Final Report Summary - CELL-MATRIX (Biophysics of the cell-matrix interface)
Multicellular organisms, such as humans, are composed of collections of cells that are organized at different levels. In tissues and organs, cells are embedded in a complex matrix (the extracellular matrix) that provides structural integrity as well as functionally important information about the environment that surrounds the cells (microenvironment). For example, it has been shown that the extracellular matrix has sufficient information to specify proper recellularization of tissues from which cell have been removed. That finding has opened up exciting new vistas in tissue engineering. Abnormalities in the extracellular matrix are also associated with a wide range of diseases, such as cancer, heart failure and emphysema. Thus there is a great deal of interest in how cells interact with the extracellular matrix, and a better understanding of cell-matrix interactions has the potential to improve medical devices and therapeutics for many conditions.
The problem of cell-matrix interactions cannot be addressed to any significant extent when working with animal models, because it is experimentally not possible to control the individual aspects of the matrix in vivo in ways needed. Thus much of the research on cell-matrix interactions is performed on isolated cells in culture, where the substrate on which cells grow can more readily be manipulated. In the past 20 years or so, microfabrication tools that were originally developed for the semiconductor industry have increasingly been employed to fabricated cell culture systems in which specific aspects of the environment can be precisely controlled. These engineered microenvironments are being used to study a number of aspects of how cells interact with their environment for the purpose of understanding cell-matrix interactions. For example, by etching features with defined dimensions onto the surface on which cells grow it has been shown that some cells will respond to topographic features on the order of 10 nanometers. By patterning cell culture substrates, it has also been shown that the spatial organization of specific proteins normally found in the extracellular matrix can influence cell shape and function. But as those types of experiments become more routine – the question of how cells respond to combinations of signals comes to the fore. That in turn raises the question of how to integrate the responses from multiple signals when studies of that type are analyzed. These are the broad question that underpins the present project.
In the long-term one will want to combine as many signals as possible. But the first step is clearly to combine pairs of signals, and we elected to start with biochemistry and topography. The basic idea was to use electron beam lithography to produce nanometer scale lines of the extracellular matrix protein fibronectin, and use focused ion beam milling to produce small topographic features (lines). Cells would be expected to respond to each of these signals individually by becoming oriented in the direction of the lines. Then by controlling the orientation of the topographic lines relative to the fibronectin lines, one would be able to establish how these two signals compete for controlling cell shape.
The first step here was to produce lines of fibronectin that were order 100 nanometers wide on cell culture compatible substrates using electron beam lithography, which was accomplished. Further, we showed that cells respond to these lines – albeit more weakly than hoped. The second step was to fabricate topographic lines on cell culture compatible substrates using focused ion beam milling, which was also accomplished. But here a problem emerged. It became clear that it would not be possible to make the biochemical patterns and the topographic patterns completely independent of each other. The topographic features would alter the binding properties of the substrate in a way that one could not prevent some binding of fibronectin to the topographic lines during the subsequent protein patterning. That is, the topographic features would probably always influence the biochemical features. Thus there would be coupling between the two, and it would not be possible to fully separate their contributions to cell shape control.
The coupling problem lead us to initiate an alternative approach to the second signal, diffusive gradients. In the tissues, small molecules such as oxygen, glucose and lactate are not uniformly distributed. Instead they are present as gradients relative the capillaries, and the structure of function of cells in the tissues depend on these gradients. Thus we pursued the aim of combining diffusive gradients with fibronectin organization. To capture the gradients we have developed a new type of cell culture chamber, restricted exchange environment chambers, in which diffusive gradients are captured by restricting diffusive access to the medium above the cells using a glass coverslip placed just above the cells with a small opening that connects to the bulk medium. Cells grown in these chambers were shown to grow in a radially dependent fashion, relative to the opening in the chamber. Further, some cell types, such as fibroblasts, aligned relative to the axis of diffusion. We also showed experimentally that there are oxygen gradients in these chambers.
