Immobilization of animal cells using macroporous gelatin microcarriers and its application for virus production

Abstract

Immobilization of anchorage-dependent animal cells and virus production were investigated using macroporous gelatin microcarriers (K-beads) developed in our laboratory. For the observation of the distribution of cells in K-beads, Vero (Af. green monkey kidney) cells were cultured in spinner. Vero cells reached a final cell concentration of $5.6\times10^6$ cells/ml. The cell density on K-beads was 2-3 times higher than that on Cytodex-3, conventional solid microcarriers, by previous experiment. It was found that Vero cells appeared to be growing and filling up available pores both on the outside and inside of the K-beads by observation with confocal microscope. As a result, K-beads could provide the protection effect of cells against hydrodynamic shear and direct air sparging. Based on a function of Kolmogorov eddy length scale, the smaller eddies (80 $\mu$m) showed the detrimental effect on cells in K-beads as compared with 160$\mu$m of eddies in conventional solid microcarriers. When direct air sparging was introduced into culture vessels, the relative growth extent of cells on K-beads was about 90% up to 1.8cm/sec superficial velocity. However, cells on conventional solid microcarriers were rapidly damaged at 0.3cm/sec superficial velocity. Since K-beads promised the mass production of anchorage-dependent animal cells, K-beads was applied for the maximization of virus production. In case of Vesicular stomatitis virus (VSV) production system, pH ranged from 6.6 to 8.0 could not show the any difference of VSV production and optimal multiplicity of index (MOI) for virus production was appeared to be 0. 1 and 1.0. K-beads among various matrices tested provided the higest VSV production ($5.7\times10^{13}$ pfu/ml). To increase the virus production, culture media containing various concentrations of serum were added to the culture vessels just before virus infection. The maximum virus production was appeared at 10\% FBS. The addition of polycations into virus produ...