Development of a new macorporous gelatin microcarrier and its characterization for mammalian cell culture

Abstract

A new type of macroporous gelatin microcarrier was prepared for the cultivation of anchorage-dependent animal cells. For the development of macroporous gelatin microcarrier, the effects of various parameters in control of the sizes of microcarrier and macropores were investigated. The optimal conditions were determined as follows; (a) the emulsion system with organic solvents (toluene: chloroform=73:27) containing emulsifier. 1\% (v/v) Tweed 80, and stirring rate between 700 and 800 rpm with magnetic stirrer, (b) the concentration (above 15\%) and the particle size (15-25 $\mu{m}$) of calcium carbonate, (c) the incubation time more than 12 hours and freezing temperature at $-20\,^\circ\!C$ for recrystallization. The microcarriers formed by the proposed method show the spherical shape and sponge-like structure. The physical properties were characterized as follows; bead size, 125-500$\mu{m}$: pore size, 25-55$\mu{m}$ density, 1.14 g/ml : surface area, 11,000 cm$^2$/g: numbers of carriers: $1.4\times10^6$ ea/g. The attachment and growth of Vero-6 cell were examined with the gelatin macroporous microcarrier. The growth rate and the maximum density on this microcarrier were compared with those on commercial microcarrier (cytodex3). The attachment rate on macroporous microcarrier was similar with that on cytodex-3. In spinner culture with 3 g/l of microcariers, the maximum cell concentration was achieved to $2.8\times 10^6$ cells/ml ($1.2\times10^8$ cells/ml-bead vol.) which was almost twice higher than that on cytodex-3 ($1.6\times10^6$ cells/ml, $3.8\times10^7$ c ells/ml-bead vol.). The cell growth was dependent on the inoculum density. The higher cell growth rate and maximum cell concentration were obtained by the centrifugal inoculation. The even distribution of cells in macroporous microcarrier was suggested by the centrifugal inoculation.