cells.

    PubMed

    Bienaimé, Christophe; Barbotin, Jean-Noël; Nava-Saucedo, José-Edmundo

    2003-11-01

    Alginates are increasingly being used as medical materials (matrices for tissue regeneration, surgical sponges, hemostatic bandages, microbial and cell encapsulation, artificial bacterial biofilms, etc.). The constitution of alginate gel networks is a complex phenomenon. A great number of different kinds of polysaccharidic frameworks can come to existence depending on the conditions used for their attainment. For instance, the degree of heterogeneity and porosity of Ca-alginate beads rely on this molecular organization. The formation of structural irregularities (superficial crust, cavities, shafts, dense or light gel frameworks, ordered or chaotic domains, etc.) within the alginate gel beads are inherent to this skeletal design. Several specific staining molecules (e.g. calcon carboxylic acid, murexide, methylene blue) that are negatively or positively charged interact with the gel network. These molecules allowed us to reveal a great variety of chemical interactions shown by the pattern coloration of the internal structure of the gel. The results observed are very different for the several matrices analyzed, which could explain to a great extent the singular behavior that cells confined in these kind of matrices exhibit.

  16. [Immobilization of E. coli cells in polyacrylamide-based microporous cryogels].

    PubMed

    Lusta, K A; Starostina, N G; Gorkina, N B; Fikhte, B A; Lozinskiĭ, V I

    1988-01-01

    E. coli cells were immobilized in polyacrylamide cryogel by three ways: (1) introduction of cells in the reaction mixture followed by cryopolymerization; (2) the filling of the cryogel pores followed by cell fixation with diluted glutaric dialdehyde (GDA), and (3) the filling of the macropores of the polymeric matrix with modified surface. The ultrastructure of the gels and immobilized cells as well as distribution of attachment of the cells immobilized by different techniques were studied. The first type of immobilization was characterized by the highest quantity of the biomass in the gel (by protein) and by a sharp decrease of the cell viability. The second failed to retain the cells in the pores, and the GDA treatment significantly decreased the viability index. The latter technique was the mildest and completely maintained the viability of the population. However, the biomass content was lower as compared to the first type of immobilization, but could be considerably increased by the GDA treatment.

  17. [The injured and the immobilized muscle cell: ultrastructural observations].

    PubMed

    Lüthi, J M; Gerber, C; Claassen, H; Hoppeler, H

    1989-06-01

    Muscle soreness is a common feature among athletes and untrained individuals who engage in unusual, especially intense eccentric exercise. Various biochemical markers as for example elevated CK demonstrate a damage of muscle cells. The most prominent structural finding is a varying degree of disruption of the contractile material up to cell degeneration. These morphological findings reach their maximum 2 or 3 days after exercise. Signs of regeneration, however, are seen even weeks after exercise. In a prospective study we investigated the structures of the quadriceps muscle in 41 patients with chronic symptomatic instability of the anterior cruciate ligament before, 9 and 26 weeks after operation using the needle biopsy technique. The immobilized muscle showed a rapid and large atrophy which markedly reduced aerobic capacity as well as maximal strength. Preoperative values weren't attained 26 weeks postoperative despite intense physiotherapeutic exercise. The control leg showed an atrophy as well, but only aerobic capacity was reduced, maximal strength remained about the same.

  18. Alginate-modifying enzymes: biological roles and biotechnological uses

    PubMed Central

    Ertesvåg, Helga

    2015-01-01

    Alginate denotes a group of industrially important 1-4-linked biopolymers composed of the C-5-epimers β-D-mannuronic acid (M) and α-L-guluronic acid (G). The polysaccharide is manufactured from brown algae where it constitutes the main structural cell wall polymer. The physical properties of a given alginate molecule, e.g., gel-strength, water-binding capacity, viscosity and biocompatibility, are determined by polymer length, the relative amount and distribution of G residues and the acetyl content, all of which are controlled by alginate modifying enzymes. Alginate has also been isolated from some bacteria belonging to the genera Pseudomonas and Azotobacter, and bacterially synthesized alginate may be O-acetylated at O-2 and/or O-3. Initially, alginate is synthesized as polymannuronic acid, and some M residues are subsequently epimerized to G residues. In bacteria a mannuronan C-5-epimerase (AlgG) and an alginate acetylase (AlgX) are integral parts of the protein complex necessary for alginate polymerization and export. All alginate-producing bacteria use periplasmic alginate lyases to remove alginate molecules aberrantly released to the periplasm. Alginate lyases are also produced by organisms that utilize alginate as carbon source. Most alginate-producing organisms encode more than one mannuronan C-5 epimerase, each introducing its specific pattern of G residues. Acetylation protects against further epimerization and from most alginate lyases. An enzyme from Pseudomonas syringae with alginate deacetylase activity has been reported. Functional and structural studies reveal that alginate lyases and epimerases have related enzyme mechanisms and catalytic sites. Alginate lyases are now utilized as tools for alginate characterization. Secreted epimerases have been shown to function well in vitro, and have been engineered further in order to obtain enzymes that can provide alginates with new and desired properties for use in medical and pharmaceutical applications

  19. Sustained release of vascular endothelial growth factor from calcium-induced alginate hydrogels reinforced by heparin and chitosan.

    PubMed

    Lee, K W; Yoon, J J; Lee, J H; Kim, S Y; Jung, H J; Kim, S J; Joh, J W; Lee, H H; Lee, D S; Lee, S K

    2004-10-01

    A possible alternative for immunosuppression is a microencapsulation technique using hydrogels, which have been utilized for cell immobilization and drug delivery systems. Angiogenesis is crucial for delivery of the metabolic products to the host tissues as well as to supply oxygen and nutrients to cells. The local delivery of angiogenic growth factors, such as VEGF and basic FGF, has been recently studied to enhance angiogenesis on peripheral tissue of graft. In this study, we evaluated sustained VEGF release with a model using hydrogels coated with chitosan and heparin in vitro. We fabricated calcium alginate gels and chitosan-coated calcium alginate gels. Heparinized chitosan-coated calcium-induced alginate hydrogel beads were prepared by soaking chitosan-coated calcium alginate gels in heparin solution. We compared the stability and VEGF release manner between three kinds of hydrogels. To compare the stability, 5 mL of each hydrogel was incubated with 20 mL PBS under the rotational culture. Compression forces were measured using a rheometer. The amount of VEGF released from the gels was measured by ELISA. The heparin-coated chitosan alginate hydrogels showed the highest surface stability among the three hydrogels. VEGF from the heparinized gel was released in sustained manner up to 10 days in vitro. Chitosan-coated alginate gels released 90% of loaded VEGF within 5 days. These results suggest that local delivery of VEGF using a heparinized hydrogel may provide a long-term supply of angiogenic growth factor that might induce new vessel formation in vivo.

  20. Catalytical Properties of Free and Immobilized Aspergillus niger Tannase

    PubMed Central

    Flores-Maltos, Abril; Rodríguez-Durán, Luis V.; Renovato, Jacqueline; Contreras, Juan C.; Rodríguez, Raúl; Aguilar, Cristóbal N.

    2011-01-01

    A fungal tannase was produced, recovered, and immobilized by entrapment in calcium alginate beads. Catalytical properties of the immobilized enzyme were compared with those of the free one. Tannase was produced intracellularly by the xerophilic fungus Aspergillus niger GH1 in a submerged fermentation system. Enzyme was recovered by cell disruption and the crude extract was partially purified. The catalytical properties of free and immobilized tannase were evaluated using tannic acid and methyl gallate as substrates. KM and Vmax values for free enzyme were very similar for both substrates. But, after immobilization, KM and Vmax values increased drastically using tannic acid as substrate. These results indicated that immobilized tannase is a better biocatalyst than free enzyme for applications on liquid systems with high tannin content, such as bioremediation of tannery or olive-mill wastewater. PMID:21918717

  1. Cell growth on immobilized cell growth factor. 7. Protein-free cell culture by using growth-factor-immobilized polymer membrane.

    PubMed

    Liu, S Q; Ito, Y; Imanishi, Y

    1993-02-01

    A protein-free culture of anchorage-dependent cells, mouse fibroblast cells, STO and 3T3-L1 and fibroic sarcoma cells, Swiss albino HSDM1C1, grown on a cell-growth protein, insulin, and/or a cell-adhesion protein, collagen, which are immobilized or coimmobilized on surface-hydrolyzed poly(methyl methacrylate) membrane, was investigated. By adding metal ions and lipids to the culture medium, a protein-free culture medium was composed, which was potent in promoting cell proliferation similarly to serum-containing culture medium. In particular, with insulin/collagen-coimmobilized membrane, a protein-free culture was established without detachment of growing cells over a long period. These protein-immobilized membranes could be used repeatedly. PMID:7763456

  2. Cell-Laden Poly(ɛ-caprolactone)/Alginate Hybrid Scaffolds Fabricated by an Aerosol Cross-Linking Process for Obtaining Homogeneous Cell Distribution: Fabrication, Seeding Efficiency, and Cell Proliferation and Distribution

    PubMed Central

    Lee, HyeongJin; Ahn, SeungHyun; Bonassar, Lawrence J.; Chun, Wook

    2013-01-01

    Generally, solid-freeform fabricated scaffolds show a controllable pore structure (pore size, porosity, pore connectivity, and permeability) and mechanical properties by using computer-aided techniques. Although the scaffolds can provide repeated and appropriate pore structures for tissue regeneration, they have a low biological activity, such as low cell-seeding efficiency and nonuniform cell density in the scaffold interior after a long culture period, due to a large pore size and completely open pores. Here we fabricated three different poly(ɛ-caprolactone) (PCL)/alginate scaffolds: (1) a rapid prototyped porous PCL scaffold coated with an alginate, (2) the same PCL scaffold coated with a mixture of alginate and cells, and (3) a multidispensed hybrid PCL/alginate scaffold embedded with cell-laden alginate struts. The three scaffolds had similar micropore structures (pore size=430–580 μm, porosity=62%–68%, square pore shape). Preosteoblast cells (MC3T3-E1) were used at the same cell density in each scaffold. By measuring cell-seeding efficiency, cell viability, and cell distribution after various periods of culturing, we sought to determine which scaffold was more appropriate for homogeneously regenerated tissues. PMID:23469894

  3. Cell-laden poly(ɛ-caprolactone)/alginate hybrid scaffolds fabricated by an aerosol cross-linking process for obtaining homogeneous cell distribution: fabrication, seeding efficiency, and cell proliferation and distribution.

    PubMed

    Lee, HyeongJin; Ahn, SeungHyun; Bonassar, Lawrence J; Chun, Wook; Kim, GeunHyung

    2013-10-01

    Generally, solid-freeform fabricated scaffolds show a controllable pore structure (pore size, porosity, pore connectivity, and permeability) and mechanical properties by using computer-aided techniques. Although the scaffolds can provide repeated and appropriate pore structures for tissue regeneration, they have a low biological activity, such as low cell-seeding efficiency and nonuniform cell density in the scaffold interior after a long culture period, due to a large pore size and completely open pores. Here we fabricated three different poly(ɛ-caprolactone) (PCL)/alginate scaffolds: (1) a rapid prototyped porous PCL scaffold coated with an alginate, (2) the same PCL scaffold coated with a mixture of alginate and cells, and (3) a multidispensed hybrid PCL/alginate scaffold embedded with cell-laden alginate struts. The three scaffolds had similar micropore structures (pore size = 430-580 μm, porosity = 62%-68%, square pore shape). Preosteoblast cells (MC3T3-E1) were used at the same cell density in each scaffold. By measuring cell-seeding efficiency, cell viability, and cell distribution after various periods of culturing, we sought to determine which scaffold was more appropriate for homogeneously regenerated tissues.

  4. Proliferation and enrichment of CD133+ glioblastoma cancer stem cells on 3D chitosan-alginate scaffolds

    PubMed Central

    Kievit, Forrest M.; Florczyk, Stephen J.; Leung, Matthew C.; Wang, Kui; Wu, Jennifer D.; Silber, John R.; Ellenbogen, Richard G.; Lee, Jerry S.H.; Zhang, Miqin

    2014-01-01

    Emerging evidence implicates cancer stem cells (CSCs) as primary determinants of the clinical behavior of human cancers, representing an ideal target for next-generation anticancer therapies. However CSCs are difficult to propagate in vitro, severely limiting the study of CSC biology and drug development. Here we report that growing cells from glioblastoma (GBM) cell lines on three dimensional (3D) porous chitosan-alginate (CA) scaffolds dramatically promotes the proliferation and enrichment of cells possessing the hallmarks of CSCs. CA scaffold-grown cells were found more tumorigenic in nude mouse xenografts than cells grown from monolayers. Growing in CA scaffolds rapidly promoted expression of genes involved in the epithelial-to-mesenchymal transition that has been implicated in the genesis of CSCs. Our results indicate that CA scaffolds have utility as a simple and inexpensive means to cultivate CSCs in vitro in support of studies to understand CSC biology and develop more effective anti-cancer therapies. PMID:25109438

  5. Identification of IL-1β and LPS as optimal activators of monolayer and alginate-encapsulated mesenchymal stromal cell immunomodulation using design of experiments and statistical methods.

    PubMed

    Gray, Andrea; Maguire, Timothy; Schloss, Rene; Yarmush, Martin L

    2015-01-01

    Induction of therapeutic mesenchymal stromal cell (MSC) function is dependent upon activating factors present in diseased or injured tissue microenvironments. These functions include modulation of macrophage phenotype via secreted molecules including prostaglandin E2 (PGE2). Many approaches aim to optimize MSC-based therapies, including preconditioning using soluble factors and cell immobilization in biomaterials. However, optimization of MSC function is usually inefficient as only a few factors are manipulated in parallel. We utilized fractional factorial design of experiments to screen a panel of 6 molecules (lipopolysaccharide [LPS], polyinosinic-polycytidylic acid [poly(I:C)], interleukin [IL]-6, IL-1β, interferon [IFN]-β, and IFN-γ), individually and in combinations, for the upregulation of MSC PGE2 secretion and attenuation of macrophage secretion of tumor necrosis factor (TNF)-α, a pro-inflammatory molecule, by activated-MSC conditioned medium (CM). We used multivariable linear regression (MLR) and analysis of covariance to determine differences in functions of optimal factors on monolayer MSCs and alginate-encapsulated MSCs (eMSCs). The screen revealed that LPS and IL-1β potently activated monolayer MSCs to enhance PGE2 production and attenuate macrophage TNF-α. Activation by LPS and IL-1β together synergistically increased MSC PGE2, but did not synergistically reduce macrophage TNF-α. MLR and covariate analysis revealed that macrophage TNF-α was strongly dependent on the MSC activation factor, PGE2 level, and macrophage donor but not MSC culture format (monolayer versus encapsulated). The results demonstrate the feasibility and utility of using statistical approaches for higher throughput cell analysis. This approach can be extended to develop activation schemes to maximize MSC and MSC-biomaterial functions prior to transplantation to improve MSC therapies.

  6. Biodegradation of phenol by using free and immobilized cells of Acinetobacter sp. BS8Y.

    PubMed

    Jiang, Lichun; Ruan, Qiping; Li, Rulan; Li, Tiandong

    2013-03-01

    Strain BS8Y with high biodegradation activity and high tolerance of phenol was isolated from activated sludge in an insulating material plant of China. This strain was capable of removing 99.2% of the initial 600 mg/l phenol in liquid minimal medium within 24 h and tolerating phenol at concentrations of up to 1,200 mg/ml. DNA sequencing and homologous analysis of the 16S rRNA gene identified that the strain BS8Y belonged to an Acinetobacter species. Polyvinyl alcohol was used as gel matrix to immobilize the strain BS8Y. The factors affecting the phenol degradation by immobilized cells and the phenol removal efficiency of free and immobilized cells were investigated; the stability of the immobilized cells is also reported. The results show that the immobilized cells could tolerate a higher phenol level and protected the bacteria much more effectively against changes in temperature and pH. The phenol degradation efficiency was high at up to 96% within 30 h, with an initial concentration of 800 mg/l phenol, and the immobilized cells showed better performance than the suspended cells. Reusability tests revealed that the immobilized cells were stable enough even after reuse for ten times or storing at 4°C for 35 d. These results demonstrate that immobilized Acinetobacter sp. BS8Y possesses a good application potential in the treatment of phenol-containing wastewater.

  7. In vitro evaluation of alginate encapsulated adipose-tissue stromal cells for use as injectable bone graft substitute

    SciTech Connect

    Abbah, S.A.; Lu, W.W. . E-mail: wwlu@hkusua.hku.hk; Chan, D.; Cheung, K.M.C.; Liu, W.G.; Zhao, F.; Li, Z.Y.; Leong, J.C.Y.; Luk, K.D.K.

    2006-08-18

    This study aims to investigate the survival and osteogenic behavior of murine-derived adipose-tissue stromal cells (ATSCs) encapsulated in alginate microcapsules thereby instigating further studies in this cell delivery strategy for in vivo osteogenesis. Cell viability was quantified using a tetrazolium-based assay and osteogenic differentiation was evaluated by both alkaline-phosphatase (ALP) histochemistry and osteocalcin mRNA analysis. Following microencapsulation, cell numbers increased from 3.9 x 10{sup 3} on day 1 to 7.8 x 10{sup 3} on day 7 and maintained excellent viability in the course of 21-day culture. ALP was 6.9, 5.5, and 3.2 times higher than monolayer cultures on days 7, 14, and 21, respectively. In addition, osteocalcin mRNA was detectable in encapsulated cultures earlier (day 14) than monolayer cultures. We conclude that alginate microcapsules can act as three-dimensional matrix for ATSC proliferation and has potential for use as injectable, biodegradable scaffold in bone tissue engineering.

  8. Enhanced trophic factor secretion by mesenchymal stem/stromal cells with Glycine-Histidine-Lysine (GHK)-modified alginate hydrogels

    PubMed Central

    Jose, Soumia; Hughbanks, Marissa L.; Binder, Bernard Y.K.; Ingavle, Ganesh C.; Leach, J. Kent

    2014-01-01

    Recombinant proteins and cytokines are under broad preclinical and clinical investigation to promote angiogenesis, but their success is limited by ineffective delivery, lack of long-term stability, and excessive cost. Mesenchymal stem/stromal cells (MSC) secrete bioactive trophic factors, and thus, may provide an effective alternative to address these challenges. Glycine-Histidine-Lysine (GHK) is a peptide fragment of osteonectin (SPARC), a matricellular protein with reported proangiogenic potential. We examined the capacity of GHK to upregulate secretion of proangiogenic factors from human MSC in culture and when covalently coupled to alginate hydrogels. GHK had no apparent cytotoxic effects on MSC in culture over a wide range of concentrations. We detected a dose-dependent increase in vascular endothelial growth factor (VEGF) concentration in media conditioned by GHK-treated MSC, which increased endothelial cell proliferation, migration, and tubule formation. We covalently coupled GHK to alginate using carbodiimide chemistry, and human MSC were entrapped in alginate hydrogels to assess VEGF secretion. Similar to monolayer culture, MSC responded to GHK-modified gels by secreting increased concentrations of VEGF and basic fibroblast growth factor (bFGF) compared to unmodified gels. The pre-treatment of MSC with antibodies to α6 and β1 integrins prior to entrapment in GHK-modified gels abrogated VEGF secretion, suggesting that the proangiogenic response of MSC was integrin-mediated. These data demonstrate that the proangiogenic potential of MSC can be significantly increased by the presentation of GHK with a biodegradable carrier, therefore increasing their clinical potential when used for tissue repair. PMID:24468583

  9. Formaldehyde degradation by Ralstonia eutropha in an immobilized cell bioreactor.

    PubMed

    Habibi, Alireza; Vahabzadeh, Farzaneh

    2013-01-01

    The formaldehyde (FA) degradation ability of the loofa-immobilized Ralstonia eutropha cells in a packed bed reactor was modeled using a statistically based design of the experiment (DOE) considering application of response surface methodology (RSM). The simultaneous effects of four operative test factors on the cells performance in terms of FA degradation rate and extent of the chemical oxygen demand (COD) removal were monitored. The combination of factors at initial FA concentration of 629.7 mg L(-1)h(-1), recycling substrate flow rate of 4.4 mL min(-1), aeration rate of 1.05 vvm, and the system's temperature of 28.8°C resulted the optimal conditions for the FA biodegradation rate and COD removal efficiency. Loofa porous structure was found to be a protective environment for the cells in exposing to the toxic substances and the scanning electron microscopy (SEM) images revealed extensive cells penetration within this support. Oxygen transfer analysis in the form of evaluating K la value was also carried out and at the optimum conditions of the DOE was equaled to 9.96 h(-1)and oxygen uptake rate was 35.6 mg L(-1)h(-1).

  10. Gingival Mesenchymal Stem Cell (GMSC) Delivery System Based on RGD-Coupled Alginate Hydrogel with Antimicrobial Properties: A Novel Treatment Modality for Peri-Implantitis

    PubMed Central

    Diniz, Ivana M. A.; Chen, Chider; Ansari, Sahar; Zadeh, Homayoun H.; Moshaverinia, Maryam; Chee, Daniel; Marques, Márcia M.; Shi, Songtao; Moshaverinia, Alireza

    2015-01-01

    Purpose Peri-implantitis is one of the most common inflammatory complications in dental implantology. Similar to periodontitis, in peri-implantitis, destructive inflammatory changes take place in the tissues surrounding a dental implant. Bacterial flora at the failing implant sites resemble the pathogens in periodontal disease and consist of Gram-negative anaerobic bacteria including Aggregatibacter actinomycetemcomitans (Aa). Here we demonstrate the effectiveness of a silver lactate (SL)-containing RGD-coupled alginate hydrogel scaffold as a promising stem cell delivery vehicle with antimicrobial properties. Materials and Methods Gingival mesenchymal stem cells (GMSCs) or human bone marrow mesenchymal stem cells (hBMMSCs) were encapsulated in SL-loaded alginate hydrogel microspheres. Stem cell viability, proliferation, and osteo-differentiation capacity were analyzed. Results Our results showed that SL exhibited antimicrobial properties against Aa in a dose-dependent manner, with 0.50 mg/ml showing the greatest antimicrobial properties while still maintaining cell viability. At this concentration, SL-containing alginate hydrogel was able to inhibit Aa on the surface of Ti discs and significantly reduce the bacterial load in Aa suspensions. Silver ions were effectively released from the SL-loaded alginate microspheres for up to 2 weeks. Osteogenic differentiation of GMSCs and hBMMSCs encapsulated in the SL-loaded alginate microspheres were confirmed by the intense mineral matrix deposition and high expression of osteogenesis-related genes. Conclusion Taken together, our findings confirm that GMSCs encapsulated in RGD-modified alginate hydrogel containing SL show promise for bone tissue engineering with antimicrobial properties against Aa bacteria in vitro. PMID:26216081

  11. Impact of alginate composition: from bead mechanical properties to encapsulated HepG2/C3A cell activities for in vivo implantation.

    PubMed

    Capone, Stephanie H; Dufresne, Murielle; Rechel, Mathias; Fleury, Marie-José; Salsac, Anne-Virginie; Paullier, Patrick; Daujat-Chavanieu, Martine; Legallais, Cecile

    2013-01-01

    Recently, interest has focused on hepatocytes' implantation to provide end stage liver failure patients with a temporary support until spontaneous recovery or a suitable donor becomes available. To avoid cell damage and use of an immunosuppressive treatment, hepatic cells could be implanted after encapsulation in a porous biomaterial of bead or capsule shape. The aim of this study was to compare the production and the physical properties of the beads, together with some hepatic cell functions, resulting from the use of different material combinations for cell microencapsulation: alginate alone or combined with type I collagen with or without poly-L-lysine and alginate coatings. Collagen and poly-L-lysine increased the bead mechanical resistance but lowered the mass transfer kinetics of vitamin B12. Proliferation of encapsulated HepG2/C3A cells was shown to be improved in alginate-collagen beads. Finally, when the beads were subcutaneously implanted in mice, the inflammatory response was reduced in the case of alginate mixed with collagen. This in vitro and in vivo study clearly outlines, based on a systematic comparison, the necessity of compromising between material physical properties (mechanical stability and porosity) and cell behavior (viability, proliferation, functionalities) to define optima hepatic cell microencapsulation conditions before implantation.

  12. Impact of Alginate Composition: From Bead Mechanical Properties to Encapsulated HepG2/C3A Cell Activities for In Vivo Implantation

    PubMed Central

    Capone, Stephanie H.; Dufresne, Murielle; Rechel, Mathias; Fleury, Marie-José; Salsac, Anne-Virginie; Paullier, Patrick; Daujat-Chavanieu, Martine; Legallais, Cecile

    2013-01-01

    Recently, interest has focused on hepatocytes’ implantation to provide end stage liver failure patients with a temporary support until spontaneous recovery or a suitable donor becomes available. To avoid cell damage and use of an immunosuppressive treatment, hepatic cells could be implanted after encapsulation in a porous biomaterial of bead or capsule shape. The aim of this study was to compare the production and the physical properties of the beads, together with some hepatic cell functions, resulting from the use of different material combinations for cell microencapsulation: alginate alone or combined with type I collagen with or without poly-L-lysine and alginate coatings. Collagen and poly-L-lysine increased the bead mechanical resistance but lowered the mass transfer kinetics of vitamin B12. Proliferation of encapsulated HepG2/C3A cells was shown to be improved in alginate-collagen beads. Finally, when the beads were subcutaneously implanted in mice, the inflammatory response was reduced in the case of alginate mixed with collagen. This in vitro and in vivo study clearly outlines, based on a systematic comparison, the necessity of compromising between material physical properties (mechanical stability and porosity) and cell behavior (viability, proliferation, functionalities) to define optima hepatic cell microencapsulation conditions before implantation. PMID:23637958

  13. Alginate Biosynthesis Factories in Pseudomonas fluorescens: Localization and Correlation with Alginate Production Level

    PubMed Central

    Maleki, Susan; Almaas, Eivind; Zotchev, Sergey; Valla, Svein

    2015-01-01

    Pseudomonas fluorescens is able to produce the medically and industrially important exopolysaccharide alginate. The proteins involved in alginate biosynthesis and secretion form a multiprotein complex spanning the inner and outer membranes. In the present study, we developed a method by which the porin AlgE was detected by immunogold labeling and transmission electron microscopy. Localization of the AlgE protein was found to depend on the presence of other proteins in the multiprotein complex. No correlation was found between the number of alginate factories and the alginate production level, nor were the numbers of these factories affected in an algC mutant that is unable to produce the precursor needed for alginate biosynthesis. Precursor availability and growth phase thus seem to be the main determinants for the alginate production rate in our strain. Clustering analysis demonstrated that the alginate multiprotein complexes were not distributed randomly over the entire outer cell membrane surface. PMID:26655760

  14. Optimization of date syrup for enhancement of the production of citric acid using immobilized cells of Aspergillus niger

    PubMed Central

    Mostafa, Yasser S.; Alamri, Saad A.

