RESUMO
The process of cell seeding on biocompatible scaffolds has a major impact on the morphological evolution of an engineered tissue because it involves all the key factors of tissue formation: cells, matrix, and their mutual interactions. In order to characterize the efficiency of cell seeding techniques, mainly static parameters are used such as cell density, cell distribution, and cell viability. Here, we present an experimental model that incorporates an optical density meter providing real-time information on the cell seeding velocity, a relevant dynamic parameter of cell-matrix interaction. Our setup may be adapted to fit various cell seeding protocols. A modified fluorimetric cuvette is used as bioreactor culture flask. The optical density of the magnetically stirred cell suspension is recorded by a digital optoelectronic device. We performed calibration experiments in order to prove that, in our experimental conditions, optical density depends linearly on the number of cells in the unit volume of suspension. Control studies showed that, during the time course of a typical experiment (up to 10 h), the cells (murine 3T3 fibroblasts) neither aggregated nor adhered significantly to the walls of the cuvette. Hence, our setup yields the number of cells attached to the scaffold as a function of time. In order to analyze the experimental seeding curves, we built a kinetic model based on Langmuir's adsorption theory, which was extended to include a preliminary step of integrin function recovery. We illustrate the proposed approach by two sets of experiments that involved trypsin-EDTA or only EDTA treatment (no trypsin) used to detach the cells from the culture flasks. The data indicate that in both cases cell-matrix adhesion has a sequential, two-step dynamics, but kinetic parameters and attachment site availability depend on the experimental protocol.
