Pancreatic cell immobilization in alginate beads produced by emulsion and internal gelation

Alginate has been used to protect transplanted pancreatic islets from immune rejection and as a matrix to increase the insulin content of islet progenitor cells. The throughput of alginate bead generation by the standard extrusion and external gelation method is limited by the rate of droplet formation from nozzles. Alginate bead generation by emulsion and internal gelation is a scaleable alternative that has been used with biological molecules and microbial cells, but not mammalian cells. We describe the novel adaptation of this process to mammalian cell immobilization. After optimization, the emulsion process yielded 90 ± 2% mouse insulinoma 6 (MIN6) cell survival, similar to the extrusion process. The MIN6 cells expanded at the same rate in both bead types to form pseudo‐islets with increased glucose stimulation index compared to cells in suspension. The emulsion process was suitable for primary pancreatic exocrine cell immobilization, leading to 67 ± 32 fold increased insulin expression after 10 days of immobilized culture. Due to the scaleability and broad availability of stirred mixers, the emulsion process represents an attractive option for laboratories that are not equipped with extrusion‐based cell encapsulators, as well as for the production of immobilized or encapsulated cellular therapeutics on a clinical scale. Biotechnol. Bioeng. 2011;108: 424–434. © 2010 Wiley Periodicals, Inc.     

A novel alginate hollow fiber bioreactor process for cellular therapy applications

Gel‐matrix culture environments provide tissue engineering scaffolds and cues that guide cell differentiation. For many cellular therapy applications such as for the production of islet‐like clusters to treat Type 1 diabetes, the need for large‐scale production can be anticipated. The throughput of the commonly used nozzle‐based devices for cell encapsulation is limited by the rate of droplet formation to ～0.5 L/h. This work describes a novel process for larger‐scale batch immobilization of mammalian cells in alginate‐filled hollow fiber bioreactors (AHFBRs). A methodology was developed whereby (1) alginate obstruction of the intra‐capillary space medium flow was negligible, (2) extra‐capillary alginate gelling was complete and (3) 83 ± 4% of the cells seeded and immobilized were recovered from the bioreactor. Chinese hamster ovary (CHO) cells were used as a model aggregate‐forming cell line that grew from mostly single cells to pancreatic islet‐sized spheroids in 8 days of AHFBR culture. CHO cell growth and metabolic rates in the AHFBR were comparable to small‐scale alginate slab controls. Then, the process was applied successfully to the culture of primary neonatal pancreatic porcine cells, without significant differences in cell viability compared with slab controls. As expected, alginate‐immobilized culture in the AHFBR increased the insulin content of these cells compared with suspension culture. The AHFBR process could be refined by adding matrix components or adapted to other reversible gels and cell types, providing a practical means for gel‐matrix assisted cultures for cellular therapy. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Embryo development from discrete cell aggregates inIpomoea Batatas (L.) Lam. in response to structural polarity

Summary High numbers of embryos are difficult to obtain in liquid cultures of sweet potato (Ipomoea batatas (L.) Lam.) because discrete cell aggregates, produced through calli fragmentation, do not support embryo growth. In an effort to demonstrate that embryo development is possible from discrete cell aggregates, we compared embryo formation from cell aggregates 250–355 μm in diameter cultured either in suspension in liquid medium, on agar solidified medium, or immobilized on alginate beads floated in liquid medium. Embryos were initiated but remained arrested in their globular stage on cell aggregates cultured in suspension. Embryos developed to the torpedo stage from cell aggregates cultured on solidified medium and from cell aggregates anchored on alginate beads. Thus, embryos continued to develop beyond the globular stage when a structural polarity, which led probably to the establishment of a physiological polarity, was created. The production of sweet potato embryos in liquid culture can be improved by using alginate beads or culture conditions and protocols leading to the release during calli fragmentation of polarized individual cell aggregates. This work was supported in part by a IFAS/Gas Research Institute cooperative grant. Florida Agriculture Experiment Station Journal Series 9297     

Paclitaxel and baccatin III production induced by methyl jasmonate in free and immobilized cells of Taxus baccata

The effects of 100 and 200 μM methyl jasmonate (MJA) on cell proliferation and paclitaxel and baccatin III production were investigated in free and alginate immobilized cells of Taxus baccata growing in a selected product formation culture medium. The greatest accumulation of paclitaxel (13.20 mg dm⁻³) and baccatin III (4.62 mg dm⁻³) occurred when 100 μM MJA was added to the culture medium of cells entrapped using a 1.5 and 2.5 % alginate solution. The effects of different treatments on the viability of cultured cells and their capacity to excrete both taxanes into the surrounding medium were considered.     