    2012-01-01

    Date syrup as an economical source of carbohydrates and immobilized Aspergillus niger J4, which was entrapped in calcium alginate pellets, were employed for enhancing the production of citric acid. Maximum production was achieved by pre-treating date syrup with 1.5% tricalcium phosphate to remove heavy metals. The production of citric acid using a pretreated medium was 38.87% higher than an untreated one that consumed sugar. The appropriate presence of nitrogen, phosphate and magnesium appeared to be important in order for citric acid to accumulate. The production of citric acid and the consumed sugar was higher when using 0.1% ammonium nitrate as the best source of nitrogen. The production of citric acid increased significantly when 0.1 g/l of KH2PO4 was added to the medium of date syrup. The addition of magnesium sulfate at the rate of 0.20 g/l had a stimulating effect on the production of citric acid. Maximum production of citric acid was obtained when calcium chloride was absent. One of the most important benefits of immobilized cells is their ability and stability to produce citric acid under a repeated batch culture. Over four repeated batches, the production of citric acid production was maintained for 24 days when each cycle continued for 144 h. The results obtained in the repeated batch cultivation using date syrup confirmed that date syrup could be used as a medium for the industrial production of citric acid. PMID:23961184

  15. Optimization of date syrup for enhancement of the production of citric acid using immobilized cells of Aspergillus niger.

    PubMed

    Mostafa, Yasser S; Alamri, Saad A

    2012-04-01

    Date syrup as an economical source of carbohydrates and immobilized Aspergillus niger J4, which was entrapped in calcium alginate pellets, were employed for enhancing the production of citric acid. Maximum production was achieved by pre-treating date syrup with 1.5% tricalcium phosphate to remove heavy metals. The production of citric acid using a pretreated medium was 38.87% higher than an untreated one that consumed sugar. The appropriate presence of nitrogen, phosphate and magnesium appeared to be important in order for citric acid to accumulate. The production of citric acid and the consumed sugar was higher when using 0.1% ammonium nitrate as the best source of nitrogen. The production of citric acid increased significantly when 0.1 g/l of KH2PO4 was added to the medium of date syrup. The addition of magnesium sulfate at the rate of 0.20 g/l had a stimulating effect on the production of citric acid. Maximum production of citric acid was obtained when calcium chloride was absent. One of the most important benefits of immobilized cells is their ability and stability to produce citric acid under a repeated batch culture. Over four repeated batches, the production of citric acid production was maintained for 24 days when each cycle continued for 144 h. The results obtained in the repeated batch cultivation using date syrup confirmed that date syrup could be used as a medium for the industrial production of citric acid.

  16. Enhanced cellular activities of polycaprolactone/alginate-based cell-laden hierarchical scaffolds for hard tissue engineering applications.

    PubMed

    Lee, HyeongJin; Kim, GeunHyung

    2014-09-15

    Biomedical scaffolds have been widely investigated because they are essential for support and promotion of cell adhesion, proliferation and differentiation in three-dimensional (3D) structures. An ideal scaffold should be highly porous to enable efficient nutrient and oxygen transfer and have a 3D structure that provides optimal micro-environmental conditions for the seeded cells to obtain homogeneous growth after a long culture period. In this study, new hierarchical osteoblast-like cell (MG-63)-laden scaffolds consisting of micro-sized struts/inter-layered micro-nanofibres and cell-laden hydrogel struts with mechanically stable and biologically superior properties were introduced. Poly(ethylene oxide) (PEO) was used as a sacrificial component to generate pores within the cell-laden hydrogel struts to attain a homogeneous cell distribution and rapid cell growth in the scaffold interior. The alginate-based cell-laden struts with PEO induced fast/homogeneous cell release, in contrast to nonporous cell-laden struts. Various weight fractions (0.5, 1, 2, 3 and 3.5 wt%) of PEO were used, of which 2 wt% PEO in the cell-laden strut resulted in the most appropriate cell release and enhanced biological activities (cell proliferation and calcium deposition), compared to nonporous cell-laden struts.

  17. Phospholipid polymer-based antibody immobilization for cell rolling surfaces in stem cell purification system.

    PubMed

    Mahara, Atsushi; Chen, Hao; Ishihara, Kazuhiko; Yamaoka, Tetsuji

    2014-01-01

    We previously developed an antibody-conjugated cell rolling column that successfully separates stem cell subpopulations depending on the cell surface marker density, but a large amount of the injected cells were retained in the column because of non-specific interactions. In this study, an amphiphilic copolymer, poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (nBMA)-co-N-vinyl formamide (NVf)], with phospholipid polar side groups was designed as a novel antibody-immobilizing modifier. The formamide groups in NVf units were converted to active maleimide groups. A plastic flow microfluidic chamber was coated with the copolymers, and a reduced anti-CD90 antibody was immobilized. The adipose tissue-derived stem cells isolated from the rat were injected into the flow chamber, and their rolling behavior was observed under a microscope with a high-speed camera. Non-specific cell adhesion was reduced strongly by means of this immobilization method because of the MPC unit, resulting in a high percentage of rolling cells. These results demonstrate that a surface coated with phospholipid polar groups can be used in an effective stem cell separation system based on the cell rolling process.

  18. Beta-glucanase productivity improvement via cell immobilization of recombinant Escherichia coli cells in different matrices.

    PubMed

    Beshay, Usama; El-Enshasy, Hesham; Ismail, I M K; Moawad, Hassan; Abd-El-Ghany, Sawsan

    2011-01-01

    The studies have been performed to analyze the production of beta-glucanase by a recombinant strain of Escherichia coli immobilized in different matrices. Porous sintered glass SIRAN, Ceramic supporting matrices and Broken Pumice stone as well as SIRAN Raschig-rings were examined for the immobilization of whole bacterial cells. The beta-glucanase activity of bacteria immobilized in CeramTec PST 5 (4-5 mm) was very low. CeramTec PST 5 (1.5-2.5 mm) was found to be the best carrier compared to all other matrices regarding glucanase production (630 U/ml) and compared to enzyme activity produced by free cells (500 U/ml). Different doses of matrices were applied (2, 5, 7, 10 g/lask) in the form of "matrix weight". Using 2 g/flask of CeramTec PST 5 (1.5-2.5 mm) yielded enzyme activity of 630 U/ml). CeramTec gives highest operational stability of beta-glucanase by repeated batch fermentation to 5 cycles, and activity reached 660 U/ml. Scanning electron microscopy observations showed a high number of vegetative cells that continued growth inside the matrices, indicating that beta-glucanase activity improvement was due to the immobilization of the cells.

  19. Comparison of some biochemical properties of artichoke polyphenol oxidase entrapped in alginate-carrageenan and alginate gels.

    PubMed

    Yagar, Hulya; Kocaturk, Selin

    2014-08-01

    Polyphenol oxidase (PPO, EC.1.14.18.1) isolated from artichoke (Cynara scolymus) was entrapped within alginate and alginate+ carrageenan beads, and the catecholase and cresolase activities of both entrapped enzymes were determined. Some properties of these immobilized enzymes such as optimum pH and temperature, kinetic parameters (Km and Vmax), thermal, and storage stability were determined and compared to each other. The highest catecholase activity was observed in alginate gel (370 U/g bead) while the highest cresolase activity was in alginate+ carrageenan gel (90 U/g bead). For catecholase and cresolase activities, optimum pHs of alginate and alginate+ carrageenan beads were determined to be 7.0 and 4.0, respectively. Optimum temperatures for catecholase activity were determined to be 40°C for both entrapped enzymes. These values for cresolase activity were 30°C and 20°C, respectively. Immobilized artichoke PPOs greatly preserved their thermal stability which exists anyway. The catalytic efficiency value (Vmax/Km) of the alginate beads is approximately high as two-and-a-half folds of that of alginate+κ-carrageenan beads for cresolase activity. These values were very close for catecholase activity. Immobilized beads saved their both activities after 30 days of storage at 4°C. PMID:23795723

  20. Comparison of some biochemical properties of artichoke polyphenol oxidase entrapped in alginate-carrageenan and alginate gels.

    PubMed

    Yagar, Hulya; Kocaturk, Selin

    2014-08-01

    Polyphenol oxidase (PPO, EC.1.14.18.1) isolated from artichoke (Cynara scolymus) was entrapped within alginate and alginate+ carrageenan beads, and the catecholase and cresolase activities of both entrapped enzymes were determined. Some properties of these immobilized enzymes such as optimum pH and temperature, kinetic parameters (Km and Vmax), thermal, and storage stability were determined and compared to each other. The highest catecholase activity was observed in alginate gel (370 U/g bead) while the highest cresolase activity was in alginate+ carrageenan gel (90 U/g bead). For catecholase and cresolase activities, optimum pHs of alginate and alginate+ carrageenan beads were determined to be 7.0 and 4.0, respectively. Optimum temperatures for catecholase activity were determined to be 40°C for both entrapped enzymes. These values for cresolase activity were 30°C and 20°C, respectively. Immobilized artichoke PPOs greatly preserved their thermal stability which exists anyway. The catalytic efficiency value (Vmax/Km) of the alginate beads is approximately high as two-and-a-half folds of that of alginate+κ-carrageenan beads for cresolase activity. These values were very close for catecholase activity. Immobilized beads saved their both activities after 30 days of storage at 4°C.

  1. Kinetic analysis of dihydroxyacetone production from crude glycerol by immobilized cells of Gluconobacter oxydans MTCC 904.

    PubMed

    Dikshit, Pritam Kumar; Moholkar, Vijayanand S

    2016-09-01

    The present study has investigated kinetic features of bioconversion of biodiesel-derived crude glycerol to dihydroxyacetone with immobilized Gluconobacter oxydans cells using modified Haldane substrate-inhibition model. The results have been compared against free cells and pure glycerol. Relative variations in the kinetic parameters KS, KI, Vmax, n and X reveal that immobilized G. oxydans cells (on PU foam substrate) with crude glycerol as substrate give higher order of inhibition (n) and lower maximum reaction velocities (Vmax). These results are essentially implications of substrate transport restrictions across immobilization matrix, which causes retention of substrate in the matrix and reduction in fractional available substrate (X) for the cells. This causes reduction in both KS (substrate concentration at Vmax/2) and KI (inhibition constant) as compared to free cells. For immobilized cells, substrate concentration (Smax) corresponding to Vmax is practically same for both pure and crude glycerol as substrate. PMID:27343447

  2. CellMAC: a novel technology for encapsulation of mammalian cells in cellulose sulfate/pDADMAC capsules assembled on a transient alginate/Ca2+ scaffold.

    PubMed

    Weber, Wilfried; Rinderknecht, Matthias; Daoud-El Baba, Marie; de Glutz, François-Nicolas; Aubel, Dominique; Fussenegger, Martin

    2004-11-01

    Microencapsulation of desired mammalian cell phenotypes in biocompatible polymer matrices represents a powerful technology for cell-based therapies and biopharmaceutical manufacturing of protein therapeutics. We have pioneered a novel jet break-up-compatible process for encapsulation of mammalian cells in cellulose sulfate (CS)/poly-diallyl-dimethyl-ammoniumchloride (pDADMAC) (CellMAC) capsules. CS and pDADMAC polymerize on a transient ad hoc co-assembled Ca2+/alginate scaffold and form homogenous capsules following dissolution of the alginate core by Ca2+ chelating agents. CellMAC capsules exhibited excellent mechanical properties and showed a molecular weight cut-off between 43 and 77kDa. Chinese hamster ovary cells engineered for constitutive production of the glycohormone erythropoietin reached high viable cell densities when grown inside CellMAC capsules, while specific erythropoietin (EPO) productivities matched those of conventional non-encapsulated control cultures. CellMAC-encapsulated EPO-production cell lines induced increased EPO serum levels when implanted intraperitoneally into mice and provided robust glycoprotein production during standard stirred-tank bioreactor operation. We expect the CellMAC technology to foster advances in therapeutic encapsulation of engineered cell lines as well as manufacturing of protein pharmaceuticals.

  3. Encapsulation in alginate and alginate coated-chitosan improved the survival of newly probiotic in oxgall and gastric juice.

    PubMed

    Trabelsi, Imen; Bejar, Wacim; Ayadi, Dorra; Chouayekh, Hichem; Kammoun, Radhouane; Bejar, Samir; Ben Salah, Riadh

    2013-10-01

    This study was undertaken to develop an optimum composition model for the microencapsulation of a newly probiotic on sodium alginate using response surface methodology. The individual and interactive effects of three independent variables, namely sodium alginate concentration, biomass concentration, and hardening time, were investigated using Box-Behnken design experiments. A second ordered polynomial model was fitted and optimum conditions were estimated. The optimal conditions identified were 2% for sodium alginate, 10(10)UFC/ml for biomass, and 30 min for hardening time. The experimental value obtained for immobilized cells under these conditions was about 80.98%, which was in close agreement with the predicted value of 82.6%. Viability of microspheres (96%) was enhanced with chitosan as coating materials. The survival rates of free and microencapsulated Lactobacillus plantarum TN8 during exposure to artificial gastrointestinal conditions were compared. The results revealed that the encapsulated cells exhibited significantly higher resistances to artificial intestinal juice (AIJ) and artificial gastric juice (AGJ). Microencapsulation was also noted to effectively protect the strain from heating at 65 °C and refrigerating at 4 °C. Taken together, the findings indicated that microencapsulation conferred important protective effects to L. plantarum against the gastrointestinal conditions encountered during the transit of food.

  4. Engineering alginate as bioink for bioprinting.

    PubMed

    Jia, Jia; Richards, Dylan J; Pollard, Samuel; Tan, Yu; Rodriguez, Joshua; Visconti, Richard P; Trusk, Thomas C; Yost, Michael J; Yao, Hai; Markwald, Roger R; Mei, Ying

    2014-10-01

    Recent advances in three-dimensional (3-D) printing offer an excellent opportunity to address critical challenges faced by current tissue engineering approaches. Alginate hydrogels have been used extensively as bioinks for 3-D bioprinting. However, most previous research has focused on native alginates with limited degradation. The application of oxidized alginates with controlled degradation in bioprinting has not been explored. Here, a collection of 30 different alginate hydrogels with varied oxidation percentages and concentrations was prepared to develop a bioink platform that can be applied to a multitude of tissue engineering applications. The authors systematically investigated the effects of two key material properties (i.e. viscosity and density) of alginate solutions on their printabilities to identify a suitable range of material properties of alginates to be applied to bioprinting. Further, four alginate solutions with varied biodegradability were printed with human adipose-derived stem cells (hADSCs) into lattice-structured, cell-laden hydrogels with high accuracy. Notably, these alginate-based bioinks were shown to be capable of modulating proliferation and spreading of hADSCs without affecting the structure integrity of the lattice structures (except the highly degradable one) after 8days in culture. This research lays a foundation for the development of alginate-based bioink for tissue-specific tissue engineering applications. PMID:24998183

  5. Engineering alginate as bioink for bioprinting

    PubMed Central

    Jia, Jia; Richards, Dylan J.; Pollard, Samuel; Tan, Yu; Rodriguez, Joshua; Visconti, Richard P.; Trusk, Thomas C.; Yost, Michael J.; Yao, Hai; Markwald, Roger R.; Mei, Ying

    2015-01-01

    Recent advances in 3D printing offer an excellent opportunity to address critical challenges faced by current tissue engineering approaches. Alginate hydrogels have been extensively utilized as bioinks for 3D bioprinting. However, most previous research has focused on native alginates with limited degradation. The application of oxidized alginates with controlled degradation in bioprinting has not been explored. Here, we prepared a collection of 30 different alginate hydrogels with varied oxidation percentages and concentrations to develop a bioink platform that can be applied to a multitude of tissue engineering applications. We systematically investigated the effects of two key material properties (i.e. viscosity and density) of alginate solutions on their printabilities to identify a suitable range of material properties of alginates to be applied to bioprinting. Further, four alginate solutions with varied biodegradability were printed with human adipose-derived stem cells (hADSCs) into lattice-structured, cell-laden hydrogels with high accuracy. Notably, these alginate-based bioinks were shown to be capable of modulating proliferation and spreading of hADSCs without affecting structure integrity of the lattice structures (except the highly degradable one) after 8 days in culture. This research lays a foundation for the development of alginate-based bioink for tissue-specific tissue engineering applications. PMID:24998183

  6. Continuous cider fermentation with co-immobilized yeast and Leuconostoc oenos cells.

    PubMed

    Nedovic; Durieuxb; Van Nedervelde L; Rosseels; Vandegans; Plaisant; Simon

    2000-06-01

    Ca-alginate matrix was used to co-immobilize Saccharomyces bayanus and Leuconostoc oenos in one integrated biocatalytic system in order to perform simultaneously alcoholic and malo-lactic fermentation of apple juice to produce cider, in a continuous packed bed bioreactor. The continuous process permitted much faster fermentation compared with the traditional batch process. The flavor formation was also better controlled. By adjusting the flow rate of feeding substrate through the bioreactor, i.e. its residence time, it was possible to obtain either "soft" or "dry" cider. However, the profile of volatile compounds in the final product was modified comparatively to the batch process, especially for higher alcohols, isoamylacetate, and diacetyl. This modification is due to different physiology states of yeast in two processes. Nevertheless, the taste of cider was quite acceptable.

  7. An acid/alkaline stress and the addition of amino acids induce a prolonged viability of Lactobacillus plantarum loaded into alginate gel.

    PubMed

    Bevilacqua, Antonio; Sinigaglia, Milena; Corbo, Maria Rosaria

    2010-08-15

    This study reports on the investigation on the effects of the conditions used throughout the step of biomass production on the survival of Lactobacillus plantarum loaded into alginate gels. L. plantarum was grown under different conditions (MRS or a laboratory medium-LB(2)-at acidic or alkaline pHs, with NaCl, phenols, vitamins or amino acids) and immobilized in sodium alginate; cell number was evaluated throughout the storage and death (delta(stand)) and first-reduction times (delta) were calculated. The storage of alginate gels at 4 degrees C prolonged cell viability up to 60 days (ca. 20 days for cells produced in MRS and stored at 30 degrees C); however, a similar prolongation was achieved for cells produced in LB(2) adjusted to pH 5.0 and 9.0 or added with amino acids (death time>50-60 days).

  8. Progress on implantable biofuel cell: Nano-carbon functionalization for enzyme immobilization enhancement.

    PubMed

    Babadi, Arman Amani; Bagheri, Samira; Hamid, Sharifah Bee Abdul

    2016-05-15

    Biofuel cells are bio-electrochemical devices, which are suitable for the environmentally friendly generation of energy. Enzymatic biofuel cell (EBFC) operates at ambient temperature and pH. Biofuel cells utilize vegetable and animal fluids (e.g. glucose) as a biofuel to produce energy. Fundamental part of each Glucose biofuel cell (GBFC) is two bioelectrodes which their surface utilizes as an enzyme immobilized site. Glucose oxidase (GOx) or glucose dehydrogenase (GDH) were immobilized on bioanode and oxidize glucose while oxygen reduced in biocathode using immobilized laccase or bilirubin oxidase in order to generate sufficient power. Glucose biofuel cells are capable to generate sufficient power for implanted devices. The key step of manufacturing a bioelectrode is the effective enzyme immobilization on the electrode surface. Due to the thin diameter of carbon nanomaterials, which make them accessible to the enzyme active sites, they are applicable materials to establish electronic communication with redox enzymes. Carbon nanomaterials regenerate the biocatalysts either by direct electron transfer or redox mediators which serve as intermediated for the electron transfer. Nano-carbon functionalization is perfectly compatible with other chemical or biological approaches to enhance the enzyme functions in implantable biofuel cells. Efficient immobilization of enzyme using the functionalized nano-carbon materials is the key point that greatly increases the possibilities of success. Current review highlights the progress on implantable biofuel cell, with focus on the nano-carbon functionalization for enzyme immobilization enhancement in glucose/O2 biofuel cells. PMID:26785309

  9. “Fish-in-Net”, a Novel Method for Cell Immobilization of Zymomonas mobilis

    PubMed Central

    Niu, Xuedun; Wang, Zhi; Li, Yang; Zhao, Zijian; Liu, Jiayin; Jiang, Li; Xu, Haoran; Li, Zhengqiang

    2013-01-01

    Background Inorganic mesoporous materials exhibit good biocompatibility and hydrothermal stability for cell immobilization. However, it is difficult to encapsulate living cells under mild conditions, and new strategies for cell immobilization are needed. We designed a “fish-in-net” approach for encapsulation of enzymes in ordered mesoporous silica under mild conditions. The main objective of this study is to demonstrate the potential of this approach in immobilization of living cells. Methodology/Principal Findings Zymomonas mobilis cells were encapsulated in mesoporous silica-based materials under mild conditions by using a “fish-in-net” approach. During the encapsulation process, polyethyleneglycol was used as an additive to improve the immobilization efficiency. After encapsulation, the pore size, morphology and other features were characterized by various methods, including scanning electron microscopy, nitrogen adsorption-desorption analysis, transmission electron microscopy, fourier transform infrared spectroscopy, and elemental analysis. Furthermore, the capacity of ethanol production by immobilized Zymomonas mobilis and free Zymomonas mobilis was compared. Conclusions/Significance In this study, Zymomonas mobilis cells were successfully encapsulated in mesoporous silica-based materials under mild conditions by the “fish-in-net” approach. Encapsulated cells could perform normal metabolism and exhibited excellent reusability. The results presented here illustrate the enormous potential of the “fish-in-net” approach for immobilization of living cells. PMID:24236145

  10. Human embryonic stem cell encapsulation in alginate microbeads in macroporous calcium phosphate cement for bone tissue engineering.

    PubMed

    Tang, M; Chen, W; Weir, M D; Thein-Han, W; Xu, H H K

    2012-09-01

    Human embryonic stem cells (hESC) are promising for use in regenerative medicine applications because of their strong proliferative ability and multilineage differentiation capability. To date there have been no reports on hESC seeding with calcium phosphate cement (CPC). The objective of this study was to investigate hESC-derived mesenchymal stem cell (hESCd-MSC) encapsulation in hydrogel microbeads in macroporous CPC for bone tissue engineering. hESC were cultured to form embryoid bodies (EB), and the MSC were then migrated out of the EB. hESCd-MSC had surface markers characteristic of MSC, with positive alkaline phosphatase (ALP) staining when cultured in osteogenic medium. hESCd-MSC were encapsulated in alginate at a density of 1millioncellsml(-1), with an average microbead size of 207μm. CPC contained mannitol porogen to create a porosity of 64% and 218-μm macropores, with 20% absorbable fibers for additional porosity when the fibers degrade. hESCd-MSC encapsulated in microbeads in CPC had good viability from 1 to 21days. ALP gene expression at 21days was 25-fold that at 1day. Osteocalcin (OC) at 21days was two orders of magnitude of that at 1day. ALP activity in colorimetric p-nitrophenyl phosphate assay at 21days was fivefold that at 1day. Mineral synthesis by the encapsulated hESCd-MSC at 21days was sevenfold that at 1day. Potential benefits of the CPC-stem cell paste include injectability, intimate adaptation to complex-shaped bone defects, ease in contouring to achieve esthetics in maxillofacial repairs, and in situ setting ability. In conclusion, hESCd-MSC were encapsulated in alginate microbeads in macroporous CPC, showing good cell viability, osteogenic differentiation and mineral synthesis for the first time. The hESCd-MSC-encapsulating macroporous CPC construct is promising for bone regeneration in a wide range of orthopedic and maxillofacial applications.

  11. DETOXIFICATION OF ORGANOPHOSPHATE PESTICIDES BY IMMOBILIZED ESCHERICHIA COLI EXPRESSING ORGANOPHOSPHORUS HYDROLASE ON CELL SURFACE. (R823663)

    EPA Science Inventory

    An improved whole-cell technology for detoxifying organophosphate nerve agents was recently developed based on genetically engineered Escherichia coli with organophosphorus hydrolase anchored on the surface. This article reports the immobilization of these novel biocatalys...

  12. Analysis of secondary cells with lithium anodes and immobilized fused-salt electrolytes

    NASA Technical Reports Server (NTRS)

    Cairns, E. J.; Rogers, G. L.; Shimotake, H.

    1969-01-01

    Secondary cells with liquid lithium anodes, liquid bismuth or tellurium cathodes, and fused lithium halide electrolytes immobilized as rigid pastes operate between 380 and 485 degrees. Applications include power sources in space, military vehicle propulsion and special commercial vehicle propulsion.

