Cell culture maintenance and stretching:
A cell’s mechanical environment is becoming increasingly recognized as a central determinant of cellular fate. (Hoffman, 2006) Cell culture experiments have recently begun probing the effects of stretching on cells. Cells in the body experience mechanical forces which create complicated changes, both mechanical and biochemical. Mechanical changes can include shape, orientation and cytoskeletal properties. Changes in biochemistry and gene expression usually accompany these structural changes. External forces have been shown to alter cell migration (Lo, 2000; Mesuda, 1993), growth (Miller, 2006), stem cell differentiation (Engler, 2006; Engler, 2008; McBeath, 2004) and disease states. (Ingler, 2003; Hahn, 2008; Paszek, 2005) Recreating the in vivo mechanical forces acting on a cell has implications across most fields of cellular biology.
Cell cultures provide a vital tool for biological research. Isolating specific cells allows many properties to be isolated and studied in depth. The researcher can control many parameters of the cellular environment, affording the ability to determine the influences of specific variables in biological processes. The simplest cell culture experiments culture cells in a passive environment, simply involving a rigid substrate coated in an adhesive protein. These experiments can provide a wealth of information about the behaviors of various cells in the body.
Cell culture experiments often omit a vital factor in cellular development: force. Most cells in the body exist in a mechanically active environment, wherein they are subject to external forces. Physical signals can mitigate or even override chemical signals in influencing morphology, growth rates and transcription, and are consequently vital to include in laboratory cell cultures. (Janmey, 2004) In particular, cells in the lungs, heart and vascular system are exposed to a regular, repeating pattern of stretching forces over their lifespans. Cellular fate is significantly altered bythese mechanical components. Recent cell culture experiments have begun stretching cells during development, and have relevance in most fields of cellular biology.
Current applications of cell stretching:
Mechanical forces are significant in nearly every cell, and the results of mechanical loading vary extensively with cell type. Forces are most important in cells which experience a regular, repeated pattern of stress, as in the lungs, heart and blood vessels. Cell stretching has therefore been vital in experiments with fetal lung cells, skin tissue, and cardiomyocytes, among others. Cell stretching experiments may elucidate the complex biological responses to mechanical loading.
Our client’s research is focused on the the heart, where the myocardium and the valves undergo stress with every heartbeat. Understanding how the constitutive properties of these cells and how they respond to the cyclical strain is the primary research focus of our client.
Determining the stress-strain relationship of the mitral valve using a biaxial tissue stretcher elucidated the unique nonlinear stress-strain relationship for the mitrall valve and the differences between the anterior and posterior valve tissue. (May-Newman, 1995) Knowing these properties is essential for the design of prosthetic valve replacements.
Aortic endothelial cells produce distinct and precise responses to stretching and compression, each dependent on the magnitude of the deformation. (Wille, 2004) Strain differences as small as one percent induce statistically significant changes in cellular responses such as realignment. Rearrangement of actin fibers accompanies realignment through a mechanism which remains unclear. To clarify this relationship requires further biaxial testing.
Cell stretching is also relevant in lung cells, where each breath exposes cells to a stretch cycle. This cycle is essential for normal lung cell development, in particular alveolar type II cells. Pulmonary surfactant secretion is drastically altered by subjecting cells to a cyclic strain regimen. (Sanchez-Esteban 1998) The ability of these cells to maintaining fluid homeostasis in the alveolar lumen and injury repair is also modulated by strain. Developing therapies for deficiencies in these cells requires knowledge of the exact molecular and cellular mechanisms at play in the cellular responses to force. Biaxial testing is critical in examining the specific receptors and signaling proteins involved in fetal lung mechanotransduction. (Wang, 2012)
The mechanical properties of skin have been vastly explored, and are dependent on both force and temperature. Mechanical behavior also varies with the rate of loading. (Potts, 1983) Simultaneously varying temperature and mechanical stretching rates can probe mechanical behavior. (Zhou, 2009) Sinusoidal stretching in a thermally controlled environment enabled clarification of the microscopic structural causes of macroscopic changes. This has significant implications for treatment strategies.
Need for biaxial tests:
Cells under strain experience both mechanical and biochemical responses. Specific cellular structures experience significant load under stress, eliciting significant mechanical responses. Actin and myosin fibers form much of the cytoskeleton and are hypothesized to bear much of the force. (Sabass, 2008; Balland, 2006) The mechanical loading of these elements elicits diverse biochemical responses. The details of these responses are not yet understood, due both to the complicated nature of force propagation through a cell and to the complexity of the mechanisms themselves. Cellular responses to stretching are diverse and complex, and the specific pathways of rearrangement remain unclear.
The response of cells to mechanical forces is complicated. Applying a shear force to endothelial cells elicits expressional changes in over 100 genes of 12 classes. (Chen, 2001) Various signaling pathways are involved in force transduction, including MAP kinases, (Giger, 2002), small GTPases (Giannone, 2006), and tyrosine kinases/phosphatases. (Sawada, 2001) The mechanisms of response are complex and diverse, requiring a wide range of experiments to be thoroughly understood.
Biological tissue is complex and heterogeneous, and consequently its mechanical behavior exhibits significant anisotropy, viscoelasticity, and non-linearity. Anisotropy is a particularly important obstacle to analyzing results. One critical shortcoming of uniaxial testing is inadequate resolution of anisotropy. (Yin, 1986) The incompressibility assumption of biological tissue enables development of two-dimensional stress-strain relationships from planar biaxial tests, resulting in far more accurate constitutive models. Biaxial testing is vital to most accurately model cellular behavior.
