Visualization of alginate-poly-L-lysine-alginate microcapsules by confocal laser scanning microscopy

Confocal laser scanning microscopy (CLSM) was used to study the distribution of polymers and cross-linking ions in alginate-poly-L-lysine (PLL) -alginate microcapsules made by fluorescent-labeled polymers. CLSM studies of Ca-alginate gel beads made in the presence and absence of non-gelling sodium ions revealed a more inhomogeneous distribution of alginate in beads formed in the absence of non-gelling ions. In the formation of alginate-PLL capsules, the polymer gradients in the preformed gel core were destabilized by the presence of non-gelling ions in the washing step and in the PLL solution. Ca-alginate gels preserved the inhomogeneous structure by exposure to ion-free solution in contrast to exposure to non-gelling ions (Na(+)). By exchanging Ca(2+) with Ba(2+) (10 mM), extremely inhomogeneous gel beads were formed that preserved their structure during the washing and exposure to PLL in saline. PLL was shown to bind at the very surface of the alginate core, forming a shell-like membrane. The thickness of the PLL-layer increased about 100% after 2 weeks of storage, but no further increase was seen after 2 years of storage. The coating alginate was shown to overlap the PLL layer. No difference in binding could be observed among coating alginates of different composition. This paper shows an easy and novel method to study the distribution of alginate and PLL in intact microcapsules. As the labeling procedures are easy to perform, the method can also be used for a variety of other polymers in other microencapsulation systems.     

Evaluation of Chaperone-like Activity of Alginate: Microcapsule and Water-soluble Forms

To evaluate the chaperone-like activity of alginate stabilization and refolding of alkaline phosphatase (ALP) was investigated in the presence of alginate through two different approaches, the soluble form and microcapsule assisted methods. It was found that in the presence of microcapsules, ALP can be stabilized to a higher degree compared with the water-soluble form, whereas the denatured ALP is refolded with a higher yield through latter method. Lower refolding yields of alginate beads compared with its soluble form may be the result of lower refolding rate of ALP upon elution of the bound enzyme by dispersing the precipitate in NaCl which left the unfolded protein in an unsuitable environment, providing enough time for protein aggregation and leading finally to lower recovered activity compared with application of soluble form of alginate. In addition in the case of alginate capsules, the choice of suitable divalent ion is essential for stability and assistance in refolding.     

Calcium alginate beads as a slow-release system for delivering angiogenic molecules in vivo and in vitro.

A method previously used in this laboratory for entrapment of tumor cells in alginate beads has been extended to provide a slow release delivery system for growth factors with known in vivo angiogenic activity. Protein growth factors were entrapped in alginate beads in amounts sufficient to cause incorporation of 3H-thymidine by COMMA-D cells in vitro, and in vivo neovascularization when injected subcutaneously into Balb/c mice. Entrapment of 125I-labelled growth factors showed that the amount of molecule entrapped in alginate beads may vary with the charge of the molecule. In vitro cell proliferation studies showed that entrapment in alginate beads may provide a slow-release system or a stabilizing environment for the protein. In some cases biological activity of the growth factor in solution was increased by the presence of control alginate beads. When alginate-entrapped growth factors were injected into Balb/c mice, induction of new blood vessels could be monitored qualitatively by macroscopic photography and assessed quantitatively by measuring the pooling of radiolabelled red blood cells at the experimental site. Subcutaneous injection of purified angiogenic factors not entrapped in alginate beads did not cause neovascularization. Diffusion of 125I-labelled growth factors from alginate beads in the animal showed that release in vivo may depend on the charge of the protein molecule. These results indicate that injection of purified molecules entrapped in alginate beads provides an effective localized and slow-release delivery of biologically active molecules. This delivery system may extend the time of effectiveness of biologically active molecules in vivo compared to direct injection without alginate entrapment. The method of entrapment and injection has potential for identifying active factors in tumor-induced angiogenesis and testing new compounds as modulators of neovascularization.     

Gel beads composed of collagen reconstituted in alginate

Spherical gel beads of collagen/alginate were prepared by discharging droplets of a mixture containing collagen (1.07-1.9 mg/ml) and alginate (1.2-1.5% w/v) into 1.5% w/v CaCl2 solution at 4°C. Collagen in the gel beads was reconstituted by raising the temperature to 37°C after alginate was liquefied by citrate. Scanning electron microscopy of the beads revealed the characteristic fibrous structure of collagen. To demonstrate the application of this new technique in cell culture, GH3 rat pituitary tumor cells were entrapped and grown in the gel beads. The immobilized cells proliferated to a density of 1.95 x 106 cell/ml which is about an order of magnitude higher than that grown in the alginate beads.     

