Electrostatic Droplet Generator System [18]. 

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... many bio-processing laboratories, major focus has been placed on attempting to find cell culture methods which can increase the concentration of cells and cell products and permit cost- effective large-scale production. Methods of animal cell culture have been developed (mainly for mammalian cells) and involve the use of hollow fibers, gel entrapment, ceramic cartridges and microcarriers [1]. It is generally recognized that compared to microbial systems, large-scale mammalian cell suspension culture has been limited, to some extent, by relatively low cell densities. The concentration of extracellular proteins such as monoclonal antibodies and growth factors produced by this method is low and purification from growth media is difficult. The same problems are also encountered in insect culture. Inlow and co-workers [2], for example, 6 reported maximum insect cell densities of 5.5 x 10 cells/mL in a spinner flask. Hink [3], 8 working with T. ni cells, obtained maximum polyhedra (AcNPV) concentrations of ca. 2.2 x 10 6 polyhedra/mL medium at a cell density of 3.8 x 10 cells/mL medium (i.e. ca. 60 polyhedra/cell). Knudson and Tinsley [4], using S. frugiperda cells, reported 12-40 polyhedra (150-500 IFU)/cell. Microencapsulation, an alternative cell immobilization technique originally developed for use as an artificial pancreas for the treatment of diabetes, has been employed industrially for the enhanced production and recovery of high-value biologicals from animal cells. The encapsulation technique entraps viable cells within semipermeable polysaccharide-polycation microcapsules [for example, alginate/poly-L-lysine (PLL)]. The capsule membrane selectively allows small molecules such as nutrients and oxygen to freely diffuse through, but prevents the passage of large molecules and cells. Posillico [5] reported the use of microencapsulation for the commercial production of monoclonal antibodies. However, while cell densities of ca. 1 x 8 10 cells/mL capsules were obtained after three weeks of culture, they reported that the cells appeared to grow preferentially near the interior surface of microcapsule membrane and speculated that this could have been due to mass transfer limitations during culture and/or to the presence of a viscous intracapsular alginate solution. The first step in the making of a microcapsule is droplet formation. Let us consider one technique, electrostatic droplet generation (i.e. electrified liquid jet), which has become the primary method used in our laboratory. Electrified liquid jets (i.e. electrostatic atomisation) and electrostatically assisted atomization have been employed in a variety of areas, including paint spraying [6], electrostatic printing [7], and cell immobilization [1, 8]. Recently, micro and nano capsules as small as 0.15 micrometers, for example, have been produced using 0.7 mm ID needles [9]. The effect of electrostatic forces on mechanically atomized liquid droplets was first studied in detail by Lord Rayleigh [10, 11] who investigated the hydrodynamic stability of a jet of liquid with and without an applied electric field. When a liquid is subjected to an electric field, a charge is induced on the surface of the liquid. Mutual charge repulsion results in an outwardly directed force. Under suitable conditions, for example, extrusion of a liquid through a needle, the electrostatic pressure at the surface forces the liquid drop into a cone shape. Surplus charge is ejected by the emission of charged droplets from the tip of the liquid. The emission process depends on such factors as the needle diameter, distance from the collecting solution, and applied voltage (strength of electrostatic field [12]. Under most circumstances, the electrical spraying process is random and irregular, resulting in drops of varying size and charge that are emitted from the capillary tip over a wide range of angles. However, when the electrostatic generator configuration has been adjusted for liquid pressure, applied voltage, electrode spacing and charge polarity, the spraying process can become quite regular and periodic. 