  13. Microwave-synthesized magnetic chitosan microparticles for the immobilization of yeast cells.

    PubMed

    Safarik, Ivo; Pospiskova, Kristyna; Maderova, Zdenka; Baldikova, Eva; Horska, Katerina; Safarikova, Mirka

    2015-01-01

    An extremely simple procedure has been developed for the immobilization of Saccharomyces cerevisiae cells on magnetic chitosan microparticles. The magnetic carrier was prepared using an inexpensive, simple, rapid, one-pot process, based on the microwave irradiation of chitosan and ferrous sulphate at high pH. Immobilized yeast cells have been used for sucrose hydrolysis, hydrogen peroxide decomposition and the adsorption of selected dyes. PMID:24753015

  14. Recent insights into the cell immobilization technology applied for dark fermentative hydrogen production.

    PubMed

    Kumar, Gopalakrishnan; Mudhoo, Ackmez; Sivagurunathan, Periyasamy; Nagarajan, Dillirani; Ghimire, Anish; Lay, Chyi-How; Lin, Chiu-Yue; Lee, Duu-Jong; Chang, Jo-Shu

    2016-11-01

    The contribution and insights of the immobilization technology in the recent years with regards to the generation of (bio)hydrogen via dark fermentation have been reviewed. The types of immobilization practices, such as entrapment, encapsulation and adsorption, are discussed. Materials and carriers used for cell immobilization are also comprehensively surveyed. New development of nano-based immobilization and nano-materials has been highlighted pertaining to the specific subject of this review. The microorganisms and the type of carbon sources applied in the dark hydrogen fermentation are also discussed and summarized. In addition, the essential components of process operation and reactor configuration using immobilized microbial cultures in the design of varieties of bioreactors (such as fixed bed reactor, CSTR and UASB) are spotlighted. Finally, suggestions and future directions of this field are provided to assist the development of efficient, economical and sustainable hydrogen production technologies. PMID:27561626

  15. Recent insights into the cell immobilization technology applied for dark fermentative hydrogen production.

    PubMed

    Kumar, Gopalakrishnan; Mudhoo, Ackmez; Sivagurunathan, Periyasamy; Nagarajan, Dillirani; Ghimire, Anish; Lay, Chyi-How; Lin, Chiu-Yue; Lee, Duu-Jong; Chang, Jo-Shu

    2016-11-01

    The contribution and insights of the immobilization technology in the recent years with regards to the generation of (bio)hydrogen via dark fermentation have been reviewed. The types of immobilization practices, such as entrapment, encapsulation and adsorption, are discussed. Materials and carriers used for cell immobilization are also comprehensively surveyed. New development of nano-based immobilization and nano-materials has been highlighted pertaining to the specific subject of this review. The microorganisms and the type of carbon sources applied in the dark hydrogen fermentation are also discussed and summarized. In addition, the essential components of process operation and reactor configuration using immobilized microbial cultures in the design of varieties of bioreactors (such as fixed bed reactor, CSTR and UASB) are spotlighted. Finally, suggestions and future directions of this field are provided to assist the development of efficient, economical and sustainable hydrogen production technologies.

  16. Fabrication, characterization and in vitro profile based interaction with eukaryotic and prokaryotic cells of alginate-chitosan-silica biocomposite.

    PubMed

    Balaure, Paul Catalin; Andronescu, Ecaterina; Grumezescu, Alexandru Mihai; Ficai, Anton; Huang, Keng-Shiang; Yang, Chih-Hui; Chifiriuc, Carmen Mariana; Lin, Yung-Sheng

    2013-01-30

    This work is focused on the fabrication of a new drug delivery system based on polyanionic matrix (e.g. sodium alginate), polycationic matrix (e.g. chitosan) and silica network. The FT-IR, SEM, DTA-TG, eukaryotic cell cycle and viability, and in vitro assay of the influence of the biocomposite on the efficacy of antibiotic drugs were investigated. The obtained results demonstrated the biocompatibility and the ability of the fabricated biocomposite to maintain or improve the efficacy of the following antibiotics: piperacillin-tazobactam, cefepime, piperacillin, imipenem, gentamicin, ceftazidime against Pseudomonas aeruginosa ATCC 27853 and cefazolin, cefaclor, cefuroxime, ceftriaxone, cefoxitin, trimethoprim/sulfamethoxazole against Escherichia coli ATCC 25922 reference strains.

  17. Fabrication, characterization and in vitro profile based interaction with eukaryotic and prokaryotic cells of alginate-chitosan-silica biocomposite.

    PubMed

    Balaure, Paul Catalin; Andronescu, Ecaterina; Grumezescu, Alexandru Mihai; Ficai, Anton; Huang, Keng-Shiang; Yang, Chih-Hui; Chifiriuc, Carmen Mariana; Lin, Yung-Sheng

    2013-01-30

    This work is focused on the fabrication of a new drug delivery system based on polyanionic matrix (e.g. sodium alginate), polycationic matrix (e.g. chitosan) and silica network. The FT-IR, SEM, DTA-TG, eukaryotic cell cycle and viability, and in vitro assay of the influence of the biocomposite on the efficacy of antibiotic drugs were investigated. The obtained results demonstrated the biocompatibility and the ability of the fabricated biocomposite to maintain or improve the efficacy of the following antibiotics: piperacillin-tazobactam, cefepime, piperacillin, imipenem, gentamicin, ceftazidime against Pseudomonas aeruginosa ATCC 27853 and cefazolin, cefaclor, cefuroxime, ceftriaxone, cefoxitin, trimethoprim/sulfamethoxazole against Escherichia coli ATCC 25922 reference strains. PMID:23178215

  18. Bone regeneration potential of stem cells derived from periodontal ligament or gingival tissue sources encapsulated in RGD-modified alginate scaffold.

    PubMed

    Moshaverinia, Alireza; Chen, Chider; Xu, Xingtian; Akiyama, Kentaro; Ansari, Sahar; Zadeh, Homayoun H; Shi, Songtao

    2014-02-01

    Mesenchymal stem cells (MSCs) provide an advantageous alternative therapeutic option for bone regeneration in comparison to current treatment modalities. However, delivering MSCs to the defect site while maintaining a high MSC survival rate is still a critical challenge in MSC-mediated bone regeneration. Here, we tested the bone regeneration capacity of periodontal ligament stem cells (PDLSCs) and gingival mesenchymal stem cells (GMSCs) encapsulated in a novel RGD- (arginine-glycine-aspartic acid tripeptide) coupled alginate microencapsulation system in vitro and in vivo. Five-millimeter-diameter critical-size calvarial defects were created in immunocompromised mice and PDLSCs and GMSCs encapsulated in RGD-modified alginate microspheres were transplanted into the defect sites. New bone formation was assessed using microcomputed tomography and histological analyses 8 weeks after transplantation. Results confirmed that our microencapsulation system significantly enhanced MSC viability and osteogenic differentiation in vitro compared with non-RGD-containing alginate hydrogel microspheres with larger diameters. Results confirmed that PDLSCs were able to repair the calvarial defects by promoting the formation of mineralized tissue, while GMSCs showed significantly lower osteogenic differentiation capability. Further, results revealed that RGD-coupled alginate scaffold facilitated the differentiation of oral MSCs toward an osteoblast lineage in vitro and in vivo, as assessed by expression of osteogenic markers Runx2, ALP, and osteocalcin. In conclusion, these results for the first time demonstrated that MSCs derived from orofacial tissue encapsulated in RGD-modified alginate scaffold show promise for craniofacial bone regeneration. This treatment modality has many potential dental and orthopedic applications. PMID:24070211

  19. Bone Regeneration Potential of Stem Cells Derived from Periodontal Ligament or Gingival Tissue Sources Encapsulated in RGD-Modified Alginate Scaffold

    PubMed Central

    Chen, Chider; Xu, Xingtian; Akiyama, Kentaro; Ansari, Sahar; Zadeh, Homayoun H.; Shi, Songtao

    2014-01-01

    Mesenchymal stem cells (MSCs) provide an advantageous alternative therapeutic option for bone regeneration in comparison to current treatment modalities. However, delivering MSCs to the defect site while maintaining a high MSC survival rate is still a critical challenge in MSC-mediated bone regeneration. Here, we tested the bone regeneration capacity of periodontal ligament stem cells (PDLSCs) and gingival mesenchymal stem cells (GMSCs) encapsulated in a novel RGD- (arginine-glycine-aspartic acid tripeptide) coupled alginate microencapsulation system in vitro and in vivo. Five-millimeter-diameter critical-size calvarial defects were created in immunocompromised mice and PDLSCs and GMSCs encapsulated in RGD-modified alginate microspheres were transplanted into the defect sites. New bone formation was assessed using microcomputed tomography and histological analyses 8 weeks after transplantation. Results confirmed that our microencapsulation system significantly enhanced MSC viability and osteogenic differentiation in vitro compared with non-RGD-containing alginate hydrogel microspheres with larger diameters. Results confirmed that PDLSCs were able to repair the calvarial defects by promoting the formation of mineralized tissue, while GMSCs showed significantly lower osteogenic differentiation capability. Further, results revealed that RGD-coupled alginate scaffold facilitated the differentiation of oral MSCs toward an osteoblast lineage in vitro and in vivo, as assessed by expression of osteogenic markers Runx2, ALP, and osteocalcin. In conclusion, these results for the first time demonstrated that MSCs derived from orofacial tissue encapsulated in RGD-modified alginate scaffold show promise for craniofacial bone regeneration. This treatment modality has many potential dental and orthopedic applications. PMID:24070211

  20. Engineering cholesterol-based fibers for antibody immobilization and cell capture

    NASA Astrophysics Data System (ADS)

    Cohn, Celine

    In 2015, the United States is expected to have nearly 600,000 deaths attributed to cancer. Of these 600,000 deaths, 90% will be a direct result of cancer metastasis, the spread of cancer throughout the body. During cancer metastasis, circulating tumor cells (CTCs) are shed from primary tumors and migrate through bodily fluids, establishing secondary cancer sites. As cancer metastasis is incredibly lethal, there is a growing emphasis on developing "liquid biopsies" that can screen peripheral blood, search for and identify CTCs. One popular method for capturing CTCs is the use of a detection platform with antibodies specifically suited to recognize and capture cancer cells. These antibodies are immobilized onto the platform and can then bind and capture cells of interest. However, current means to immobilize antibodies often leave them with drastically reduced function. The antibodies are left poorly suited for cell capture, resulting in low cell capture efficiencies. This body of work investigates the use of lipid-based fibers to immobilize proteins in a way that retains protein function, ultimately leading to increased cell capture efficiencies. The resulting increased efficiencies are thought to arise from the retained three-dimensional structure of the protein as well as having a complete coating of the material surface with antibodies that are capable of interacting with their antigens. It is possible to electrospin cholesterol-based fibers that are similar in design to the natural cell membrane, providing proteins a more natural setting during immobilization. Such fibers have been produced from cholesterol-based cholesteryl succinyl silane (CSS). These fibers have previously illustrated a keen aptitude for retaining protein function and increasing cell capture. Herein the work focuses on three key concepts. First, a model is developed to understand the immobilization mechanism used by electrospun CSS fibers. The antibody immobilization and cell capturing

  1. Sustained Delivery of Bioactive GDNF from Collagen and Alginate-Based Cell-Encapsulating Gel Promoted Photoreceptor Survival in an Inherited Retinal Degeneration Model.

    PubMed

    Wong, Francisca S Y; Wong, Calvin C H; Chan, Barbara P; Lo, Amy C Y

    2016-01-01

    Encapsulated-cell therapy (ECT) is an attractive approach for continuously delivering freshly synthesized therapeutics to treat sight-threatening posterior eye diseases, circumventing repeated invasive intravitreal injections and improving local drug availability clinically. Composite collagen-alginate (CAC) scaffold contains an interpenetrating network that integrates the physical and biological merits of its constituents, including biocompatibility, mild gelling properties and availability. However, CAC ECT properties and performance in the eye are not well-understood. Previously, we reported a cultured 3D CAC system that supported the growth of GDNF-secreting HEK293 cells with sustainable GDNF delivery. Here, the system was further developed into an intravitreally injectable gel with 1x104 or 2x105 cells encapsulated in 2mg/ml type I collagen and 1% alginate. Gels with lower alginate concentration yielded higher initial cell viability but faster spheroid formation while increasing initial cell density encouraged cell growth. Continuous GDNF delivery was detected in culture and in healthy rat eyes for at least 14 days. The gels were well-tolerated with no host tissue attachment and contained living cell colonies. Most importantly, gel-implanted in dystrophic Royal College of Surgeons rat eyes for 28 days retained photoreceptors while those containing higher initial cell number yielded better photoreceptor survival. CAC ECT gels offers flexible system design and is a potential treatment option for posterior eye diseases. PMID:27441692

  2. Sustained Delivery of Bioactive GDNF from Collagen and Alginate-Based Cell-Encapsulating Gel Promoted Photoreceptor Survival in an Inherited Retinal Degeneration Model

    PubMed Central

    Chan, Barbara P.; Lo, Amy C. Y.

    2016-01-01

    Encapsulated-cell therapy (ECT) is an attractive approach for continuously delivering freshly synthesized therapeutics to treat sight-threatening posterior eye diseases, circumventing repeated invasive intravitreal injections and improving local drug availability clinically. Composite collagen-alginate (CAC) scaffold contains an interpenetrating network that integrates the physical and biological merits of its constituents, including biocompatibility, mild gelling properties and availability. However, CAC ECT properties and performance in the eye are not well-understood. Previously, we reported a cultured 3D CAC system that supported the growth of GDNF-secreting HEK293 cells with sustainable GDNF delivery. Here, the system was further developed into an intravitreally injectable gel with 1x104 or 2x105 cells encapsulated in 2mg/ml type I collagen and 1% alginate. Gels with lower alginate concentration yielded higher initial cell viability but faster spheroid formation while increasing initial cell density encouraged cell growth. Continuous GDNF delivery was detected in culture and in healthy rat eyes for at least 14 days. The gels were well-tolerated with no host tissue attachment and contained living cell colonies. Most importantly, gel-implanted in dystrophic Royal College of Surgeons rat eyes for 28 days retained photoreceptors while those containing higher initial cell number yielded better photoreceptor survival. CAC ECT gels offers flexible system design and is a potential treatment option for posterior eye diseases. PMID:27441692

  3. Bioconversion of l-glutamic acid to α-ketoglutaric acid by an immobilized whole-cell biocatalyst expressing l-amino acid deaminase from Proteus mirabilis.

    PubMed

    Hossain, Gazi Sakir; Li, Jianghua; Shin, Hyun-dong; Chen, Rachel R; Du, Guocheng; Liu, Long; Chen, Jian

    2014-01-01

    The goal of this work was to develop an immobilized whole-cell biocatalytic process for the environment-friendly synthesis of α-ketoglutaric acid (α-KG) from l-glutamic acid. We compared the suitability of Escherichia coli and Bacillus subtilis strains overexpressing Proteus mirabilisl-amino acid deaminase (l-AAD) as potential biocatalysts. Although both recombinant strains were biocatalytically active, the performance of B. subtilis was superior to that of E. coli. With l-glutamic acid as the substrate, α-KG production levels by membranes isolated from B. subtilis and E. coli were 55.3±1.73 and 21.7±0.39μg/mg protein/min, respectively. The maximal conversion ratio of l-glutamic acid to α-KG was 31% (w/w) under the following optimal conditions: 15g/L l-glutamic acid, 20g/L whole-cell biocatalyst, 5mM MgCl2, 40°C, pH 8.0, and 24-h incubation. Immobilization of whole cells with alginate increased the recyclability by an average of 23.33% per cycle. This work established an efficient one-step biotransformation process for the production of α-KG using immobilized whole B. subtilis overexpressing P. mirabilisl-AAD. Compared with traditional multistep chemical synthesis, the biocatalytic process described here has the advantage of reducing environmental pollution and thus has great potential for the large-scale production of α-KG.

  4. Study of Carbon Nano-Tubes Effects on the Chondrogenesis of Human Adipose Derived Stem Cells in Alginate Scaffold

    PubMed Central

    Valiani, Ali; Hashemibeni, Batool; Esfandiary, Ebrahim; Ansar, Malek Masoud; Kazemi, Mohammad; Esmaeili, Nafiseh

    2014-01-01

    Background: Osteoarthritis is one of the most common diseases in middle-aged populations in the World and could become the fourth principal cause of disability by the year 2020. One of the critical properties for cartilage tissue engineering (TE) is the ability of scaffolds to closely mimic the extracellular matrix and bond to the host tissue. Therefore, TE has been presented as a technique to introduce the best combination of cells and biomaterial scaffold and to stimulate growth factors to produce a cartilage tissue resembling natural articular cartilage. The aim of study is to improve differentiation of adipose derived stem cells (ADSCs) into chondrocytes in order to provide a safe and modern treatment for patients suffering from cartilage damages. Methods: After functionalization, dispersions and sterilizing carbon nano-tubes (CNTs), a new type of nanocomposite gel was prepared from water-soluble CNTs and alginate. ADSCs seeded in 1.5% alginate scaffold and cultured in chondrogenic media with and without transforming growth factor-β1 (TGF-β1) for 7 and 14 days. The genes expression of sex determining region Y-box 9 (SOX9), types II and X collagens was assessed by real-time polymerase chain reaction and the amount of aggrecan (AGC) and type I collagen was measured by ELISA. Results: Our findings showed that the expression of essential cartilage markers, SOX9, type II collagen and AGC, in differentiated ADSCs at the concentration of 1 μg/ml CNTs in the presence of TGF-β1 were significantly increased in comparison with the control group (P < 0.001). Meanwhile, type X collagen expression and also type I collagen production were significantly decreased (P < 0.001). Conclusions: The results showed that utilized three-dimensional scaffold had a brilliant effect in promoting gene expression of chondrogenesis. PMID:25104993

  5. Comparing different methods to fix and to dehydrate cells on alginate hydrogel scaffolds using scanning electron microscopy.

    PubMed

    Santana, Bianca Palma; Nedel, Fernanda; Perelló Ferrúa, Camila; Marques e Silva, Ricardo; da Silva, Adriana Fernandes; Demarco, Flávio Fernando; Lenin Villarreal Carreño, Neftali

    2015-07-01

    Scanning electron microscopy (SEM) is commonly used in the analysis of scaffolds morphology, as well as cell attachment, morphology and spreading on to the scaffolds. However, so far a specific methodology to prepare the alginate hydrogel (AH) scaffolds for SEM analysis has not been evaluated. This study compared different methods to fix/dehydrate cells in AH scaffolds for SEM analysis. AH scaffolds were prepared and seeded with NIH/3T3 cell line; fixed with glutaraldehyde, osmium tetroxide, or the freeze drying method and analyzed by SEM. Results demonstrated that the freeze dried method interferes less with cell morphology and density, and preserves the scaffolds structure. The fixation with glutaraldehyde did not affect cells morphology and density; however, the scaffolds morphology was affected in some level. The fixation with osmium tetroxide interfered in the natural structure of cells and scaffold. In conclusion the freeze drying and glutaraldehyde are suitable methods for cell fixation in AH scaffold for SEM, although scaffolds structure seems to be affected by glutaraldehyde.

  6. Comparing different methods to fix and to dehydrate cells on alginate hydrogel scaffolds using scanning electron microscopy.

    PubMed

    Santana, Bianca Palma; Nedel, Fernanda; Perelló Ferrúa, Camila; Marques e Silva, Ricardo; da Silva, Adriana Fernandes; Demarco, Flávio Fernando; Lenin Villarreal Carreño, Neftali

    2015-07-01

    Scanning electron microscopy (SEM) is commonly used in the analysis of scaffolds morphology, as well as cell attachment, morphology and spreading on to the scaffolds. However, so far a specific methodology to prepare the alginate hydrogel (AH) scaffolds for SEM analysis has not been evaluated. This study compared different methods to fix/dehydrate cells in AH scaffolds for SEM analysis. AH scaffolds were prepared and seeded with NIH/3T3 cell line; fixed with glutaraldehyde, osmium tetroxide, or the freeze drying method and analyzed by SEM. Results demonstrated that the freeze dried method interferes less with cell morphology and density, and preserves the scaffolds structure. The fixation with glutaraldehyde did not affect cells morphology and density; however, the scaffolds morphology was affected in some level. The fixation with osmium tetroxide interfered in the natural structure of cells and scaffold. In conclusion the freeze drying and glutaraldehyde are suitable methods for cell fixation in AH scaffold for SEM, although scaffolds structure seems to be affected by glutaraldehyde. PMID:25871651

  7. [Study on immobilized cells for producing alpha-amylase by using polyving alcohol as the carrier(II): The effect of fermentating conditions on the ability producing alpha-amylase of the cells immobilized with polyving alcohol as the corrier and continuous fermentation of the immobilized cells in CSTR].

    PubMed

    Liu, Z; Wang, J; Li, Z

    1998-03-01

    The effects of fermentating conditions on the ability of immobilized cells with PVA as carrier for producing alpha-amylase were studied. The continuous fermentation with the immobilized cells were tested in continuous flow stirred tank reactor (CSTR). The results showed that the adaptability of the immobilized Bacillus substilis to pH increased after immobilization. In CSTR, the immobilized cells can be fermentated continuously for 360 hrs and the activity of alpha-amylase can be kept on the level of about 170 u/ml.

  8. Role of Calcium Alginate and Mannitol in Protecting Bifidobacterium

    PubMed Central

    Dianawati, Dianawati; Mishra, Vijay

    2012-01-01

    Fourier transform infrared (FTIR) spectroscopy was carried out to ascertain the mechanism of Ca-alginate and mannitol protection of cell envelope components and secondary proteins of Bifidobacterium animalis subsp. lactis Bb12 after freeze-drying and after 10 weeks of storage at room temperature (25°C) at low water activities (aw) of 0.07, 0.1, and 0.2. Preparation of Ca-alginate and Ca-alginate-mannitol as microencapsulants was carried out by dropping an alginate or alginate-mannitol emulsion containing bacteria using a burette into CaCl2 solution to obtain Ca-alginate beads and Ca-alginate-mannitol beads, respectively. The wet beads were then freeze-dried. The aw of freeze-dried beads was then adjusted to 0.07, 0.1, and 0.2 using saturated salt solutions; controls were prepared by keeping Ca-alginate and Ca-alginate-mannitol in aluminum foil without aw adjustment. Mannitol in the Ca-alginate system interacted with cell envelopes during freeze-drying and during storage at low aws. In contrast, Ca-alginate protected cell envelopes after freeze-drying but not during 10-week storage. Unlike Ca-alginate, Ca-alginate-mannitol was effective in retarding the changes in secondary proteins during freeze-drying and during 10 weeks of storage at low aws. It appears that Ca-alginate-mannitol is more effective than Ca-alginate in preserving cell envelopes and proteins after freeze-drying and after 10 weeks of storage at room temperature (25°C). PMID:22843535

  9. Role of calcium alginate and mannitol in protecting Bifidobacterium.

    PubMed

    Dianawati, Dianawati; Mishra, Vijay; Shah, Nagendra P

    2012-10-01

    Fourier transform infrared (FTIR) spectroscopy was carried out to ascertain the mechanism of Ca-alginate and mannitol protection of cell envelope components and secondary proteins of Bifidobacterium animalis subsp. lactis Bb12 after freeze-drying and after 10 weeks of storage at room temperature (25°C) at low water activities (a(w)) of 0.07, 0.1, and 0.2. Preparation of Ca-alginate and Ca-alginate-mannitol as microencapsulants was carried out by dropping an alginate or alginate-mannitol emulsion containing bacteria using a burette into CaCl(2) solution to obtain Ca-alginate beads and Ca-alginate-mannitol beads, respectively. The wet beads were then freeze-dried. The a(w) of freeze-dried beads was then adjusted to 0.07, 0.1, and 0.2 using saturated salt solutions; controls were prepared by keeping Ca-alginate and Ca-alginate-mannitol in aluminum foil without a(w) adjustment. Mannitol in the Ca-alginate system interacted with cell envelopes during freeze-drying and during storage at low a(w)s. In contrast, Ca-alginate protected cell envelopes after freeze-drying but not during 10-week storage. Unlike Ca-alginate, Ca-alginate-mannitol was effective in retarding the changes in secondary proteins during freeze-drying and during 10 weeks of storage at low a(w)s. It appears that Ca-alginate-mannitol is more effective than Ca-alginate in preserving cell envelopes and proteins after freeze-drying and after 10 weeks of storage at room temperature (25°C). PMID:22843535

  10. Cells (MC3T3-E1)-laden alginate scaffolds fabricated by a modified solid-freeform fabrication process supplemented with an aerosol spraying.

    PubMed

    Ahn, SeungHyun; Lee, HyeongJin; Bonassar, Lawrence J; Kim, GeunHyung

    2012-09-10

    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.

  11. Proliferation and enrichment of CD133(+) glioblastoma cancer stem cells on 3D chitosan-alginate scaffolds.

    PubMed

    Kievit, Forrest M; Florczyk, Stephen J; Leung, Matthew C; Wang, Kui; Wu, Jennifer D; Silber, John R; Ellenbogen, Richard G; Lee, Jerry S H; Zhang, Miqin

    2014-11-01

    Emerging evidence implicates cancer stem cells (CSCs) as primary determinants of the clinical behavior of human cancers, representing an ideal target for next-generation anti-cancer therapies. However CSCs are difficult to propagate in vitro, severely limiting the study of CSC biology and drug development. Here we report that growing cells from glioblastoma (GBM) cell lines on three dimensional (3D) porous chitosan-alginate (CA) scaffolds dramatically promotes the proliferation and enrichment of cells possessing the hallmarks of CSCs. CA scaffold-grown cells were found more tumorigenic in nude mouse xenografts than cells grown from monolayers. Growing in CA scaffolds rapidly promoted expression of genes involved in the epithelial-to-mesenchymal transition that has been implicated in the genesis of CSCs. Our results indicate that CA scaffolds have utility as a simple and inexpensive means to cultivate CSCs in vitro in support of studies to understand CSC biology and develop more effective anti-cancer therapies.

