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

M. A. C. HUERGO; M. A. PASQUALE; A. E. BOLZAN; A. J. ARVIA; P.H. GONZALEZ

Brasilia, Brasil

Simposio; “9th International Symposium on Mathematical and Computational Biology”,; 2009

BIOMAT Institute for Advanced Studies of Biosystems

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 are observed during colony growth. At the first stage, the growth pattern is uniform and compact. At the second stage, the cell density at the edge front decreases and  few large multinuclear cells are observed.  Eventually, at the third stage, most of the cells located at the edge of the growing front tend to acquire a fibroblastic-like shape, trying to keep their anchorage to the main colony by means a emerging filaments. At this stage a number of cells tend to grow isolated from the bulk colony.    For the linear growth regime, the roughness R increases with   time with an exponent b = 0.5±0.05, reaching a saturation value in the long-time range.  The mean height <h> increases linearly with time and the fractal dimension, determined from the counting box method, increases with the size of the system from 1.5 to 1.9.  For the system under  radial growth  regime,   no roughness saturation is attained,  and the mean radius <r> increases according to <r> µ exp(kt) , with a time constant rate k =  2 x 10-6 s-1. In this case, the fractal dimension   increases with <r>, i.e. as  the size of the system (colony) increases.