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

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 ofgelation. 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 microm 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 (5x10(8) and 1x10(9) 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 resulting gel. The cell presence decreased the rate of network formation as well as the strength of the obtained Ca-alginate hydrogel.     

Enhanced production of plumbagin in immobilized cells of Plumbago rosea by elicitation and in situ adsorption

Cell cultures of Plumbago rosea were immobilized in calcium alginate and cultured in Murashige and Skoog's basal medium containing 10 mM CaCl(2) for the production of plumbagin, an important medicinal compound. Studies were carried to find out the impact of immobilization on the increased accumulation of this secondary metabolite. Immobilization in calcium alginate enhanced the production of plumbagin by three, two and one folds compared to that of control, un-crosslinked alginate and CaCl(2) treated cells respectively. Cell loading at a level of 20% to the polymer volume (Na-alginate) was optimal and maximum plumbagin was obtained. At higher cell loading (40-50%), lower plumbagin accumulation was noticed. Addition of 200 mg l(-1) chitosan as an elicitor to the immobilized cells resulted in eight and two folds higher accumulation of plumbagin over control and immobilized cells. Also, more than 70% of the plumbagin was released into the medium, which is highly desirable for easy recovery of the product. Sucrose utilization rate of the cells was higher when cells were subjected to in situ product removal using Amberlite XAD-7. This may indicate that the toxicity of plumbagin was reduced on cells when it was removed from the medium. Cells subjected to combined treatments of chitosan, immobilization and in situ extraction showed a synergistic effect and yielded 92.13 mg g(-1) DCW of plumbagin which is 21, 5.7, 2.5 times higher than control, immobilized, immobilized and elicited cells respectively.     

固相化HEK细胞在无血清培养基中高效表达重组人活性蛋白C

研究固相化HEK工程细胞的培养和产生rhAPC的水平。先将HEK细胞由贴壁适应为无血清悬浮培养,再用海藻酸钙固定,并在无血清培养基中悬浮培养(最高约为27mg/L)。固相细胞动态培养的表达水平高于细胞静态悬浮培养的表达水平(最高约为50mg/L)。固相细胞动态培养可实现细胞的高密度培养,得到较高的表达水平。     

Resolution of N-(2-ethyl-6-methylphenyl) alanine by using microgel beads containing Pseudomonas cepacia lipase

Abstract

Pseudomonas cepacia lipase (PCL) was immobilized in alginate microgel beads by electrostatic dispersion. The high electrical potential applied in the immobilization process could significantly decrease the droplet size. The optimum conditions for lipase immobilization were 2% (w/v) alginate, 100 mM CaCl₂, 8 mg/mL enzyme, 4 kV electrical potential and 200 μm mean bead size. Under these conditions, 78.2 U/g of immobilized PCL activity was obtained with 39.1% retained activity and 57.2% immobilization efficiency. The immobilized PCL (PCL-CA) was subsequently used in the enantioselective hydrolysis of (R, S)-N-(2-ethyl-6-methylphenyl) alanine methyl ester. Although PCL-CA exhibited slightly lower activity than free PCL, it preserved the high enantioselectivity (E-value >?200), which afforded enantiomerically pure (R)-acid (99% e.e._p). Furthermore, PCL-CA exhibited higher thermal stability, storage and medium stability than that of free PCL. Batch-wise operational stability studies demonstrated that PCL-CA retained its initial activity for at least 10 cycles of hydrolysis.     

Penicillin G production by immobilized fungal vesicles

Summary The activity of penicillin G production was compared in a well defined medium by native vesicles as well as by calcium alginate gel immobilized vesicles isolated from the protoplasts of Penicillium chrysogenum PQ-96. The activity yield of the immobilized vesicles was 44% in comparison with native vesicles. After 60 h of storage at 4° C the native vesicles showed a rapid decrease in penicillin G production. The storage stability of these vesicles was improved after entrapment inside the calcium alginate gel. After 240 h of storage 1 mg of immobilized vesicular protein catalyzed the production of about 140 nmoles of penicillin G in 1 h.     

