Desenvolvimento de Protocolo Passível de Ampliação de Escala para Propagação de Células Vero em Microcarregadores

Due to the increasing need of developing production processes for viral vaccines using mammalian cell lines in the absence of animal components, as well as a new method for the production of an inactivated vaccine against yellow fever, it is very important to develop a new process based on more controlled conditions. One way to achieve such demand is the propagation of yellow fever virus in cell lines grown in bioreactors using microcarriers and under monitored and controlled conditions in order to enable greater reproducibility. For this reason, the expansion of the production scale is an important step in the development of a product, since it must be performed in such a way that all the results obtained at laboratory scale can be reproduced at industrial scale. However, this procedure presents challenges to be faced, for instance, when using microcarriers, precisely in the cell transfer operation among microcarriers at industrial scale. For this purpose, the commonly used technique of trypsinization was evaluated aiming the removal of fetal bovine serum during the process of inactivation of trypsin, substituting it by conditioned medium, since Vero cells adapted to serum-free medium were used The metabolic and growth profile of Vero cell cultures in static T-flasks and stirred spinner flasks using microcarriers was also evaluated. Knowing the growth profile, it was possible to use the technique of bead-to-bead transfer, under intermittent stirring in different intervals. It was observed that only with 1 hour of stirring and 15 minutes of resting, there was significant growth and that bridges among cells on beads were formed, as shown by microscopic analyses. Alternatively, trypsinization of the cells on microcarriers was tested aiming the releasing of the cells and their inoculation to cultures containing new and old microcarriers. Different concentrations of trypsin were tested and it was observed that trypsinized cells showed ability to adhere to microcarriers, but without significant proliferation. For better results, a pretreatment of the medium with an alkalizing agent was added to the trypsinization step until it reached a pH between 8-9. Under these conditions the cells showed ability to adhere again to the microcarriers and to proliferate, indicating that such pretreatment is valid and applicable on a seed train strategy in microcarrier based processes.