  12. Gelling concept combining chitosan and alginate-proof of principle.

    PubMed

    Khong, Thang Trung; Aarstad, Olav A; Skjåk-Bræk, Gudmund; Draget, Kurt I; Vårum, Kjell M

    2013-08-12

    Biocompatible hydrogels are very interesting for applications in, e.g., tissue engineering and for immobilization of cells, such as calcium-alginate gels where the calcium ions form specific interactions with the guluronic acid units. We here report on a new gelling system of chitosan and alginate containing only mannuronic acid (poly-M), which are prepared using the following steps: (i) mixing at a pH well above 7 where the chitosan is mainly uncharged; (ii) controlled lowering of the pH by adding the slowly hydrolyzing d-glucono-δ-lactone (GDL); (iii) formation of a homogeneous chitosan-alginate gel upon leaving the mixture at room temperature. Some properties of the new gelling system are demonstrated herein by adding controlled amounts of GDL to (i) a mixture of a polymeric and neutral-soluble chitosan with poly-M oligomers (MO) and (ii) a mixture of poly-M and neutral-soluble chitosan oligomers. The neutral-solubility of the polymeric chitosan is achieved by selecting a polymeric chitosan with an intermediate degree of acetylation of 40%, while the neutral-solubility of the fully de-N-acetylated chitosan oligomers (CO) is obtained by selecting oligomers with a chain length below 10. A proof of concept of the new gelling system is demonstrated by measuring the gel strengths of the polymeric chitosan-MO, and a poly-M-CO. The results show that the gel strength increases with decreasing the pH from neutral to 5, and that the gel strength decreases with increasing ionic strength, indicative of an ionic gel formation. Poly-M formed relatively strong gels with CO while an alginate highly enriched in Guluronic acid formed gels of very limited mechanical strength, suggesting the importance of the match in charge distances in the poly-M and chitosan, both with diequatorially linked sugar units in the (4)C1 conformation.

  13. Engineering cholesterol-based fibers for antibody immobilization and cell capture

    NASA Astrophysics Data System (ADS)

    Cohn, Celine

    In 2015, the United States is expected to have nearly 600,000 deaths attributed to cancer. Of these 600,000 deaths, 90% will be a direct result of cancer metastasis, the spread of cancer throughout the body. During cancer metastasis, circulating tumor cells (CTCs) are shed from primary tumors and migrate through bodily fluids, establishing secondary cancer sites. As cancer metastasis is incredibly lethal, there is a growing emphasis on developing "liquid biopsies" that can screen peripheral blood, search for and identify CTCs. One popular method for capturing CTCs is the use of a detection platform with antibodies specifically suited to recognize and capture cancer cells. These antibodies are immobilized onto the platform and can then bind and capture cells of interest. However, current means to immobilize antibodies often leave them with drastically reduced function. The antibodies are left poorly suited for cell capture, resulting in low cell capture efficiencies. This body of work investigates the use of lipid-based fibers to immobilize proteins in a way that retains protein function, ultimately leading to increased cell capture efficiencies. The resulting increased efficiencies are thought to arise from the retained three-dimensional structure of the protein as well as having a complete coating of the material surface with antibodies that are capable of interacting with their antigens. It is possible to electrospin cholesterol-based fibers that are similar in design to the natural cell membrane, providing proteins a more natural setting during immobilization. Such fibers have been produced from cholesterol-based cholesteryl succinyl silane (CSS). These fibers have previously illustrated a keen aptitude for retaining protein function and increasing cell capture. Herein the work focuses on three key concepts. First, a model is developed to understand the immobilization mechanism used by electrospun CSS fibers. The antibody immobilization and cell capturing

  14. Detection of bioavailable heavy metals in EILATox-Oregon samples using whole-cell luminescent bacterial sensors in suspension or immobilized onto fibre-optic tips.

    PubMed

    Hakkila, Kaisa; Green, Tal; Leskinen, Piia; Ivask, Angela; Marks, Robert; Virta, Marko

    2004-01-01

    At the EILATox-Oregon Workshop, nine luminescent whole-cell bacterial sensors were used for the determination of bioavailable metals in blind samples (17 synthetic and 3 environmental). A non-inducible luminescent control strain was used to determine sample matrix effects and bacterial toxicity. Whole-cell bacterial sensors capable of determining arsenic, inorganic mercury and its organic derivatives, cadmium, lead or copper were used in suspensions and a bacterial sensor for the detection of inorganic mercury was immobilized onto fibre-optic tips using calcium alginate. Bioavailable amounts of metals were estimated using calibration plots, that were constructed to determine the range of metals giving rise to a linear relationship between luminescence and the amount of metals present in the standard solutions. EILATox-Oregon sample 5, which contained 74 mg l(-1) of Hg, gave a significant response with both formats of the mercury sensor. The bioavailable amounts of mercury according to the measurement of bacterial sensor in suspension and immobilized onto a fibre-optic tip were 76 and 93 mg l(-1), respectively. The bacterial sensor for arsenic and copper showed a response with sample 6 (58 mg l(-1) of As) and sample 8 (400 mg l(-1) of metham sodium), respectively. This study showed that the bacterial sensors in suspension or immobilized onto optical fibres are capable of quantifying bioavailable metals from unknown samples. The measurement protocol of bacterial sensors is simple and possible to perform in the field. Moreover, the samples do not need any pretreatment before analysis. Construction and characterization of the strain for the detection of bioavailable copper are described.

  15. Viability, ultrastructure and cytokinin metabolism of free and immobilized tobacco chloroplasts.

    PubMed

    Polanská, Lenka; Vicánková, Anna; Dobrev, Petre I; Cková, Ivana Macháv; Vanková, Radomíra

    2004-10-01

    Cytokinins play a decisive role in regulation of plastid development and differentiation, but their metabolism in plastids is not known. Metabolic studies using intact chloroplasts are prevented by their instability once they are isolated from leaf cells. Chloroplasts of Nicotiana tabacum L. cv. Petit Havana SR1 were therefore immobilized into low-viscosity alginate. Their intactness was assessed by a glyceraldehyde-3-phosphate dehydrogenase assay which indicated that free chloroplasts totally disintegrated within 7 h, while more than 50% of immobilized chloroplasts remained intact after 24 h. The immobilization had no marked impact on ultrastructure and postponed final destruction. The metabolite profile was similar in free and immobilized chloroplasts after 4 h incubation with tritiated zeatin. Nevertheless, the yield of conversion products decreased twice in immobilized chloroplasts, which was probably the outcome of mass transfer limitations and/or the sorption to polysaccharide matrix. PMID:15604795

  16. Intensified nitrogen removal in immobilized nitrifier enhanced constructed wetlands with external carbon addition.

    PubMed

    Wang, Wei; Ding, Yi; Wang, Yuhui; Song, Xinshan; Ambrose, Richard F; Ullman, Jeffrey L

    2016-10-01

    Nitrogen removal performance response of twelve constructed wetlands (CWs) to immobilized nitrifier pellets and different influent COD/N ratios (chemical oxygen demand: total nitrogen in influent) were investigated via 7-month experiments. Nitrifier was immobilized on a carrier pellet containing 10% polyvinyl alcohol (PVA), 2.0% sodium alginate (SA) and 2.0% calcium chloride (CaCl2). A batch experiment demonstrated that 73% COD and 85% ammonia nitrogen (NH4-N) were degraded using the pellets with immobilized nitrifier cells. In addition, different carbon source supplement strategies were applied to remove the nitrate (NO3-N) transformed from NH4-N. An increase in COD/N ratio led to increasing reduction in NO3-N. Efficient nitrification and denitrification promoted total nitrogen (TN) removal in immobilized nitrifier biofortified constructed wetlands (INB-CWs). The results suggested that immobilized nitrifier pellets combined with high influent COD/N ratios could effectively improve the nitrogen removal performance in CWs.

  17. Cell-on-hydrogel platform made of agar and alginate for rapid, low-cost, multidimensional test of antimicrobial susceptibility.

    PubMed

    Sun, Han; Liu, Zhengzhi; Hu, Chong; Ren, Kangning

    2016-08-01

    Antimicrobial resistance (AMR) is a rapidly increasing threat to the effective treatment of infectious diseases worldwide. The two major remedies include: (1) using narrow-spectrum antibiotics based on rapid diagnosis; and (2) developing new antibiotics. A key part of both remedies is the antimicrobial susceptibility test (AST). However, the current standard ASTs that monitor colony formation are costly and time-consuming and the new strategies proposed are not yet practical to be implemented. Herein, we report a strategy to fabricate whole-hydrogel microfluidic chips using alginate-doped agar. This agar-based microfabrication makes it possible to prepare inexpensive hydrogel devices, and allows a seamless link between microfluidics and conventional agar-based cell culture. Different from common microfluidic systems, in our system the cells are cultured on top of the device, similar to normal agar plate culture; on the other hand, the microfluidic channels inside the hydrogel allow precise generation of linear gradient of drugs, thus giving a better performance than the conventional disk diffusion method. Cells in this system are not exposed to any shear flow, which allows the reliable tracking of individual cells and AST results to be obtained within 2-3 hours. Furthermore, our system could test the synergistic effect of drugs through two-dimensional gradient generation. Finally, the platform could be directly implemented to new drug discovery and other applications wherein a fast, cost-efficient method for studying the response of microorganisms upon drug administration is desirable. PMID:27452345

  18. Alginate cryogel based glucose biosensor

    NASA Astrophysics Data System (ADS)

    Fatoni, Amin; Windy Dwiasi, Dian; Hermawan, Dadan

    2016-02-01

    Cryogel is macroporous structure provides a large surface area for biomolecule immobilization. In this work, an alginate cryogel based biosensor was developed to detect glucose. The cryogel was prepared using alginate cross-linked by calcium chloride under sub-zero temperature. This porous structure was growth in a 100 μL micropipette tip with a glucose oxidase enzyme entrapped inside the cryogel. The glucose detection was based on the colour change of redox indicator, potassium permanganate, by the hydrogen peroxide resulted from the conversion of glucose. The result showed a porous structure of alginate cryogel with pores diameter of 20-50 μm. The developed glucose biosensor was showed a linear response in the glucose detection from 1.0 to 5.0 mM with a regression of y = 0.01x+0.02 and R2 of 0.994. Furthermore, the glucose biosensor was showed a high operational stability up to 10 times of uninterrupted glucose detections.

  19. Development of functionalized multi-walled carbon-nanotube-based alginate hydrogels for enabling biomimetic technologies

    NASA Astrophysics Data System (ADS)

    Joddar, Binata; Garcia, Eduardo; Casas, Atzimba; Stewart, Calvin M.

    2016-08-01

    Alginate is a hydrogel commonly used for cell culture by ionically crosslinking in the presence of divalent Ca2+ ions. However these alginate gels are mechanically unstable, not permitting their use as scaffolds to engineer robust biological bone, breast, cardiac or tumor tissues. This issue can be addressed via encapsulation of multi-walled carbon nanotubes (MWCNT) serving as a reinforcing phase while being dispersed in a continuous phase of alginate. We hypothesized that adding functionalized MWCNT to alginate, would yield composite gels with distinctively different mechanical, physical and biological characteristics in comparison to alginate alone. Resultant MWCNT-alginate gels were porous, and showed significantly less degradation after 14 days compared to alginate alone. In vitro cell-studies showed enhanced HeLa cell adhesion and proliferation on the MWCNT-alginate compared to alginate. The extent of cell proliferation was greater when cultured atop 1 and 3 mg/ml MWCNT-alginate; although all MWCNT-alginates lead to enhanced cell cluster formation compared to alginate alone. Among all the MWCNT-alginates, the 1 mg/ml gels showed significantly greater stiffness compared to all other cases. These results provide an important basis for the development of the MWCNT-alginates as novel substrates for cell culture applications, cell therapy and tissue engineering.

  20. Development of functionalized multi-walled carbon-nanotube-based alginate hydrogels for enabling biomimetic technologies

    PubMed Central

    Joddar, Binata; Garcia, Eduardo; Casas, Atzimba; Stewart, Calvin M.

    2016-01-01

    Alginate is a hydrogel commonly used for cell culture by ionically crosslinking in the presence of divalent Ca2+ ions. However these alginate gels are mechanically unstable, not permitting their use as scaffolds to engineer robust biological bone, breast, cardiac or tumor tissues. This issue can be addressed via encapsulation of multi-walled carbon nanotubes (MWCNT) serving as a reinforcing phase while being dispersed in a continuous phase of alginate. We hypothesized that adding functionalized MWCNT to alginate, would yield composite gels with distinctively different mechanical, physical and biological characteristics in comparison to alginate alone. Resultant MWCNT-alginate gels were porous, and showed significantly less degradation after 14 days compared to alginate alone. In vitro cell-studies showed enhanced HeLa cell adhesion and proliferation on the MWCNT-alginate compared to alginate. The extent of cell proliferation was greater when cultured atop 1 and 3 mg/ml MWCNT-alginate; although all MWCNT-alginates lead to enhanced cell cluster formation compared to alginate alone. Among all the MWCNT-alginates, the 1 mg/ml gels showed significantly greater stiffness compared to all other cases. These results provide an important basis for the development of the MWCNT-alginates as novel substrates for cell culture applications, cell therapy and tissue engineering. PMID:27578567

  1. Development of functionalized multi-walled carbon-nanotube-based alginate hydrogels for enabling biomimetic technologies.

    PubMed

    Joddar, Binata; Garcia, Eduardo; Casas, Atzimba; Stewart, Calvin M

    2016-01-01

    Alginate is a hydrogel commonly used for cell culture by ionically crosslinking in the presence of divalent Ca(2+) ions. However these alginate gels are mechanically unstable, not permitting their use as scaffolds to engineer robust biological bone, breast, cardiac or tumor tissues. This issue can be addressed via encapsulation of multi-walled carbon nanotubes (MWCNT) serving as a reinforcing phase while being dispersed in a continuous phase of alginate. We hypothesized that adding functionalized MWCNT to alginate, would yield composite gels with distinctively different mechanical, physical and biological characteristics in comparison to alginate alone. Resultant MWCNT-alginate gels were porous, and showed significantly less degradation after 14 days compared to alginate alone. In vitro cell-studies showed enhanced HeLa cell adhesion and proliferation on the MWCNT-alginate compared to alginate. The extent of cell proliferation was greater when cultured atop 1 and 3 mg/ml MWCNT-alginate; although all MWCNT-alginates lead to enhanced cell cluster formation compared to alginate alone. Among all the MWCNT-alginates, the 1 mg/ml gels showed significantly greater stiffness compared to all other cases. These results provide an important basis for the development of the MWCNT-alginates as novel substrates for cell culture applications, cell therapy and tissue engineering. PMID:27578567

  2. Hydrophilic PCU scaffolds prepared by grafting PEGMA and immobilizing gelatin to enhance cell adhesion and proliferation.

    PubMed

    Shi, Changcan; Yuan, Wenjie; Khan, Musammir; Li, Qian; Feng, Yakai; Yao, Fanglian; Zhang, Wencheng

    2015-05-01

    Gelatin contains many functional motifs which can modulate cell specific adhesion, so we modified polycarbonate urethane (PCU) scaffold surface by immobilization of gelatin. PCU-g-gelatin scaffolds were prepared by direct immobilizing gelatins onto the surface of aminated PCU scaffolds. To increase the immobilization amount of gelatin, poly(ethylene glycol) methacrylate (PEGMA) was grafted onto PCU scaffolds by surface initiated atom transfer radical polymerization. Then, following amination and immobilization, PCU-g-PEGMA-g-gelatin scaffolds were obtained. Both modified scaffolds were characterized by chemical and biological methods. After immobilization of gelatin, the microfiber surface became rough, but the original morphology of scaffolds was maintained successfully. PCU-g-PEGMA-g-gelatin scaffolds were more hydrophilic than PCU-g-gelatin scaffolds. Because hydrophilic PEGMA and gelatin were grafted and immobilized onto the surface, the PCU-g-PEGMA-g-gelatin scaffolds showed low platelet adhesion, perfect anti-hemolytic activity and excellent cell growth and proliferation capacity. It could be envisioned that PCU-g-PEGMA-g-gelatin scaffolds might have potential applications in tissue engineering artificial scaffolds.

  3. Production of Poly-γ-Glutamate (PGA) Biopolymer by Batch and Semicontinuous Cultures of Immobilized Bacilluslicheniformis strain-R

    PubMed Central

    Berekaa, Mahmoud M.; El Aassar, Samy A.; El-Sayed, Samia M.; EL Borai, Aliaa M.

    2009-01-01

    Production of Polyglutamate (PGA) biopolymer by immobilized Bacillus licheniformis strain-R was intensively investigated. Preliminary experiments were carried out to address the most suitable immobilization methodology. Entrapment of Bacillus cells in alginate–agar led optimal PGA production (36.75 g/l), with 1.32-and 2.18-fold increase in comparison with alginate-or K-carrageenan-immobilized cells, respectively. During semicontinuous cultivation of agar-alginate gel-cell mixture, production of PGA by 10 ml mixture was increased from 2nd to 3rd run whereas, increased till the 4th run using 15ml mixture. Adsorption was the most suitable immobilization technique for production of PGA and the sponge cubes was the preferred matrix recording 43.2 g/l of PGA with the highest cell adsorption. Furthermore, no PGA was detected when B. licheniformis cells were adsorbed on wood and pumice. Although luffa pulp-adsorbed cells recorded the highest PGA production (50.4 g/l), cell adsorption was the lowest. Semicontinuous cultivation of B. licheniformis cells adsorbed on sponge led to increase of PGA production till the 3rd run and reached 55.5 g/l then slightly decreased in the 4th run. The successful use of fixed-bed bioreactor for semicontinuous cultivation of B. licheniformis cells held on sponge cubes (3 runs, 96 hours/run) provides insight for the potential biotechnological production of PGA by immobilized cells. PMID:24031418

  4. Practical reactor systems for yeast cell immobilization using biomass support particles

    SciTech Connect

    Black, G.M.; Webb, C.; Mattews, T.M.; Atkinson, B.

    1984-01-01

    The technique of cell immobilization using porous support particles (biomass support particles) has been successfully applied to yeast cells. Two reactor configurations exploiting the use of these particles have been developed and assessed for use in aseptic yeast fermentations. A liquid-fluidized bed fermenter has been devised for use with particles denser than the fermentation liquor whilst a gas-stirred circulating bed fermenter proved suitable for particles of essentially neutral buoyancy. Both systems have been operated successfully for extended periods of continuous operation. The utilization of biomass support particle technology in such reactors provides a practical and robust system for immobilized cell reactors. This technology offers significant opportunities for further development.

  5. Propagation of human iPS cells in alginate-based microcapsules prepared using reactions catalyzed by horseradish peroxidase and catalase.

    PubMed

    Ashida, Tomoaki; Sakai, Shinji; Taya, Masahito

    2016-09-01

    Cell encapsulation has been investigated as a bioproduction system in the biomedical and pharmaceutical fields. We encaps-ulated human induced pluripotent stem (hiPS) cells in duplex microcapsules prepared from an alginate derivative possessing phenolic hydroxyl moieties, in a single-step procedure based on two competing enzymatic reactions catalyzed by horseradish peroxidase (HRP) and catalase. The encapsulated cells maintained 91.4% viability and proliferated to fill the microcapsules following 19 days of culture. Encapsulated hiPS cells showed pluripotency comparable to that of unencapsulated cells during the cultures, as demonstrated by the expression of the SSEA-4 marker. PMID:26148179

  6. Lindane removal by pure and mixed cultures of immobilized actinobacteria.

    PubMed

    Saez, Juliana M; Benimeli, Claudia S; Amoroso, María J

    2012-11-01

    Lindane (γ-HCH) is an organochlorine insecticide that has been widely used in developing countries. It is known to persist in the environment and can cause serious health problems. One of the strategies adopted to remove lindane from the environment is bioremediation using microorganisms. Immobilized cells present advantages over free suspended cells, like their high degradation efficiency and protection against toxins. The aims of this work were: (1) To evaluate the ability of Streptomyces strains immobilized in four different matrices to remove lindane, (2) To select the support with optimum lindane removal by pure cultures, (3) To assay the selected support with consortia and (4) To evaluate the reusability of the immobilized cells. Four Streptomyces sp. strains had previously shown their ability to grow in the presence of lindane. Lindane removal by microorganisms immobilized was significantly higher than in free cells. Specifically immobilized cells in cloth sachets showed an improvement of around 25% in lindane removal compared to the abiotic control. Three strains showed significantly higher microbial growth when they were entrapped in silicone tubes. Strains immobilized in PVA-alginate demonstrated lowest growth. Mixed cultures immobilized inside cloth sachets showed no significant enhancement compared to pure cultures, reaching a maximum removal of 81% after 96 h for consortium I, consisting of the four immobilized strains together. Nevertheless, the cells could be reused for two additional cycles of 96 h each, obtaining a maximum removal efficiency of 71.5% when each of the four strains was immobilized in a separate bag (consortium III). PMID:22840534

  7. Perfluorinated alginate for cellular encapsulation.

    PubMed

    Gattás-Asfura, Kerim M; Fraker, Christopher A; Stabler, Cherie L

    2012-08-01

    Molecules of pentadecafluorooctanoyl chloride (PFC) were grafted onto alginate (Alg) using a linear poly(ethylene glycol) linker and amide bonds. The resulting Alg-PFC material was characterized by proton nuclear magnetic resonance and infrared spectroscopies. The degree of PFC functionalization significantly influenced the physical and chemical properties of Alg-PFC, particularly when the resulting polymer was ionically crosslinked into hydrogels. Alg-PFC hydrogel beads fabricated via Ba(2+) crosslinking were found to match the permeability properties of control alginate beads, except upon swelling over time in culture media. When used to encapsulate MIN6 cells, a beta cell line, Alg-PFC beads demonstrated enhanced cell proliferation over alginate control beads. These results indicate that Alg-PFC hydrogels retain some of the PFC's biological-relevant benefits, such as enhancement of mass transport and bioinertness, to enhance cellular viability within alginate three-dimensional hydrogel environments. We envision these functionalized hydrogels to be particularly useful in the encapsulation of cells with a high metabolic demand, such as pancreatic islets.

  8. Chondrogenesis of human bone marrow mesenchymal stromal cells in highly porous alginate-foams supplemented with chondroitin sulfate.

    PubMed

    Huang, Zhao; Nooeaid, Patcharakamon; Kohl, Benjamin; Roether, Judith A; Schubert, Dirk W; Meier, Carola; Boccaccini, Aldo R; Godkin, Owen; Ertel, Wolfgang; Arens, Stephan; Schulze-Tanzil, Gundula

    2015-05-01

    To overcome the limited intrinsic cartilage repair, autologous chondrocyte or bone-marrow-derived mesenchymal stromal cell (BM-MSC) was implanted into cartilage defects. For this purpose suitable biocompatible scaffolds are needed to provide cell retention, chondrogenesis and initial mechanical stability. The present study should indicate whether a recently developed highly porous alginate (Alg) foam scaffold supplemented with chondroitin sulfate (CS) allows the attachment, survival and chondrogenesis of BM-MSCs and articular chondrocytes. The foams were prepared using a freeze-drying method; some of them were supplemented with CS and subsequently characterized for porosity, biodegradation and mechanical profile. BM-MSCs were cultured for 1-2 weeks on the scaffold either under chondrogenic or maintenance conditions. Cell vitality assays, histology, glycosaminoglycan (sGAG) assay, and type II and I collagen immunolabelings were performed to monitor cell growth and extracellular matrix (ECM) synthesis in the scaffolds. Scaffolds had a high porosity ~93-95% with a mean pore sizes of 237±48 μm (Alg) and 197±61 μm (Alg/CS). Incorporation of CS increased mechanical strength of the foams providing gradually CS release over 7 days. Most of the cells survived in the scaffolds. BM-MSCs and articular chondrocytes formed rounded clusters within the scaffold pores. The BM-MSCs, irrespective of whether cultured under non/chondrogenic conditions and chondrocytes produced an ECM containing sGAGs, and types II and I collagen. Total collagen and sGAG contents were higher in differentiated BM-MSC cultures supplemented with CS than in CS-free foams after 14 days. The cell cluster formation induced by the scaffolds might stimulate chondrogenesis via initial intense cell-cell contacts.

  9. Development of a morphogenetically active scaffold for three-dimensional growth of bone cells: biosilica-alginate hydrogel for SaOS-2 cell cultivation.