Production of lysine by using immobilized livingCorynebacterium sp cells

Summary Lysine production by immobilizedCorynebacterium sp cells in alginate gel beads was investigated in flasks. ImmobilizedCorynebacterium sp cells exhibited a slightly greater lysine production than free cells and accumulated 60 g/l of L-lysine at maximum, when cultured for 120h in a medium containing 200g/l glucose as carbon source. Several factors, such as inoculum size, incubation time and alginate gel concentration were examined in order to improve lysine production by immobilized growing cells.     

The production of ethanol by immobilized yeast cells

Saccharomyces cerevisiae cells were immobilized in calcium alginate beads for use in the continuous production of ethanol. Yeasts were grown in medium supplemented with ethanol to selectively screen for a culture which showed the greatest tolerance to ethanol inhibition. Yeast beads were produced from a yeast slurry containing 1.5% alginate (w/v) which was added as drops to 0.05M CaCl₂ solution. To determine their optimum fermentation parameters, ethanol production using glucose as a substrate was monitored in batch systems at varying physiological conditions (temperature, pH, ethanol concentration), cell densities, and gel concentration. The data obtained were compared to optimum free cell ethanol fermentation parameters. The immobilized yeast cells examined in a packed-bed reactor system operated under optimized parameters derived from batch-immobilized yeast cell experiments. Ethanol production rates, as well as residual sugar concentration were monitored at different feedstock flow rates.     

Effects of Cellular Metabolism and Viability on Metal Ion Accumulation by Cultured Biomass from a Bloom of the Cyanobacterium Microcystis aeruginosa

The sorption of nickel, cadmium, and copper by cultured biomass from a naturally occurring bloom of Microcystis aeruginosa was demonstrated in two systems: cells suspended in culture medium and cells immobilized in alginate. Incubation in the absence of light, in the presence of metabolic inhibitors, and at 4°C did not substantially decrease the copper accumulation by cells in culture medium. Heat-killed, formaldehyde-treated, and air-dried biomass samples sorbed nearly as much (or in some cases slightly more) copper as did viable samples.     

Induction of stress metabolites in immobilized Glycyrrhiza echinata cultured cells

Transfer into a fresh medium or immobilization by entrapment in calcium alginate gels of cultured Glycyrrhiza echinata cells caused a rapid and transient accumulation of a retrochalcone, echinatin, in both the cells and the medium. The higher level and longer duration of echinatin production was observed in the immobilized cells than in freely suspended cells. Transfer of the cells into the medium containing either sodium alginate or calcium chloride, and the addition of sodium alginate into the suspension culture, caused the same effect as observed in cell immobilization. A novel metabolite was also detected in the induced cells. Activities of the enzymes involved in echinatin biosynthesis were shown to rapidly increase by immobilization of the cells.Abbreviations IAA indole-3-acetic acid - LMT S-adenosylmethionine: licodione 2-O-methyltransferase - CHS chalcone synthase     

Microcallus growth from maize protoplasts

Maize (Zea mays L.) protoplasts obtained from Type I and Type II calli from several genotypes were shown to be capable of synthesizing cell walls and forming small clusters of cells. The medium used also supported cluster formation from protoplasts obtained from root tips. The effects of various additions to the medium (such as casein hydrolysate, coconut water, amino acids, sugars, phytohormones, nitrate, calcium, and dimethylsulfoxide as well as pH variations on cellcluster formation were determined. The method of culture (protoplasts plated in agarose or supported in alginate beads in liquid medium) as well as several components of the medium were found to be critical for microcallus formation. Protoplasts obtained from embryogenic Type I callus and cultured in the medium of C. Nitsch and J.P. Nitsch (1967, Planta 72, 355–370) modified by various additions (NN 67-mod medium) were affected most by various sugars, casein hydrolysate, coconut water, and a combination of the auxins napthalene-1-acetic acid (2 mg/l) and 2,4-dichlorophenoxyacetic acid (0.1 mg/l), and the cytokinin N⁶-benzylaminopurine (0.5 mg/l). Cluster size in the agarose culture system was from 0.1 to 0.5 mm diameter and in the alginate culture system, up to 2.0 mm diameter.     

