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Development of 3-D Microbioreactor Systems for Cell-Based High Throughput Screening

Zang, Ru
http://rave.ohiolink.edu/etdc/view?acc_num=osu1338338425

Abstract Details

2012, Doctor of Philosophy, Ohio State University, Chemical and Biomolecular Engineering.
Drug screening is a long and costly process confronted with low productivity and challenges in using animals. 3-D cell-based high-throughput platforms have been developed to improve drug-screening efficiency and minimize animal testing. However, online monitoring of cell proliferation, pH, and dissolved oxygen (DO) has been a challenge in 3-D cell-based assays. In this work, a 40 micro-well plate bioreactor (40-MBR) system was developed as a high-throughput platform for 3-D cell cultures. This 40-MBR has similar dimensions of a 384-well plate (384-MWP) can provide real-time and noninvasive monitoring of cell proliferation, pH, and DO in 3-D microenvironments. A colon cancer cell line expressing enhanced green fluorescent protein (EGFP) under the control of a constitutive CMV promoter was tested with two potential cancer drugs using the 40-MBR and 384-MWP. Compared to the 384-MWP, the 40-MBR gave more reliable and highly reproducible growth kinetic data with reduced experimental errors. This study demonstrated the potential application of the 40-MBR as a high-throughput platform for screening potential cancer drugs and evaluating their cytotoxic effects in the early-stage drug discovery. Cytotoxicity and embryotoxicity of chemicals were also investigated in the 40-MBR using EGFP-expressing embryonic stem cells (ESCs). Embryonic stem cell test (EST) has been used as an in vitro model for assessing embryotoxicity. However, the current EST can only provide end-point data and cannot predict embryotoxicity of chemicals affecting organs such as bone. In this study, a novel high-throughput embryotoxicity assay was developed using EGFP-expressing ESCs under the control of a survivin promoter. Survivin expression is closely associated with embryo development and cell differentiation. For control, ESCs expressing EGFP under the control of a CMV promoter were used to monitor cytotoxicity of chemicals. Using survivin as a diagnostic marker for predicting embryotoxicity was first tested and validated with various chemicals with known developmental toxicity. Then, the potential embryotoxicity of three Chinese herbal medicines, Ginkgo biloba (GB), Ganoderma lucidum spore (GLS), and Epimedium brevicornum (EB), was tested. The results showed that the survivin-based assay has great potential in screening compounds with different degrees of embryotoxicity. Compared to conventional 2-D cultures, cells cultured in 3-D scaffolds generally can better recapitulate in vivo cellular responses to drugs. However, the next-generation 3-D scaffolds should also provide tunable ‘surface’ properties similar to those found in native microenvironments. Recently, carbon nanotubes (CNTs) have been used in several tissue-engineering studies for their unique physical properties at the nanoscale. The potential of CNTs in promoting in vitro differentiation of ESCs into neuronal cells was investigated in both 2-D and 3-D cultures. Using polyethylene terephthalate (PET) membranes as the base substrate, coating the PET surface with CNTs promoted neuronal differentiation, exhibiting a high degree of neurites extension and outgrowth while maintaining good cell viability. Similarly, ESCs cultured in 3-D nonwoven fibrous PET matrices coated with CNTs showed enhanced cellular functions, including proliferation, morphology and differentiation, compared to cells cultured in the uncoated matrices. With increased biological relevance, cells cultured in 3-D PET matrices coated with CNTs can provide more representative cellular responses to drugs.
Shang-Tian Yang (Advisor)
Jeffery Chalmers (Committee Member)
Andre Palmer (Committee Member)
Chemical Engineering
microbioreactor; dissolved oxygen; cell proliferation; three-dimensional; cytotoxicity; embryotoxicity; developmental toxicity; survivin; green fluorescence protein; carbon nanotubes; embryonic stem cells

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Citations

  • Zang, R. (2012). Development of 3-D Microbioreactor Systems for Cell-Based High Throughput Screening [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1338338425

    APA Style (7th edition)

  • Zang, Ru. Development of 3-D Microbioreactor Systems for Cell-Based High Throughput Screening. 2012. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1338338425.

    MLA Style (8th edition)

  • Zang, Ru. "Development of 3-D Microbioreactor Systems for Cell-Based High Throughput Screening." Doctoral dissertation, Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1338338425

    Chicago Manual of Style (17th edition)

osu1338338425
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© 2012, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.