Microfluidic and microscale cell cultures for high-throughput cell-based assays and bioprocess development

Cells can be cultured in different formats, such as in three-dimensional (3D) tissue constructs, in free suspension, in microcarriers, or in attached monolayer. Microscale and microfluidic cell culture devices are potentially useful in future pharmaceutical development and biomedical applications. However, culturing cells at microscale or in a microfluidic device imposes unique challenges and deserves further investigations.;For high-throughput cytotoxicity studies of anticancer drugs, increasing evidence indicates that 3D cell cultures create superior in vitro models to conventional monolayer cultures in multi-well plates. Due to high cell density in 3D culture, mass transport is critical. Perfusion culture via microfluidic operation provides continuous nutrient inflow and waste removal, significantly improving mass transport. To make it even more advantageous, 3D perfusion microbioreactors may help create an in-vivo like hydrodynamic microenvironment. In this study, a perfusion microbioreactor array was designed and fabricated for 3D culture of human colon cancer cells in modular fibrous matrix. Proliferation was monitored non-invasively with auto-fluorescence from green fluorescent protein expressed by engineered cells. Dose dependent response of the 3D cell proliferation to 5-fluorouracil was observed. The modular design allowed direct post-culture access to intact tissue constructs for various analyses. The perfusion microbioreactor array is a platform technology, which has a potential to be used as an in-vitro cancer model and in high throughput drug screening device.;One of the challenges for microfluidic cell culture in an array was parallel flow control. Flow control in multiple hydrophobic microchannels with a common inlet was thus investigated. Based on a series of designs of perfusion microbioreactor arrays, theoretical analysis and experimental validation were developed. It was found that capillary pressure played a key role in parallel flow control in the startup phase. The theory and the experiments had an excellent match. A novel method of achieving parallel flow control at significantly reduced flowrates was developed by using fibrous matrices in the microchannels. This study provided a rational approach to achieving parallel flow control by design, material selection and operations.;Another high-throughput cell culture platform developed in this study was a 24-well microbioreactor array for suspension culture. A central static mixer was fabricated to effectively break poor mixing patterns in multi-well-plate-based cell cultures, minimizing cell aggregate formation. Furthermore, evaporation was reduced with non-polar gas permeable membranes for both the bottom and the lid. Therefore, cell culture data quality was improved with the microbioreactor array. In addition, its mixing time and maximum oxygen transfer rate were characterized with optical methods. With design of experiment using the microbioreactor array, active factors for serum-free media were identified for Chinese hamster ovary cells producing monoclonal antibody (MAb).;Microcarriers are useful in culturing anchorage-dependent cells in conventional stirred tank bioreactors. However, microcarrier cell cultures are very sensitive to shear stress and microcarrier collision in a stirred tank. Furthermore, high cost of commercial microcarriers also limits large scale use of this technology. In the perfusion microbioreactor array project, dynamic cell seeding onto small fibrous matrices was studied. Due to the similarity in operation, macroporous fibrous microcarriers were thus developed. In a comparative study, the fibrous microcarriers had comparable performances with several commercial microcarriers on cell attachment, cell growth and MAb production. Excellent protection of cells against high shear stress was achieved. In addition, the fibrous microcarriers provide over 1000-fold cost savings. These could be especially meaningful for large-scale microcarrier cell cultures, such as in viral vaccine production.