    PubMed

    Müller, Werner E G; Schröder, Heinz C; Feng, Qingling; Schlossmacher, Ute; Link, Thorben; Wang, Xiaohong

    2015-11-01

    Polymeric silica is formed from ortho-silicate during a sol-gel formation process, while biosilica is the product of an enzymatically driven bio-polycondensation reaction. Both polymers have recently been described as a template that induces an increased expression of the genes encoding bone morphogenetic protein 2 (BMP-2) and osteoprotegerin in osteoblast-related SaOS-2 cells; simultaneously or subsequently the cells respond with enhanced hydroxyapatite formation. In order to assess whether the biocompatible polymeric silica/biosilica can serve as a morphogenetically active matrix suitable for three-dimensional (3D) cell growth, or even for 3D cell bioprinting, SaOS-2 cells were embedded into a Na-alginate-based hydrogel. Four different gelatinous hydrogel matrices were used for suspending SaOS-2 cells: (a) the hydrogel alone; (b) the hydrogel with 400 μM ortho-silicate; (c) the hydrogel supplemented with 400 μM ortho-silicate and recombinant silicatein to allow biosilica synthesis to occur; and (d) the hydrogel with ortho-silicate and BSA. The SaOS-2 cells showed an increased growth if silica/biosilica components were present in the hydrogel. Likewise intensified was the formation of hydroxyapatite nodules in the silica-containing hydrogels. After an incubation period of 2 weeks, cells present in silica-containing hydrogels showed a significantly higher expression of the genes encoding the cytokine BMP-2, the major fibrillar structural protein collagen 1 and likewise of carbonic anhydrase. It is concluded that silica, and to a larger extent biosilica, retains its morphogenetic/osteogenic potential after addition to Na-alginate-based hydrogels. This property might qualify silica hydrogels to be also used as a matrix for 3D cell printing.

  10. Live cell imaging compatible immobilization of Chlamydomonas reinhardtii in microfluidic platform for biodiesel research.

    PubMed

    Park, Jae Woo; Na, Sang Cheol; Nguyen, Thanh Qua; Paik, Sang-Min; Kang, Myeongwoo; Hong, Daewha; Choi, Insung S; Lee, Jae-Hyeok; Jeon, Noo Li

    2015-03-01

    This paper describes a novel surface immobilization method for live-cell imaging of Chlamydomonas reinhardtii for continuous monitoring of lipid droplet accumulation. Microfluidics allows high-throughput manipulation and analysis of single cells in precisely controlled microenvironment. Fluorescence imaging based quantitative measurement of lipid droplet accumulation in microalgae had been difficult due to their intrinsic motile behavior. We present a simple surface immobilization method using gelatin coating as the "biological glue." We take advantage of hydroxyproline (Hyp)-based non-covalent interaction between gelatin and the outer cell wall of microalgae to anchor the cells inside the microfluidic device. We have continuously monitored single microalgal cells for up to 6 days. The immobilized microalgae remain viable (viability was comparable to bulk suspension cultured controls). When exposed to wall shear stress, most of the cells remain attached up to 0.1 dyne/cm(2) . Surface immobilization allowed high-resolution, live-cell imaging of mitotic process in real time-which followed previously reported stages in mitosis of suspension cultured cells. Use of gelatin coated microfluidics devices can result in better methods for microalgae strain screening and culture condition optimization that will help microalgal biodiesel become more economically viable. PMID:25220860

  11. On chip single-cell separation and immobilization using optical tweezers and thermosensitive hydrogel.

    PubMed

    Arai, Fumihito; Ng, Chinaik; Maruyama, Hisataka; Ichikawa, Akihiko; El-Shimy, Haitham; Fukuda, Toshio

    2005-12-01

    A novel approach appropriate for rapid separation and immobilization of a single cell by concomitantly utilizing laser manipulation and locally thermosensitive hydrogelation is proposed in this paper. We employed a single laser beam as optical tweezers for separating a target cell and locating it adjacent to a fabricated, transparent micro heater. Simultaneously, the target cell is immobilized or partially entrapped by heating the thermosensitive hydrogel with the micro heater. The state of the thermosensitive hydrogel can be switched from sol to gel and gel to sol by controlling the temperature through heating and cooling by the micro heater. After other unwanted cells are removed by the high-speed cleaning flow in the microchannel, the entrapped cell is successfully isolated. It is possible to collect the immobilized target cell for analysis or culture by switching off the micro heater and releasing the cell from the entrapment. We demonstrated that the proposed approach is feasible for rapid manipulation, immobilization, cleaning, isolation and extraction of a single cell. The experimental results are shown here. PMID:16286972

  12. Immobilization of bioluminescent Escherichia coli cells using natural and artificial fibers treated with polyethyleneimine.

    PubMed

    Chu, Yi-Fang; Hsu, Chia-Hua; Soma, Pavan K; Lo, Y Martin

    2009-07-01

    Biosensors based on whole-cell bioluminescence have the potential to become a cost-effective alternative to conventional detection methods upon validation of target selectivity and sensitivity. However, quantitative analysis of bioluminescence is greatly hindered due to lack of control over the total number of cells in a suspending culture. In this study, the effect of surface properties of genetically engineered luminous E. coli cells and fibrous matrices on the immobilization capacity and effectiveness under various environmental conditions were characterized. Four different fibers, including cotton, polyester, viscose rayon, and silk, were investigated. Although cell adhesion was observed on untreated viscose and cotton fibers, viscose fiber pretreated with 0.667% polyethyleneimine (PEI) was found capable of immobilizing the most viable E. coli DPD2234 cells, followed by viscose treated with 0.33% and 1% PEI. The cells immobilized on PEI-treated viscose remained viable and yielded 20% or more bioluminescence signals immediately upon contact with the inducer up to 72 h without feeding nutrients to the cells, suggesting that viscose treated with 0.667% PEI could provide a stable immobilization mechanism for bioluminescent E. coli cells with long sensing period, quick response time, and good signal reproducibility.

  13. Determination of diffusion coefficients and diffusion characteristics for chlorferon and diethylthiophosphate in Ca-alginate gel beads.

    PubMed

    Ha, Jiyeon; Engler, Cady R; Lee, Seung Jae

    2008-07-01

    Diffusion characteristics of chlorferon and diethylthiophosphate (DETP) in Ca-alginate gel beads were studied to assist in designing and operating bioreactor systems. Diffusion coefficients for chlorferon and DETP in Ca-alginate gel beads determined at conditions suitable for biodegradation studies were 2.70 x 10(-11) m(2)/s and 4.28 x 10(-11) m(2)/s, respectively. Diffusivities of chlorferon and DETP were influenced by several factors, including viscosity of the bulk solution, agitation speed, and the concentrations of diffusing substrate and immobilized cells. Diffusion coefficients increased with increasing agitation speed, probably due to poor mixing at low speed and some attrition of beads at high speeds. Diffusion coefficients also increased with decreasing substrate concentration. Increased cell concentration in the gel beads caused lower diffusivity. Theoretical models to predict diffusivities as a function of cell weight fraction overestimated the effective diffusivities for both chlorferon and DETP, but linear relations between effective diffusivity and cell weight fraction were derived from experimental data. Calcium-alginate gel beads with radii of 1.65-1.70 mm used in this study were not subject to diffusional limitations: external mass transfer resistances were negligible based on Biot number calculations and effectiveness factors indicated that internal mass transfer resistance was negligible. Therefore, the degradation rates of chlorferon and DETP inside Ca-alginate gel beads were reaction-limited. PMID:18080347

  14. Combination of Controllably Released Platelet Rich Plasma Alginate Beads and Bone Morphogenic Protein-2 Gene-Modified Mesenchymal Stem Cells for Bone Regeneration

    PubMed Central

    Fernandes, Gabriela; Wang, Changdong; Yuan, Xue; Liu, Zunpeng; Dziak, Rosemary; Yang, Shuying

    2016-01-01

    Background Platelet rich plasma (PRP) consists of platelet derived growth factor (PDGF) and Transforming growth factor-beta (TGF-β) that increase cell proliferation of mesenchymal stem cells (MSCs), whereas, bone morphogenic Protein-2 (BMP2) promotes osteogenic differentiation of MSCs. However, the high degradation rate of fibrin leads to the dissociation of cytokines even before the process of bone regeneration has begun. Hence, for the first time, we studied the combined effect of sustained released PRP from alginate beads on BMP2 modified MSCs osteogenic differentiation in vitro and of sustained PRP alone on a fracture defect model ex vivo as well as its effect on the calvarial suture closure. Methods After optimizing the concentration of alginate for the microspheres, the osteogenic and mineralization effect of PRP and BMP2 in combinations on MSCs was studied. A self-setting alginate hydrogel carrying PRP was tested on a femur defect model ex-vivo. The effect of PRP was studied on the closure of the embryonic (E15) mouse calvaria sutures ex vivo. Results Increase of PRP concentration promoted cellular proliferation of MSCs. 2.5%–10% of PRP displayed gradually increased ALP activity on the cells in a dose dependent manner. Sustained release PRP and BMP2 demonstrated a significantly higher ALP and mineralization activity (p<0.05). The radiographs of alginate hydrogel with PRP treated bone demonstrated a nearly complete healing of the fracture and the histological sections of the embryonic calvaria revealed that PRP leads to suture fusion. Conclusions Sustained release of PRP along with BMP2 gene modified MSCs can significantly promote bone regeneration. PMID:26745613

  15. [Comparison of fibroblastic cell compatibility of type I collagen-immobilized titanium between electrodeposition and immersion].

    PubMed

    Kyuragi, Takeru

    2014-03-01

    Titanium is widely used for medical implants. While many techniques for surface modification have been studied for optimizing its biocompatibility with hard tissues, little work has been undertaken to explore ways of maximizing its biocompatibility with soft tissues. We investigated cell attachment to titanium surfaces modified with bovine Type I collagen immobilized by either electrodeposition or a conventional immersion technique. The apparent thickness and durability of the immobilized collagen layer were evaluated prior to incubation of the collagen-immobilized titanium surfaces with NIH/3T3 mouse embryonic fibroblasts. The initial cell attachment and expression of actin and vinculin were evaluated. We determined that the immobilized collagen layer was much thicker and more durable when placed using the electrodeposition technique than the immersion technique. Both protocols produced materials that promoted better cell attachment, growth and structural protein expression than titanium alone. However, electrodeposition was ultimately superior to immersion because it is quicker to perform and produces a more durable collagen coating. We conclude that electrodeposition is an effective technique for immobilizing type I collagen on titanium surfaces, thus improving their cytocompatibility with fibroblasts.

  16. Continuous separation of cells of high osteoblastic differentiation potential from mesenchymal stem cells on an antibody-immobilized column.

    PubMed

    Mahara, Atsushi; Yamaoka, Tetsuji

    2010-05-01

    Here, we report that two distinctive cell populations with osteoblastic differentiation ability were found in adherent cell populations from bone marrow. Mesenchymal stem cells (MSCs) were conventionally isolated by using adherent property of bone marrow cells onto a plastic culture dish. MSCs enriched on the basis of their adherent property were considered phenotypically and functionally heterogeneous. We developed a ligand-immobilized surface for separating subpopulation of adherent cells derived from bone marrow by the cell rolling process. We successfully isolate two cell populations with high differentiation ability for osteoblasts in adherent bone marrow cells by using the anti-CD34 antibody-immobilized column. The antibody was covalently conjugated with polyacrylic acid and introduced onto the inner surface of a silicone tube. When cell suspension of MSCs was injected into the antibody-immobilized column, different cell populations were isolated. After the cultivation of isolated cells in the osteoblastic differentiation medium for 1 week, few sub-populations were strongly induced to form osteoblastic cells. This study revealed that the ligand-immobilized surface can be used to continually separate cell populations under a labeling-free condition. PMID:20185169

  17. Hydrophobic modification of sodium alginate and its application in drug controlled release.

    PubMed

    Yao, Bolong; Ni, Caihua; Xiong, Cheng; Zhu, Changping; Huang, Bo

    2010-05-01

    Sodium alginate was hydrophobically modified by coupling of polybutyl methacrylate onto the alginate. The polybutyl methacrylate was previously prepared through polymerization of butyl methacrylate in the presence of 2-amino-ethanethiol as a chain transfer agent. The structure of the product was characterized by Fourier-transformed infrared spectrometry, nuclear magnetic resonance ((1)HNMR) and thermogravimetry. The result of fluorescence analysis showed that the hydrophobicity of the modified alginate was obviously increased. The modified alginate conjugate was used for immobilization of bovine serum albumin in the presence of calcium chloride. In addition, the release behavior of the drug-loaded alginate in deionized water and Tris-HCl buffer solution (pH 7.2) was investigated. It was found that the modified sodium alginate possessed prolonged release behavior compared to unmodified sodium alginate, and it had potential application in controlled release as a drug carrier.

  18. Immobilization of Rubia tinctorum L. Suspension Cultures and Biomass Production.

    PubMed

    Nartop, Pınar

    2016-01-01

    Plants are natural sources of valuable secondary metabolites used as pharmaceuticals, agrochemicals, flavors, fragrances, colors, biopesticides, and food additives. There is an increasing demand to obtain these metabolites through more productive plant tissue applications and cell culture methods due to the importance of secondary metabolites.Immobilization of plant cells is a method used in plant cell cultures to induce secondary metabolite production. In this method, plant cells are fixed in or on a supporting material or matrix such as agar, agarose, calcium alginate, glass, or polyurethane foam. In the present study, three natural lignocellulosic materials, loofah sponge, and the long fibers of sisal and jute, were used to immobilize suspended R. tinctorum cells. PMID:27108315

  19. Immobilization of Rubia tinctorum L. Suspension Cultures and Biomass Production.

    PubMed

    Nartop, Pınar

    2016-01-01

    Plants are natural sources of valuable secondary metabolites used as pharmaceuticals, agrochemicals, flavors, fragrances, colors, biopesticides, and food additives. There is an increasing demand to obtain these metabolites through more productive plant tissue applications and cell culture methods due to the importance of secondary metabolites.Immobilization of plant cells is a method used in plant cell cultures to induce secondary metabolite production. In this method, plant cells are fixed in or on a supporting material or matrix such as agar, agarose, calcium alginate, glass, or polyurethane foam. In the present study, three natural lignocellulosic materials, loofah sponge, and the long fibers of sisal and jute, were used to immobilize suspended R. tinctorum cells.

  20. Development of bioreactors for the culture of surface immobilized plant cells.

    PubMed

    Archambault, J; Volesky, B; Kurz, W G

    1990-03-25

    The scaleup of the technique of plant cell surface immobilization was performed successfully in specifically designed laboratory size bioreactors. The immobilizing matrix was formed into a vertically wound spiral providing for a high immobilizing area-to-volume ratio (0.8-1.2 cm(-1)). A modified airlift and a mechanically stirred vessel delivered a best bioreactor performance characterized by low biomass frothing and highly efficient plant cell attachment and retention (>or=96%). The growth of Catharanthus roseus cells investigated in these bioreactors was found not to be mass transfer limited. It required mild mixing and aeration levels (k(L)a approximately 10-15 h(-1)). The biomass formation pattern of surface immobilized plant cells generally exhibited a linear growth phase followed by a stationary phase characterized by the presence of residual carbohydrates in the medium, contrary to suspension cultures. This behavior was found to depend on the plant cell type and/or line cultured, as well as on the inoculum age. The space restriction and unidirectional growth of the SIPC biofilm combined with the limited availability of essential intracellular nutrients rapidly accumulated from the medium by the stationary phase inoculated plant cells all likely contributed to the culture behavior.

  1. Impact of substrates and cell immobilization on siderophore activity by Pseudomonads in a Fe and/or Cr, Hg, Pb containing-medium.

    PubMed

    Braud, Armelle; Jézéquel, Karine; Lebeau, Thierry

    2007-06-01

    To increase the amount of bioavailable metals in phytoextraction purposes, soil bioaugmentation with Pseudomonads, as siderophore producers with high metal complexation levels, could be relevant. Unfortunately, siderophore synthesis may be inhibited by soluble iron in soil and bacteria can suffer at the same time from the toxicity of some other metals, predation and oligotrophy. To overcome these drawbacks, we attempted to co-locate a carbon substrate and Pseudomonas aeruginosa or P. fluorescens in Ca-alginate beads. First, free-cell cultures showed that glycerol, fructose, mannitol and skim milk enhanced the siderophore activity which was the highest in the medium with neither Fe or TM (toxic metal) (Cr, Hg and Pb) and the lowest in the Fe-containing medium without TM. The negative effect of iron was partly offset when TM was added to the medium. In a second part, co-location of microorganisms and substrates was only feasible with skim milk. By comparison with free cells, siderophore activity by immobilized cells was higher in culture media containing Fe with or without TM (up to a ratio of 9), and varied in a narrow margin, according to the medium composition. PMID:17112663

  2. Wheat Bran Enhances the Cytotoxicity of Immobilized Alcaligenes aquatilis F8 against Microcystis aeruginosa.

    PubMed

    Sun, Pengfei; Lin, Hui; Wang, Guan; Zhang, Ximing; Zhang, Qichun; Zhao, Yuhua

    2015-01-01

    Algicidal bacteria offer a promising option for killing cyanobacteria. Therefore, a new Alcaligenes aquatilis strain F8 was isolated to control Microcystis aeruginosa in this study. The algicidal activity of strain F8 was dependent on the cell density of M. aeruginosa, and the maximal algicidal rate of the free bacterium reached 88.45% within 72 h. With a view to its application to the control of M. aeruginosa in the natural environment, strain F8 was immobilized in sodium alginate beads, but immobilization of the strain decreased its algicidal rate compared to that of the free bacterium. However, addition of wheat bran to the sodium alginate matrix used to immobilize strain F8 not only eliminated the adverse effects of immobilization on the bacteria but also resulted in an 8.83% higher algicidal rate of the immobilized than free bacteria. Exclusion and recovery methods were used to identify key ingredients of wheat bran and gain insight into the mechanism underlying the observed enhancement of algicidal activity. This analysis indicated that certain factors in wheat bran, including vitamins B1, B2, B9, and E were responsible for promoting bacterial growth and thereby improving the algicidal rate of immobilized strain F8. Our findings indicate that wheat bran is able to improve the algicidal efficiency of A. aquatilis strain F8 for killing M. aeruginosa and is a good source of not only carbon and nitrogen but also vitamins for bacteria. PMID:26295573

  3. The removal of thermo-tolerant coliform bacteria by immobilized waste stabilization pond algae.

    PubMed

    Pearson, H W; Marcon, A E; Melo, H N

    2011-01-01

    This study investigated the potential of laboratory- scale columns of immobilized micro-algae to disinfect effluents using thermo-tolerant coliforms (TTC) as a model system. Cells of a Chlorella species isolated from a waste stabilization pond complex in Northeast Brazil were immobilized in calcium alginate, packed into glass columns and incubated in contact with TTC suspensions for up to 24 hours. Five to six log removals of TTC were achieved in 6 hours and 11 log removals in 12 hours contact time. The results were similar under artificial light and shaded sunlight. However little or no TTC removal occurred in the light in columns of alginate beads without immobilized algae present or when the immobilized algae were incubated in the dark suggesting that the presence of both algae and light were necessary for TTC decay. There was a positive correlation between K(b) values for TTC and increasing pH in the effluent from the immobilized algal columns within the range pH 7.2 and 8.9. The potential of immobilized algal technology for wastewater disinfection may warrant further investigation.

  4. Wheat Bran Enhances the Cytotoxicity of Immobilized Alcaligenes aquatilis F8 against Microcystis aeruginosa

    PubMed Central

    Sun, Pengfei; Lin, Hui; Wang, Guan; Zhang, Ximing; Zhang, Qichun; Zhao, Yuhua

    2015-01-01

    Algicidal bacteria offer a promising option for killing cyanobacteria. Therefore, a new Alcaligenes aquatilis strain F8 was isolated to control Microcystis aeruginosa in this study. The algicidal activity of strain F8 was dependent on the cell density of M. aeruginosa, and the maximal algicidal rate of the free bacterium reached 88.45% within 72 h. With a view to its application to the control of M. aeruginosa in the natural environment, strain F8 was immobilized in sodium alginate beads, but immobilization of the strain decreased its algicidal rate compared to that of the free bacterium. However, addition of wheat bran to the sodium alginate matrix used to immobilize strain F8 not only eliminated the adverse effects of immobilization on the bacteria but also resulted in an 8.83% higher algicidal rate of the immobilized than free bacteria. Exclusion and recovery methods were used to identify key ingredients of wheat bran and gain insight into the mechanism underlying the observed enhancement of algicidal activity. This analysis indicated that certain factors in wheat bran, including vitamins B1, B2, B9, and E were responsible for promoting bacterial growth and thereby improving the algicidal rate of immobilized strain F8. Our findings indicate that wheat bran is able to improve the algicidal efficiency of A. aquatilis strain F8 for killing M. aeruginosa and is a good source of not only carbon and nitrogen but also vitamins for bacteria. PMID:26295573

  5. Alginate composites for bone tissue engineering: a review.

    PubMed

    Venkatesan, Jayachandran; Bhatnagar, Ira; Manivasagan, Panchanathan; Kang, Kyong-Hwa; Kim, Se-Kwon

    2015-01-01

    Bone is a complex and hierarchical tissue consisting of nano hydroxyapatite and collagen as major portion. Several attempts have been made to prepare the artificial bone so as to replace the autograft and allograft treatment. Tissue engineering is a promising approach to solve the several issues and is also useful in the construction of artificial bone with materials including polymer, ceramics, metals, cells and growth factors. Composites consisting of polymer-ceramics, best mimic the natural functions of bone. Alginate, an anionic polymer owing enormous biomedical applications, is gaining importance particularly in bone tissue engineering due to its biocompatibility and gel forming properties. Several composites such as alginate-polymer (PLGA, PEG and chitosan), alginate-protein (collagen and gelatin), alginate-ceramic, alginate-bioglass, alginate-biosilica, alginate-bone morphogenetic protein-2 and RGD peptides composite have been investigated till date. These alginate composites show enhanced biochemical significance in terms of porosity, mechanical strength, cell adhesion, biocompatibility, cell proliferation, alkaline phosphatase increase, excellent mineralization and osteogenic differentiation. Hence, alginate based composite biomaterials will be promising for bone tissue regeneration. This review will provide a broad overview of alginate preparation and its applications towards bone tissue engineering.

  6. Cells immobilized on patterns printed in DNA by an inkjet printer.

    PubMed

    Sakurai, Kengo; Teramura, Yuji; Iwata, Hiroo

    2011-05-01

    The ability to two-dimensionally align various kinds of cells freely onto substrate would be a useful tool for analysis of cell-cell interactions. In this study, we aimed to establish a method for attaching cells to the substrate, in which the pattern is drawn by an inkjet printer. Poly-deoxyribonucleic acid (DNA) was immobilized onto the cell surface by use of DNA-conjugated poly(ethylene) glycol-phospholipid (DNA-PEG-lipid), which is the amphiphilic conjugate of PEG-lipid and single-stranded DNA. The surface of the substrate was then modified with the complementary DNA using an inkjet printer. Finally, DNA-immobilized cells were attached onto the substrate through DNA hybridization. The use of the inkjet printer enabled us to draw the DNA pattern accurately on the substrate with a resolution of a few hundred micrometers. DNA-immobilized cells could be attached precisely along the DNA pattern on the substrate. In addition, various kinds of cells could be attached simultaneously by using various sequences of DNA. Our technique is promising for analysis of cell-cell interactions and differentiation induction in stem cell research.

  7. Fabrication of cross-linked alginate beads using electrospraying for adenovirus delivery.

    PubMed

    Park, Hongkwan; Kim, Pyung-Hwan; Hwang, Taewon; Kwon, Oh-Joon; Park, Tae-Joon; Choi, Sung-Wook; Yun, Chae-Ok; Kim, Jung Hyun

    2012-05-10

    Cross-linked alginate beads containing adenovirus (Ad) were successfully fabricated using an electrospraying method to achieve the protection and release of Ad in a controlled manner. An aqueous alginate solution containing Ad was electrosprayed into an aqueous phase containing a cross-linking agent (calcium chloride) at different process variables (voltages, alginate concentrations, and flow rates). Alginate beads containing Ad were used for transduction of U343 glioma cells and the transduction efficiency of the alginate beads was measured by quantification of gene expression using a fluorescence-activated cell sorter at different time points. In vitro results of gene expression revealed that the Ad encapsulated in the alginate beads with 0.5 wt% of alginate concentration exhibited a high activity for a long period (over 7 days) and was released in a sustained manner from the alginate beads. The Ad-encapsulating alginate beads could be promising materials for local delivery of Ad at a high concentration into target sites.

  8. Entrapment of cross-linked cellulase colloids in alginate beads for hydrolysis of cellulose.

    PubMed

    Nguyen, Le Truc; Lau, Yun Song; Yang, Kun-Lin

    2016-09-01

    Entrapment of enzymes in calcium alginate beads is a popular enzyme immobilization method. However, leaching of immobilized enzymes from the alginate beads is a common problem because enzyme molecules are much smaller than the pore size of alginate beads (∼200nm). To address this issue, we employ a millifluidic reactor to prepare cross-linked cellulase aggregate (XCA) colloids with a uniform size (∼300nm). Subsequently, these colloids are immobilized in calcium alginate beads as biocatalysts to hydrolyze cellulose substrates. By using fluorescent microscopy, we conclude that the immobilized XCA colloids distribute uniformly inside the beads and do not leach out from the beads after long-term incubation. Meanwhile, the pore size of the alginate beads is big enough for the cellulose substrates and fibers to diffuse into the beads for hydrolysis. For example, palm oil fiber and microcrystalline cellulose can be hydrolyzed within 48h and release reducing sugar concentrations up to 2.48±0.08g/l and 4.99±0.09g/l, respectively. Moreover, after 10 cycles of hydrolysis, 96.4% of the XCA colloids remain inside the alginate beads and retain 67% of the original activity. In contrast, free cellulase immobilized in the alginate beads loses its activity completely after 10 cycles. The strategy can also be used to prepare other types of cross-linked enzyme aggregates with high uniformity.

