Surface immobilization of anchorage-dependent mammalian cells

Anchorage-dependent HeLa cells were successfully cultured on two fibrous materials (A07 and R100) with porosities of 75-125 and 40 mum, void fractions of 92% and 81%, and fiber diameters of 7.6 and 10.2 mum, respectively, in 100-mL spinner flasks and 2-L stirred tank bioreactors. The matrix was formed into a fixed vertical spiral configuration. All cultures displayed rapid (/=95%) to the matrix, uniform coverage of the immobilizing area with viable cells, and no significant amount of cell debris in the medium. Spinner flask cultures indicated that the denser material R100 showed better results in terms of final cell density. The growth of HeLa cells on material R100 in both culture systems was similar to that observed in tissue culture dishes (specific growth rate approximately 0.03-0.04 h(-1), maximum cell density of 8 x 10(6)-9 x 10(6) cells . mL(-1), and yields of 0.4 x 10(8) cells . mM(-1) on glucose and 2 x 10(8)-3 x 10(8) cells . mM(-1) on glutamine). Scale-up of this culture technique in a 2-L bioreactor under perfusion with pH and dissolved oxygen (DO) control yielded cell densities of up to 1.6 x 10(6) cells . mL(-1). Two other anchorage-dependent mammalian cells (ADC) known to be cultured with difficulty in roller bottles or with micro carriers were easily grown on material R100 in spinner flasks. The performance of this culture technique was compared to other ADC culture systems.     

Fluorometric estimation of viable animal cells immobilized in alginate gel

Fluorescein diacetate (FDA) is hydrolyzed to a fluorescent compound, fluorescein (FRC) which is retained in viable animal cells having an intact plasma membrane. The FDA-FRC system was applied to analysis of viable cells for a mouse hybridoma 16-3F cell entrapped in alginate gel. As a result, visualization of the viable cell distribution in the gel matrix became possible. Moreover, a linear relationship was confirmed between the viable cell concentration determined by microscopic counting and the fluorescence intensity derived from FRC in the viable cells. Thus, cell growth could be quantitatively estimated on the basis of the fluorometry for the immobilized-cell culture.     

Optimization of critical parameters for immobilization of yeast cells to alginate gel matrix

The optimum concentrations of sodium alginate (wt. %), calcium chloride (M) and yeast cells (wt. %), and curing time (h) for enhanced gel stability were obtained employing a full factorial search. The results indicate that the concentrations of sodium alginate and CaCl₂, and the curing time of the beads were found to have a pronounced effect on the stability of the beads. The cell concentration, on the other hand, has an adverse influence either individually or in combination with other variables. The path of steepest ascent method has been used to optimize the variables and the resultant gel beads were evaluated for fermentation ability.     

Silica nanotubes-doped alginate gel for yeast alcohol dehydrogenase immobilization

A novel alginate–silica nanotubes (ALG–SiNTs) composite was prepared through the incorporation of silica nanotubes (SiNTs) into the alginate (ALG) gel followed by Ca²⁺ cross-linking for encapsulating yeast alcohol dehydrogenase (YADH, EC 1.1.1.1) from Saccharomyces cerevisiae. Pre-adsorption of YADH onto the surface of SiNTs before encapsulating in alginate gel was adopted to circumvent the enzyme leakage. AFM and SEM characterization confirmed that YADH molecules were substantially adsorbed on the SiNTs. SEM and EDX studies showed that the SiNTs homogenously distributed in alginate matrix. The enzyme leakage from ALG–SiNTs–YADH composite was remarkably reduced about 50% compared to that of ALG–YADH composite. Meanwhile, the optimum reaction condition, catalytic activity and kinetic parameters of immobilized YADH in ALG–SiNTs composite were studied. The results showed that stronger affinity between substrates and enzyme, higher activity retention, improved storage and operational stability were achieved when YADH was immobilized in ALG–SiNTs composite instead of ALG–YADH composite.     

Silica nanotubes-doped alginate gel for yeast alcohol dehydrogenase immobilization

A novel alginate–silica nanotubes (ALG–SiNTs) composite was prepared through the incorporation of silica nanotubes (SiNTs) into the alginate (ALG) gel followed by Ca²⁺ cross-linking for encapsulating yeast alcohol dehydrogenase (YADH, EC 1.1.1.1) from Saccharomyces cerevisiae. Pre-adsorption of YADH onto the surface of SiNTs before encapsulating in alginate gel was adopted to circumvent the enzyme leakage. AFM and SEM characterization confirmed that YADH molecules were substantially adsorbed on the SiNTs. SEM and EDX studies showed that the SiNTs homogenously distributed in alginate matrix. The enzyme leakage from ALG–SiNTs–YADH composite was remarkably reduced about 50% compared to that of ALG–YADH composite. Meanwhile, the optimum reaction condition, catalytic activity and kinetic parameters of immobilized YADH in ALG–SiNTs composite were studied. The results showed that stronger affinity between substrates and enzyme, higher activity retention, improved storage and operational stability were achieved when YADH was immobilized in ALG–SiNTs composite instead of ALG–YADH composite.     

