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- All Subjects: Synthetic Biology
- All Subjects: engineering
- All Subjects: Regenerative Medicine
- Creators: Wang, Xiao
- Member of: Barrett, The Honors College Thesis/Creative Project Collection
A variety of different genes have been associated with cell fate. For example, the Nanog/Oct-4/Sox2 network forms the core interaction of a gene network that maintains stem cell pluripotency, and Oct-4 and Sox2 also play a role in the tissue types that stem cells eventually differentiate into. Using the CRISPR/cas9 based homology independent targeted integration (HITI) method developed by Suzuki et al., we can integrate fluorescent tags behind genes with reasonable efficiency via the non-homologous end joining (NHEJ) DNA repair pathway. With human embryonic kidney (HEK) 293T cells, which can be transfected with high efficiencies, we aim to create a three-parameter reporter cell line with fluorescent tags for three different genes related to cell fate. This cell line would provide several advantages for the study of cell fate, including the ability to quantitatively measure cell state, observe expression heterogeneity among a population of genetically identical cells, and easily monitor fluctuations in expression patterns.
The project is partially complete at this time. This report discusses progress thus far, as well as the challenges faced and the future steps for completing the reporter line.
Regenerative medicine utilizes living cells as therapeutics to replace or repair damaged or diseased tissue, but the manufacturing processes to produce cell-based tissue products require customized biounit operations that do not currently exist as conventional biochemical and biopharma manufacturing processes. Living cells are constantly changing and reacting to their environment, which in the case of cells isolated from their hosts, are utilized as living bioreactor components that, by themselves, are manipulated to biomanufacturer selected tissue products. Therefore, specialized technology is required to assure that cellular products produce the phenotypical tissue characteristics that the final product is designated to have, while also maintaining sterility of the culture. Because of this, FDA guidelines encourage the use of Process Analytical Technology (PAT – see Ref ) to be integrated into manufacturing systems of biologics to ensure quality and safety. To address the need for evaluation of sensor technologies for potential use in PAT, a literature review of both existing sensing technologies and biomarkers was conducted. After a thorough assessment of the sensor technologies that were most applicable to biomanufacturing, spectrophotometry was selected to monitor the metabolic components glucose and lactate of living cells in culture in real time. Initially, spectrophotometric measurements were taken of mock solutions of glucose and lactate solutions at concentrations relevant to human cell culture and physiology. With that data, a mathematical model was developed to predict a solution’s glucose and lactate concentration. This model was then integrated into a Matlab program that was used to continuously monitor and estimate solutions of glucose and lactate concentrations in real time. After testing the accuracy of this program in different solutions, it was determined that calibration curves and models must be made for each media type and estimates of glucose and lactate were found accurate only at higher concentrations. This program was successfully utilized to monitor in real time glucose and lactate production and consumption trends of Mesenchymal Stem Cells (MSCs) in culture, demonstrating proof-of-concept of the proposed bioprocess monitoring schema.