  9. Entrapment of cross-linked cellulase colloids in alginate beads for hydrolysis of cellulose.

    PubMed

    Nguyen, Le Truc; Lau, Yun Song; Yang, Kun-Lin

    2016-09-01

    Entrapment of enzymes in calcium alginate beads is a popular enzyme immobilization method. However, leaching of immobilized enzymes from the alginate beads is a common problem because enzyme molecules are much smaller than the pore size of alginate beads (∼200nm). To address this issue, we employ a millifluidic reactor to prepare cross-linked cellulase aggregate (XCA) colloids with a uniform size (∼300nm). Subsequently, these colloids are immobilized in calcium alginate beads as biocatalysts to hydrolyze cellulose substrates. By using fluorescent microscopy, we conclude that the immobilized XCA colloids distribute uniformly inside the beads and do not leach out from the beads after long-term incubation. Meanwhile, the pore size of the alginate beads is big enough for the cellulose substrates and fibers to diffuse into the beads for hydrolysis. For example, palm oil fiber and microcrystalline cellulose can be hydrolyzed within 48h and release reducing sugar concentrations up to 2.48±0.08g/l and 4.99±0.09g/l, respectively. Moreover, after 10 cycles of hydrolysis, 96.4% of the XCA colloids remain inside the alginate beads and retain 67% of the original activity. In contrast, free cellulase immobilized in the alginate beads loses its activity completely after 10 cycles. The strategy can also be used to prepare other types of cross-linked enzyme aggregates with high uniformity. PMID:27318817

  10. Development of immobilized cellulase

    SciTech Connect

    Luther, M.A.; Halbert, D.J.

    1982-08-01

    The immobilization of cellulase from the fungus Trichoderma reesei on the surface of calcium alginate gel spheres was investigated. The immobilized cellulase catalyzed the hydrolysis of cellulose to glucose. The linking agents, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and glutaraldehyde, decreased free-enzyme activity by 95%, and the maximum observed retention of cellulase activity after immobilization was 2%. Leakage of enzyme from the support was observed. A fivefold increase in glucose production was seen after the addition of ..beta..-glucosidase-impregnated spheres to the cellulase spheres, suggesting that cellobiose may be accumulating during the reaction. A simple economic analysis suggested that the immobilized-enzyme activity per unit volume might have to be increased by a factor of fifty to become competitive with the free-enzyme process.

  11. Continuous Operation of Fluidized Bed Bioreactor for Biogenic Sulfide Oxidation Using Immobilized Cells of Thiobacillus sp.

    NASA Astrophysics Data System (ADS)

    Ravichandra, P.; Mugeraya, Gopal; Gangagni Rao, A.; Ramakrishna, M.; Jetty, Annapurna

    In the present study, obligate autotrophic Thiobacillus sp. was isolated from aerobic sludge distillery effluent treatment plant and the experiments were conducted in a fluidized bed bioreactor for the biological oxidation of sulfide using Ca-alginate immobilized Thiobacillus sp. All the experiments were conducted in continuous mode at different sulfide loading rates 0.018, 0.02475, 0.03375, 0.03825 and 0.054 and different hydraulic retention times 5, 3.67, 2.67, 2.35 and 1.67 h by varying flow rates 2.4x10-4, 3.3x10-4, 4.5x10-4, 5.1x10-4 and 7.2x10-4. Sulfide conversions higher than 90% were obtained at almost all sulfide loading rates and hydraulic retention times. All the experiments were conducted at constant pH of around 6 and temperature of 30±5°C.

  12. Improving the controlled delivery formulations of caffeine in alginate hydrogel beads combined with pectin, carrageenan, chitosan and psyllium.

    PubMed

    Belščak-Cvitanović, Ana; Komes, Draženka; Karlović, Sven; Djaković, Senka; Spoljarić, Igor; Mršić, Gordan; Ježek, Damir

    2015-01-15

    Alginate-based blends consisting of carrageenan, pectin, chitosan or psyllium husk powder were prepared for assessment of the best formulation aimed at encapsulation of caffeine. Alginate-pectin blend exhibited the lowest viscosity and provided the smallest beads. Alginate-psyllium husk blend was characterised with higher viscosity, yielding the largest bead size and the highest caffeine encapsulation efficiency (83.6%). The release kinetics of caffeine indicated that the porosity of alginate hydrogel was not reduced sufficiently to retard the diffusion of caffeine from the beads. Chitosan coated alginate beads provided the most retarded release of caffeine in water. Morphological characteristics of beads encapsulating caffeine were adversely affected by freeze drying. Bitterness intensity of caffeine-containing beads in water was the lowest for alginate-psyllium beads and chitosan coated alginate beads. Higher sodium alginate concentration (3%) for production of hydrogel beads in combination with psyllium or chitosan coating would present the most favourable carrier systems for immobilization of caffeine.

  13. The production of sulfonated chitosan-sodium alginate found in brown algae (Sargassum sp.) composite membrane as proton exchange membrane fuel cell (PEMFC)

    NASA Astrophysics Data System (ADS)

    Wafiroh, Siti; Pudjiastuti, Pratiwi; Sari, Ilma Indana

    2016-03-01

    The majority of energy was used in this period is from fossil fuel, which getting decreased in the future. The objective of this research is production and characterization of sulfonated chitosan-sodium alginate found in brown algae (Sargassum sp.) composite membrane as Proton Exchange Membrane Fuel Cell (PEMFC) for alternative energy. PEMFC was produced with 4 variations (w/w) ratio between chitosan and sodium alginate, 8 : 0, 8 : 1, 8 : 2, 8 : 4 (w/w). The production of membrane was mixed sodium alginate solution into chitosan solution and sulfonated with H2SO4 0.72 N. The characterization of the PEM was uses Modulus Young analysis, water swelling, ion exchange capacity, FTIR, SEM, DTA, methanol permeability and proton conductivity. The result of the research, showed that the optimum membrane was with ratio 8 : 2 (w/w) that the Modulus Young 8564 kN/m2, water swelling 31.86%, ion exchange capacity 1.020 meq/g, proton conductivity 8,8 × 10-6 S/cm, methanol permeability 1.90 × 10-8 g/cm2s and glass transition temperature (Tg) 100.9 °C, crystalline temperature (Tc) 227.6 °C, and the melting temperature (Tm) 267.9 °C.

  14. Immobilization of Cell-Adhesive Laminin Peptides in Degradable PEGDA Hydrogels Influences Endothelial Cell Tubulogenesis

    PubMed Central

    Ali, Saniya; Saik, Jennifer E.; Gould, Dan J.; Dickinson, Mary E.

    2013-01-01

    Abstract Attachment, spreading, and organization of endothelial cells into tubule networks are mediated by interactions between cells in the extracellular microenvironment. Laminins are key extracellular matrix components and regulators of cell adhesion, migration, and proliferation. In this study, laminin-derived peptides were conjugated to poly(ethylene glycol) (PEG) monoacrylate and covalently incorporated into degradable PEG diacrylate (PEGDA) hydrogels to investigate the influence of these peptides on endothelial cellular adhesion and function in organizing into tubule networks. Degradable PEGDA hydrogels were synthesized by incorporating a matrix metalloproteinase (MMP)–sensitive peptide, GGGPQGIWGQGK (abbreviated PQ), into the polymer backbone. The secretion of MMP-2 and MMP-9 by endothelial cells promotes polymer degradation and consequently cell migration. We demonstrate the formation of extensive networks of tubule-like structures by encapsulated human umbilical vein endothelial cells in hydrogels with immobilized synthetic peptides. The resulting structures were stabilized by pericyte precursor cells (10T1/2s) in vitro. During tubule formation and stabilization, extracellular matrix proteins such as collagen IV and laminin were deposited. Tubules formed in the matrix of metalloproteinase sensitive hydrogels were visualized from 7 days to 4 weeks in response to different combination of peptides. Moreover, hydrogels functionalized with laminin peptides and transplanted in a mouse cornea supported the ingrowth and attachment of endothelial cells to the hydrogel during angiogenesis. Results of this study illustrate the use of laminin-derived peptides as potential candidates for modification of biomaterials to support angiogenesis. PMID:23914330

  15. Immobilization of microbial cells on inner epidermis of onion bulb scale for biosensor application.

    PubMed

    Kumar, Jitendra; D'Souza, S F

    2011-07-15

    Inner epidermis of onion bulb scales was used as a natural support for immobilization of microbial cells for biosensor application. A bacterium Sphingomonas sp. that hydrolyzes methyl parathion into a chromophoric product, p-nitrophenol (PNP), has been isolated and identified in our laboratory. PNP can be detected by electrochemical and colorimetric methods. Whole cells of Sphingomonas sp. were immobilized on inner epidermis of onion bulb scale by adsorption followed by cross-linking methods. Cells immobilized onion membrane was directly placed in the wells of microplate and associated with the optical transducer. Methyl parathion is an organophosphorus pesticide that has been widely used in the field of agriculture for insect pest control. This pesticide causes environmental pollution and ecological problem. A detection range 4-80 μM of methyl parathion was estimated from the linear range of calibration plot of enzymatic assay. A single membrane was reused for 52 reactions and was found to be stable for 32 days with 90% of its initial hydrolytic activity. The applicability of the cells immobilized onion membrane was also demonstrated with spiked samples.

  16. Effects of immobilized glycosaminoglycans on the proliferation and differentiation of mesenchymal stem cells.

    PubMed

    Uygun, Basak E; Stojsih, Sarah E; Matthew, Howard W T

    2009-11-01

    Mesenchymal stem cells (MSCs) are adult stem cells with potential for multilineage differentiation. They represent an attractive cell source alternative to embryonic stem cells for therapeutic applications. Optimal utilization of MSCs for tissue engineering requires improved biomaterials that can enhance their growth and direct differentiation. The biological activity of glycosaminoglycans (GAGs) has been previously exploited for use in tissue engineering applications. In this study, MSC proliferation and differentiation was studied on GAG-derivatized chitosan membranes. The GAGs included heparin, heparan sulfate, dermatan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate, and hyaluronic acid. The covalent GAG immobilization method and amount of immobilized GAG were varied. It was found that MSC growth increased as much as fivefold on GAG-immobilized surfaces compared to tissue culture plastic and chitosan-only controls. The MSC growth rates increased significantly with increasing GAG density on the culture surfaces. The MSC proliferation rates on heparin, heparan sulfate, dermatan sulfate, and chondroitin 6-sulfate exhibited nonlinear increases with the level of fibronectin binding on these surfaces. In contrast, MSC proliferation on hyaluronic acid and chondroitin 4-sulfate was found to be independent of fibronectin or vitronectin binding on the surfaces, suggesting that these GAGs influenced MSC proliferation through different mechanisms. In conclusion, the results indicate that GAG immobilization on chitosan scaffolds provides an effective means of manipulating MSC proliferation and has promising potential for directing MSC differentiation in tissue engineering applications employing chitosan. PMID:19456238

  17. Pentachlorophenol contaminated groundwater bioremediation using immobilized Sphingomonas cells inoculation in the bioreactor system.

    PubMed

    Yang, Chu-Fang; Lee, Chi-Mei

    2008-03-21

    Pentachlorophenol (PCP) has been used as a wood preservative for more than 100 years. The extensive use of PCP has widely contaminated soil and groundwater. PCP is toxic to living organisms. The main objective of this research was to inoculate the pure PCP-degrading bacterium strain Sphingomonas chlorophenolica PCP-1, isolated from PCP-contaminated soils, into PCP-contaminated groundwater for remediation purposes. The factors that influenced the bioremediation were explored with batch experiments using the inoculated immobilized and suspended cells as inoculation. A biological treatment system inoculated with immobilized cells was set up to estimate the microbial capability to degrade PCP. The results indicated that the suspended and immobilized cells could be inoculated into PCP-contaminated groundwater without adding other supplementary nitrogen and phosphate sources in batch conditions. Moreover, PCP decomposition was accompanied with released Cl- and decreasing pH value. The optimum HRT in the bioreactor system was 12.6h. PCP removal in the bioreactor remained stable and PCP removal efficiency was higher than 92% at this phase. Furthermore, PCP concentration in the biotreatment system effluent remained undetectable. It is possible to bioremediate PCP-contaminated groundwater using immobilized S. chlorophenolica PCP-1 cells in a bioreactor system. The proposed biological treatment system could be maintained for at least for 2 months.

  18. Matrix molecule influence on chondrocyte phenotype and proteoglycan 4 expression by alginate-embedded zonal chondrocytes and mesenchymal stem cells.

    PubMed

    Coates, Emily E; Riggin, Corinne N; Fisher, John P

    2012-12-01

    Articular cartilage resists load and provides frictionless movement at joint surfaces. The tissue is organized into the superficial, middle, deep, and calcified zones throughout its depth, each which serve distinct functions. Proteoglycan 4 (PRG4), found in the superficial zone, is a critical component of the joint's lubricating mechanisms. Maintenance of both the chondrocyte and zonal chondrocyte phenotype remain challenges for in vitro culture and tissue engineering. Here we investigate the expression of PRG4 mRNA and protein by primary bovine superficial zone chondrocytes, middle/deep zone chondrocytes, and mesenchymal stem cells encapsulated in alginate hydrogels with hyaluronic acid (HA) and chondroitin sulfate (CS) additives. Chondrogenic phenotype and differentiation markers are evaluated by mRNA expression, histochemical, and immunohistochemical staining. Results show middle/deep cells express no measurable PRG4 mRNA by day 7. In contrast, superficial zone cells express elevated PRG4 mRNA throughout culture time. This expression can be significantly enhanced up to 15-fold by addition of both HA and CS to scaffolds. Conversely, PRG4 mRNA expression is downregulated (up to 5-fold) by CS and HA in differentiating MSCs, possibly due to build up of entrapped protein. HA and CS demonstrate favorable effects on chondrogenesis by upregulating transcription factor Sox9 mRNA (up to 4.6-fold) and downregulating type I collagen mRNA (up to 18-fold). Results highlight the important relationship between matrix components and expression of critical lubricating proteins in an engineered cartilage scaffold. PMID:22674584

  19. A nanoscale characterization of the interaction of a novel alginate oligomer with the cell surface and motility of Pseudomonas aeruginosa.

    PubMed

    Powell, Lydia C; Pritchard, Manon F; Emanuel, Charlotte; Onsøyen, Edvar; Rye, Philip D; Wright, Chris J; Hill, Katja E; Thomas, David W

    2014-03-01

    Pseudomonas aeruginosa (PA) biofilm-associated infections are a common cause of morbidity in chronic respiratory disease and represent a therapeutic challenge. Recently, the ability of a novel alginate oligomer (OligoG) to potentiate the effect of antibiotics against gram-negative, multi-drug-resistant bacteria and inhibit biofilm formation in vitro has been described. Interaction of OligoG with the cell surface of PA was characterized at the nanoscale using atomic force microscopy (AFM), zeta potential measurement (surface charge), and sizing measurements (dynamic light scattering). The ability of OligoG to modify motility was studied in motility assays. AFM demonstrated binding of OligoG to the bacterial cell surface, which was irreversible after exposure to hydrodynamic shear (5,500 × g). Zeta potential analysis (pH 5-9; 0.1-0.001 M NaCl) demonstrated that binding was associated with marked changes in the bacterial surface charge (-30.9 ± 0.8 to -47.0 ± 2.3 mV; 0.01 M NaCl [pH 5]; P < 0.001). Sizing analysis demonstrated that alteration of surface charge was associated with cell aggregation with a 2- to 3-fold increase in mean particle size at OligoG concentrations greater than 2% (914 ± 284 to 2599 ± 472 nm; 0.01 M NaCl [pH 5]; P < 0.001). These changes were associated with marked dose-dependent inhibition in bacterial swarming motility in PA and Burkholderia spp. The ability of OligoG to bind to a bacterial surface, modulate surface charge, induce microbial aggregation, and inhibit motility represents important direct mechanisms by which antibiotic potentiation and biofilm disruption is affected. These results highlight the value of combining multiple nanoscale technologies to further our understanding of the mechanisms of action of novel antibacterial therapies.

  20. Inhibiting Smooth Muscle Cell Proliferation via Immobilization of Heparin/Fibronectin Complexes on Titanium Surfaces.

    PubMed

    Li, Gui Cai; Xu, Qi Fei; Yang, Ping

    2015-05-01

    The aim of this study was to investigate the inhibitory effect of heparin/fibronectin (Hep/Fn) complexes on neointimal hyperplasia following endovascular intervention. Hep/Fn complexes were immobilized onto titanium (Ti) surfaces, with subsequent X-ray photoelectron spectroscopy (XPS), Toluidine Blue O (TBO) and immunohistochemistry methods were used to characterize surface properties. Smooth muscle cell (SMC) cultures were used to evaluate the effect of Hep/Fn complexes on SMC proliferation. Results showed that Hep/Fn complexes successfully immobilized onto Ti surfaces and resulted in an inhibition of SMC proliferation. This study suggests that Hep/Fn surface-immobilized biomaterials develop as a new generation of biomaterials to prevent neointimal hyperplasia, particularly for use in cardiovascular implants. PMID:26055566

  1. Stability of alginate microbead properties in vitro

    PubMed Central

    Moya, Monica L.; Morley, Michael; Khanna, Omaditya; Opara, Emmanuel C.

    2013-01-01

    Alginate microbeads have been investigated clinically for a number of therapeutic interventions, including drug delivery for treatment of ischemic tissues, cell delivery for tissue regeneration, and islet encapsulation as a therapy for type I diabetes. The physical properties of the microbeads play an important role in regulating cell behavior, protein release, and biological response following implantation. In this research alginate microbeads were synthesized, varying composition (mannuronic acid to guluronic acid ratio), concentration of alginate and needle gauge size. Following synthesis, the size, volume fraction, and morphometry of the beads were quantified. In addition, these properties were monitored over time in vitro in the presence of varying calcium levels in the microenvironment. The initial volume available for solute diffusion increased with alginate concentration and mannuronic (M) acid content, and bead diameter decreased with M content but increased with needle diameter. Interestingly, microbeads eroded completely in saline in less than 3 weeks regardless of synthesis conditions much faster than what has been observed in vivo. However, microbead stability was increased by the addition of calcium in the culture medium. Beads synthesized with low alginate concentration and high G content exhibited a more rapid change in physical properties even in the presence of calcium. These data suggest that temporal variations in the physical characteristics of alginate microbeads can occur in vitro depending on synthesis conditions and microbead environment. The results presented here will assist in optimizing the design of the materials for clinical application in drug delivery and cell therapy. PMID:22350778

  2. Evaluation of biocompatible alginate- and deferoxamine-coated ternary composites for magnetic resonance imaging and gene delivery into glioblastoma cells

    PubMed Central

    Sham, Kathy W. Y.; Chak, Chun-Pong; Lai, Josie M. Y.; Lee, Siu-Fung

    2015-01-01

    Background This paper describes comparative studies in cytotoxicities, magnetic resonance imaging (MRI), and gene delivery into glioblastoma U87MG or U138MG cells with ternary composites that are consist of superparamagnetic iron oxide (SPIO) nanoparticles (NPs) (size: 8-10 nm) with different surface coatings, circular plasmid DNA (pDNA) (~4 kb) equipped with fluorescent/luminescent probe, and branched polyethylenimine (25 kDa, PDI 2.5). Methods Three types of SPIO-NPs were used, including: (I) naked iron oxide NPs with Fe-OH surface group (Bare-NP); (II) iron oxide NPs with a coating of alginate (Alg-NPs); and (III) iron oxide NPs with a coating of deferoxamine (Def-NPs). By tuning the polyethylenimine (PEI)/NP ratios and with a fixed DNA amount, different ternary composites were employed for NP/gene transfection into glioblastoma U87MG or U138MG cells, which were then characterized by Prussian blue staining, in vitro MRI, green fluorescence protein (GFP) fluorescence and luciferase assay. Results Among the composites prepared, 0.2 ng PEI/0.5 µg DNA/1.0 µg Bare-NP ternary composite possessed the best cellular uptake efficiency of NP to the cytoplasm, following the trend Bare-NP > Alg-NP > Def-NP. This observation was consistent to the MRI assessments with in vitro T2 relaxivity (r2) values of 46.0, 35.5, and 23.7 s−1·µM−1·Fe, respectively. For cellular uptake efficiency of the pDNA, all variations of PEI/NP ratios of the composites did not yield significant differences. However, cellular uptake efficiencies of pDNA in the ternary composites in U138MG cells were generally higher than that of U87MG cells by an order of magnitude. Exceptionally, the ternary composite 0.2 ng PEI/0.5 µg DNA/1.0 µg Bare-NP possessed a lowered luciferase activity RLU for gene expression in U138MG cells. A total of 0.2 ng PEI/0.5 µg DNA/0.1 µg Bare-NP would be uptaken to the cell nucleus with the highest luciferase activity. A working concentration range of PEI with at least

  3. Alginate Oligosaccharides Inhibit Fungal Cell Growth and Potentiate the Activity of Antifungals against Candida and Aspergillus spp

    PubMed Central

    Tøndervik, Anne; Sletta, Håvard; Klinkenberg, Geir; Emanuel, Charlotte; Powell, Lydia C.; Pritchard, Manon F.; Khan, Saira; Craine, Kieron M.; Onsøyen, Edvar; Rye, Phil D.; Wright, Chris; Thomas, David W.; Hill, Katja E.

    2014-01-01

    The oligosaccharide OligoG, an alginate derived from seaweed, has been shown to have anti-bacterial and anti-biofilm properties and potentiates the activity of selected antibiotics against multi-drug resistant bacteria. The ability of OligoG to perturb fungal growth and potentiate conventional antifungal agents was evaluated using a range of pathogenic fungal strains. Candida (n = 11) and Aspergillus (n = 3) spp. were tested using germ tube assays, LIVE/DEAD staining, scanning electron microscopy (SEM), atomic force microscopy (AFM) and high-throughput minimum inhibition concentration assays (MICs). In general, the strains tested showed a significant dose-dependent reduction in cell growth at ≥6% OligoG as measured by optical density (OD600; P<0.05). OligoG (>0.5%) also showed a significant inhibitory effect on hyphal growth in germ tube assays, although strain-dependent variations in efficacy were observed (P<0.05). SEM and AFM both showed that OligoG (≥2%) markedly disrupted fungal biofilm formation, both alone, and in combination with fluconazole. Cell surface roughness was also significantly increased by the combination treatment (P<0.001). High-throughput robotic MIC screening demonstrated the potentiating effects of OligoG (2, 6, 10%) with nystatin, amphotericin B, fluconazole, miconazole, voriconazole or terbinafine with the test strains. Potentiating effects were observed for the Aspergillus strains with all six antifungal agents, with an up to 16-fold (nystatin) reduction in MIC. Similarly, all the Candida spp. showed potentiation with nystatin (up to 16-fold) and fluconazole (up to 8-fold). These findings demonstrate the antifungal properties of OligoG and suggest a potential role in the management of fungal infections and possible reduction of antifungal toxicity. PMID:25409186

  4. Bioflavour production from orange peel hydrolysate using immobilized Saccharomyces cerevisiae.

    PubMed

    Lalou, Sofia; Mantzouridou, Fani; Paraskevopoulou, Adamantini; Bugarski, Branko; Levic, Steva; Nedovic, Victor

    2013-11-01

    The rising trend of bioflavour synthesis by microorganisms is hindered by the high manufacturing costs, partially attributed to the cost of the starting material. To overcome this limitation, in the present study, dilute-acid hydrolysate of orange peel was employed as a low-cost, rich in fermentable sugars substrate for the production of flavour-active compounds by Saccharomyces cerevisiae. With this purpose, the use of immobilized cell technology to protect cells against the various inhibitory compounds present in the hydrolysate was evaluated with regard to yeast viability, carbon and nitrogen consumption and cell ability to produce flavour active compounds. For cell immobilization the encapsulation in Ca alginate beads was used. The results were compared with those obtained using free-cell system. Based on the data obtained immobilized cells showed better growth performance and increased ability for de novo synthesis of volatile esters of "fruity" aroma (phenylethyl acetate, ethyl hexanoate, octanoate, decanoate and dodecanoate) than those of free cells. The potential for in situ production of new formulations containing flavour-active compounds derive from yeast cells and also from essential oil of orange peel (limonene, α-terpineol) was demonstrated by the fact that bioflavour mixture was found to accumulate within the beads. Furthermore, the ability of the immobilized yeast to perform efficiently repeated batch fermentations of orange peel hydrolysate for bioflavour production was successfully maintained after six consecutive cycles of a total period of 240 h. PMID:23995224

  5. A strategic study using mutant-strain entrapment in calcium alginate for the production of Saccharomyces cerevisiae cells with high invertase activity.

    PubMed

    Rossi-Alva, Juan Carlos; Rocha-Leão, Maria Helena Miguez

    2003-08-01

    Entrapped cells and entrapped cells grown inside of a calcium alginate matrix as well as free cells have been investigated using Saccharomyces cerevisiae mutant strains with regard to their pattern of growth and invertase activity. The repression of invertase by glucose and glucose-consumption ability were considered in the selection process of the mutants. Efficient sucrose bioconversion due to high invertase activity was obtained when entrapped mutant strain Q6R2 cells were grown within calcium alginate gel beads using sucrose plus glucose as the carbon source. Under these conditions, 1 mg (dry weight) of entrapped cells is able to produce 20 micromol of inverted sugar in 3 min (the maximum activity obtained was 20 units x mg(-1)). The experiments were carried out for 6 months without appreciable loss of either bead integrity or invertase activity. The biocatalyst was also stored at 4 degrees C for 6 months without appreciable loss of the invertase activity. This work shows that entrapped yeast cells with a weak ability to consume sugar may be used to produce inverted sugar.

