Date of Award

8-2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Bioengineering

Committee Member

Dr. Sarah Harcum, Committee Chair

Committee Member

Dr. Susan Chapman

Committee Member

Dr. Robert Latour

Committee Member

Dr. Dan Simionescu

Abstract

In the past several decades, advances in our understanding of cells that comprise our bodies have been remarkable. This is due in large part to the development of mammalian cell culture practices that allow researchers to mimic conditions in the body. Cell culture techniques have allowed researchers to study the behavior of cells under various circumstances that would be impossible to study in humans. While there are some drawbacks to the reductionism inherent in studying cells in a dish, many medical treatments have resulted from cell culture experiments. Additionally, many new therapeutics are only possible due to these improved cell culture techniques. To that end, this research has encompassed the full spectrum of cell culture techniques and applications. For example, DNA microarrays can be used to study glycosylation gene expression in mammalian cells, where glycosylation is an important quality attribute of recombinant human therapeutic proteins. Specifically, a method is described to conduct glycosylation experiments. Next, research is described where DNA microarrays were used to investigate the response to elevated ammonium levels for a mammalian cell line often used to produce human therapeutics, because it has been show that elevated ammonium decreases product quality for these recombinant therapeutics. So, understanding if gene expression controlled the response was important. It was determined, through DNA microarray and PCR analysis, that glycosylation genes in these cells are unaffected by the elevated ammonium levels commonly experienced in end-stage cultures. Therefore, it was concluded that the effects of ammonium on protein quality appear to be due to translational or substrate-level effects. This research transitioned from analyzing the behavior of NS0 cell cultures to analyzing the behavior of stem cell cultures. For NS0 cells, the recombinant proteins are the product of interest. For stem cells, the products are the actual cells, which are anticipated to serve as starting material for future cell-based regenerative therapies. For NS0 cells, the goal is to produce high quality protein products. For stem cells, the goal is to produce robust multipotent stem cells. To that end, we developed a novel method for buffering the pH of mesenchymal stem cell (MSCs) cultures, such that it might be easier to culture the MSCs in large volumes without requiring carbon dioxide sparging. Through the use of PCR and histological staining, it was demonstrated that MSCs were capable of growth in the absence of an elevated CO2 environment and differentiation into adipogenic, chondrogenic, and osteogenic lineages. These results provide a route for simplifying bioreactor control and paving the way for efficient, large-scale generation of high-quality multipotent stem cells. Just as elevated ammonium affects the protein quality for NS0 cells and pH governs the behavior of stem cells, metabolites within tumors affect the behavior of cancer cells. One such metabolite – lactate – accumulates in tumors and adversely affects patient outcomes. Interestingly, lactate accumulation is also a major challenge in therapeutic protein production and cell-based therapy cell production. Thus, gaining a better understanding of elevated extracellular lactate’s effects on the metabolic behavior of breast cancer cells was examined. Three breast cancer cell lines were used. A comprehensive technique called 13C-metabolic flux analysis (MFA) was used to estimate intracellular fluxes. All three cell lines has decreased glucose consumption and decreased lactate production under the elevated lactate compared to the control condition. Under the elevated lactate conditions, all three cell lines also consumed lactate, an effect seen in stage late cultures of cells used for recombinant protein production, which might represent a general cellular mechanism used by a variety of cell lines to deal with extracellular lactate accumulation. At the same time, all three cell lines increased reductive carboxylation in the TCA cycle under elevated lactate compared to control, and this effect has also been observed in cells – both normal and cancerous - under hypoxia. These results suggest a previously unreported link between hypoxia and lactate, which demands further investigation.

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