Ryota Umegaki

Evaluation of attachment and growth of anchorage-dependent cells on culture surfaces with type I collagen coating.

The effects of coating the culture surface with bovine type I collagen on the culture properties of anchorage-dependent cells were investigated. When human fibroblasts were cultured on a surface coated with collagen at 5.8 x 10(-3) mg/cm2, cell attachment and subsequent cell growth were both enhanced compared to the culture on an uncoated surface. The degrees of cell attachment and growth enhancement were numerically characterized using the time constant of cell adhesion (tau) and doubling time (t(d)) as kinetic parameters. These parameters applied to cultures of human keratinocytes and rabbit chondrocytes allowed the effects of collagen coating on the respective culture properties of both types of cells to be evaluated. In addition, the relative parameters R(tau) and R(t(d)) (defined as the ratios of the tau and t(d) values at a given collagen concentration against those without collagen coating, respectively) were employed to estimate the effects of collagen based on a standardized criterion. Similar R(tau) and R(t(d)) profiles were obtained for collagen concentrations ranging from 5.8 x 10(-13) to 5.8 x 10(-3) mg/cm2, whether the cells were fibroblasts, keratinocytes or chondrocytes. It was also revealed that coating the surface with collagen at a concentration over 5.8 x 10(-7) mg/cm2 led to reductions in both the R(tau) and R(t(d)) values, i.e. the promotion of cell attachment and growth, in the culture of each type of cells examined.

Valuation of growth parameters in monolayer keratinocyte cultures based on a two-dimensional cell placement model.

The influence of inoculum size on the growth of keratinocyte cells was investigated in a monolayer culture with serum-free medium. A growth model of cell placement was applied to the expression of the cell adhesion phase after the inoculation, lag phase, exponential growth phase, and stationary phase because of contact inhibition at high cell densities. Based on the model, the lag time until the onset of cell division was shortened in proportion to the logarithm of the inoculum cell size, resulting in the enhancement of overall cell propagation. It was verified that the proposed model is valid for the determination of the optimal inoculum size to realize the efficient growth of keratinocytes, indicating that the model is a useful tool to predict an optimal culture scheme for the production of skin grafts.

Correlation of cellular life span with growth parameters observed in successive cultures of human keratinocytes

In serial cultures of keratinocytes isolated from human neonatal foreskin, the potential for cell attachment and cell division decreased with an increase in the number of culture passages. The specific growth rate of the cells (mu) reduced gradually with elapsed culture time and an appreciable drop in the rate was observed below mu=0.02 h(-1) owing to cellular senescence. Then, a critical point in the serial cultures was determined as the last data point keeping the mu value at more than 0.02 h(-1). In serial cultures of keratinocytes isolated from the breast skin of 22 and 70-year-old individuals, the time profiles of the mu value displayed critical points, as observed in the neonatal foreskin cultures. Plots of the mu value and average cell area against the remaining numbers of population doublings (N(dF)/t-N(d)) were found to give a relationship which overlapped irrespective of the three kinds of keratinocytes, and the propensity for a depression of growth potential [estimated as a ratio of differential value of mu to that of (N(dF)-N(d))] was much higher after the critical point than before. In addition, a data map in terms of the specific growth rate and average cell area, which could be classified into proliferative, senescent and transitional phases, was proposed for the discrimination of cellular senescence in serial cultures for the production of epithelial sheets.

Characterization of cellular motions through direct observation of individual cells at early stage in anchorage-dependent culture.

In the monolayer culture of murine fibroblasts, the characterization of cellular motions was performed by morphological observation using a tool consisting of an optical assembly connected to a computer-aided image analysis system. This tool enabled the estimation of the projected area of the cells with sufficient accuracy, being able to follow the serial behavior of the cells on the culture surface. Trypsinization was chosen as an external factor for altering the state of the cells, and the rate of cell spreading (r(S)) and time of first cell division (t1) were evaluated by subjecting the cells to trypsin treatment for t(T)=3 and 15 min. During culture of the cells treated for t(T)=3 min, the r(S) values of individual cells exhibited a broad variation, ranging from 41 to 321 microm2/h, and the average r(S) was 165+/-78 microm2/h , which was 1.5 times larger than that of the cells treated for t(T)=15 min. On the contrary, the average t1 of the cells treated for t(T)=3 min was 9.5 h which was 60% reduced as compared with that of the cells treated for t (T)=15 min. The prolonged time of trypsin treatment was considered to induce the decrease in r(S) and delay of the first cell division due to the requirement for the recovery from cell surface damage caused by trypsinization. The logarithmic plots of r(S) and t1 were found to have an inverse relation regardless of the state of the individual cells.

Characterization of Growth Properties through Morphological Observation in Culture of Anchorage-Dependent Cells

The focus on the end point of the sequence of intracellular reactions, namely, the behavioral phenotypes has been dedicated to emerging technologies for characterizing motility-related parameters. In the present study, the cell attachment and spreading on culture surface during the adhesion phase after inoculation were examined and a morphological rate parameter was proposed for the evaluation of growth potential during the culture of mouse fibroblast cells (3T3) using the tool developed for the observation of cell morphology. To ensure the validity of the tool, the adherent area of an individual cell was measured during the culture. After inoculation, it was observed that cells attached and then spread on the bottom surface of T-flask. The adherent areas of cells on the surface, which were determined using our system, expanded with elapsed time, approaching to a saturated value of the area. After that, cell division occurred with a sharp decrease in the area. In addition, re-spreading of the individual cell was observed, indicating that our system was available for measurement of the cell spreading and cell division. By using the system, the variation in adherent area of an individual cell was investigated with the cells that underwent after the treatment for cell detachment with trypsin solution for given periods. The excessive treatment resulted in a decrease in spreading rate of cells. Moreover, the time of first cell division decreased with increasing spreading rate, suggesting that the spreading rate was an available parameter to evaluate the growth potential.