Effect of ice nucleation by droplet of immobilized silver iodide on freezing of rabbit and bovine embryos

Silver iodide was immobilized by applying the insoluble reaction between sodium alginate and calcium chloride. The immobilized silver iodide was immersed into a freezing solution in order to trigger ice nucleation. Temperature change during cooling and postthaw in vitro development of embryos were examined in order to evaluate the effectiveness of the immobilized silver iodide (AgI alginate-gel droplet) on embryo development. Samples containing the AgI alginate-gel droplets released the latent heat of fusion at a higher subzero temperature than samples without the AgI alginate-gel droplets. When the AgI alginate-gel droplet was added to the freezing solution of rabbit and bovine embryos, they were successfully preserved in liquid nitrogen.     

Development of hepatocyte spheroids immobilization technique using alternative encapsulation method

Primary hepatocytes of small animals such as rat and rabbit were often used for the study of extracorporeal liver support systems. Freshly isolated rat hepatocytes form spheroids within two days when cultivated as suspension in spinner vessels. These spheroids showed enhanced liver specific functions and more differentiated morphology compared to hepatocytes cultured as monolayers. However, shear stress caused by continuous agitation deteriorated spheroids gradually. In this work we immobilized spheroids to prolong liver specific activities. First, hepatocyte spheroids were suspended in collagen solution containing calcium chloride and then dropped into alginate solution. A thin layer of calcium alginate was formed around the droplet and then was removed after the inner collagen was gelled by treatment of sodium citrate buffer. Spheroids embedded in collagen-gel bead maintained liver specific functions such as albumin secretion rate longer than hepatocyte spheroids exposed to shear stress. Therefore, we suggest that this immobilization technique may offer an effective long-term hepatocyte cultivation and facilitate the development of a bioartificial liver support device.     

Studies on the application of immobilization technique for the production of cyclosporin A by a local strain of Aspergillus terreus

The formation of cyclosporin A (Cy A) by immobilized spores and mycelia of Aspergillus terreus was investigated. Different carriers were tested as immobilizing carriers, whereby Ca-alginate was selected for further experimentation. The role of alginate concentration, biomass weight, pH value of the cultivation medium, repeated utilization of the immobilized fungus as well as the supplementation of different amino acid precursors were studied. Best Cy A outputs were attained with Ca-alginate 3% (w/v), mycelial weight 15% (w/v), pH 4.5 and four repeated cycles. Similarly, the Cy A productivity was markedly accelerated in the presence of L-valine and L-valine and L-leucine mixture.     

Influence of pH and ionic strength on formation and stability of emulsions containing oil droplets coated by beta-lactoglobulin-alginate interfaces

Emulsions of 0.1 wt % corn oil-in-water containing oil droplets coated by beta-lactoglobulin (0.009 wt % beta-Lg, 5 mM phosphate buffer, pH 7.0) were prepared in the absence and presence of sodium alginate (0 or 0.004 wt %). The pH (3-7) and ionic strength (0-250 mM NaCl) of these emulsions were adjusted, and the particle charge, particle size, and creaming stability were measured. Alginate adsorbed to the beta-Lg-coated droplets from pH 3 to 6, which was attributed to electrostatic attraction between the anionic polymer and cationic patches on the droplet surfaces. Droplets coated by beta-Lg-alginate had better stability to flocculation than those coated by beta-Lg alone, especially around the isoelectric point of the adsorbed proteins and at low ionic strengths (< 100 mM NaCl). At pH 5, alginate molecules desorbed from the droplet surfaces at high salt concentrations due to weakening of the electrostatic attraction.     

l-Glutamate production by protoplasts immobilized in carrageenan gel

Protoplastization of Brevibacterium flavum cultured in a medium containing 50 μg l⁻¹ and 5 units penicillin per ml was performed by lysozyme treatment. The protoplasts were immobilized in various polymer matrices, such as agar, polyacrylamide, calcium alginate, and κ-carrageenan and then used for l-glutamate production from glucose and urea in a batch system. The protoplasts immobilized in κ-carrageenan gels showed the highest productivity of l-glutamate being twice that of immobilized whole cells under optimum conditions. The maximum productivity reached 2.3 mg ml⁻¹ initially. The immobilized B. flavum protoplasts could be used 8 times (192 h) for l-glutamate production retaining about 22% of the initial productivity during the last reaction.