Development of a Substrate with Photo-Modulatable Rigidity for Probing Spatial and Temporal Responses of Cells to Mechanical Signals: A Dissertation

Topographical and mechanical properties of adhesive substrates provide important biological cues that affect cell spreading, migration, growth, and differentiation. The phenomenon has led to the increased use of topographically patterned and flexible substrates in studying cultured cells. However, these studies may be complicated by various limitations. For example, the effects of ligand distribution and porosity are affected by topographical features of 3D biological constructs. Similarly, many studies of mechanical cues are compounded with cellular deformation from external forces, or limited by comparative studies of separate cells on different substrates. Furthermore, understanding cell responses to mechanical input is dependent upon reliable measurements of mechanical properties. This work addresses each of these issues. To determine how substrate topography and focal adhesion kinase (FAK) affect cell shape and movement, I studied FAK-null (FAK -/-) and wild type mouse 3T3 fibroblasts on chemically identical polystyrene substrates with either flat surfaces or micron-sized pillars, I found that, compared to cells on flat surfaces, those on pillar substrates showed a more branched shape, an increased linear speed, and a decreased directional stability, which were dependent on both myosin-II and FAK. To study the dynamic responses to changes in substrate stiffness without other confounding effects, I developed a UV-modulatable substrate that softens upon UV irradiation. As atomic force microscopy (AFM) proved inadequate to detect microscale changes in stiffness, I first developed and validated a microsphere indentation method that is compatible with fluorescence microscopy. The results obtained with this method were comparable to those obtained with AFM. The UV-modulatable substrates softened by ~20-30% with an intensity of irradiation that has no detectable effect on 3T3 cells on control surfaces. Cells responded to global softening of the substrate with an initial retraction followed by a gradual reduction in spread area. Precise spatial control of softening is also possible - while there was little response to posterior softening, anterior softening elicited a pronounced retraction and either a reversal of cell polarity or a significant decrease in spread area if the cells move into the softened region. In conclusion, these techniques provide advances in gaining mechanistic insight into cellular responses to topographical and mechanical cues. Additionally, there are various other potential applications of the novel UV-softening substrate, particularly in regenerative medicine and tissue engineering

Development of a substrate with photo-modulatable rigidity for probing spatial and temporal responses of cells to mechanical signals

Topographical and mechanical properties of adhesive substrates provide important biological cues that affect cell spreading, migration, growth, and differentiation. The phenomenon has led to the increased use of topographically patterned and flexible substrates in studying cultured cells. However, these studies may be complicated by various limitations. For example, the effects of ligand distribution and porosity are affected by topographical features of 3D biological constructs. Similarly, many studies of mechanical cues are compounded with cellular deformation from external forces, or limited by comparative studies of separate cells on different substrates. Furthermore, understanding cell responses to mechanical input is dependent upon reliable measurements of mechanical properties. This work addresses each of these issues. To determine how substrate topography and focal adhesion kinase (FAK) affect cell shape and movement, I studied FAK-null (FAK -/-) and wild type mouse 3T3 fibroblasts on chemically identical polystyrene substrates with either flat surfaces or micron-sized pillars, I found that, compared to cells on flat surfaces, those on pillar substrates showed a more branched shape, an increased linear speed, and a decreased directional stability, which were dependent on both myosin-II and FAK. To study the dynamic responses to changes in substrate stiffness without other confounding effects, I developed a UV-modulatable substrate that softens upon UV irradiation. As atomic force microscopy (AFM) proved inadequate to detect microscale changes in stiffness, I first developed and validated a microsphere indentation method that is compatible with fluorescence microscopy. The results obtained with this method were comparable to those obtained with AFM. The UV-modulatable substrates softened by ~20-30% with an intensity of irradiation that has no detectable effect on 3T3 cells on control surfaces. Cells responded to global softening of the substrate with an initial retraction followed by a gradual reduction in spread area. Precise spatial control of softening is also possible - while there was little response to posterior softening, anterior softening elicited a pronounced retraction and either a reversal of cell polarity or a significant decrease in spread area if the cells move into the softened region. In conclusion, these techniques provide advances in gaining mechanistic insight into cellular responses to topographical and mechanical cues. Additionally, there are various other potential applications of the novel UV-softening substrate, particularly in regenerative medicine and tissue engineering

Micro- and nanoengineering approaches to control stem cell-biomaterial interactions.

