A Multiparameter Approach to Separation and Clonal Analysis of Mammalian Cells
Amaya, Peter. "A Multiparameter Approach to Separation and Clonal Analysis of Mammalian Cells." PhD diss., The Ohio State University, 2017.
Mammalian cell bioprocessing is a critical part of both diagnostic and therapeutic applications. In terms of diagnostics, blood based biomarkers can be isolated from the peripheral blood and analyzed to help clinicians make informed decisions regarding treatment. Three blood based biomarkers that have gained a lot of attention recently are circulating tumor cells (CTCs), circulating tumor DNA (ctDNA) and extracellular vesicles (EVs). Viable CTCs are able to provide information about living tumor cells in circulation and how they survive, while ctDNA is able to provide information about tumor cells that are apoptotic, maybe due to treatment. EVs are produced by living cells and found in several types of bodily fluids including urine, making it a promising diagnostic.
Refinement of a negative immunomagnetic depletion approach to CTC separation revealed a population of cells known as efferocytes that can masquerade as CTCs in the peripheral blood. That is, they can appear to be negative for CD45, positive for cytokeratins (CK) and positive for vimentin a profile that would be expected of a CTC that has undergone epithelial to mesenchymal transition (EMT). However, flow cytometric analysis revealed that these cells are a combination of neutrophils and monocytes suggesting that CK positivity is not a random artifact, but rather the result of phagocytosis. Efferocytes are responsible for cleaning up apoptotic tumor cells in the peripheral blood and tumor tissue and have clinical significance as they consist of M2 polarized monocytes (macrophages) that are associated with EMT, angiogenesis and tumor progression.
EVs show potential as a delivery vehicle for nucleic acids. However, their manufacture and scale-up is not nearly as well established as production of monoclonal antibodies from Chinese Hamster Ovary (CHO) cells. Using similar approaches to CHO, an upstream bioprocess for EV production from HEK293T cells is presented consisting of cell engineering, clone selection, scale-up and scale-down. The clone selection and scale-down approaches involved growing three dimensional engineered HEK293T colonies on electrospun nanofibers while scale-up involved growing engineered HEK293T on microcarriers in a stirred tank environment.
Microcarriers can also be used to grow human mesenchymal stem cells (hMSCs) for transplant; however, protocols for their scale-up and manufacturing are not well established and there is concern that hMSCs will be damaged when manufactured according to processes developed for commonly used cell lines (e.g. vero cells). Shear sensitivity analysis of hMSCs using a contractional flow device suggested that they are just as robust as vero cells provided they are not extensively passaged. Therefore, hMSCs should be scaled-up on microcarriers with high attachment efficiency and consistent transfer to fresh microcarriers to avoid extensive amounts of cell doublings. Keeping the number of cell doublings low may have greater implications when considering the sublethal effects of hMSC senescence.