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High Throughput 3D Hydrogel Cell and Tissue Encapsulation Assay to Measure Matrix Metalloproteinase and Metabolic Activity

Fakhouri, Abdulaziz Saud W.
http://orcid.org/0000-0002-9856-3412
http://rave.ohiolink.edu/etdc/view?acc_num=osu1555518371350346

Abstract Details

2019, Doctor of Philosophy, Ohio State University, Biomedical Engineering.
Currently, most assays for cancer chemotherapeutic screening and development utilize two dimensional (2D) culturing systems. These 2D systems lack aspects of the in vivo microenvironment creating a poor in vitro representation that significantly affects cellular responses. To recapitulate the in vivo cellular microenvironment more closely, three dimensional (3D) cell culture systems have been developed and utilized. However, 3D cell culture systems are more complex, making analysis of cellular responses more difficult. Therefore, most high throughput (HT) 3D assays have been limited to measurements of cell viability. Yet other cellular functions play critical role in diseases and are promising pharmacological targets. There is a need for a HT 3D culturing system that enables the measurement of cellular functions, other than viability, for drug testing applications. To address this need, we developed, characterized, validated and demonstrated the utility of a HT 3D culturing system to measure matrix metalloproteinase (MMP) and metabolic activity, simultaneously. MMPs are critical regulators of tissue homeostasis and are upregulated in many diseases, such as arthritis and cancer. The developed assay produced edge effect, drift, Z’-factor, %CV, inter-plate, and inter-day fold shifts of the signals that were within the acceptable range for HT applications, designating it suitable for screening applications. Human MMP-1, -2 and -9 resulted in a significant increase in signal intensity. Encapsulation of several cell types, utilizing two different MMP-degradable peptides, produced robust signals above background noise and within the linear range of the assay. Finally, the utility of the system to measure cellular MMP activity in response to chemotherapeutics was demonstrated. Fibrosarcoma cell line (HT1080) was treated with several drugs, known to alter MMP activity, over a range of concentrations. Interestingly, sorafenib (SOR), a small molecule multi-kinase inhibitor utilized in clinical trials, increased MMP activity in a dose-dependent. This assay combines 3D cellular encapsulation and MMP activity detection in a HT format, which makes it suitable for drug screening and development applications. Historically, immortalized cancer cell lines have been used for the vast majority of in vitro cancer studies due to their unlimited proliferation and ease of use. However, cell lines lack the tumor heterogeneity and native microenvironment encountered in vivo, factors that also affect cellular responses. To more closely approximate the heterogeneity observed with in vivo tumor cells, human tissue samples have been used in patient derived xenografts (PDX) and tumor organotypic slice culture models. However, PDX and organotypic slice culture models are unsuitable for HT applications because they are expensive and have long turnaround time to produce results. A simple and efficient system that enables culturing human tissue is needed. To address this need, we developed a 3D ex vivo culture system which maintained viability of human tissue samples derived from patients undergoing surgeries. Moreover, MMP and metabolic activity were measured in tissue size dependent manner. After further characterization, we envision that this system will enable drug screening utilizing human tissue samples for drug development and personalized medicine applications.
Jennifer Leight, Ph.D. (Advisor)
Jessica Winter, Ph.D. (Committee Member)
Keith Gooch, Ph.D. (Committee Member)
178 p.
Biomedical Engineering
Biosensors; Fluorescence; Biomaterials; Hydrogels; 3D Cell Encapsulation; High Throughput Assays; Assay Development; Matrix Metalloproteinases; Metabolic Activity

Recommended Citations

Citations

  • Fakhouri, A. S. W. (2019). High Throughput 3D Hydrogel Cell and Tissue Encapsulation Assay to Measure Matrix Metalloproteinase and Metabolic Activity [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1555518371350346

    APA Style (7th edition)

  • Fakhouri, Abdulaziz. High Throughput 3D Hydrogel Cell and Tissue Encapsulation Assay to Measure Matrix Metalloproteinase and Metabolic Activity. 2019. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1555518371350346.

    MLA Style (8th edition)

  • Fakhouri, Abdulaziz. "High Throughput 3D Hydrogel Cell and Tissue Encapsulation Assay to Measure Matrix Metalloproteinase and Metabolic Activity." Doctoral dissertation, Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1555518371350346

    Chicago Manual of Style (17th edition)

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This open access ETD is published by The Ohio State University and OhioLINK.