Real time in situ microscopy for animal cell-concentration monitoring during high density culture in bioreactor.

An in situ microscope (ISM) device is utilised in this study to monitor hybridoma cells concentration in a stirred bioreactor. It generates images by using pulsed illumination of the liquid broth synchronised with the camera frame generation to avoid blur from the cell's motion. An appropriate image processing isolates the sharp objects from the blurred ones that are far from the focal plane. As image processing involves several parameters, this paper focuses on the robustness of the results of the cells counting. This stage determines the applicability of the measuring device and has seldom been tackled in the presentations of ISM devices. Calibration is secondly performed for assessing the cell-concentration from the cell automated numeration provided by the ISM. Flow cytometry and hemacytometer chamber were used as reference analytical methods. These measures and the output of the image processing allow estimating a single calibration parameter: the reference volume per image equal to 1.08 x 10(-6) mL. In these conditions, the correlation coefficient between both reference and ISM data sets becomes equal to 0.99. A saturation of this system during an ultrasonic wave perfusion phase that deeply changes the culture conditions is observed and discussed. Principal component analysis (PCA) is used to undergo the robustness study and the ISM calibration step.