Phenomenological study of the growth rate of transformed cells, transitions in the growth mode and fractal behavior of colony contours.

M.A. HUERGO; M.A.PASQUALE; A. E. BOLZÁN; A.J. ARVÍA; P.H. GONZÁLEZ

BIOMAT 2009. International Symposium on Mathematical and Computational Biology

World Scientific Publishing Co. Pte. Ltd.

Lugar: Rio de Janeiro; Año: 2010; p. 32 - 44

Vero cell colonies were studied following the shape evolution of the growing front in contact with the medium to determine the growth kinetics, the fractal characteristics of the colony contour, and the appearance of transitions in the growth mode. Growth dynamics and morphology were investigated using   dynamic scaling and AFM techniques, respectively.  Cell colonies were grown in Petri dishes by shedding disaggregated cells at low density (500-1000 cell/ml) in a culture medium (RPMI with 10% fetal bovine serum). Cultures were maintained in a 5% carbon dioxide and 95% humidity atmosphere at 37o C, changing one half of the culture medium every two days.  Two different experimental approaches were utilised. In the  first one, several individual clones,   each containing about 3-8 cells,  were chosen after 48 h culture. These cell colonies exhibited a quasi-bidimensional (2D) radial growth on the plate surface (“radial growth regime”).  In the second one,   cells were first plated on 5 mm2 square glasses   located in Petri dishes.  After 48 hours culture, with a 100% confluence,  the glasses were removed and re-plated into new Petri dishes, containing fresh culture medium, and the evolution of the linear growth fronts at the edges of the glasses, was followed (“linear growth regime”). Snapshots of the colonies, either under radial or linear growth regime, were taken on a daily basis using a digital camera coupled to an inverted phase-contrast microscope. Images were processed and analysed using computational programs developed in the laboratory to obtain   the mean radius (for radial growths), the mean height, (for linear growths), the front roughness, and the fractal dimension. Three growth stages were observed during colony growth. At the first stage, the growth pattern was uniform and compact. At the second stage, the cell density at the edge front decreased and  few large multinuclear cells were observed.  Eventually, at the third stage, most of the cells located at the edge of the growing front tended to acquire a fibroblastic-like shape, trying to keep their anchorage to the main colony by means of  emerging filaments. At this stage a number of cells tended to grow isolated from the bulk colony.    For the linear growth regime, the roughness R increased with   time with an exponent b = 0.5±0.05, reaching a saturation value in the long-time range.  The mean height <h> increased linearly with time, and the fractal dimension, determined from the counting box method, increased with the size of the system from 1.5 to 1.9.  For the system under  radial growth  regime,   no roughness saturation was attained,  and the mean radius <r> increased according to <r> µ exp(kt)  with a time constant rate k =  2 x 10-6 s-1.  In this case, the fractal dimension   increased with <r>, i.e. as  the size of the system (colony) increased.