UBC Theses and Dissertations
Microfluidic cell culture arrays for clonal expansion and characterization of mammalian cells Lecault, Véronique
Single-cell culture provides a unique means to reveal the heterogeneity within mammalian cell populations. Advances in multilayer soft lithography have enabled the development of high-throughput nanoliter-volume cell culture platforms with integrated and programmable fluidic control to precisely modulate the microenvironment. Coupled with time-lapse imaging, these microfluidic systems allow hundreds of single cells to be monitored simultaneously while providing analytical advantages to characterize each clone. However, there are many challenges associated with the miniaturization of mammalian cell cultures and even greater difficulties for non-adherent cell types. This work shows how microfluidic devices and their control system can be designed to gently trap suspension cells and enable robust clonal expansion. Mouse hematopoietic stem cell (HSC) populations were chosen for their sensitivity and stringent cell culture requirements to demonstrate that normal cell growth and function could be sustained in the microfluidic system. Using microfluidic clonal analysis and image processing it was observed that cells from HSC-enriched populations had highly heterogeneous growth profiles. Automated medium exchange and temporal stimulation were then exploited to show that a high Steel factor (SF) concentration was needed for survival of primary HSCs specifically at the time of exit from quiescence. The ability to perform live immunostaining was combined with genealogical tracing to identify distinct characteristics, such as long cell cycle times and frequent asynchrony of daughter cells, associated with HSC clones exhibiting persistent endothelial protein C receptor expression (EPCR) after in vitro culture. Finally, the flexibility of this microfluidic system was demonstrated with the culture of Chinese hamster ovary (CHO) cells, the most widely used suspension-adapted mammalian cell type for the production of therapeutic recombinant proteins. In this system, the high cell density and the rapid concentration of cell-secreted products in nanoliter-volume chambers were exploited to measure the amount of secreted monoclonal antibodies from single cells and to increase their cloning efficiency. The ability to recover clones from the microfluidic system has allowed the selection and expansion of high-producing cell lines. This thesis demonstrates the potential and adaptability of high-throughput microfluidic single-cell culture systems for both research and therapeutic applications.
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