  6. Production and estimation of alkaline protease by immobilized Bacillus licheniformis isolated from poultry farm soil of 24 Parganas and its reusability

    PubMed Central

    Chatterjee, Shamba

    2015-01-01

    Microbial alkaline protease has become an important industrial and commercial biotech product in the recent years and exerts major applications in food, textile, detergent, and pharmaceutical industries. By immobilization of microbes in different entrapment matrices, the enzyme produced can be more stable, pure, continuous, and can be reused which in turn modulates the enzyme production in an economical manner. There have been reports in support of calcium alginate and corn cab as excellent matrices for immobilization of Bacillus subtilis and Bacillus licheniformis, respectively. This study has been carried out using calcium alginate, κ-carrageenan, agar-agar, polyacrylamide gel, and gelatin which emphasizes not only on enzyme activity of immobilized whole cells by different entrapment matrices but also on their efficiency with respect to their reusability as first attempt. Gelatin was found to be the best matrix among all with highest enzyme activity (517 U/ml) at 24 h incubation point and also showed efficiency when reused. PMID:25709962

  7. Application of magnetic immobilized microorganisms. Ethanol production by saccharomyces cerevisiae

    SciTech Connect

    Birnbaum, S.; Larsson, P.O.

    1982-01-01

    Magnetic Ca alginate yeast beads, made by incorporation of magnetite or the colloidal magnetic liquid ferrofluid, exhibited catalytic behavior similar to that of their nonmagnetic counterparts. The magnetic immobilized preparations short-term performance, long-term operational stability, and capacity for in-situ activation were unaffected by the inclusion of magnetic material. The magnetic quality of the alginate beads provides manipulatory advantages.

  8. Gibberellic acid production by free and immobilized cells in different culture systems.

    PubMed

    Durán-Páramo, Enrique; Molina-Jiménez, Héctor; Brito-Arias, Marco A; Robles-Martínez, Fabián

    2004-01-01

    Gibberellic acid production was studied in different fermentation systems. Free and immobilized cells of Gibberella fujikuroi cultures in shake-flask, stirred and fixed-bed reactors were evaluated for the production of gibberellic acid (GA3). Gibberellic acid production with free cells cultured in a stirred reactor reached 0.206 g/L and a yield of 0.078 g of GA3/g biomass.

  9. Matrix-driven formation of mesenchymal stem cell-extracellular matrix microtissues on soft alginate hydrogels.

    PubMed

    Maia, F Raquel; Fonseca, Keila B; Rodrigues, Gabriela; Granja, Pedro L; Barrias, Cristina C

    2014-07-01

    Mesenchymal stem cells (MSCs) can be made to rearrange into microtissues in response to specific matrix cues, a process that depends on a balance between cell-matrix and cell-cell interactions. The effect of such cues, and especially their interplay, is still not fully understood, particularly in three-dimensional (3-D) systems. Here, the behaviour of human MSCs cultured within hydrogel matrices with tailored stiffness and composition was evaluated. MSC aggregation occurred only in more compliant matrices (G'≤ 120 Pa), when compared to stiffer ones, both in the presence and in the absence of matrix-bound arginine-glycine-aspartic acid cell-adhesion ligands (RGD; 0, 100 and 200 μM). Fibronectin assembly stabilized cell-cell contacts within aggregates, even in non-adhesive matrices. However, MSCs were able to substantially contract the artificial matrix only when RGD was present. Moreover, compliant matrices facilitated cell proliferation and provided an environment conducive for MSC osteogenic differentiation, even without RGD. Cell interactions with the original matrix became less important as time progressed, while the de novo-produced extracellular matrix became a more critical determinant of cell fate. These data provide further insights into the mechanisms by which MSCs sense their microenvironment to organize into tissues, and provide new clues to the design of cell-instructive 3-D matrices.

  10. Matrix-driven formation of mesenchymal stem cell-extracellular matrix microtissues on soft alginate hydrogels.

    PubMed

    Maia, F Raquel; Fonseca, Keila B; Rodrigues, Gabriela; Granja, Pedro L; Barrias, Cristina C

    2014-07-01

    Mesenchymal stem cells (MSCs) can be made to rearrange into microtissues in response to specific matrix cues, a process that depends on a balance between cell-matrix and cell-cell interactions. The effect of such cues, and especially their interplay, is still not fully understood, particularly in three-dimensional (3-D) systems. Here, the behaviour of human MSCs cultured within hydrogel matrices with tailored stiffness and composition was evaluated. MSC aggregation occurred only in more compliant matrices (G'≤ 120 Pa), when compared to stiffer ones, both in the presence and in the absence of matrix-bound arginine-glycine-aspartic acid cell-adhesion ligands (RGD; 0, 100 and 200 μM). Fibronectin assembly stabilized cell-cell contacts within aggregates, even in non-adhesive matrices. However, MSCs were able to substantially contract the artificial matrix only when RGD was present. Moreover, compliant matrices facilitated cell proliferation and provided an environment conducive for MSC osteogenic differentiation, even without RGD. Cell interactions with the original matrix became less important as time progressed, while the de novo-produced extracellular matrix became a more critical determinant of cell fate. These data provide further insights into the mechanisms by which MSCs sense their microenvironment to organize into tissues, and provide new clues to the design of cell-instructive 3-D matrices. PMID:24607421

  11. Capture of endothelial cells under flow using immobilized vascular endothelial growth factor

    PubMed Central

    Smith, Randall J.; Koobatian, Maxwell T.; Shahini, Aref; Swartz, Daniel D.; Andreadis, Stelios T.

    2015-01-01

    We demonstrate the ability of immobilized vascular endothelial growth factor (VEGF) to capture endothelial cells (EC) with high specificity under fluid flow. To this end, we engineered a surface consisting of heparin bound to poly-L-lysine to permit immobilization of VEGF through the C-terminal heparin-binding domain. The immobilized growth factor retained its biological activity as shown by proliferation of EC and prolonged activation of KDR signaling. Using a microfluidic device we assessed the ability to capture EC under a range of shear stresses from low (0.5 dyne/cm2) to physiological (15 dyne/cm2). Capture was significant for all shear stresses tested. Immobilized VEGF was highly selective for EC as evidenced by significant capture of human umbilical vein and ovine pulmonary artery EC but no capture of human dermal fibroblasts, human hair follicle derived mesenchymal stem cells, or mouse fibroblasts. Further, VEGF could capture EC from mixtures with non-EC under low and high shear conditions as well as from complex fluids like whole human blood under high shear. Our findings may have far reaching implications, as they suggest that VEGF could be used to promote endothelialization of vascular grafts or neovascularization of implanted tissues by rare but continuously circulating EC. PMID:25771020

  12. Amperometric determination of nitric oxide derived from pulmonary artery endothelial cells immobilized in a microchip channel.

    PubMed

    Spence, Dana M; Torrence, Nicholas J; Kovarik, Michelle L; Martin, R Scott

    2004-11-01

    A simple method for immobilizing a confluent layer of bovine pulmonary artery endothelial cells (bPAECs) in microchip-based channels is described. The microchips are prepared from poly(dimethylsiloxane) and have channel dimensions that approximate resistance vessels in vivo. The reversibly sealed channels were coated with fibronectin (100 microg ml(-1)) by aspiration. The bPAECs, which were introduced in the same manner, became attached to the fibronectin coating in about 2 h. The microchip could then be resealed over a micromolded carbon ink electrode (24 microm width x 6 microm height). Coating the carbon microelectrode with a 0.05% Nafion solution selectively blocked nitrite (10 microM) from being transported to the electrode surface while nitric oxide (NO, 10 microM) was amperometrically measured. Upon stimulation with adenosine triphosphate (ATP, 100 microM) the immobilized bPAECs produced and released micromolar amounts of NO. This NO production was effectively inhibited when the immobilized cells were incubated with L-nitro-arginine methyl ester (L-NAME), a competitive inhibitor for nitric oxide synthase. Moreover, once the immobilized bPAECs were no longer able to produce NO, incubation with L-arginine allowed for further ATP-stimulated NO production.

  13. PLGA/alginate composite microspheres for hydrophilic protein delivery.

    PubMed

    Zhai, Peng; Chen, X B; Schreyer, David J

    2015-11-01

    Poly(lactic-co-glycolic acid) (PLGA) microspheres and PLGA/alginate composite microspheres were prepared by a novel double emulsion and solvent evaporation technique and loaded with bovine serum albumin (BSA) or rabbit anti-laminin antibody protein. The addition of alginate and the use of a surfactant during microsphere preparation increased the encapsulation efficiency and reduced the initial burst release of hydrophilic BSA. Confocal laser scanning microcopy (CLSM) of BSA-loaded PLGA/alginate composite microspheres showed that PLGA, alginate, and BSA were distributed throughout the depths of microspheres; no core/shell structure was observed. Scanning electron microscopy revealed that PLGA microspheres erode and degrade more quickly than PLGA/alginate composite microspheres. When loaded with anti-laminin antibody, the function of released antibody was well preserved in both PLGA and PLGA/alginate composite microspheres. The biocompatibility of PLGA and PLGA/alginate microspheres were examined using four types of cultured cell lines, representing different tissue types. Cell survival was variably affected by the inclusion of alginate in composite microspheres, possibly due to the sensitivity of different cell types to excess calcium that may be released from the calcium cross-linked alginate.

  14. Enzyme-entrapping behaviors in alginate fibers and their papers

    SciTech Connect

    Kobayashi, Y.; Matsuo, R.; Ohya, T.; Yokoi, N.

    1987-01-01

    Enzyme immobilization in the form of fiber and paper was easily achieved by wet spinning of aqueous admixture of sodium alginate and enzymes into divalent metallic ion solution as a coagulating bath, followed by paper making of resultant shortly cut fibers. Entrapment yields of enzymes used, e.g., glucoamylase, cyclodextrin glucanotransferase, endo-polygalacturonase, and protease, were always higher in calcium alginate fibers and their papers than those in corresponding beads. It was found that the yields increased with an increase of the discharge rate through the spinning nozzle because the higher discharge rate could provide more highly oriented metal-chelate linear polymer molecules along the fiber axis for preventing leakage of entrapped enzymes. Divalent metallic ions affected greatly the entrapment of glucoamylase in alginate fibers, the order of which followed rougly the ionotropic series of Thiele. Entrapment of glucoamylase in bicomponent systems comprising alginate and other water-soluble polymers was also investigated. (Refs. 41).

  15. Improved lectin-mediated immobilization of human red blood cells in superporous agarose beads.

    PubMed

    Gottschalk, Ingo; Gustavsson, Per Erik; Ersson, Bo; Lundahl, Per

    2003-01-25

    A new type of agarose bead, superporous agarose, was used as a gel support for immobilization of human red blood cells (RBCs) mediated by wheat germ lectin. The number of immobilized cells was similar to that obtained with commercial wheat germ lectin-agarose but the cell stability appeared to be superior. This allowed improved frontal affinity chromatographic analyses of cytochalasin B (CB)-binding to the glucose transporter GLUT1 which established a ratio of one CB-binding site per GLUT1 dimer for both plain RBCs or those treated with different poly amino acids. The measured dissociation constants, 70+/-14 nM for CB and 12+/-3 mM for glucose binding to GLUT1, are similar to those reported earlier.

  16. Biofunctionalized Calcium Phosphate Cement to Enhance the Attachment and Osteodifferentiation of Stem Cells Released from Fast-Degradable Alginate-Fibrin Microbeads

    PubMed Central

    Chen, Wenchuan; Weir, Michael D.

    2012-01-01

    Stem cell-encapsulating microbeads could be mixed into a paste such as calcium phosphate cement (CPC), where the microbeads could protect the cells from the mixing and injection forces. After being placed, the microbeads could quickly degrade to release the cells throughout the scaffold, while creating macropores. The objectives of this study were to (1) construct alginate-fibrin microbeads encapsulating human umbilical cord mesenchymal stem cells (hUCMSCs) embedded in the surface of novel biofunctionalized CPC and (2) investigate microbead degradation, cell release, and osteodifferentiation on CPC. Hydrogel microbeads were fabricated that encapsulated hUCMSCs at 1×106 cells/mL. CPC was biofunctionalized with fibronectin (Fn) and Arg-Gly-Asp (RGD). Four scaffolds were tested: CPC control, CPC mixed with Fn, CPC mixed with RGD, and CPC grafted with RGD. The degradable microbeads released hUCMSCs at 7 days, which attached to CPC. Adding Fn or RGD to CPC greatly improved cell attachment. CPC grafted with RGD showed the fastest cell proliferation, with cell density being ninefold that on CPC control. The released hUCMSCs underwent osteodifferentiation. Alkaline phosphatase, osteocalcin, collagen 1, and runt-related transcription factor 2 (Runx2) gene expression increased by 10 to 30 fold at 7–21 days, compared with day 1. The released cells on CPC synthesized bone minerals, with the mineralization amount at 21 days being two orders of magnitude higher than that at 7 days. In conclusion, alginate-fibrin microbeads embedded in CPC surface were able to quickly release the hUCMSCs that attached to biofunctionalized CPC. Incorporating Fn and RGD into CPC greatly improved cell function, and CPC grafted with RGD had the fastest cell proliferation. The released cells on CPC differentiated into the osteogenic lineage and synthesized bone minerals. The new biofunctionalized CPC with hUCMSC-encapsulating microbeads is promising for bone regeneration applications. PMID:22435653

  17. Growth characteristics of Sanguinaria canadensis L. cell suspensions and immobilized cultures for production of benzophenanthridine alkaloids.

    PubMed

    Rho, D; Chauret, N; Laberge, N; Archambault, J

    1992-02-01

    Sanguinaria canadensis L. plants were harvested from a local forest and calli were initiated from leaf explants. The production of benzophenanthridine alkaloids (i.e. sanguinarine, sanguilutine, sanguirubine, chelerythrine, chelilutine and chelirubine) by S. canadensis cell grown in modified B5 and IM2 media was compared to the alkaloid content of rhizomes. Sanguinarine accounted for approximately 80% of the total alkaloid content of cultured cells (1.3%, g g-1) while sanguinarine and sanguirubine accounted for 70% of rhizome alkaloids (9.0%, g g-1). Sanguinarine, chelirubine and chelerythrine were the only known alkaloids detected in cultured S. canadensis cells. Maximum alkaloid production of cultures performed using B5 medium, containing half the original nitrate concentration, was observed following extracellular nitrate and sugar depletion. The scale-up of this culture was successfully performed in a 2-1 immobilization bioreactor. The consumption of sugar and nitrate as well as the oxygen (OTR) and carbon dioxide (CTR) transfer rates of the immobilized cell culture were monitored for 15 days. The maximum sugar and nitrate consumption rates were 1.8 g l-1 per day and 2.3 mM per day respectively. The maximum OTR and CTR of the immobilized cell culture were 0.8 mmol O2 l-1 h-1 and 0.95 mmol CO2 l-1 h-1 respectively. The sanguinarine yield of this culture reached 1.0% based on biomass dry weight (g g-1 dw) by day 15.

  18. Optimizing Immobilized Enzyme Performance in Cell-Free Environments to Produce Liquid Fuels

    SciTech Connect

    Belfort, Georges; Grimaldi, Joseph J.

    2015-01-27

    Limitations on biofuel production using cell culture (Escherichia coli, Clostridium, Saccharomyces cerevisiae, brown microalgae, blue-green algae and others) include low product (alcohol) concentrations (≤0.2 vol%) due to feedback inhibition, instability of cells, and lack of economical product recovery processes. To overcome these challenges, an alternate simplified biofuel production scheme was tested based on a cell-free immobilized enzyme system. Using this cell free system, we were able to obtain about 2.6 times higher concentrations of iso-butanol using our non-optimized system as compared with live cell systems. This process involved two steps: (i) converts acid to aldehyde using keto-acid decarboxylase (KdcA), and (ii) produces alcohol from aldehyde using alcohol dehydrogenase (ADH) with a cofactor (NADH) conversion from inexpensive formate using a third enzyme, formate dehydrogenase (FDH). To increase stability and conversion efficiency with easy separations, the first two enzymes were immobilized onto methacrylate resin. Fusion proteins of labile KdcA (fKdcA) were expressed to stabilize the covalently immobilized KdcA. Covalently immobilized ADH exhibited long-term stability and efficient conversion of aldehyde to alcohol over multiple batch cycles without fusions. High conversion rates and low protein leaching were achieved by covalent immobilization of enzymes on methacrylate resin. The complete reaction scheme was demonstrated by immobilizing both ADH and fKdcA and using FDH free in solution. The new system without in situ removal of isobutanol achieved a 55% conversion of ketoisovaleric acid to isobutanol at a concentration of 0.5 % (v/v). Further increases in titer will require continuous removal of the isobutanol using our novel brush membrane system that exhibits a 1.5 fold increase in the separation factor of isobutanol from water versus that obtained for commercial silicone rubber membranes. These bio-inspired brush membranes are based on the

  19. Three immobilized-cell columnar bioreactors for enhanced production of commodity chemicals

    SciTech Connect

    Davison, B.H.; Scott, C.D.; Kaufman, E.N.

    1993-12-31

    Immobilized-cell fluidized-bed bioreactors (FBRs) can be used with a variety of fermentations to increase production of fuels, solvents, organic acids, and other fermentation products. Part of the increased rates and yields are due to the immobilization of the biocatalyst at high concentrations. This FBR system with immobilized Zymomonas mobilis increased ethanol productivity more than tenfold with 99% conversion and near stoichiometric yields. FBRs also offer several additional modes of operation for simultaneous fermentation and separation to further increase production by removing the inhibitory products directly from the continuous fermentation. The production of lactic acid by immobilized Lactobacillus was augmented with the addition and removal of solid adsorbent particles to the FBR. An immiscible organic extractant also was used to extract butanol from the acetone-butanol fermentation by Clostridium acetobutylicum. Demonstrations with these FBR systems have already shown definite advantages by improved overall product yields (decreasing feed costs) and by increased rates (decreasing capital and operating costs). Further demonstration and scale-up continue.

  20. Three immobilized-cell columnar bioreactors for enhanced production of commodity chemicals

    SciTech Connect

    Davison, B.H.; Scott, C.D.; Kaufman, E.N.

    1993-07-01

    Immobilized-cell fluidized-bed bioreactors (FBRS) can be used with a variety of fermentations to increase production of fuels, solvents, organic acids, and other fermentation products. Part of the increased rates and yields are due to the immobilization of the biocatalyst at high concentrations. This FBR system with immobilized Zymomonas mobiles increased ethanol productivity more than tenfold with 99% conversion and near stoichiometric yields. FBRs also offer several additional modes of operation for simultaneous fermentation and separation to further increase production by removing the inhibitory products directly from the continuous fermentation. The production of lactic acid by immobilized Lactobacillus was augmented with the addition and removal of solid adsorbent particles to the FBR. An immiscible organic extractant also was used to extract butanol from the acetone-butanol fermentation by Clostridium acetobutylicum. Demonstrations with these FBR systems have already shown definite advantages by improved overall product yields (decreasing feed costs) and by increased rates (decreasing capital and operating costs). Further demonstration and scale-up continue.

  1. A novel cell weighing method based on the minimum immobilization pressure for biological applications

    SciTech Connect

    Zhao, Qili; Shirinzadeh, Bijan; Cui, Maosheng; Sun, Mingzhu; Liu, Yaowei; Zhao, Xin

    2015-07-28

    A novel weighing method for cells with spherical and other regular shapes is proposed in this paper. In this method, the relationship between the cell mass and the minimum aspiration pressure to immobilize the cell (referred to as minimum immobilization pressure) is derived for the first time according to static theory. Based on this relationship, a robotic cell weighing process is established using a traditional micro-injection system. Experimental results on porcine oocytes demonstrate that the proposed method is able to weigh cells at an average speed of 16.3 s/cell and with a success rate of more than 90%. The derived cell mass and density are in accordance with those reported in other published results. The experimental results also demonstrated that this method is able to detect less than 1% variation of the porcine oocyte mass quantitatively. It can be conducted by a pair of traditional micropipettes and a commercial pneumatic micro-injection system, and is expected to perform robotic operation on batch cells. At present, the minimum resolution of the proposed method for measuring the cell mass can be 1.25 × 10{sup −15 }kg. Above advantages make it very appropriate for quantifying the amount of the materials injected into or moved out of the cells in the biological applications, such as nuclear enucleations and embryo microinjections.

  2. Internalization: acute apoptosis of breast cancer cells using herceptin-immobilized gold nanoparticles

    PubMed Central

    Rathinaraj, Pierson; Al-Jumaily, Ahmed M; Huh, Do Sung

    2015-01-01

    Herceptin, the monoclonal antibody, was successfully immobilized on gold nanoparticles (GNPs) to improve their precise interactions with breast cancer cells (SK-BR3). The mean size of the GNPs (29 nm), as determined by dynamic light scattering, enlarged to 82 nm after herceptin immobilization. The in vitro cell culture experiment indicated that human skin cells (FB) proliferated well in the presence of herceptin-conjugated GNP (GNP–Her), while most of the breast cancer cells (SK-BR3) had died. To elucidate the mechanism of cell death, the interaction of breast cancer cells with GNP–Her was tracked by confocal laser scanning microscopy. Consequently, GNP–Her was found to be bound precisely to the membrane of the breast cancer cell, which became almost saturated after 6 hours incubation. This shows that the progression signal of SK-BR3 cells is retarded completely by the precise binding of antibody to the human epidermal growth factor receptor 2 receptor of the breast cancer cell membrane, causing cell death. PMID:25709498

  3. A novel cell weighing method based on the minimum immobilization pressure for biological applications

    NASA Astrophysics Data System (ADS)

    Zhao, Qili; Shirinzadeh, Bijan; Cui, Maosheng; Sun, Mingzhu; Liu, Yaowei; Zhao, Xin

    2015-07-01

    A novel weighing method for cells with spherical and other regular shapes is proposed in this paper. In this method, the relationship between the cell mass and the minimum aspiration pressure to immobilize the cell (referred to as minimum immobilization pressure) is derived for the first time according to static theory. Based on this relationship, a robotic cell weighing process is established using a traditional micro-injection system. Experimental results on porcine oocytes demonstrate that the proposed method is able to weigh cells at an average speed of 16.3 s/cell and with a success rate of more than 90%. The derived cell mass and density are in accordance with those reported in other published results. The experimental results also demonstrated that this method is able to detect less than 1% variation of the porcine oocyte mass quantitatively. It can be conducted by a pair of traditional micropipettes and a commercial pneumatic micro-injection system, and is expected to perform robotic operation on batch cells. At present, the minimum resolution of the proposed method for measuring the cell mass can be 1.25 × 10-15 kg. Above advantages make it very appropriate for quantifying the amount of the materials injected into or moved out of the cells in the biological applications, such as nuclear enucleations and embryo microinjections.

  4. Continuous conversion of sweet sorghum juice to ethanol using immobilized yeast cells

    SciTech Connect

    Mohite, U.; SivaRaman, H.

    1984-01-01

    While extensive work has been reported on sugarcane and sugarcane molasses for ethanol production, relatively few reports are available on ethanol production from sweet sorghum juice. With the advent of immobilized cell technology, an attempt has been made to utilize this technology for the production of ethanol from sweet sorghum juice. The species was Sorghum bicolar (Moench). The maximum productivity obtained at 30/sup 0/C with Saccharomyces uvarum cells immobilized in gelatin was 168 g/L h at an ethanol concentration of 2.4 g (w/v) using sweet sorghum juice having 11.5% fermentable sugars. The calculated value for full conversion was 86 g/L at an ethanol concentration of 5.5 g (w/v). The low concentration of total sugars in the juice, however, would make ethanol recovery expensive unless a uniformly high concentration of 16% or more of total sugars can be obtained.

  5. Production of biofuels from pretreated microalgae biomass by anaerobic fermentation with immobilized Clostridium acetobutylicum cells.

    PubMed

    Efremenko, E N; Nikolskaya, A B; Lyagin, I V; Senko, O V; Makhlis, T A; Stepanov, N A; Maslova, O V; Mamedova, F; Varfolomeev, S D

    2012-06-01

    The purpose of this work was to study the possible use of pretreated biomass of various microalgae and cyanobacteria as substrates for acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum cells immobilized into poly(vinyl alcohol) cryogel. To this end, the biochemical composition of photosynthetic microorganisms cultivated under various conditions was studied. The most efficient technique for pretreating microalgal biomass for its subsequent conversion into biofuels appeared to be thermal decomposition at 108 °C. For the first time the maximum productivity of the ABE fermentation in terms of hydrogen (8.5 mmol/L medium/day) was obtained using pretreated biomass of Nannochloropsis sp. Maximum yields of butanol and ethanol were observed with Arthrospira platensis biomass used as the substrate. Immobilized Clostridium cells were demonstrated to be suitable for multiple reuses (for a minimum of five cycles) in ABE fermentation for producing biofuels from pretreated microalgal biomass.

  6. Continuous Ethanol Production Using Immobilized-Cell/Enzyme Biocatalysts in Fluidized-Bed Bioreactor (FBR)

    SciTech Connect

    Nghiem, NP

    2003-11-16

    The immobilized-cell fluidized-bed bioreactor (FBR) was developed at Oak Ridge National Laboratory (ORNL). Previous studies at ORNL using immobilized Zymomonas mobilis in FBR at both laboratory and demonstration scale (4-in-ID by 20-ft-tall) have shown that the system was more than 50 times as productive as industrial benchmarks (batch and fed-batch free cell fermentations for ethanol production from glucose). Economic analysis showed that a continuous process employing the FBR technology to produce ethanol from corn-derived glucose would offer savings of three to six cents per gallon of ethanol compared to a typical batch process. The application of the FBR technology for ethanol production was extended to investigate more complex feedstocks, which included starch and lignocellulosic-derived mixed sugars. Economic analysis and mathematical modeling of the reactor were included in the investigation. This report summarizes the results of these extensive studies.