The final step of the project, combining the two signals, has not yet been completed. However, the results from the research carried out provides important new tools for studying cells in engineered microenvironments and cell matrix interactions, and a further understanding of how cells behave in metabolic gradients. In particular, the restricted exchange environment chamber has potential applications to a wide range of biological and biomedical problems, and we believe will find widespread use in applications such as improved drug screening.
The problem of cell-matrix interactions cannot be addressed to any significant extent when working with animal models, because it is experimentally not possible to control the individual aspects of the matrix in vivo in ways needed. Thus much of the research on cell-matrix interactions is performed on isolated cells in culture, where the substrate on which cells grow can more readily be manipulated. In the past 20 years or so, microfabrication tools that were originally developed for the semiconductor industry have increasingly been employed to fabricated cell culture systems in which specific aspects of the environment can be precisely controlled. These engineered microenvironments are being used to study a number of aspects of how cells interact with their environment for the purpose of understanding cell-matrix interactions. For example, by etching features with defined dimensions onto the surface on which cells grow it has been shown that some cells will respond to topographic features on the order of 10 nanometers. By patterning cell culture substrates, it has also been shown that the spatial organization of specific proteins normally found in the extracellular matrix can influence cell shape and function. But as those types of experiments become more routine – the question of how cells respond to combinations of signals comes to the fore. That in turn raises the question of how to integrate the responses from multiple signals when studies of that type are analyzed. These are the broad question that underpins the present project.
In the long-term one will want to combine as many signals as possible. But the first step is clearly to combine pairs of signals, and we elected to start with biochemistry and topography. The basic idea was to use electron beam lithography to produce nanometer scale lines of the extracellular matrix protein fibronectin, and use focused ion beam milling to produce small topographic features (lines). Cells would be expected to respond to each of these signals individually by becoming oriented in the direction of the lines. Then by controlling the orientation of the topographic lines relative to the fibronectin lines, one would be able to establish how these two signals compete for controlling cell shape.
The first step here was to produce lines of fibronectin that were order 100 nanometers wide on cell culture compatible substrates using electron beam lithography, which was accomplished. Further, we showed that cells respond to these lines – albeit more weakly than hoped. The second step was to fabricate topographic lines on cell culture compatible substrates using focused ion beam milling, which was also accomplished. But here a problem emerged. It became clear that it would not be possible to make the biochemical patterns and the topographic patterns completely independent of each other. The topographic features would alter the binding properties of the substrate in a way that one could not prevent some binding of fibronectin to the topographic lines during the subsequent protein patterning. That is, the topographic features would probably always influence the biochemical features. Thus there would be coupling between the two, and it would not be possible to fully separate their contributions to cell shape control.
The coupling problem lead us to initiate an alternative approach to the second signal, diffusive gradients. In the tissues, small molecules such as oxygen, glucose and lactate are not uniformly distributed. Instead they are present as gradients relative the capillaries, and the structure of function of cells in the tissues depend on these gradients. Thus we pursued the aim of combining diffusive gradients with fibronectin organization. To capture the gradients we have developed a new type of cell culture chamber, restricted exchange environment chambers, in which diffusive gradients are captured by restricting diffusive access to the medium above the cells using a glass coverslip placed just above the cells with a small opening that connects to the bulk medium. Cells grown in these chambers were shown to grow in a radially dependent fashion, relative to the opening in the chamber. Further, some cell types, such as fibroblasts, aligned relative to the axis of diffusion. We also showed experimentally that there are oxygen gradients in these chambers.
The final step of the project, combining the two signals, has not yet been completed. However, the results from the research carried out provides important new tools for studying cells in engineered microenvironments and cell matrix interactions, and a further understanding of how cells behave in metabolic gradients. In particular, the restricted exchange environment chamber has potential applications to a wide range of biological and biomedical problems, and we believe will find widespread use in applications such as improved drug screening.