Scale-up of suspension and anchorage-dependent animal cells

Alternative culture processes for laboratory scale-up (to 20 L) are described for both suspension and anchorage-dependent cells. Systems range from simple multiple culture units such as the roller bottle, through stirred suspension and microcarrier unit bioreactors, to highly sophisticated perfusion culture capable of maintaining cells at densities of about 10⁸/mL. Critical parameters in scale-up are discussed, and the advantages and disadvantages of each culture system are critically evaluated.     

Cultivation of anchorage-dependent animal cells in microsphere-induced aggregate culture

Summary Diethylaminoethyl-derivatized dextran microspheres were used to cultivate Chinese hamster ovary, 293, Vero and swine testicular cells. Cells became attached to the microspheres but did not spread out. Instead, they grew in a more spherical shape and eventually formed multiple-cell-layer aggregates. Viability in these aggregates remained high after the cultures reached high cell concentrations. This cultivation method allows a high cell density to be achieved with a low microsphere concentration.Offprint requests to: W.-S. Hu     

Evaluation of some gelling agents for immobilization of aerobic microbial cells in alginate and carrageenan gel beads

Summary Different gelling agents were used to immobilized viable cells in either alginate or -carrageenan gel beads. Based on cell leakage from the gel beads, oxygen and glucose diffusion coefficients and toxicity of the gelling agents, SrCl₂ was found to be the best for immobilization of aerobic microbial cells in, not only alginate but also carrageenan gel beads.     

Optimization of critical parameters for immobilization of Streptomyces clavuligerus on alginate gel matrix for cephamycin C production

The optimum critical parameters for immobilization of Streptomyces clavuligerus on alginate gel matrix for cephamycin C production, i.e. sodium alginate (wt. %), CaCl₂ (M) and cell concentration (wt. %), curing time (h.), for enhanced gel stability, were obtained employing a full factorial search. The results indicate that the concentrations of CaCl₂ and inoculum size were found to have a pronounced effect on cephamycin C fermentation. On the other hand, the higher concentration of sodium alginate exerted an adverse influence either individually or in combination with other variables. The path steepest ascent method has been used to optimize the variables. The optimum concentrations of matrix components were 3.218% sodium alginate, 0.996 M CaCl2, 19.06% cell concentration and 17.16 h. of curing time supported higher cephamycin C production, at 48 h. of fermentation.     

High density culture of anchorage-dependent animal cells by polyurethane foam packed-bed culture systems

Summary Monkey kidney cells (Vero) and Chinese hamster ovary cells (CHO-K1) attached to the internal surface of polyurethane foam (PUF) and grew to a high cell density (1.1 × 10⁸ cells/cm³ PUF and 4.2 × 10⁷ cells/cm³ PUF, respectively) in a PUF-plates packed-bed culture system. This density of Vero cells was twice that obtained previously with a PUF-particles packed-bed culture system. A maximum cell density of 6.7 × 10⁷ cells/cm³ culture vessel volume was obtained in a PUF-disc packed-bed culture of Vero cells. From the cell density of CHO-K1, growing in a monolayer on the surface of PUF and a petri dish, per bulk volume of PUF, we estimated that a surface area to volume ratio of PUF plates effective for cell growth was about 109 cm²/cm³.Offprint requests to: K. Funatsu     

Growth and morphology of anchorage-dependent animal cells in a liquid/liquid interface system

In general, anchorage-dependent animal cells cultivated on a solid culture substrate, such as polystyrene, are collected by trypsin treatment. This treatment may have detrimental effects such as the proteolysis of the cell membrane proteins. To avoid these effects, cell cultivation using a liquid/liquid interface system has been investigated. In this cultivation method, the cells grow at the interface between a culture medium and a hydrophobic liquid. In this study, various fluorocarbons (FC-40, FC-70, KPF-91, KPF-102, and KPF-142) were used as substrates for the interface, and the cultivation of fibroblast cells (L-929; the mouse-derived cell line) at the interfaces was investigated. Early in the cultivation period, the growth of L-929 cells depended on the substrate type. Although cell cultivation at the interfaces was possible, it was slower than that at the polystyrene surface. Cell spreading at the interfaces was relatively small, which indicates that cell adhesion at the interfaces may be weak. In particular, the cells at the MEM/FC-70 interface anchored with one another and formed multicellular hemispherical aggregations shaped like spheroids. The difference in the adhesions to the interfaces appears to be dependent on the contaminants contained in the fluorocarbons because the physical properties of the fluorocarbon did not affect the cell growth and adhesion. Moreover, subcultivation from the interfaces to the same interface was possible without trypsin treatment. In this case, the delay of the growth at the interfaces did not occur because the cells were not affected by trypsin treatment.     