  7. Chondrocyte Culture in Three Dimensional Alginate Sulfate Hydrogels Promotes Proliferation While Maintaining Expression of Chondrogenic Markers

    PubMed Central

    Mhanna, Rami; Kashyap, Aditya; Palazzolo, Gemma; Vallmajo-Martin, Queralt; Becher, Jana; Möller, Stephanie; Schnabelrauch, Matthias

    2014-01-01

    The loss of expression of chondrogenic markers during monolayer expansion remains a stumbling block for cell-based treatment of cartilage lesions. Here, we introduce sulfated alginate hydrogels as a cartilage biomimetic biomaterial that induces cell proliferation while maintaining the chondrogenic phenotype of encapsulated chondrocytes. Hydroxyl groups of alginate were converted to sulfates by incubation with sulfur trioxide–pyridine complex (SO3/pyridine), yielding a sulfated material cross-linkable with calcium chloride. Passage 3 bovine chondrocytes were encapsulated in alginate and alginate sulfate hydrogels for up to 35 days. Cell proliferation was five-fold higher in alginate sulfate compared with alginate (p=0.038). Blocking beta1 integrins in chondrocytes within alginate sulfate hydrogels significantly inhibited proliferation (p=0.002). Sulfated alginate increased the RhoA activity of chondrocytes compared with unmodified alginate, an increase that was blocked by β1 blocking antibodies (p=0.017). Expression and synthesis of type II collagen, type I collagen, and proteoglycan was not significantly affected by the encapsulation material evidenced by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry. Alginate sulfate constructs showed an opaque appearance in culture, whereas the unmodified alginate samples remained translucent. In conclusion, alginate sulfate provides a three dimensional microenvironment that promotes both chondrocyte proliferation and maintenance of the chondrogenic phenotype and represents an important advance for chondrocyte-based cartilage repair therapies providing a material in which cell expansion can be done in situ. PMID:24320935

  8. Polyglycerol dendrimers immobilized on radiation grafted poly-HEMA hydrogels: Surface chemistry characterization and cell adhesion

    NASA Astrophysics Data System (ADS)

    Higa, Olga Z.; Faria, Henrique Antonio Mendonça; de Queiroz, Alvaro A. A.

    2014-05-01

    Radiation induced grafting of poly(2-hydroxyethylmethacrylate) (PHEMA) on low density polyethylene (LDPE) films and subsequent immobilization of poly(glycerol) dendrimer (PGLD) has been performed with the aim to improve cell adhesion and proliferation on the surface of the polymer, in order to enhance their properties for bone tissue engineering scaffolding applications. Radiation grafting of PHEMA onto LDPE was promoted by γ-ray radiation. The covalent immobilization of PGLD on LDPE-g-PHEMA surface was performed by using a dicyclohexyl carbodiimide (DCC)/N,N-dimethylaminopyridine (DMAP) method. The occurrence of grafting polymerization of PHEMA and further immobilization of PGLD was quantitatively confirmed by photoelectron spectroscopy (XPS) and fluorescence, respectively. The LDPE-g-PHEMA surface topography after PGLD coupling was studied by atomic force microscopy (AFM). The hydrophilicity of the LDPE-g-PHEMA film was remarkably improved compared to that of the ungrafted LDPE. The core level XPS ESCA spectrum of PHEMA-grafted LDPE showed two strong peaks at 286.6 eV (from hydroxyl groups and ester groups) and 289.1 eV (from ester groups) due to PHEMA brushes grafted onto LDPE surfaces. The results from the cell adhesion studies show that MCT3-E1 cells tended to spread more slowly on the LDPE-g-PHEMA than on the LDPE-g-PHEMA-i-PGLD.

  9. Raspberry wine fermentation with suspended and immobilized yeast cells of two strains of Saccharomyces cerevisiae.

    PubMed

    Djordjević, Radovan; Gibson, Brian; Sandell, Mari; de Billerbeck, Gustavo M; Bugarski, Branko; Leskošek-Čukalović, Ida; Vunduk, Jovana; Nikićević, Ninoslav; Nedović, Viktor

    2015-01-01

    The objectives of this study were to assess the differences in fermentative behaviour of two different strains of Saccharomyces cerevisiae (EC1118 and RC212) and to determine the differences in composition and sensory properties of raspberry wines fermented with immobilized and suspended yeast cells of both strains at 15 °C. Analyses of aroma compounds, glycerol, acetic acid and ethanol, as well as the kinetics of fermentation and a sensory evaluation of the wines, were performed. All fermentations with immobilized yeast cells had a shorter lag phase and faster utilization of sugars and ethanol production than those fermented with suspended cells. Slower fermentation kinetics were observed in all the samples that were fermented with strain RC212 (suspended and immobilized) than in samples fermented with strain EC1118. Significantly higher amounts of acetic acid were detected in all samples fermented with strain RC212 than in those fermented with strain EC1118 (0.282 and 0.602 g/l, respectively). Slightly higher amounts of glycerol were observed in samples fermented with strain EC1118 than in those fermented with strain RC212.

  10. Raspberry wine fermentation with suspended and immobilized yeast cells of two strains of Saccharomyces cerevisiae.

    PubMed

    Djordjević, Radovan; Gibson, Brian; Sandell, Mari; de Billerbeck, Gustavo M; Bugarski, Branko; Leskošek-Čukalović, Ida; Vunduk, Jovana; Nikićević, Ninoslav; Nedović, Viktor

    2015-01-01

    The objectives of this study were to assess the differences in fermentative behaviour of two different strains of Saccharomyces cerevisiae (EC1118 and RC212) and to determine the differences in composition and sensory properties of raspberry wines fermented with immobilized and suspended yeast cells of both strains at 15 °C. Analyses of aroma compounds, glycerol, acetic acid and ethanol, as well as the kinetics of fermentation and a sensory evaluation of the wines, were performed. All fermentations with immobilized yeast cells had a shorter lag phase and faster utilization of sugars and ethanol production than those fermented with suspended cells. Slower fermentation kinetics were observed in all the samples that were fermented with strain RC212 (suspended and immobilized) than in samples fermented with strain EC1118. Significantly higher amounts of acetic acid were detected in all samples fermented with strain RC212 than in those fermented with strain EC1118 (0.282 and 0.602 g/l, respectively). Slightly higher amounts of glycerol were observed in samples fermented with strain EC1118 than in those fermented with strain RC212. PMID:25418076

  11. Mobile and immobile calcium buffers in bovine adrenal chromaffin cells.

    PubMed Central

    Zhou, Z; Neher, E

    1993-01-01

    1. The calcium binding capacity (kappa S) of bovine chromaffin cells preloaded with fura-2 was measured during nystatin-perforated-patch recordings. 2. Subsequently, the perforated patch was ruptured to obtain a whole-cell recording situation, and the time course of kappa S was monitored during periods of up to one hour. 3. No rapid change (within 10-20 s) of kappa S was observed upon transition to whole-cell recording, as would be expected, if highly mobile organic anions contributed significantly to calcium buffering. However, approximately half of the cells investigated displayed a drop in kappa S within 2-5 min, indicative of the loss of soluble Ca2+ binding proteins in the range of 7-20 kDa. 4. The average Ca2+ binding capacity (differential ratio of bound calcium over free calcium) was 9 +/- 7 (mean +/- S.E.M.) for the poorly mobile component and 31 +/- 10 for the fixed component. It was concluded that a contribution of 7 from highly mobile buffer would have been detected, if present. Thus, this value can be considered as an upper bound to highly mobile Ca2+ buffer. 5. Both mobile and fixed calcium binding capacity appeared to have relatively low Ca2+ affinity, since kappa S did not change in the range of Ca2+ concentrations between 0.1 and 3 microM. 6. It was found that cellular autofluorescence and contributions to fluorescence of non-hydrolysed or compartmentalized dye contribute a serious error in estimation of kappa S. 'Balanced loading', a degree of fura-2 loading such that the calcium binding capacity of fura-2 equals cellular calcium binding capacity, minimizes these errors. Also, changes in kappa S at the transition from perforated-patch to whole-cell recording can be most faithfully recorded for similar degrees of loading in both situations. 7. Nystatin was found unable to make pores from inside of the plasma membrane of chromaffin cells. With careful preparation and storage the diluted nystatin solution maintained its high activity of membrane

  12. Bioactive apatite incorporated alginate microspheres with sustained drug-delivery for bone regeneration application.

    PubMed

    Li, Haibin; Jiang, Fei; Ye, Song; Wu, Yingying; Zhu, Kaiping; Wang, Deping

    2016-05-01

    The strontium-substituted hydroxyapatite microspheres (SrHA) incorporated alginate composite microspheres (SrHA/Alginate) were prepared via adding SrHA/alginate suspension dropwise into calcium chloride solution, in which the gel beads were formed by means of crosslinking reaction. The structure, morphology and in vitro bioactivity of the composite microspheres were studied by using XRD, SEM and EDS methods. The biological behaviors were characterized and analyzed through inductively coupled plasma optical emission spectroscopy (ICP-OES), CCK-8, confocal laser microscope and ALP activity evaluations. The experimental results indicated that the synthetic SrHA/Alginate showed similar morphology to the well-known alginate microspheres (Alginate) and both of them possessed a great in vitro bioactivity. Compared with the control Alginate, the SrHA/Alginate enhanced MC3T3-E1 cell proliferation and ALP activity by releasing osteoinductive and osteogenic Sr ions. Furthermore, vancomycin was used as a model drug to investigate the drug release behaviors of the SrHA/Alginate, Alginate and SrHA. The results suggested that the SrHA/Alginate had a highest drug-loading efficiency and best controlled drug release properties. Additionally, the SrHA/Alginate was demonstrated to be pH-sensitive as well. The increase of the pH value in phosphate buffer solution (PBS) accelerated the vancomycin release. Accordingly, the multifunctional SrHA/Alginate can be applied in the field of bioactive drug carriers and bone filling materials. PMID:26952484

  13. Layer by layer chitosan/alginate coatings on poly(lactide-co-glycolide) nanoparticles for antifouling protection and Folic acid binding to achieve selective cell targeting.

    PubMed

    Zhou, Jie; Romero, Gabriela; Rojas, Elena; Ma, Lie; Moya, Sergio; Gao, Changyou

    2010-05-15

    Polyelectrolyte multilayers (PEMs) composed of two natural polysaccharides-chitosan (Chi) and alginate (Alg) were deposited by Layer by layer (LbL) assembly on top of biocompatible poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs). Folic acid (FA) or FA grafted poly(ethylene glycol) (PEG-FA) were covalently bounded to the PEMs via carbodiimide chemistry. The assembly of biocompatible PEMs was monitored on planar surfaces by means of the quartz crystal microbalance with dissipation (QCM-D) technique and on top of PLGA NPs by means of ζ-potential measurements. BSA was used as model protein to characterize protein adsorption on PEMs. QCM-D showed protein deposition could not be observed on the Chi/Alg multilayer, for both Chitosan and Alginate as top layers. Finally, cellular uptake experiments were carried out by co-culture of HepG2 cells in presence of NPs. Flow Cytometry and confocal laser scanning microscopy (CLSM) were used to investigate the influence of the surface chemistry of the NPs on uptake. For the HepG2 cell line significantly less uptake of PLGA NPs coated with Chi/Alg than the bare NPs was observed but the uptake increased after attachment of FA molecules.

  14. Bioethanol Production from Uncooked Raw Starch by Immobilized Surface-engineered Yeast Cells

    NASA Astrophysics Data System (ADS)

    Chen, Jyh-Ping; Wu, Kuo-Wei; Fukuda, Hideki

    Surface-engineered yeast Saccharomyces cerevisiae codisplaying Rhizopus oryzae glucoamylase and Streptococcus bovis α-amylase on the cell surface was used for direct production of ethanol from uncooked raw starch. By using 50 g/L cells during batch fermentation, ethanol concentration could reach 53 g/L in 7 days. During repeated batch fermentation, the production of ethanol could be maintained for seven consecutive cycles. For cells immobilized in loofa sponge, the concentration of ethanol could reach 42 g/L in 3 days in a circulating packed-bed bioreactor. However, the production of ethanol stopped thereafter because of limited contact between cells and starch. The bioreactor could be operated for repeated batch production of ethanol, but ethanol concentration dropped to 55% of its initial value after five cycles because of a decrease in cell mass and cell viability in the bioreactor. Adding cells to the bioreactor could partially restore ethanol production to 75% of its initial value.

  15. Efficiently engineered cell sheet using a complex of polyethylenimine–alginate nanocomposites plus bone morphogenetic protein 2 gene to promote new bone formation

    PubMed Central

    Jin, Han; Zhang, Kai; Qiao, Chunyan; Yuan, Anliang; Li, Daowei; Zhao, Liang; Shi, Ce; Xu, Xiaowei; Ni, Shilei; Zheng, Changyu; Liu, Xiaohua; Yang, Bai; Sun, Hongchen

    2014-01-01

    Regeneration of large bone defects is a common clinical problem. Recently, stem cell sheet has been an emerging strategy in bone tissue engineering. To enhance the osteogenic potential of stem cell sheet, we fabricated bone morphogenetic protein 2 (BMP-2) gene-engineered cell sheet using a complex of polyethylenimine–alginate (PEI–al) nanocomposites plus human BMP-2 complementary(c)DNA plasmid, and studied its osteogenesis in vitro and in vivo. PEI–al nanocomposites carrying BMP-2 gene could efficiently transfect bone marrow mesenchymal stem cells. The cell sheet was made by culturing the cells in medium containing vitamin C for 10 days. Assays on the cell culture showed that the genetically engineered cells released the BMP-2 for at least 14 days. The expression of osteogenesis-related gene was increased, which demonstrated that released BMP-2 could effectively induce the cell sheet osteogenic differentiation in vitro. To further test the osteogenic potential of the cell sheet in vivo, enhanced green fluorescent protein or BMP-2-producing cell sheets were treated on the cranial bone defects. The results indicated that the BMP-2-producing cell sheet group was more efficient than other groups in promoting bone formation in the defect area. Our results suggested that PEI–al nanocomposites efficiently deliver the BMP-2 gene to bone marrow mesenchymal stem cells and that BMP-2 gene-engineered cell sheet is an effective way for promoting bone regeneration. PMID:24855355

  16. Ethanol production by fermentation using immobilized cells of Saccharomyces cerevisiae in cashew apple bagasse.

    PubMed

    Pacheco, Alexandre Monteiro; Gondim, Diego Romão; Gonçalves, Luciana Rocha Barros

    2010-05-01

    In this work, cashew apple bagasse (CAB) was used for Saccharomyces cerevisiae immobilization. The support was prepared through a treatment with a solution of 3% HCl, and delignification with 2% NaOH was also conducted. Optical micrographs showed that high populations of yeast cells adhered to pre-treated CAB surface. Ten consecutive fermentations of cashew apple juice for ethanol production were carried out using immobilized yeasts. High ethanol productivity was observed from the third fermentation assay until the tenth fermentation. Ethanol concentrations (about 19.82-37.83 g L(-1) in average value) and ethanol productivities (about 3.30-6.31 g L(-1) h(-1)) were high and stable, and residual sugar concentrations were low in almost all fermentations (around 3.00 g L(-1)) with conversions ranging from 44.80% to 96.50%, showing efficiency (85.30-98.52%) and operational stability of the biocatalyst for ethanol fermentation. Results showed that cashew apple bagasse is an efficient support for cell immobilization aiming at ethanol production.

  17. Cell Proliferation on Macro/Nano Surface Structure and Collagen Immobilization of 3D Polycaprolactone Scaffolds.

    PubMed

    Park, Young-Ouk; Myung, Sung-Woon; Kook, Min-Suk; Jung, Sang-Chul; Kim, Byung-Hoon

    2016-02-01

    In this study, 3D polycaprolactone (PCL) scaffolds were fabricated by 3D printing technique. The macro/nano morphology of, 3D PCL scaffolds surface was etched with oxygen plasma. Acrylic acid (AA) plasma-polymerization was performed to functionalize the macro/nano surface with carboxyl groups and then collagen was immobilized with plasma-polymerized 3D PCL scaffolds. After O2 plasma and AA plasma-polymerization, contact angles were decreased. The FE-SEM and AFM results showed that O2 plasma is increased the surface roughness. The MTT assay results showed that proliferation of the M3CT3-E1 cells increased on the oxygen plasma treated and collagen immobilized 3D PCL scaffolds. PMID:27433597

  18. Chitooligomer-Immobilized Biointerfaces with Micropatterned Geometries for Unidirectional Alignment of Myoblast Cells

    PubMed Central

    Poosala, Pornthida; Kitaoka, Takuya

    2016-01-01

    Skeletal muscle possesses a robust capacity to regenerate functional architectures with a unidirectional orientation. In this study, we successfully arranged skeletal myoblast (C2C12) cells along micropatterned gold strips on which chitohexaose was deposited via a vectorial chain immobilization approach. Hexa-N-acetyl-d-glucosamine (GlcNAc6) was site-selectively modified at its reducing end with thiosemicarbazide, then immobilized on a gold substrate in striped micropatterns via S–Au chemisorption. Gold micropatterns ranged from 100 to 1000 µm in width. Effects of patterning geometries on C2C12 cell alignment, morphology, and gene expression were investigated. Unidirectional alignment of C2C12 cells having GlcNAc6 receptors was clearly observed along the micropatterns. Decreasing striped pattern width increased cell attachment and proliferation, suggesting that the fixed GlcNAc6 and micropatterns impacted cell function. Possibly, interactions between nonreducing end groups of fixed GlcNAc6 and cell surface receptors initiated cellular alignment. Our technique for mimicking native tissue organization should advance applications in tissue engineering. PMID:26784249

  19. Immobilization of yeast cells on hydrogel carriers obtained by radiation-induced polymerization

    NASA Astrophysics Data System (ADS)

    Xin, Lu Zhao; Carenza, Mario; Kaetsu, Isao; Kumakura, Minoru; Yoshida, Masaru; Fujimura, Takashi

    Polymer hydrogels were obtained by radiation-induced copolymerization at -78°C of aqueous solutions of acrylic and methacrylic esters. The matrices were characterized by equilibrium water content measurements, by optical microscopy observations and by scanning electron microscopy analysis. Yeast cells were immobilized on these hydrogels and the ethanol productivity by batch fermentation was determined. Matrix hydrophilicity and porosity were found to deeply influence the adhesion of yeast cells and, hence, the ethanol productivity. The latter as well as other physico-chemical properties were also affected by the presence of a crosslinking agent added in small amounts to the polymerizing mixture.

  20. Monoolein production by triglycerides hydrolysis using immobilized Rhizopus oryzae lipase.

    PubMed

    Ghattas, Nesrine; Abidi, Ferid; Galai, Said; Marzouki, M Nejib; Salah, Abderraouf Ben

    2014-07-01

    Lipase extracted from Rhizopus oryzae was immobilized in alginate gel beads. The effects of the immobilization conditions, such as, alginate concentration, CaCl2 concentration and amount of initial enzyme on retained activity (specific activity ratio of entrapped active lipase to free lipase) were investigated. The optimal conditions for lipase entrapment were determined: 2% (w/v) alginate concentration, 100mM CaCl2 and enzyme ratio of 2000IU/mL.In such conditions, immobilized lipase by inclusion in alginate showed a highest stability and activity, on olive oil hydrolysis reaction where it could be reused for 10 cycles. After 15min of hydrolysis reaction, the mass composition of monoolein, diolein and triolein were about 78%, 10% and 12%. Hydrolysis' products purification by column chromatography lead to a successful separation of reaction compounds and provide a pure fraction of monoolein which is considered as the widest used emulsifier in food and pharmaceutical industries. PMID:24755261

  1. Monoolein production by triglycerides hydrolysis using immobilized Rhizopus oryzae lipase.

    PubMed

    Ghattas, Nesrine; Abidi, Ferid; Galai, Said; Marzouki, M Nejib; Salah, Abderraouf Ben

    2014-07-01

    Lipase extracted from Rhizopus oryzae was immobilized in alginate gel beads. The effects of the immobilization conditions, such as, alginate concentration, CaCl2 concentration and amount of initial enzyme on retained activity (specific activity ratio of entrapped active lipase to free lipase) were investigated. The optimal conditions for lipase entrapment were determined: 2% (w/v) alginate concentration, 100mM CaCl2 and enzyme ratio of 2000IU/mL.In such conditions, immobilized lipase by inclusion in alginate showed a highest stability and activity, on olive oil hydrolysis reaction where it could be reused for 10 cycles. After 15min of hydrolysis reaction, the mass composition of monoolein, diolein and triolein were about 78%, 10% and 12%. Hydrolysis' products purification by column chromatography lead to a successful separation of reaction compounds and provide a pure fraction of monoolein which is considered as the widest used emulsifier in food and pharmaceutical industries.

  2. Effects of Alginate Oligosaccharide Mixture on the Bioavailability of Lysozyme as an Antimicrobial Agent.

    PubMed

    Park, Hyun Jung; Ahn, Joo-Myung; Park, Ra-Mi; Lee, Sang-Hee; Sekhon, Simranjeet Singh; Kim, Sang Yong; Wee, Ji-Hyang; Kim, Yang-Hoon; Min, Jiho

    2016-02-01

    In this study, we report an oral drug delivery system without any additional process using pH-sensitive biopolymer, alginate, and alginate oligosaccharide with lysozyme as a model drug. These biopolymers exhibited pH-sensitive characteristics such as shrinking at acidic pH and eroding with dissolution at alkaline pH. The incorporation of lysozyme and biopolymers was performed an artificial intestinal juice (pH 6.8). The immobilization efficiency and lysozyme stability in gastric juice (pH 1.2) has been tested by E coil antimicrobial activity. The lysozyme without biopolymer immobilization lost approximately 80-90% of antimicrobial activity than that of pure lysozyme. However, the pH-sensitive biopolymer-controlled lysozyme maintained similar antimicrobial activity compared to that of pure lysozyme (50-90% of cell mortality). Therefore, this simple, easy, and rapid system can be effectively and practically applied for pathogen treatment for in vivo oral drug delivery. PMID:27433602

  3. Preservation of viability and antibacterial activity of Lactobacillus spp. in calcium alginate beads.

    PubMed

    Brachkova, Mariya I; Duarte, Maria A; Pinto, João F

    2010-12-23

    The objective of the study was to produce calcium alginate beads able to deliver Lactobacillus spp. (Lactobacillus plantarum, Lactobacillus rhamnosus GG, Lactobacillus bulgaricus and Lactobacillus lactis) with preserved viability and antibacterial activity. Four types of beads, containing entrapped (E), surface and entrapped (ES), surface (S) and concentrated surface and entrapped lactobacilli (C(ES)) were prepared and physically characterized. The antibacterial activity of lactobacilli cultures before and after immobilization, freeze-drying and throughout storage was studied in relationship to the viable number of lactobacilli. Multi-resistant clinical isolates (methicillin-resistant Staphylococcus aureus, vancomycine-resistant Enterococcus faecalis, VIM-2-metalo-β-lactamase producing Pseudomonas aeruginosa and CTX-M-15-β-lactamase producing strains: Escherichia coli and Klebsiella pneumoniae) were used as indicator strains. Alginate beads in which lactobacilli proliferated to the beads surface (ES and C(ES)) differed significantly from the other types of beads in their physicochemical properties, showing smoother surface morphology, more spherical shape, bigger weight, lower calcium content, density and crushing force. Lactobacilli cultures, at high cell concentrations (10(8)cfu/ml) were active against both Gram-positive and negative multi-resistant bacteria. Beads containing both entrapped and surface lactobacilli (ES) resulted in viability and antibacterial activity most similar to non-processed lactobacilli cultures. The viability and antibacterial activity of the immobilized lactobacilli remained stable after 6 months storage.

  4. The use of covalently immobilized stem cell factor to selectively affect hematopoietic stem cell activity within a gelatin hydrogel.

    PubMed

    Mahadik, Bhushan P; Pedron Haba, Sara; Skertich, Luke J; Harley, Brendan A C

    2015-10-01

    Hematopoietic stem cells (HSCs) are a rare stem cell population found primarily in the bone marrow and responsible for the production of the body's full complement of blood and immune cells. Used clinically to treat a range of hematopoietic disorders, there is a significant need to identify approaches to selectively expand their numbers ex vivo. Here we describe a methacrylamide-functionalized gelatin (GelMA) hydrogel for in vitro culture of primary murine HSCs. Stem cell factor (SCF) is a critical biomolecular component of native HSC niches in vivo and is used in large dosages in cell culture media for HSC expansion in vitro. We report a photochemistry based approach to covalently immobilize SCF within GelMA hydrogels via acrylate-functionalized polyethylene glycol (PEG) tethers. PEG-functionalized SCF retains the native bioactivity of SCF but can be stably incorporated and retained within the GelMA hydrogel over 7 days. Freshly-isolated murine HSCs cultured in GelMA hydrogels containing covalently-immobilized SCF showed reduced proliferation and improved selectivity for maintaining primitive HSCs. Comparatively, soluble SCF within the GelMA hydrogel network induced increased proliferation of differentiating hematopoietic cells. We used a microfluidic templating approach to create GelMA hydrogels containing gradients of immobilized SCF that locally direct HSC response. Together, we report a biomaterial platform to examine the effect of the local presentation of soluble vs. matrix-immobilized biomolecular signals on HSC expansion and lineage specification. This approach may be a critical component of a biomaterial-based artificial bone marrow to provide the correct sequence of niche signals to grow HSCs in the laboratory.

  5. The use of covalently immobilized stem cell factor to selectively affect hematopoieti