As our population ages, there is a greater need for a suitable supply of engineered tissues to address a range of debilitating ailments. Stem cell based therapies are envisioned to meet this emerging need. Despite significant progress in controlling stem cell differentiation, it is still difficult to engineer human tissue constructs for transplantation. Recent advances in micro- and nanofabrication techniques have enabled the design of more biomimetic biomaterials that may be used to direct the fate of stem cells. These biomaterials could have a significant impact on the next generation of stem cell based therapies. Here, we highlight the recent progress made by micro- and nanoengineering techniques in the biomaterials field in the context of directing stem cell differentiation. Particular attention is given to the effect of surface topography, chemistry, mechanics and micro- and nanopatterns on the differentiation of embryonic, mesenchymal and neural stem cells

Probing Cellular Response to Heterogeneous Rigidity at the Micro- and Nanoscale

Physical factors in the environment of a cell regulate cell function and behavior and are involved in the formation and maintenance of tissue. There is strong evidence that substrate rigidity plays a key role in determining cell response in culture. Previous studies have demonstrated the importance of rigidity in numerous cellular processes including migration and adhesion and stem cell differentiation. Immune cells have been shown to respond differently to surfaces having different rigidities. Atypical response to rigidity is also a characteristic of cancerous cells. Understanding the mechanisms that support cellular rigidity sensing can lead to new tissue engineering strategies and potential new therapies based on rigidity modulation. A new technique was developed for the creation of biomimetic surfaces comprising regions of heterogeneous rigidity on the micro- and nanoscale. The surfaces are formed by exposing an elastomeric film of polydimethylsiloxane (PDMS) to a focused electron beam to form patterned regions of micro- and nanoscale spots. This thesis involves the formation of theses surfaces, characterization of their physical and chemical properties as a consequence of the electron beam exposure and investigation of how cells behave when plated on these surfaces. Cellular response to different patterns of heterogeneous rigidity is performed for several cell types. Human mesenchymal stem cells plated upon electron beam-exposed PDMS in a pattern of spots with diameters ranging from 2 µm to 100 nm display differential focal adhesion co-localization to the exposed features, depending on both rigidity and feature size. This behavior persists as the area of the exposed regions is reduced below ~1 µm. On spots with diameters of ~ 250 nm and smaller, focal adhesion co-localization is lost. This supports the notion that there is a length scale for cellular rigidity sensing, with the critical length in the range of a few hundred nanometers. When the heterogeneous rigidity surfaces are applied to CD4+ T cells, accumulations of proteins including TCR and pCasL on the exposed features are observed as a function of feature size. The pCasL appeared to significantly accumulate on 2 µm spots; For spots ~ 1 µm and below, cells appeared unable to identify the rigid regions. Further, Ca2+ release, a functional indicator of immunoresponse, is significantly enhanced on mixed-rigidity patterned PDMS relative to both soft and hard PDMS. Possible signaling pathways of TCR activation have been verified on e-beam exposed PDMS substrates with heterogeneous rigidity. These results are suggestive of possible new approaches to adoptive immunotherapy based on rigidity modulation. Studies on breast cancer cells indicate that on patterned substrates, sub-cellular processes are also significantly modulated. Integrin recruitment is enhanced on the rigid regions. Understanding the role of geometry in cellular rigidity response will point the way toward revealing its functional response and will shed light on the mechanistic underpinnings of this process

Designing stem cell niches for differentiation and self-renewal

Mesenchymal stem cells, characterized by their ability to differentiate into skeletal tissues and self-renew, hold great promise for both regenerative medicine and novel therapeutic discovery. However, their regenerative capacity is retained only when in contact with their specialized microenvironment, termed the stem cell niche. Niches provide structural and functional cues that are both biochemical and biophysical, stem cells integrate this complex array of signals with intrinsic regulatory networks to meet physiological demands. Although, some of these regulatory mechanisms remain poorly understood or difficult to harness with traditional culture systems. Biomaterial strategies are being developed that aim to recapitulate stem cell niches, by engineering microenvironments with physiological-like niche properties that aim to elucidate stem cell-regulatory mechanisms, and to harness their regenerative capacity in vitro. In the future, engineered niches will prove important tools for both regenerative medicine and therapeutic discoveries

