Differential effect of the shape of calcium alginate matrices on the physiology of immobilized neuroblastoma N2a and Vero cells: A comparative study (original) (raw)
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Elucidation of optimum conditions for immobilization of viable cells by using calcium alginate
Journal of Fermentation and Bioengineering, 1989
Different factors which affect the stability of calcium alginate gel beads entrapping viable cells during fermentation were investigated. It was found that among others, the initial population of cells per ml of gel beads, the length of period of incubation in CaCi2 solution, and the concentration of sodium alginate used for the immobilization were the most important factors affecting the stability of the gel beads during fermentation. By using an initial cell population of about l0 scelis per mi of 2.0% sodium alginate, and incubating the beads for at least 22 h in a CaCI2 solution after immobilization, the percentage of beads which developed cracks during fermentation was highly reduced. Also, without the addition of CaCi2 into the fermenting broth, the gel beads were stable for nine consecutive batch fermentations.
Culture of Neural Stem Cells in Calcium Alginate Beads
Biotechnology Progress, 2006
Neural stem cells (NSCs) with the capacity of extensive self-renewal and multilineage differentiation have attracted more and more attention in research as NSCs will play an important role in the nerve disease treatment and nerve injury repair. The shortage of NSCs, both their sources and their numbers, however, is the biggest challenge for their clinic application, and hence, in vitro culture and expansion of NSCs is vitally important to realize their potentials. In this work, mouse-derived NSCs were cultured in three-dimensional calcium alginate beads (Ca-Alg-Bs). Gelling conditions, cell density, and cell harvest were determined by the exploration of formation and dissociation parameters for Ca-Alg-Bs. Additionally, the recovered and the subsequent induced cells were identified by immunofluorescence staining of Nestin, -tubulin, and GFAP. The results show that the 2-mm diameter Ca-Alg-Bs, prepared with 1.5% sodium alginate solution and 3.5% CaCl 2 solution and with gelling for 10 min, is suitable for the NSCs culture. The seeding density of 0.8 × 10 5 cells‚mL -1 for the encapsulation of NSCs resulted in the most expansion, and the NSCs almost doubled during the experiment. The average cell recovery rate is over 88.5%, with the Ca-Alg-Bs dissolving in 55 mM sodium citrate solution for 10 min. The recovered cells cultured in the Ca-Alg-Bs still expressed Nestin and had the capacity of multilineage differentiation into neurons and glial cells and, thus, remained to be NSCs. These results demonstrate that NSC expansion within Ca-Alg-Bs is feasible and provides further possibilities for NSC expansion in bioreactors of the scale of clinical relevance.
Materials Science and Engineering: C, 2014
Neural stem cells (NSCs) forming neurospheres in a conventional culture tend to develop necrotic/apoptotic centers due to mass transport limitations. In this study, the internal pore structure of calcium-alginate/gelatin (CAG) microbeads was tuned and controlled to provide a suitable three-dimensional environment supporting NSC proliferation. Direct impact of three-dimensional space availability was quantified by oxygen consumption rates of NSCs and cells were cultured in three different methods: neurospheres, single cell suspension of NSCs, and encapsulated NSCs in microbeads. Our results showed that encapsulated NSCs in CAG microbeads maintained higher cell viability than in conventional culture. In addition, NSCs encapsulated in CAG microbeads preserved their original stemness and continued to express nestin, CNPase, GFAP and β-tubulin-III post-encapsulation. Oxygen consumption rates of encapsulated NSCs in CAG microbeads were the lowest as compared to the other two culture methods. The optimal cell density supporting high cell proliferation in CAG microbeads was found to be 1.5 × 10 5 cells/mL. The glucose consumption curve suggests that encapsulated NSCs in microbeads had a slower growth profile. This study presents an alternative method in hybrid microbead preparation to generate a highly favorable three-dimensional cell carrier for NSCs and was successfully applied for its effective in vitro expansion.
