Micropatterned cell cultures on elastic membranes as an in vitro model of myocardium

We describe here a new in vitro protocol for structuring cardiac cell cultures to mimic important aspects of the in vivo ventricular myocardial phenotype by controlling the location and mechanical environment of cultured cells. Microlithography is used to engineer microstructured silicon metal wafers. Those are used to fabricate either microgrooved silicone membranes or silicone molds for microfluidic application of extracellular matrix proteins onto elastic membranes (involving flow control at micrometer resolution). The physically or microfluidically structured membranes serve as a cell culture growth substrate that supports cell alignment and allows the application of stretch. The latter is achieved with a stretching device that can deliver isotropic or anisotropic stretch. Neonatal ventricular cardiomyocytes, grown on these micropatterned membranes, develop an in vivo-like morphology with regular sarcomeric patterns. The entire process from fabrication of the micropatterned silicon metal wafers to casting of silicone molds, microfluidic patterning and cell isolation and seeding takes approximately 7 days.     

Featured Article: Temporal responses of human endothelial and smooth muscle cells exposed to uniaxial cyclic tensile strain

The physiology of vascular cells depends on stimulating mechanical forces caused by pulsatile flow. Thus, mechano-transduction processes and responses of primary human endothelial cells (ECs) and smooth muscle cells (SMCs) have been studied to reveal cell-type specific differences which may contribute to vascular tissue integrity. Here, we investigate the dynamic reorientation response of ECs and SMCs cultured on elastic membranes over a range of stretch frequencies from 0.01 to 1 Hz. ECs and SMCs show different cell shape adaptation responses (reorientation) dependent on the frequency. ECs reveal a specific threshold frequency (0.01 Hz) below which no responses is detectable while the threshold frequency for SMCs could not be determined and is speculated to be above 1 Hz. Interestingly, the reorganization of the actin cytoskeleton and focal adhesions system, as well as changes in the focal adhesion area, can be observed for both cell types and is dependent on the frequency. RhoA and Rac1 activities are increased for ECs but not for SMCs upon application of a uniaxial cyclic tensile strain. Analysis of membrane protrusions revealed that the spatial protrusion activity of ECs and SMCs is independent of the application of a uniaxial cyclic tensile strain of 1 Hz while the total number of protrusions is increased for ECs only. Our study indicates differences in the reorientation response and the reaction times of the two cell types in dependence of the stretching frequency, with matching data for actin cytoskeleton, focal adhesion realignment, RhoA/Rac1 activities, and membrane protrusion activity. These are promising results which may allow cell-type specific activation of vascular cells by frequency-selective mechanical stretching. This specific activation of different vascular cell types might be helpful in improving strategies in regenerative medicine.     

A common mechanism for the mechanosensitive regulation of apoptosis in different cell types and for different mechanical stimuli

In all kinds of tissue cells are influenced by mechanical forces. In vivo fibroblasts are exposed to mechanical tension and endothelial cells are subjected directly to hemodynamic flow. It has been shown that disturbance of the mechanical stimulus leads to apoptosis by induction of an autocrine loop with thrombospondin-1 as ligand and an integrin/integrin associated protein (CD47) complex as receptor. In the present study the nature of the mechanical stimulus has been exchanged for these two cell types. If fibroblasts are subjected to laminar flow apoptosis decreases about 20-fold whereas turbulence leads to an significant increase compared with the static conditions. If endothelial cells grown on thin silicone membranes are exposed to permanent and pulsatile uniaxial strain, the cells are completely devoid of apoptosis. The thrombospondin-1 secretion as well as the expression of CD47 occurs exclusively under mechanical relaxation respectively turbulence. So different types of cells seem to share a common sense deciding whether a mechanical stimulus induces or suppresses apoptosis and use a common molecular machinery for the regulation of the process.     

Cyclical strain effects on production of vasoactive materials in cultured endothelial cells.

Mechanical forces due to fluid flow and cyclical strain can alter endothelial cell morphology and function, including the release of vasoactive materials endothelin, prostacyclin (PGI2), and tissue plasminogen activator (t-PA). In this study, effects of cyclical strain were modeled by culturing bovine aortic endothelial cells on fibronectin-coated elastic membranes of silicone rubber (Silastic) or poly-etherurethane urea (Mitrathane). After growing to confluence under static conditions of 37 degrees C in humidified air with 5% CO2, cells were strained cyclically at membrane elongations of 5% or 10% for 24 hours at 1 Hz. Controls were maintained under static conditions or were exposed to fluid motions similar to the strained cells but without stretching. Secretion rates were constant throughout experiments in the strain chamber with no initial burst in metabolism associated with the initiation of strain. Secretion rates were not altered by choice of elastic membrane. At a physiological level of 10% cyclical strain, prostacyclin and endothelian secretion rates were increased by 2.5-fold and 1.7-fold, respectively, above stationary controls. Endothelin production demonstrated a dose-dependent response with cyclical strain, while PGI2 appeared to require a threshold strain before an increase in secretion occurred. No significant differences in t-PA levels were seen in cyclically strained cells compared with controls. These results indicate that endothelial cells respond metabolically to cyclical strain and suggest that mechanical strain may modulate secretion of selective vasoactive materials by vascular endothelial cells.     