A cell’s mechanical environment is becoming increasingly recognized as a central determinant of cellular fate. (Hoffman, 2006) Cell culture experiments have recently begun probing the effects of stretching on cells. Cells in the body experience mechanical forces which create complicated changes, both mechanical and biochemical. Mechanical changes can include shape, orientation and cytoskeletal properties. Changes in biochemistry and gene expression usually accompany these structural changes. External forces have been shown to alter cell migration (Lo, 2000; Mesuda, 1993), growth (Miller, 2006), stem cell differentiation (Engler, 2006; Engler, 2008; McBeath, 2004) and disease states. (Ingler, 2003; Hahn, 2008; Paszek, 2005) Recreating the in vivo mechanical forces acting on a cell has implications across most fields of cellular biology.
Cell cultures provide a vital tool for biological research. Isolating specific cells allows many properties to be isolated and studied in depth. The researcher can control many parameters of the cellular environment, affording the ability to determine the influences of specific variables in biological processes. The simplest cell culture experiments culture cells in a passive environment, simply involving a rigid substrate coated in an adhesive protein. These experiments can provide a wealth of information about the behaviors of various cells in the body.
Cell culture experiments often omit a vital factor in cellular development: force. Most cells in the body exist in a mechanically active environment, wherein they are subject to external forces. Physical signals can mitigate or even override chemical signals in influencing morphology, growth rates and transcription, and are consequently vital to include in laboratory cell cultures. (Janmey, 2004) In particular, cells in the lungs, heart and vascular system are exposed to a regular, repeating pattern of stretching forces over their lifespans. Cellular fate is significantly altered bythese mechanical components. Recent cell culture experiments have begun stretching cells during development, and have relevance in most fields of cellular biology.
Current applications of cell stretching:
Mechanical forces are significant in nearly every cell, and the results of mechanical loading vary extensively with cell type. Forces are most important in cells which experience a regular, repeated pattern of stress, as in the lungs, heart and blood vessels. Cell stretching has therefore been vital in experiments with fetal lung cells, skin tissue, and cardiomyocytes, among others. Cell stretching experiments may elucidate the complex biological responses to mechanical loading.
Our client’s research is focused on the the heart, where the myocardium and the valves undergo stress with every heartbeat. Understanding how the constitutive properties of these cells and how they respond to the cyclical strain is the primary research focus of our client.
Determining the stress-strain relationship of the mitral valve using a biaxial tissue stretcher elucidated the unique nonlinear stress-strain relationship for the mitrall valve and the differences between the anterior and posterior valve tissue. (May-Newman, 1995) Knowing these properties is essential for the design of prosthetic valve replacements.
Aortic endothelial cells produce distinct and precise responses to stretching and compression, each dependent on the magnitude of the deformation. (Wille, 2004) Strain differences as small as one percent induce statistically significant changes in cellular responses such as realignment. Rearrangement of actin fibers accompanies realignment through a mechanism which remains unclear. To clarify this relationship requires further biaxial testing.
Cell stretching is also relevant in lung cells, where each breath exposes cells to a stretch cycle. This cycle is essential for normal lung cell development, in particular alveolar type II cells. Pulmonary surfactant secretion is drastically altered by subjecting cells to a cyclic strain regimen. (Sanchez-Esteban 1998) The ability of these cells to maintaining fluid homeostasis in the alveolar lumen and injury repair is also modulated by strain. Developing therapies for deficiencies in these cells requires knowledge of the exact molecular and cellular mechanisms at play in the cellular responses to force. Biaxial testing is critical in examining the specific receptors and signaling proteins involved in fetal lung mechanotransduction. (Wang, 2012)
The mechanical properties of skin have been vastly explored, and are dependent on both force and temperature. Mechanical behavior also varies with the rate of loading. (Potts, 1983) Simultaneously varying temperature and mechanical stretching rates can probe mechanical behavior. (Zhou, 2009) Sinusoidal stretching in a thermally controlled environment enabled clarification of the microscopic structural causes of macroscopic changes. This has significant implications for treatment strategies.
Need for biaxial tests:
Cells under strain experience both mechanical and biochemical responses. Specific cellular structures experience significant load under stress, eliciting significant mechanical responses. Actin and myosin fibers form much of the cytoskeleton and are hypothesized to bear much of the force. (Sabass, 2008; Balland, 2006) The mechanical loading of these elements elicits diverse biochemical responses. The details of these responses are not yet understood, due both to the complicated nature of force propagation through a cell and to the complexity of the mechanisms themselves. Cellular responses to stretching are diverse and complex, and the specific pathways of rearrangement remain unclear.
The response of cells to mechanical forces is complicated. Applying a shear force to endothelial cells elicits expressional changes in over 100 genes of 12 classes. (Chen, 2001) Various signaling pathways are involved in force transduction, including MAP kinases, (Giger, 2002), small GTPases (Giannone, 2006), and tyrosine kinases/phosphatases. (Sawada, 2001) The mechanisms of response are complex and diverse, requiring a wide range of experiments to be thoroughly understood.
Biological tissue is complex and heterogeneous, and consequently its mechanical behavior exhibits significant anisotropy, viscoelasticity, and non-linearity. Anisotropy is a particularly important obstacle to analyzing results. One critical shortcoming of uniaxial testing is inadequate resolution of anisotropy. (Yin, 1986) The incompressibility assumption of biological tissue enables development of two-dimensional stress-strain relationships from planar biaxial tests, resulting in far more accurate constitutive models. Biaxial testing is vital to most accurately model cellular behavior.