Cell properties have been extensively examined in two-dimensions (2D), yielding fruitful information regarding focal adhesions, migration, the establishment of polarity, traction forces and mechanotransductive responses. However, with few exceptions, most cells in vivo exist in a threedimensional (3D) environment, where their behavior is considerably different compared to when they are plated on a petri dish. Not only are there significant geometric differences, but there are also omnidirectional cell-cell interactions as well as cell-matrix interactions. As disease and tissue engineering research advance, it is imperative that cellular behavior and properties are established in detail. However, achieving this goal has been slow, in a large part because imaging and otherwise probing cells embedded within other cells and/or matrices is difficult due to scattering and inaccessibility. Thus there is a need for transforming techniques to derive information from three-dimensional cellular constructs.
The scientific objectives of this proposal are (1) to design and implement a microrheometer to characterize cellular properties that can remove out-of-plane noise and perform three-dimensional tracking, (2) to use the microrheometer to assess changes in cell rheological properties under resting conditions as well as under mechanical stimuli, in a variety of 2D and 3D environments, and (3) to model 2D and 3D rheological changes using a novel 9-parameter computational model based on a Monte Carlo simulation which will be used to interpret the results from the experimental parts of the project. Preliminary work demonstrates that cell behavior in layered 2D arrangements are already more complex than simple 2D plating can predict. These results further demonstrate the effectiveness of new techniques for acquiring microrheological measurements in a variety of conditions. The next logical step is to deploy more advanced techniques to 3D, and to carefully examine cell behavior and response in more realistic environments, and to create computational models to help interpret the results. Thus, this project will provide the groundwork for years of research in basic cell biomechanics, tissue engineering, as well as imaging techniques that may rely on these measurements for diagnoses or treatments.
Intellectual Merit: The driving goal of this project is to characterize cells in threedimensional environments by designing advanced imaging setups, coupled with computational models to help interpret the results. Because 3D is the next major area of cell-based research, and because tools for examining biomechanics and mechanotransduction are currently lacking for 3D experimentation, the work proposed here will help advance the field of biomechanics, tissue engineering and imaging. Ultimately, a vastly improved and more realistic model of cell behavior in vivo will be established and better models of cell regulation and physiology can be created.
The overall merit of this work lies in the ability to precisely characterize cell properties under realistic but controlled conditions, where one can vary the extracellular matrix constituents, the mechanical stresses acting on the cells, and the degree of cell-cell interactions. Thus, these results can be transformative for the field of cell mechanics and mechanotransduction, which themselves are relevant for a broad range of other fields. Because of the fast development of 3D tissue-engineered constructs and the need for characterizing cell properties, this proposal seeks to fill a large gap in current knowledge. For example, is it true that in response to mechanical stimuli, cells first fluidize their interior to permit remodeling? Do cells plated in 3D exhibit different anisotropy from cells plated in 2D? Does altering the adhesive ligand engaged by one layer of cells affect a second layer of cells plated on top of the first layer? This proposal seeks to answer these, and other, questions.
Broader Impact: Because of the breadth of this project, there is considerable opportunity for having broader impacts on many aspects of biomedical engineering education and outreach. Because knowledge of contemporary biomedical engineering techniques and stem cell biology are not yet widely available to educational programs and the public at large, part of this project is dedicated to outreach. This outreach program is based on expanding a remote lab program by creating freely available videos along the line of the "Minute Physics" videos, but with a focus on biomedical engineering topics and techniques, posted to YouTube and other high-traffic video sites. Additionally, a broader selection of undergraduate students will be provided the opportunities to engage in laboratory research. Finally, courses in tissue and molecular engineering and biomechanics will be enhanced by the topics explored in this proposal, with plans for supplementary material stemming from a textbook that is currently in press.