Probing Cellular Response to Heterogeneous Rigidity at the Micro- and Nanoscale

Physical factors in the environment of a cell regulate cell function and behavior and are involved in the formation and maintenance of tissue. There is strong evidence that substrate rigidity plays a key role in determining cell response in culture. Previous studies have demonstrated the importance of rigidity in numerous cellular processes including migration and adhesion and stem cell differentiation. Immune cells have been shown to respond differently to surfaces having different rigidities. Atypical response to rigidity is also a characteristic of cancerous cells. Understanding the mechanisms that support cellular rigidity sensing can lead to new tissue engineering strategies and potential new therapies based on rigidity modulation. A new technique was developed for the creation of biomimetic surfaces comprising regions of heterogeneous rigidity on the micro- and nanoscale. The surfaces are formed by exposing an elastomeric film of polydimethylsiloxane (PDMS) to a focused electron beam to form patterned regions of micro- and nanoscale spots. This thesis involves the formation of theses surfaces, characterization of their physical and chemical properties as a consequence of the electron beam exposure and investigation of how cells behave when plated on these surfaces. Cellular response to different patterns of heterogeneous rigidity is performed for several cell types. Human mesenchymal stem cells plated upon electron beam-exposed PDMS in a pattern of spots with diameters ranging from 2 µm to 100 nm display differential focal adhesion co-localization to the exposed features, depending on both rigidity and feature size. This behavior persists as the area of the exposed regions is reduced below ~1 µm. On spots with diameters of ~ 250 nm and smaller, focal adhesion co-localization is lost. This supports the notion that there is a length scale for cellular rigidity sensing, with the critical length in the range of a few hundred nanometers. When the heterogeneous rigidity surfaces are applied to CD4+ T cells, accumulations of proteins including TCR and pCasL on the exposed features are observed as a function of feature size. The pCasL appeared to significantly accumulate on 2 µm spots; For spots ~ 1 µm and below, cells appeared unable to identify the rigid regions. Further, Ca2+ release, a functional indicator of immunoresponse, is significantly enhanced on mixed-rigidity patterned PDMS relative to both soft and hard PDMS. Possible signaling pathways of TCR activation have been verified on e-beam exposed PDMS substrates with heterogeneous rigidity. These results are suggestive of possible new approaches to adoptive immunotherapy based on rigidity modulation. Studies on breast cancer cells indicate that on patterned substrates, sub-cellular processes are also significantly modulated. Integrin recruitment is enhanced on the rigid regions. Understanding the role of geometry in cellular rigidity response will point the way toward revealing its functional response and will shed light on the mechanistic underpinnings of this process

Tumor cell migration in complex microenvironments

Tumor cell migration is essential for invasion and dissemination from primary solid tumors and for the establishment of lethal secondary metastases at distant organs. In vivo and in vitro models enabled identification of different factors in the tumor microenvironment that regulate tumor progression and metastasis. However, the mechanisms by which tumor cells integrate these chemical and mechanical signals from multiple sources to navigate the complex microenvironment remain poorly understood. In this review, we discuss the factors that influence tumor cell migration with a focus on the migration of transformed carcinoma cells. We provide an overview of the experimental and computational methods that allow the investigation of tumor cell migration, and we highlight the benefits and shortcomings of the various assays. We emphasize that the chemical and mechanical stimulus paradigms are not independent and that crosstalk between them motivates the development of new assays capable of applying multiple, simultaneous stimuli and imaging the cellular migratory response in real-time. These next-generation assays will more closely mimic the in vivo microenvironment to provide new insights into tumor progression, inform techniques to control tumor cell migration, and render cancer more treatable.National Science Foundation (U.S.) (Graduate Research Fellowship)Charles Stark Draper Laboratory (Research and Development Program (N.DL-H-550151))National Cancer Institute (U.S.) (R21CA140096

MODELLING THE INFLUENCE OF NUCLEUS ELASTICITY ON CELL INVASION IN FIBER NETWORKS AND MICROCHANNELS

Cell migration in highly constrained extracellular matrices is exploited in scaffold-based tissue engineering and is fundamental in a wide variety of physiological and pathological phenomena, among others in cancer invasion and development. Research into the critical processes involved in cell migration has mainly focused on cell adhesion and proteolytic degradation of the external environment. However, rising evidence has recently shown that a number of cell-derived biophysical and mechanical parameters, among others nucleus stiffness and cell deformability, plays a major role in cell motility, especially in the ameboid-like migration mode in 3D confined tissue structures. We here present an extended cellular Potts model (CPM) first used to simulate a micro-fabricated migration chip, which tests the active invasive behavior of cancer cells into narrow channels. As distinct features of our approach, cells are modeled as compartmentalized discrete objects, differentiated in the nucleus and in the cytosolic region, while the migration chamber is composed of channels of different widths. We find that cell motile phenotype and velocity in open spaces (i.e., 2D flat surfaces or large channels) are not significantly influenced by cell elastic properties. On the contrary, the migratory behavior of cells within subcellular and subnuclear structures strongly relies on the deformability of the cytosol and of the nuclear cluster, respectively. Further, we characterize two migration dynamics: a stepwise way, characterized by fluctuations in cell length, within channels smaller than nucleus dimensions and a smooth sliding (i.e., maintaining constant cell length) behavior within channels larger than the nuclear cluster. These resulting observations are then extended looking at cell migration in an artificial fiber network, which mimics cell invasion in a 3D extracellular matrix. In particular, in this case, we analyze the effect of variations in elasticity of the nucleus on cell movement. In order to summarize, with our simulated migration assays, we demonstrate that the dimensionality of the environment strongly affects the migration phenotype and we suggest that the cytoskeletal and nuclear elastic characteristics correlate with the tumor cell's invasive potentia