OPTIMIZATsIYa TEKhNOLOGII KUL'TIVIROVANIYa DERMAL'NYKh FIBROBLASTOV DLYa TERAPEVTIChESKIKh TsELEY S POMOShch'Yu ROBOTIZIROVANNOY STANTsII

The potential of the automated cell culture system CompacT SelecT (TAP Biosystems, UK) allows using it for the large scale production of dermal fibroblasts for cell therapy. Automated maintenance and expansion of fibroblasts requires evaluation of the proper seeding density and defining the time for the achievement of the optimal confluency of cell monolayer for further passaging. Objective - to determine the optimal parameters of dermal fibroblasts passaging for development of standardized protocol for automated cell cultivation using CompacT SelecT system. Human dermal fibroblasts were isolated from donor skin samples by enzyme dissociation. Cells proliferation rate was evaluated by the calculation of proliferation index. The dependence of proliferation rate from cell monolayer density was determined using the value of average number of divisions of single cell per day. The dependence of fibroblasts proliferation rate from seeding density (from 50 to 1.2х104cells/cm2) in various periods of cultivation (3rd, 5th, 7th and 9th day) was investigated. The inverse correlation between proliferation rate and seeding density, together with dramatic decrease of proliferation rate at fibroblasts monolayer confluency of (3.5-4.5)х104 cells/cm2, was demonstrated. The optimal seeding density was evaluated as 3х103 cells/ cm2, the most suitable density of cell monolayer for further passaging was evaluated as 3.5х104 cells/cm2. In case of high seeding density (1.2х104 cells/cm2), on day 7, when the cell monolayer density reached the critical value, the cell extinction occured, followed by renewal of cell growth. This phenomenon may be explained by the selection of cells capable of replication under conditions of high density of monolayer.

dermal fibroblasts, automated cell cultivation, seeding density, proliferation rate