Process parameters for the high-scale production of alginate-encapsulated stem cells for storage and distribution throughout the cell therapy supply chain

With the ever-increasing clinical application of cell-based therapies, it is considered critical to develop systems that facilitate the storage and distribution of cell therapy products (CTPs) between sites of manufacture and the clinic. For such systems to be realized, it is essential that downstream bioprocessing strategies be established that are scalable, reproducible and do not influence the viability or function of the living biologic. To this end, we examined alginate-encapsulation as a method to heighten the preservation of human adipose-derived stem cells (hASCs) during hypothermic storage, and establish a scalable process for high-volume production. A drop-wise method for scalable alginate bead generation, using calcium as the cross-linker, was modified to enable the yield of up to 3500 gelled beads per minute. The effect of alginate concentration on the viscosity of non-gelled sodium alginate and the mechanical properties and internal structure of calcium-crosslinked alginate in response to different alginate and calcium concentrations were investigated. Mechanical strength was chiefly dependent on alginate concentration and 1.2% alginate cross-linked with 100 mM calcium chloride could withstand stress in the order of 35 kPa. Upon selection of appropriate parameters, we demonstrated the suitability of using this method for immobilizing human stem cells. Encapsulated hASCs demonstrated no loss in cell viability, and had a uniform distribution after high-volume production. Following storage, released cells were able to attach and recover a normal morphology upon return to culture conditions. Thus we present a scalable method for stem cell encapsulation and storage for application within the cell therapy supply chain.     

Lactococcus lactis release from calcium alginate beads.

Cell release during milk fermentation by Lactococcus lactis immobilized in calcium alginate beads was examined. Numbers of free cells in the milk gradually increased from 1 x 10(6) to 3 x 10(7) CFU/ml upon successive reutilization of the beads. Rinsing the beads between fermentations did not influence the numbers of free cells in the milk. Cell release was not affected by initial cell density within the beads or by alginate concentration, although higher acidification rates were achieved with increased cell loading. Coating alginate beads with poly-L-lysine (PLL) did not significantly reduce the release of cells during five consecutive fermentations. A double coating of PLL and alginate reduced cell release by a factor of approximately 50. However, acidification of milk with beads having the PLL-alginate coating was slower than that with uncoated beads. Immersing the beads in ethanol to kill cells on the periphery reduced cell release, but acidification activity was maintained. Dipping the beads in aluminum nitrate or a hot CaCl2 solution was not as effective as dipping them in ethanol. Ethanol treatment or heating of the beads appears to be a promising method for maintaining acidification activity while minimizing viable cell release due to loosely entrapped cells near the surface of the alginate beads.     

Lactococcus lactis release from calcium alginate beads.

Cell release during milk fermentation by Lactococcus lactis immobilized in calcium alginate beads was examined. Numbers of free cells in the milk gradually increased from 1 x 10(6) to 3 x 10(7) CFU/ml upon successive reutilization of the beads. Rinsing the beads between fermentations did not influence the numbers of free cells in the milk. Cell release was not affected by initial cell density within the beads or by alginate concentration, although higher acidification rates were achieved with increased cell loading. Coating alginate beads with poly-L-lysine (PLL) did not significantly reduce the release of cells during five consecutive fermentations. A double coating of PLL and alginate reduced cell release by a factor of approximately 50. However, acidification of milk with beads having the PLL-alginate coating was slower than that with uncoated beads. Immersing the beads in ethanol to kill cells on the periphery reduced cell release, but acidification activity was maintained. Dipping the beads in aluminum nitrate or a hot CaCl2 solution was not as effective as dipping them in ethanol. Ethanol treatment or heating of the beads appears to be a promising method for maintaining acidification activity while minimizing viable cell release due to loosely entrapped cells near the surface of the alginate beads.     

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

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.     

