Development of tissue engineered test systems to study mammary cell interactions in vitro.

摘要

The work described in this dissertation was conducted in the interdisciplinary research environment of the Clemson University Institute for Biological Interfaces of Engineering. A note at the beginning of each chapter acknowledges, as relevant, collaborating doctoral students and reminds the reader where work from each chapter has been presented or published. The overall goal of this work was to develop tissue engineered test system methodologies to allow the study of mammary cell interactions in vitro . The background, as described in Chapter 1, was published in part in Philosophical Transactions of the Royal Society A in 2010. The studies were designed to encompass both microfabrication technology as well as traditional 3D gel-based macrofabrication techniques, both of which will ultimately be necessary to design and fabricate biologically relevant 3D composite breast tissue cultures. The first step was to assess the effectiveness of microfabrication technology (a custom inkjet bioprinter) to eject cellular and acellular bio-inks into specified two-dimensional patterns on a variety of surfaces. Hence Chapter 2 addresses the overall project feasibility; studies are described wherein printing parameters are modified to identify optimal model conditions. These particular studies were designed to 1) evaluate the effect of stage height on cell viability, 2) identify the relationship between the rate of nozzle firing and the viscosity of a bio-ink, and 3) determine the accuracy of cell placement in a printed co-culture pattern. This work was presented at the 2008 Hilton Head Conference on Regenerative Medicine (stage height) and the 2009 Annual Conference of the Society For Biomaterials (nozzle firing). Chapter 3 addresses one of the major limitations of bioprinting, that of cartridge nozzle clogging, and evaluates the effectiveness of ethylenediaminetetraacetic acid as an anti-scalant and anti-aggregant in 2D high-throughput bioprinting. This work was published in 2009 in the Journal of Tissue Engineering and Regenerative Medicine. Furthermore, Chapters 4 and 5 describe the high resolution capability of the custom bioprinting system, which was demonstrated by 1) printing mono- and co-culture patterns and 2) applying thermal inkjet technology to stain histological samples and cell monolayers, which will be important in the future analysis of test systems. Work described in Chapter 4 was presented in part at the 2009 Annual Meeting and Exposition of the Society For Biomaterials and the 2009 IEEE Engineering in Medicine and Biology Society Conference, while work described in Chapter 5 was presented in part at the 2010 Annual Meeting and Exposition of the Society For Biomaterials. As a final bioprinting-based study, Chapter 6 describes the printing of high-resolution patterns of murine cells in 2D to evaluate paracrine signaling among adipocytes and cancer cells. To achieve this end, D1 and 4T1 cells were printed in co-culture patterns and the effect of 4T1 cells on the proliferation of D1 cells treated with an adipogenic cocktail was evaluated. This work was presented at the 2010 IEEE Engineering in Medicine and Biology Society Conference.;Before merging inkjet technology with traditional 3D gel-based culture techniques, 3D gels with incorporated 3D rigid substrates were developed to sustain anchorage dependent stromal cells in a breast tissue co-culture model. As described in Chapter 7, the differences in the activity of stromal cells (adipocytes) seeded on beads versus cells suspended in a gel were determined, as was the effect of adipocytes (seeded on beads and directly in a gel) on mammary epithelial cells. This work will provide a foundation on which tissue test systems with biologically relevant features may be built.;Chapter 8 presents work dedicated to education and outreach in tissue engineering. Specifically, a series of classroom teaching modules are presented that can be used to demonstrate basic tissue engineering concepts, such as the effect of the shape of a medical implant on surrounding tissue or the effect of scaffold surface texture on cell attachment. The long-term goal of this work will be to enhance science, technology, engineering, and mathematics education teaching methods and to enhance graduate student communication skills with a non-scientific audience.