Label free assessment of live cell quality with total internal reflection microscopy

Summary form only given. Cellular feedstocks underpin many Regenerative Medicine (RM) therapies. Suitable strategies for the large-scale manufacture are therefore required to ensure high quality RM products are produced consistently at an economically acceptable price. Allied to this is the need for measurement tools capable of characterising cell quality and its dependence on key manufacture parameters in-process. Currently, cell populations are routinely monitored to assess quality using conventional biological analysis (e.g. cell surface markers, gene expression). This approach is destructive, not suitable for in-process measurements and renders time course experiments impossible. Alternatively non-destructive approaches that assess cell morphology can also used, with light microscopy techniques (e.g. bright field, phase contrast imaging) being the primary methods. Often these microscopy techniques are combined with pre-treatment of cells with exogenous labels such as fluorescent markers. This can provide functional information but has the disadvantage that such cell modifications are invasive and potentially toxic to the cells. Label free approaches are also used and whilst this enables non-invasive monitoring of live cells in culture, such microscopy techniques are currently non-quantitative with characterisation fully dependent on the skills and experience of the operator. Image contrast and resolution are also often lacking making morphological assessments unreliable. Additionally, more complex parameters such as dynamic cell behaviour and cell-substrate interactions are needed to provide the necessary mechanistic insight to characterise cellular processes and as parameters for effective process design and quality control tools. This presentation will address the above issues through the development of a total internal reflection microscope (TIRM) to enable the quantitative study of cellular processes and live cell quality at high resolution and without the - se of labels. TIRM is a non-fluorescent imaging technique which is based on the principle that an object with refractive index (n₃) will scatter an evanescent field created when a light beam undergoes total internal reflection at an interface between two media with different refractive indices, such as glass (n1) and air (n₂), where n₃>;n₂. The key design considerations with respect to development of a TIRM instrument are discussed. In addition the application of TIRM as an optical imaging tool to non-invasively monitor the quality of cells in culture at higher resolution than traditional light microscopy (e.g. bright field and phase contrast imaging) to enable validation of manufacturing procedures in-process will be discussed.