Monitoring of the Viability of Cells Immobilized by Sol-Gel Process
Journal of Sol-gel Science and Technology, 2004
Three different types of cells, Pseudomonas fluorescens HK44, Saccharomyces cerevisiae strain SP4 and plant cells Nicotiana tabacum L. BY-2, were immobilized by entrapment in tetramethoxysilane prepolymer (TMOS) gel or in composite gel containing prepolymer TMOS and alginate in various ratios. Their growth and viability were monitored by bioluminescence and 2-D fluorescence spectra, which are fast and do not need the dissolution of a matrix. The resulting biocomposite gels were obtained by gelation of the mixtures of TMOS prep. or TMOS/alginate sols and the particular cells in proper media on glass supports to provide films ∼1 mm thick. The effect of the following parameters on the growth and viability of the cells was studied: (a) the composition of the biocomposites, (b) the preparation conditions of TMOS and (c) the conditions of the procedure of entrapment. All three types of cells were tested in TMOS gel and the composite TMOS/alginate = 1:1 (v/v). The sensitivity of the cells to the changes of conditions increased in the sequence: P. fluorescence HK44 < S. cerevisiae strain SP4 < N. tabacum L. BY-2. Cell viability decreased with the increasing content of Si in biocomposites. The entrapment into alginate–silica composites resulted in the leakage of microbial and yeast cells. However, it had positive effects on the growth and metabolic activity of plant cells.
Evaluation of the use of Sr 2+ in alginate immobilization of cells
Naturwissenschaften, 2001
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 Ca 2+ 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 Ca 2+ , Sr 2+ and Ba 2+ 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.
2011
This paper reports a unique approach to immobilize mammalian cell populations (mouse myeloma NS0) through in situ gelation of calcium alginate triggered by an electrical signal under physiologically relevant conditions. This is the first experimental observation to evaluate the electrically triggered assembly of calcium alginate gel for entrapping and culturing mammalian cells. Subsequent cell viabilities immediately after electrodeposition and after three days of culturing are studied. Our cell assembly strategy is applicable to fragile mammalian cells and can be used for in vitro study of dynamic cellular processes and cell-based assays under a microfluidic environment.
Cytotechnology, 1995
A murine hybridoma cell line producing a monoclonal antibody against penicillin-G-amidase and a murine transfectoma cell line secreting a monovalent chimeric human/mouse Fab-antibody fragment were cultivated in three different media (serum-containing, low protein serum-free, and iron-rich protein-free) in flask cultures, stirred reactors and a fixed bed reactor. In static batch cultures in flasks both cell lines showed similar good growth in all three media. In suspension in a stirred reactor, the hybridoma cell line could be cultivated satisfactory only in serum-containing medium. In low protein serum-free medium, Pluronic F68 had to be added to protect the hybridoma cells against shear stress. But even with this supplement only batch, not chemostat mode was possible. In iron-rich protein-free medium the hybridoma cells grew also in continuous chemostat mode, but the stability of the culture was low. The transfectoma cell line did not grow in stirred reactors in any of the three media. Good results with both cell lines were obtained in fixed bed experiments, where the cells were immobilized in macroporous Siran |-carriers. The media, which were optimized in flask cultures, could be used without any further adaptation in the fixed bed reactor. Immobilization improved the stability and reliability of cultures of non-adherent animal cells in serum-free media tremendously compared to suspension cultures in stirred reactors. The volume-specific glucose uptake rate, an indicator of the activity of the immobilized cells, was similar in all three media. Deviations in the metabolism of immobilized and suspended cells seem to be mainly due to low oxygen concentrations within the macroporous carriers, where the cells are supplied with oxygen only by diffusion.
Investigations of cell immobilization in alginate: rheological and electrostatic extrusion studies
Journal of Chemical Technology & Biotechnology, 2006
In this study, the process of electrostatic extrusion as a method for cell immobilization was investigated. We have assessed the effects of concentrations of yeast cells (as a model cell type) and Na alginate on the size of the resulting microbeads and attempted to rationalize the obtained findings by rheological characterization of the cell-alginate suspensions. Under the investigated conditions, microbeads, 50-600 µm in diameter, were produced and the increase in both alginate and cell concentrations resulted in larger microbeads with their sizes having higher standard deviations. 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 × 10 8 and 1 × 10 9 cells mL −1 ) did not significantly affect the rheological properties of the Na alginate solution. Finally, we have investigated the kinetics of alginate gelation with respect to the quantity of Ca 2+ ions and the presence of cells. The molar ratio of α-L-guluronic acid units to Ca 2+ ions of 4:1 provided complete crosslinking. The presence of cells decreased the rate of network formation as well as the strength of the obtained Ca alginate hydrogel.
International Journal of Nanomedicine, 2006
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 (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 Ca 2+ ions and cell presence. The gelation kinetics were examined under conditions of limited supply with Ca 2+ ions, which can be essential for immobilization of highly sensitive mammalian cells that require minimal exposure to CaCl 2 solution. The molar ratio of G units to Ca 2+ 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.
Journal of biomedical materials research. Part A, 2003
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 p...