Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Biomedical Engineering

Collaborative Specialization

Musculoskeletal Health Research

Supervisor

Lauren, Flynn E.

Abstract

Recognizing the cell-instructive capacity of tissue-specific extracellular matrix (ECM) to direct cell attachment, proliferation and differentiation, there is a need for the development of in vitro cell culture models that reflect the complexity of the ECM to improve stem/progenitor cell expansion and lineage-commitment. This thesis focused on the development and characterization of ECM-derived microcarriers for the in vitro dynamic culture and expansion of stromal cells for cell therapy and tissue engineering applications.

To develop novel platforms for use in dynamic culture systems, initial work focused on applying electrospraying techniques to fabricate microcarriers from decellularized dermal tissue (DDT) and decellularized myocardial tissue (DMT) and compare their properties to our previously-established decellularized adipose tissue (DAT) microcarriers. The soft and compliant microcarriers comprised exclusively of ECM were stable over a range of concentrations without the need for chemical crosslinking. The microcarriers were well tolerated in vivo and supported the growth of tissue-specific stromal cell populations within spinner flasks.

Recognizing the multilineage differentiation capacity of human adipose-derived stromal cells (hASCs), as well as the potential for the ECM to provide cell-instructive cues that can direct ASC differentiation, the effects of expanding hASCs on microcarriers derived from DAT versus decellularized cartilage tissue (DCT) were explored. More specifically, novel DCT microcarriers were fabricated and characterized. Both platforms supported hASC attachment and growth over 2 weeks in spinner flasks under proliferation conditions, with PCR array and global protein analyses suggesting that the DCT microcarriers may have predisposed the cells towards the chondrogenic lineage.

Building on previous work, the final goal was to investigate the adipogenic differentiation of hASCs cultured directly on DAT or DCT microcarriers within spinner flasks. Recognizing that both the tissue-specific ECM composition and the dynamic culture conditions may impact hASC differentiation, the hASC response was compared under varying stirring rates (20 versus 40 rpm) and oxygen tensions (~20 % versus 2 % O2). While adipogenic gene expression was not affected by the dynamic culture conditions, analysis of glycerol‑3‑phosphate dehydrogenase (GPDH) enzyme activity and intracellular lipid accumulation supported that hASC adipogenesis was enhanced on the DAT microcarriers cultured under ~20 % O2 and 20 rpm.

Summary for Lay Audience

There has been increasing interest in using mesenchymal stromal cells (MSCs) for cell therapy applications for a number of diseases and tissue regeneration strategies. The most common MSC population can be obtained from the bone marrow but obtaining these cells can be a painful process for patients and the frequency of these cells is low. Adipose tissue (fat) provides an abundant and accessible alternative source of MSCs called adipose-derived stromal cells (ASCs) that have the potential to differentiate into mature fat, cartilage and bone cells.

In order to use these cells in human clinical therapies, it is estimated that million to billions of cells will be needed per treatment, with the potential for multiple treatments required. This presents a significant challenge in translating stem cell therapies because the conventional methods used for expanding MSCs diminish their pro-regenerative properties and reduce their capacity to differentiate. The goal of this project was to make novel platforms for expanding cells within stirred bioreactor systems as an alternative approach that may better preserve the regenerative capacity of the cells.

More specifically, proteins isolated from tissues discarded as surgical waste were used to generate three-dimensional (3D) microcarriers that can support cell attachment and growth, and potentially serve as injectable cell delivery platforms. Each tissue in our body has its own unique protein composition and emerging research suggests that this tissue-specific composition can direct the differentiation of MSC populations in culture. To explore this, the work in this thesis aimed to develop and characterize novel microcarriers using proteins isolated from fat, cartilage, heart, and skin, and explore their potential as platforms for cell expansion or differentiation within a stirred bioreactor system. Long-term, this project aims to develop a strategy to expand cell populations from small biopsies to obtain clinically‑relevant cell populations, and potentially use them as tissue-specific platforms to deliver regenerative cells for a wide range of applications.

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