2.1 PRODUCTION OF ALGINATE BEADS USING ELECTRIFIED LIQUID JETS The section starts with a detailed experimental description of electrostatic droplet generation for those not familiar with the technique [12] (Fig. 1). Attach a syringe pump to a vertical stand. Use a 10 mL plastic syringe and 22- or 26- gage stainless steel needles. A variable high voltage power supply (0-30 kV) with low current (less than 0.4 mA) is required. We have used a commercial power supply model 230-30R from Bertan (Hicksville, NY). Prepare 1.5% (w/v) CaCl 2 in saline (0.85 g NaCl in 100 mL distilled water). Saline can be replaced with distilled water if an alginate solution without cells is being extruded. Place the CaCl 2 solution in a petri dish on top of an adjustable stand. The stand allows for fine tuning of the distance between the needle tip and collecting solution. Prepare 1 to 4 % (w/v) low viscosity sodium alginate by dissolving alginate powder with stirring in a warm water bath. Slowly add the 1 to 4 g sodium alginate to 100 mL warm saline solution (or distilled water), stirring continuously. It may take several hours to dissolve all of the alginate. Add about 8 mL of the alginate solution to a 10-mL plastic syringe, put back the plunger, and attach the syringe to the upright syringe pump. Make sure that the stainless steel needle, 22 gage, is firmly attached and the syringe plunger is in firm contact with the moveable bar on the pump. Position the petri dish (or beaker) containing CaCl 2 so that the needle tip is about 3 cm from the top of the CaCl 2 hardening solution. This is the primary reason for using an adjustable stand. Attach the positive electrode wire to the stainless steel needle and the ground wire to the collecting solution. The wires may need some additional support to prevent them from bending the needle. Switch on the syringe pump and wait for the first few drops to come out of the end of the needle. This could take a minute or two. Doing it this way also ensures that the needle is not plugged. After the first drop or two has been produced, switch on the voltage supply. Make sure that the voltage is set low, less than 5 kV. If this is the first time that you have tried electrostatic droplet generation, raise the voltage slowly and observe what happens to the droplets. The rate at which they are removed from the needle tip increases until only a fine stream of droplets can be seen. The changeover from individual droplets to a fine stream can be quite dramatic. The most effective electrode and charge arrangement for producing small droplets is a positively charged needle and a grounded plate. Two other arrangements are also possible; positively charged plate attached to needle, and a positively charged collecting solution. Make sure that the positive charge is always on the needle. This ensures that the smallest microbead size is produced at the lowest applied potential. With a 22-gage needle and an electrode spacing of 2.5-4.8 cm there will be a sharp drop in microbead size at about 6 kV. This can be noticed visually by observing the droplets coming from the needle tip. Standard commercially available stainless steel needles can be employed. However, when going from a 22- to 26-gage (or higher) needle, needle oscillation may be observed. This needle vibration will produce a bimodal bead size distribution with one peak around 50 m diameter beads and another around 200 μ m. If a syringe pump is not available, remove the syringe plunger and attach an air line with a regulator to the end of the syringe. Varying the air pressure on the regulator can control the alginate extrusion rate. Lumps of sodium alginate often form if the powder is added all at once to the warm saline. Sprinkle the alginate powder into the saline a small amount at a time with gentle mixing. Once it has dissolved (up to 1-2 h), allow the viscous solution to cool and then transfer it to several plastic test tubes, cap and store in the refrigerator until needed. This prevents bacterial growth. If the alginate solution is very viscous, air bubbles will be trapped during the stirring. These bubbles will disappear if the viscous solution is left to stand overnight. If the needle is plugged, place it in dilute citrate solution for a few minutes. Passing a fine wire through the needle also helps. Resuspending cells in 1 % (w/v) sodium alginate solution will dilute the alginate and could give tear-drop shaped beads when the solution is extruded. To solve this problem, increase the concentration of sodium alginate solution to 3 or 4 %. Extrusion of alginate droplets using a 5.7 KV fixed-voltage power supply showed that there is a direct relationship between the electrode distance and the bead diameter. For example, at 10-cm electrode distance, the bead diameter was 1500 μm while at 2 cm it decreased to 800 μm. The greatest effect on bead diameter was observed between 2 and 6 cm electrode distance. While there was overlap in bead sizes between 6, 8 and 10 cm electrode distance, there was a significant difference (i.e., no overlap in SD) between bead sizes at 2 and 6-cm electrode distance. An inverse relationship between needle size and microbead ...