Electronic Thesis and Dissertation Repository

Thesis Format

Monograph

Degree

Master of Engineering Science

Program

Biomedical Engineering

Collaborative Specialization

Musculoskeletal Health Research

Supervisor

Flynn, Lauren

Abstract

The cell microenvironment plays a critical role in modulating adipose-derived stromal cell (ASC) proliferation and paracrine function. The current study investigated the effects of decellularized adipose tissue (DAT) coatings, low-level oscillatory shear stress (~0.04‑0.3 dyn/cm2), hypoxia (2% O2), and pro-inflammatory cytokine priming with IFN-g and TNF-a on human ASC proliferation and paracrine factor secretion in the context of a rocking bioreactor. Culturing under 20% O2 resulted in a higher cell density after 7 days of culture. Without cytokine priming, the varying culture conditions significantly impacted the levels of the pro-angiogenic factors VEGF, HGF, and angiogenin detected in conditioned media samples. In contrast, when the cells were primed, the levels of the immunomodulatory factors IL-6 and IL-8 were most affected by the varying microenvironmental factors. Overall, a novel bioreactor system was developed for ASC expansion and preconditioning, demonstrating that the cell microenvironment could be tuned to modulate ASC paracrine factor secretion.

Summary for Lay Audience

Adipose tissue or fat is found throughout the body and has important structural and functional roles, including defining the normal body contours. Adipose tissue loss due to disease, trauma, or resection of tumors can lead to contour defects, impaired function, and decreased emotional well-being of the patient. Therefore, adipose tissue reconstruction is usually considered in cases of large adipose tissue defects. However, conventional reconstruction therapies used to restore adipose tissue after damage generally only provide temporary solutions that lack long-term stability. To support functional regeneration, tissue engineering strategies utilizing pro-regenerative cells that can be isolated from fat, termed adipose-derived stromal cells (ASCs), have gained significant interest. Promisingly, ASCs are abundant within fat and have the ability to secrete therapeutic signalling molecules that can promote blood vessel growth and modulate the inflammatory response to stimulate healing. With the goal of developing a clinically-applicable strategy for ASC expansion that augments their therapeutic properties, efforts have been made to assess how the environment in which the ASCs are cultured in the lab influences their pro-regenerative functionality. One strategy is to culture the cells on decellularized adipose tissue (DAT) scaffolds that mimic the composition of their native environment within the body. Other factors, such as mechanical stimulation, low oxygen concentration, and the addition of inflammation-triggering molecules, have also been shown to regulate ASC therapeutic properties. This study aimed to develop a rocking bioreactor system that enabled the systematic investigation of these factors and evaluated their effects on human ASC expansion and therapeutic signalling molecule secretion. In general, cell growth was well supported under all conditions studied, with a higher cell density observed at 7 days in the samples cultured under 20% O2. Without the addition of inflammation triggering molecules, the different culture environmental factors had the greatest effects on the secretion of signalling molecules that can promote blood vessel growth. In contrast, with the addition of inflammation-triggering molecules, the different culture environmental factors had the greatest effects on the secretion of signalling molecules that can modulate inflammation. Overall, this study showcases the complex relationships between the culture environment and the therapeutic properties of ASCs, and developed a novel rocking bioreactor system that may hold potential for augmenting the pro-regenerative functionality of ASCs for future clinical applications in cell-based therapies.

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