New immobilization method of mammalian cells using alginate and polyacrylate

Mouse-mouse hybridoma cells were immobilized in polyacrylate-alginate gels. The immobilized hybridoma cells were cultured semi-continuously using a fluidized bed reactor, and allowed continuous antibody production without any gel destruction for one month. It has been proved that the polyacrylate-alginate gels were tolerant against physical stress. The composition of the gels suitable for cell growth and antibody production was given as follows; viscosity of alginate at 1% solution: 60–100 cP, alginate concentration: 0.8%, and polyacrylate concentration: 0.2%. In the semi-continuous culture using gels prepared under suitable conditions, the viable cell number was estimated as 2.5×10⁷ cells/ml-gel, and the antibody production rate was 2.2 mg/ml-gel/d, at maximum.     

Elucidation of optimum conditions for immobilization of viable cells by using calcium alginate

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 CaCl₂ 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 10⁵ cells per ml of 2.0% sodium alginate, and incubating the beads for at least 22 h in a CaCl₂ solution after immobilization, the percentage of beads which developed cracks during fermentation was highly reduced. Also, without the addition of CaCl₂ into the fermenting broth, the gel beads were stable for nine consecutive batch fermentations.     

Covalent stabilization of alginate gel for the entrapment of living whole cells

Summary Three methods were developed for preparing alginate gels containing cells that are stable in phosphate containing media. In Method I preformed alginate beads containing entrapped cells were treated with polyethyl eneimine followed by glutaraldehyde. In Method II alginate sol was treated with a carbodiimide and N-hydroxysuccinimide (to form active esters), mixed with cells and extruded into calcium chloride solution. The beads were subsequently cross-linked with polyethyleneimine. In Method III alginate so] was treated with periodate (to form aldehyde groups), mixed with cells and extruded into calcium chloride solution. The beads were subsequently cross-linked with polyethyleneimine. Saccharomyces cerevisiae cells, immobilized in such stabilized gels, exhibited almost the same fermentation activity as the standard preparation. The viability of the immobilized cells was retained during the stabilization procedure as judged from their ability to multiply in the presence of nutrients.The preparations remained stable in phosphate buffer for at least ten days without substantial release of cells. The extent of cross-linking was controlled by varying the time and the concentration of reactants, thus giving preparations ranging from beads with a thin stabilized shell to beads homogeneously stabilized.     

Electrotransfection of anchorage-dependent mammalian cells

Reversible electropermeabilization (or electroporation) of cell membranes is a very efficient method for intracellular delivery of xenomolecules, particularly of DNA. In the case of anchorage-dependent cells, however, enzymatic or mechanical detachment from the substratum is required prior to electropulsing. This can damage the plasma membrane and lead to low transfection yields. Here we present an efficient method for in situ electroporation of mammalian cells while they are attached to a solid substratum. For this purpose an electroporation chamber was constructed that housed a cell culture insert with a cell monolayer grown on a porous filter. By real-time monitoring the transmonolayer resistance, the field pulse parameters resulting in transient and reversible permeabilization of cell membranes were determined for two adherent cell lines, which were found to differ markedly in their sensitivity to electropulsing. Based on the transmonolayer resistance data, the pulsing conditions for optimum electrotransfection of two murine cell lines with plasmid DNA could be established in a very short time. The transfection yield and gene expression were significantly higher in cell monolayers facing the cathode compared to those exposed to field pulses of the reverse direction. This might be due to contribution of the electrophoresis to the translocation of the polyanionic plasmid DNA across the electropermeabilized cell membrane. The experimental setup presented here appears to be a promising tool not only for rapid optimization of in situ electrotransfection of anchorage-dependent cells but also for studying the molecular/biophysical mechanisms of the membrane breakdown and resealing.     

Agar gel immobilization ofBacillus brevis cells for production of thermostable α-amylase

Growing cells of a thermophilic strain ofBacillus brevis, producer of thermostable α-amylase, were immobilized by entrapment in agar gel. Optimum immobilization conditions for effective α-amylase production in batch fermentations were established (gel concentration 3%, initial biomass concentration in the gel 0.8% (W/V), and preculture age—late exponential phase). The dynamics of α-amylase synthesis by the biocatalysts obtained under the optimal conditions was compared with that of free cells and the operational stability of the biocatalysts was studied in semicontinuous cultivation experiments. Maximum α-amylase yields (252% of the control) were achieved after the second cycle of cultivation. Scanning electron microscopy was used to characterize the bacteria entrapped in agar gel.     

Modified hydroxyalkyl methacrylate gel as a support for immobilization of yeast cells

Summary Whole cells of Zygosaccharomyces lactis have been covalently linked to fine-grained hydroxyalkyl methacrylate gel Spheron P 1000 E which was prepared by treatment with epichlorhydrin and modified by an amine spacer. Experiments on the coupling of permeabilized and non-permeabilized cells to this gel support have shown that immobilized cell agregates may be obtained by the immobilization of thermally permeabilized cells. Cell clustering can be bypassed by immobilizing non-permeabilized cells. This immobilization procedure makes additional permeabilization possible.