Evaluation of microbeads of calcium alginate as a fluidized bed medium for affinity chromatography of Aspergillus niger Pectinase

Calcium alginate microbeads (212-425 microm) were prepared by spraying 2% (w/v) alginate solution into 1 M CaCl2 solution. The fluidization behavior of these beads was studied, and the bed expansion index and terminal velocity were found to be 4.3 and 1808 cm h(-1), respectively. Residence time distribution curves showed that the dispersion of the protein was much less with these microbeads than with conventionally prepared calcium alginate macrobeads when both kinds of beads were used for chromatography in a fluidized bed format. The fluidized bed of these beads was used for the purification of pectinase from a commercial preparation. The media performed well even with diluted feedstock; 90% activity recovery with 211-fold purification was observed.     

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

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.     

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

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.     

Mutual relationships between soils and biological carrier systems

Improved viability and antagonistic activity of biocontrol agents during soil inoculation is of crucial importance to their effective application. The chitinolytic bacterium Serratia marcescens was used as a model organism to study the efficacy of freeze-dried alginate beads (in comparison to their non-dried counterparts) as possible carriers for immobilized biocontrol agents. The release of bacteria and chitinolytic enzyme from alginate beads, before and during their application in soil, was examined, and the beads' physical properties characterized. Dispersal of the alginate bead-entrapped S. marcescens in the soil resulted in high soil cell densities throughout the 35 days of the experiment. Chitin inclusion in the beads resulted in significantly higher chitinolytic activity of S. marcescens, increased dry-bead porosity and decreased stiffness. Rehydration of the dried beads (after immersion in soil) resulted in a sixfold increase in weight due to water absorption. No significant differences were found in bacterial count inside the non-dried (gel) versus dried beads. However, higher cell densities and chitinase activity were detected in soil containing dried beads with chitin than in that containing their non-dried counterparts. The biological performance of S. marcescens was examined in the greenhouse: a free cell suspension reduced bean (Phaseolus vulgaris L.) disease by 10%, while immobilized bacteria found in the dried, chitin-containing beads reduced disease by 60%.     

A new method of synthesis of iron doped calcium alginate beads and determination of iron content by radiometric method

The present study describes a new method of synthesis of an anionic biopolymer, iron doped calcium alginate beads (Fe-CA). The beads were characterized in terms of size, porosity and surface area. The adsorption of the anionic species depends mainly on the iron content of the beads. Thus, iron content in the Fe-CA beads has been determined employing radiometric technique using ⁵⁹Fe radiotracer. It has been found that Fe-CA beads contain 37.8 mg Fe/g of wet alginate beads in the proposed method, which is much higher than the earlier reported methods, keeping the mechanical stability.     

Preparation and Characterization of Silver Nanoparticles-Loaded Calcium Alginate Beads Embedded in Gelatin Scaffolds

Silver nanoparticles (AgNPs)-loaded alginate beads embedded in gelatin scaffolds were successfully prepared. The AgNPs-loaded calcium alginate beads were prepared by electrospraying method. The effect of alginate concentration and applied voltage on shape and diameter of beads was studied. The diameter of dry AgNPs-loaded calcium alignate beads at various concentrations of AgNO₃ ranged between 154 and 171 μm. The AgNPs-loaded calcium alginate beads embedded in gelatin scaffolds were fabricated by freeze-drying method. The water swelling and weight loss behaviors of the AgNPs-loaded alginate beads embedded in gelatin scaffolds increased with an increase in the submersion time. Moreover, the genipin-cross-linked gelatin scaffolds were proven to be nontoxic to normal human dermal fibroblasts, suggesting their potential uses as wound dressings.     

Enhanced production of bioethanol and ultrastructural characteristics of reused Saccharomyces cerevisiae immobilized calcium alginate beads

Yeast immobilized on alginate beads produced a higher ethanol yield more rapidly than did free yeast cells under the same batch-fermentation conditions. The optimal fermentation conditions were 30 °C, pH 5.0, and 10% initial glucose concentration with 2% sodium alginate beads. The fermentation time using reused alginate beads was 10-14 h, whereas fresh beads took 24 h, and free cells took 36 h. All bead samples resulted in nearly a 100% ethanol yield, whereas the free cells resulted in an 88% yield. Transmission electron microscopy (TEM) showed that the shortened time and higher yield with the reused beads was due to a higher yeast population per bead as well as a higher porosity. The ultrastructure of calcium alginate beads and the alginate matrix structure known as the “egg-box” model were observed using TEM.     

