Noninvasive measurement of cell/colony motion using image analysis methods to evaluate the proliferative capacity of oral keratinocytes as a tool for quality control in regenerative medicine

Abstract

Image-based cell/colony analyses offer promising solutions to compensate for the lack of quality control (QC) tools for noninvasive monitoring of cultured cells, a regulatory challenge in regenerative medicine. Here, the feasibility of two image analysis algorithms, optical flow and normalised cross-correlation, to noninvasively measure cell/colony motion in human primary oral keratinocytes for screening the proliferative capacity of cells in the early phases of cell culture were examined. We applied our software to movies converted from 96 consecutive time-lapse phase-contrast images of an oral keratinocyte culture. After segmenting the growing colonies, two indices were calculated based on each algorithm. The correlation between each index of the colonies and their proliferative capacity was evaluated. The software was able to assess cell/colony motion noninvasively, and each index reflected the observed cell kinetics. A positive linear correlation was found between cell/colony motion and proliferative capacity, indicating that both algorithms are potential tools for QC.

Keywords: Oral keratinocyte; cell/colony motion; image analysis; quality control; regenerative medicine.

Figure 1.

Basic principles of the OF algorithm used to calculate the MS: (a) A representative distribution of the displacement vectors calculated by OF analysis is shown for three extracted colonies, labelled 1, 2 and 3. All three colonies are used for explaining the following methods and results. The original video file is provided in Supplemental material 1 and 2 movies and (b) higher magnification of colony 2, surrounded by a red line in Figure 1(a). The motion speed of colony 2 was determined as the mean value of the magnitude of vectors with values greater than 1 within the region.

Figure 2.

Illustration of the template-matching procedure in the NCC. Basic principle of the NCC algorithm to calculate the normalised cross-correlation coefficient (ρ), which is finally converted into the DI.

Figure 3.

Illustration of the method of determining the proliferative capacity of the extracted colonies: (a) A representative area of the initial image of time-lapse observation, including three extracted colonies that are enclosed with individual boxes, labelled colonies 1, 2 and 3, delineating with lines coloured light green, red and blue. The number of cells is six in colony 1, six in colony 2, and two in colony 3 and (b) the entire image scanned after fixation of the culture dish, identical to Figure 3(a). After 48 h in culture, cells in all colonies proliferated and produced daughter cells, resulting in 13 cells in colony 1, 16 in colony 2, and 4 in colony 3.

Figure 4.

A step-by-step chart of the experimental design and procedures.

Figure 5.

Representative outcomes of MS and DI of colonies 1, 2 and 3 in the 96 frames: (a) Changes in MS of colonies 1, 2 and 3, as shown in Figures 3(a) and (b) during time-lapse microscopic observation; (b) changes of DI of colonies 1, 2 and 3, as shown in Figures 3(a) and (b) during time-lapse microscopic observation; and (c) changes in MS (green, red and blue solid line) are overlaid on those of DI (green, red and blue dotted line) of all three colonies, as shown in Figures 3(a) and (b).

Figure 6.

Correlation of the two indices of oral keratinocytes colonies with their proliferative capacity: (a) Scatterplot showing the correlation of MMS between individual colonies and their proliferative capacity (PD). The trend line is shown, and its equation and the correlation coefficient are shown. Pearson’s r = 0.398, p = 2.60E-10 and n = 234 colonies obtained from 13 individuals and (b) scatterplot showing the correlation of MDI between individual colonies and their proliferative capacity (PD). The trend line is shown, and its equation and the correlation coefficient are shown. Pearson’s r = 0.28, p = 0.002 and n = 120 colonies obtained from 11 individuals.