On the Permanent Life of Tissue Outside the Organism
Open Access
- Author:
- Ravi, Dhurjati
- Graduate Program:
- Materials Science and Engineering
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- October 15, 2007
- Committee Members:
- Erwin A Vogler, Committee Chair/Co-Chair
Paul Wencil Brown, Committee Member
Ronald Hedden, Committee Member
Kumble Sandeep Prabhu, Committee Member
Andrea Marie Mastro, Committee Member - Keywords:
- bioreactors
bone tissue engineering
3D cell culture models
breast cancer metastasis - Abstract:
- Growth and maintenance of cells/tissue outside the body is central to the practice of biomedical sciences and technology. In vitro culture techniques, developed almost a century ago, are fundamental enabling tools for the study of life processes and are routinely employed in many applications that impact human health care. Emergence of the field of tissue engineering, focused on developing tissue surrogates for transplantation, has renewed the emphasis on critically examining the conditions under which we culture cells/tissue outside the body. It has been well recognized that isolation of cells from the complexity of their native physiological environment results in adaptive responses that often limit cell viability and function in vitro. Bridging the gap between physiological complexity and in vitro culture environments is critical to the successful implementation of the tissue engineering strategy. We show herein that a compartmentalized bioreactor based on the principle of continuous-growth–and-dialysis provides stable culture conditions that better simulate the physiological environment. The bioreactor was used to grow mineralizing, collagenous bone tissue up to 150 thick from an inoculum of isolated murine (mouse calvaria MC3T3-E1, ATCC CRL-2593) or human (hFOB 1.19 ATCC CRL-11372) osteoblasts over uninterrupted culture periods up to a year. Proliferation and phenotypic progression of an osteogenic-cell monolayer into a tissue comprised of cell layers of mature osteoblasts in the bioreactor was compared to cell performance in conventional tissue-culture polystyrene (TCPS) controls. Cells in the bioreactor basically matched results obtained in TCPS over a 15 d culture interval, but loss of insoluble ECM (iECM) and ~2X increase in apoptosis rates in TCPS after 30 d indicated progressive instability of cultures maintained in TCPS with periodic refeeding but without subculture. By contrast, stable cultures were maintained in the bioreactor for up to a year, suggesting that extended-term tissue maintenance is feasible with little-or-no special technique. Month’s long culture interval in the bioreactor lead to progression of pre-osteoblasts to osteocyte-like cells embedded in mineralized matrix observed in normal bone and production of visually-apparent (macroscopic) bone. Challenging bioreactor-derived bone tissue at different stages of development with metastatic breast cancer cells (MDA-MB-231) known to invade the skeleton created a system-in-crisis that captured early stages breast cancer colonization of bone. In situ confocal microscopy revealed sequential steps of breast cancer cell adhesion, penetration, and degradation of the 3D bone-like osteoblast tissue over co-culture intervals ranging from 3 to 10 days. Bioreactor enabled direct observation of interactions of breast cancer cells with engineered 3D bone like tissue and simulated an important step in the progression of the disease that may be a target for therapeutic intervention. Using bone as a model tissue, this study demonstrates that complex physiological and pathological processes of great importance to human health can be simulated using engineered cellular environments for potential applications in drug discovery and toxicology.