Preliminary Study towards the Use of In‐situ Microscopy for the Online Analysis of Microcarrier Cultivations

In order to apply newly developed non‐invasive in‐situ microscope systems for the monitoring of microcarrier‐based cultivations, appropriate image analysis systems must be available. Thus a simple, but effective greyscale distribution scan algorithm was tested for the evaluation of images generated by either a standard phase‐contrast microscope or an in‐situ microscope. The images were analyzed according to their greyscale pattern in order to examine whether the greyscale distribution is a possibility to gain information about the covering ratio. The mouse fibroblast cell line (NIH–3T3) was grown on different microcarrier spheres. At first, different microcarriers were tested with respect to their suitability for microscopic observation. In a second part, the phase‐contrast pictures and in‐situ microscope pictures of the microcarrier were separately analyzed using the histogram function of CorelPhotopaint, which analyzes the greyscale distribution within the chosen area. Due to the low optical density of the polydextrin matrix, the images of Cytodex 1 microcarriers proved to be an ideal model for the image analysis. Significant differences in the greyscale distribution of this microcarrier without cells and with increased cell density were observed. Therefore a relationship between the cell density on the microcarriers and the greyscale pattern can be assumed. After automating this image analysis and calibrating the cell number/greyscale pattern relationship, it should be possible to analyze the plating efficiency/covering ratio on the microcarrier online by in‐situ microscopy.