Microencapsulation offers a unique potential for high cell density, high productivity mammalian cell cultures. However, for successful exploitation there is the need for microcapsules of defined size, properties and mechanical stability. Four types of alginate/poly-L-Lysine microcapsules, containing recombinant CHO cells, have been investigated: (a) 800 lm liquid core microcapsules, (b) 500 lm liquid core microcapsules, (c) 880 lm liquid core microcapsules with a double PLL membrane and (d) 740 lm semiliquid core microcapsules. With encapsulated cells a reduced growth rate was observed, however this was accompanied by a 2-3 fold higher specific production rate of the recombinant protein. Interestingly, the maximal intracapsular cell concentration was only 8.7 · 10 7 cell mL -1 , corresponding to a colonization of 20% of the microcapsule volume. The low level of colonization is unlikely to be due to diffusional limitations since reduction of microcapsule size had no effect.Measurement of cell leaching and mechanical properties showed that liquid core microcapsules are not suitable for continuous long-term cultures (>1 month). By contrast semi-liquid core microcapsules were stable over long periods with a constant level of cell colonization (u = 3%). This indicates that the alginate in the core plays a predominant role in determining the level of microcapsule colonization. This was confirmed by experiments showing reduced growth rates of batch suspension cultures of CHO cells in medium containing dissolved alginate. Removal of this alginate would therefore be expected to increase microcapsule colonization.
A new approach combining electrostatic and covalent bonds was established for the formation of resistant capsules with long-term stability under physiological conditions. Three kinds of interactions were generated in the same membrane: (1) electrostatic bonds between alginate and poly-L-lysine (PLL), (2) covalent bonds (amides) between propylene-glycol-alginate (PGA) and PLL, and (3) covalent bonds (amides) between BSA and PGA. Down-scaling of the capsules size (< or =1 mm diameter) with a jet break-up technology was achieved by modifying the rheological properties of the polymer solution. Viscosity of the PGA solution was reduced by 95% with four successive pH stabilizations (pH 7), while filtration (0.2 microm) and sterilization was possible. Covalent bond formation was initiated by addition of NaOH (pH 11) using a transacylation reaction. Kinetics of the chemical reaction (pH 11) were simulated by two mathematical models and adapted in order to preserve immobilization of animal cells. It was demonstrated that diffusion of NaOH in the absence of BSA resulted in gelation of 94% of the bead and death of 94% of the cells after 10 s reaction. By addition of BSA only 46% of the cells were killed within the same reaction time (10 s). Mechanical resistance of this new type of capsule could be increased 5-fold over the standard polyelectrolytic system (PLL-alginate). Encapsulated CHO cells were successfully cultivated for 1 month in a repetitive batch mode, with the mechanical resistance of the capsules decreasing by only 10% during this period. The combination of a synthetic and natural protein resulted in enhanced stability toward culture medium and proteolytic enzymes (250%).
A study of alginate lyase was carried out to determine if this enzyme could be used to remove alginate present in the core of alginate/poly-L-lysine (AG/PLL) microcapsules in order to maximize cell growth and colonization. A complete kinetic study was undertaken, which indicated an optimal activity of the enzyme at pH 7-8, 50 degrees C, in the presence of Ca2+. The buffer, not the ionic strength, influenced the alginate degradation rate. Alginate lyase was also shown to be active on gelled forms of alginate, as well as on the AG/PLL complex constituting the membrane of microcapsules. Batch cultures of CHO cells in the presence of alginate showed a decrease of the growth rate by a factor of 2, although the main metabolic flux rates were not modified. The addition of alginate lyase to cell culture medium increased the doubling time 5-7-fold and decreased the protein production rate, although cell viability was not affected. The addition of enzyme to medium containing alginate did not improve growth conditions. This suggests that alginate lyase is probably not suitable for hydrolysis of microcapsules in the presence of cells, in order to achieve high cell density and high productivity. However, the high activity may be useful for releasing cells from alginate beads or AG/PLL microcapsules.
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