Date of Graduation

5-2024

Document Type

Thesis

Degree Name

Bachelor of Science in Chemistry

Degree Level

Undergraduate

Department

Chemistry & Biochemistry

Advisor/Mentor

Julie A. Stenken

Committee Member

Neil T. Allison

Second Committee Member

Kate M. Chapman

Third Committee Member

Andrew J. Dowdle

Abstract

Co-cultures are utilized as a tool in biology to measure immune responses to pathogens. This method involves a coordinated reaction between two populations, a bacterial biofilm of S.aureus and macrophages derived from THP-1 cells. The current state of the art prevents the continuous supply of nutrients and removal of waste products which causes macrophage death. This limits the time to collect samples and therefore the ability to measure proteins within the innate immune response. The time constraint is a major issue for long term co-culturing. This thesis aims to optimize the co-culturing process by developing a two-pump system to provide a constant flow of nutrients to the macrophages and eliminate the metabolic waste to a sampling port. A dual flow model can regulate key components within the co-culture environment, including the pH. Over a 108-hour period, the pH of a culture of macrophages without the pump system was measured to be a pH of 7.27 and decreased to 6.21, at which point 76.405% of the macrophages within the culture were found dead. Based on this data, a flow rate of 50 steps/sec was chosen because it would be able to filter 3 mL of fluid over a 48-hour period. The quantitative data for 50 steps/sec measured to be 0.961 ± 0.3 𝜇L/min , n=3, and 1.09 ± 0.1 𝜇L/min, n=3, for pump 1 and 2, respectively. At this rate the pump system was able to modulate the culture medium pH of the macrophages to an average of 7.49 ± 0.1, n=7, pH units over a 72-hour period. A baseline of pH and nitric oxide (NO), a biocidal chemical emitted by the macrophages, was obtained for a co-culture of a S. aureus biofilm and macrophages. The pH decreased from a pH of 7.31 to 6.46 over 48 hours. The nitrite concentration, an indirect measurement for NO, increased from 14.5 µM to 124.3 µM over 48 hours. A two-peristaltic pump model can be used for culture work, as an increase macrophage viability was observed through pH regulation; and therefore, NO levels should increase when using the pump system.

Keywords

Macrophage; Bacterial Biofilms; S. aureus; Healthcare-associated infections

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