Encapsulation of Ketoprofen and Ketoprofen Lysinate by Prilling for Controlled Drug Release

In this paper, ketoprofen and ketoprofen lysinate were used as model drugs in order to investigate release profiles of poorly soluble and very soluble drug from sodium alginate beads manufactured by prilling. The effect of polymer concentration, viscosity, and drug/polymer ratio on bead micromeritics and drug release rate was studied. Ketoprofen and ketoprofen lysinate loaded alginate beads were obtained in a very narrow dimensional range when the Cross model was used to set prilling operative conditions. Size distribution of alginate beads in the hydrated state was strongly dependent on viscosity of drug/polymer solutions and frequency of the vibration. The release kinetics of the drugs showed that drug release rate was related with alginate concentration and solubility of the drug. Alginate solutions with concentration higher than 0.50% (w/w) were suitable to prepare ketoprofen gastro-resistant formulation, while for ketoprofen lysinate alginate, concentration should be increased to 1.50% (w/w) in order to retain the drug in gastric environment. Differential scanning calorimetry thermograms and Fourier transform infrared analyses of drug-loaded alginate beads indicated complex chemical interactions between carboxyl groups of the drug and polymer matrix in drug-loaded beads that contribute to the differences in release profile between ketoprofen and ketoprofen lysinate. Total release of the drugs in intestinal medium was dependent on the solubility of the drug and was achieved between 4 and 6 h.     

Evaluation of Chitosan/Alginate Beads Using Experimental Design: Formulation and <Emphasis Type="Italic">In Vitro</Emphasis> Characterization

Bovine serum albumin-loaded beads were prepared by ionotropic gelation of alginate with calcium chloride and chitosan. The effect of sodium alginate concentration and chitosan concentration on the particle size and loading efficacy was studied. The diameter of the beads formed is dependent on the size of the needle used. The optimum condition for preparation alginate–chitosan beads was alginate concentration of 3% and chitosan concentration of 0.25% at pH 5. The resulting bead formulation had a loading efficacy of 98.5% and average size of 1,501 μm, and scanning electron microscopy images showed spherical and smooth particles. Chitosan concentration significantly influenced particle size and encapsulation efficiency of chitosan–alginate beads (p < 0.05). Decreasing the alginate concentration resulted in an increased release of albumin in acidic media. The rapid dissolution of chitosan–alginate matrices in the higher pH resulted in burst release of protein drug.     

Determination of the radial distribution of Saccharomyces cerevisiae immobilised in calcium alginate gel beads

Summary The dissolution of alginate gel beads in 20 g sodium citrate /l produces a linear decrease in bead diameter. The rate of dissolution is dependent on the concentration of CaCl₂ within the gel beads. This method allows the controlled release of Saccharomyces cerevisiae from alginate gel beads and permits the simple and rapid determination of the radial distribution of cell concentration.     

Low-temperature electron microscopy for the study of polysaccharide ultrastructures in hydrogels. II. Effect of temperature on the structure of Ca2+-alginate beads

Calcium alginate beads were thermally treated at temperatures ranging from 25 degrees C to 130 degrees C for periods of up to 30 minutes. Important modifications to the structure of the alginate beads were shown to be a function of the temperature and period of incubation at each temperature. Modifications to the alginate beads included reduction in size, mechanical resistance, and molecular weight cut-off with increasing temperature and incubation period. Thus, heating 700 microm calcium alginate beads for 20 min at 130 degrees C resulted in a 23% reduction in diameter, 70% increase in mechanical resistance, and 67% reduction in molecular weight cut-off.Incubation of calcium alginate beads containing 2 x 10(6) kDa blue dextran for 20 min at 130 degrees C resulted in no detectable loss of either dye or alginate. This indicates the shrinkage of the beads was due to re-arrangement of the alginate chains within the beads, coupled with loss of water. This hypothesis was verified by direct visual observation of calcium alginate beads before and after thermal treatment using cryo-scanning electron microscopy (cryo-SEM). Unlike other microscopy methods cryo-SEM offers the advantage of extremely rapid freezing which preserves the original structure of the alginate network. As a result cryo-SEM is a powerful tool for studies of hydrogel and capsule structure and formation.Differential scanning calorimetry (DSC) showed that the water entrapped in 2% alginate beads was present in a single state, irrespective of the thermal treatment. This result is attributed to the low alginate concentration used to form the beads.