Dependence of alignment direction on magnitude of strain in esophageal smooth muscle cells

The response of cells in vitro to mechanical forces has been the subject of much research using devices to exert controlled mechanical stimulation on cultured cells or isolated tissue. In this study, esophageal smooth muscle cells were seeded on flexible polyurethane membranes to form a confluent cell layer. The cells were then subjected to uniform cyclic stretch of varying magnitudes at a frequency of approximately five cycles per minute in a custom made mechatronic bioreactor, providing similar strains experienced in the in vivo mechanical environment of the esophagus. The results show that the orientation response is dependent on the magnitude of cyclic stretch applied. Smooth muscle cells showed parallel alignment to the force direction at low cyclic strains (2%) compared to the hill‐valley morphology of static controls. At higher strains (5% and 10% magnitude), the cells exhibited a consistent alignment perpendicular to the strain. To our knowledge, this is the first time that the alignment direction's dependence on strain magnitude has been demonstrated. MTS analysis indicated that cell metabolism was reduced when mechanical strain was applied, and proliferation was inhibited by mechanical strain. Protein expression indicates a decrease in smooth muscle α‐actin, indicative of changes in cell phenotype, an increase in vimentin, which is associated with increased cell motility, and an increase in desmin, indicating differentiation in stimulated cells. Biotechnol. Bioeng. 2009;102: 1703–1711. © 2008 Wiley Periodicals, Inc.     

Effects of Equiaxial Strain on the Differentiation of Dental Pulp Stem Cells without Using Biochemical Reagents

During orthodontic treatments, applied mechanical forces create strain and result in tooth movement through the alveolar bone. This response to mechanical strain is a fundamental biological reaction. The present study evaluated the effect of equiaxial strain within the range of orthodontic forces on the osteogenic differentiation of human dental pulp stem cells (hDPSCs). Following isolation and culture of hDPSCs, 3rd passage cells were transferred on a silicone membrane covered with collagen. Cell adhesion to the membrane was evaluated under scanning electron microscope (SEM). Cells were divided into three groups: the first group was placed in a conventional culture medium, transferred to an equiaxial stretching device (3% strain for 2 weeks). The positive control was placed in an osteogenic medium with no mechanical strain. The negative control group was placed in the conventional culture medium with no mechanical strain either. Study groups were evaluated for expression of osteogenic markers (Alkaline phosphatase and Osteopontin) with immunofluorescence and real time PCR. SEM images revealed optimal adhesion of cells to the silicone membrane. Immunofluorescence study demonstrated that osteocalcin expression occurred after 2 weeks in the two groups under mechanical and chemical signals. After application of equiaxial strain, level of expression of osteogenic markers was significantly higher than in the negative and positive control groups. Based on the study results, static equiaxial strain which mimics the types of orthodontic forces can result in differentiation of hDPSCs to osteoblasts. The results obtained may be used in cell therapy and tissue engineering.     

Construction and Characterization of a Novel Vocal Fold Bioreactor

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To emulate the dynamic environment of vocal folds, a novel vocal fold bioreactor capable of producing vibratory stimulations at fundamental phonation frequencies is constructed and characterized. The device is composed of a function generator, a power amplifier, a speaker selector and parallel vibration chambers. Individual vibration chambers are created by sandwiching a custom-made silicone membrane between a pair of acrylic blocks. The silicone membrane not only serves as the bottom of the chamber but also provides a mechanism for securing the cell-laden scaffold. Vibration signals, generated by a speaker mounted underneath the bottom acrylic block, are transmitted to the membrane aerodynamically by the oscillating air. Eight identical vibration modules, fixed on two stationary metal bars, are housed in an anti-humidity chamber for long-term operation in a cell culture incubator. The vibration characteristics of the vocal fold bioreactor are analyzed non-destructively using a Laser Doppler Vibrometer (LDV). The utility of the dynamic culture device is demonstrated by culturing cellular constructs in the presence of 200-Hz sinusoidal vibrations with a mid-membrane displacement of 40 µm. Mesenchymal stem cells cultured in the bioreactor respond to the vibratory signals by altering the synthesis and degradation of vocal fold-relevant, extracellular matrix components. The novel bioreactor system presented herein offers an excellent in vitro platform for studying vibration-induced mechanotransduction and for the engineering of functional vocal fold tissues.     

Enhancing cell seeding of scaffolds in tissue engineering through manipulation of hydrodynamic parameters

The seeding of cells onto biocompatible scaffolds is a determinant step in the attainment of functional properties of engineered tissues. Efficient, fast and spatially uniform cell seeding can improve the clinical potential of engineered tissue templates. One way to approach these cell seeding requirements is through bioreactor design. In the present study, bovine chondrocytes were seeded (2.5, 5.0 or 10.0 million cells per scaffold) onto polyglycolic acid scaffolds within the hydrodynamic environments of wavy-walled and spinner flask bioreactors. Previous characterizations of the hydrodynamic environment in the vicinity of constructs cultivated in these bioreactors suggested decreased flow-induced shear stress as well as increased recirculation and magnitude of the axial fluid velocities in the wavy-walled bioreactor. Here we report more efficient and spatially uniform cell seeding in the wavy-walled bioreactor, and at intermediate initial cell densities (5 million cells per scaffold). This study constitutes an important step towards the achievement of functional tissue-engineered implants by (i) increasing our understanding of the influence of hydrodynamic parameters on the efficiency and spatial distribution of cell attachment to scaffolds and the production of extracellular matrix and (ii) introducing a comprehensive approach to the investigation of the effects of bioprocessing conditions on tissue morphology and composition.     

Native extracellular matrix orientation determines multipotent vascular stem cell proliferation in response to cyclic uniaxial tensile strain and simulated stent indentation

Cardiovascular disease is the leading cause of death worldwide, with multipotent vascular stem cells (MVSC) implicated in contributing to diseased vessels. MVSC are mechanosensitive cells which align perpendicular to cyclic uniaxial tensile strain. Within the blood vessel wall, collagen fibers constrain cells so that they are forced to align circumferentially, in the primary direction of tensile strain. In these experiments, MVSC were seeded onto the medial layer of decellularized porcine carotid arteries, then exposed to 10%, 1 Hz cyclic tensile strain for 10 days with the collagen fiber direction either parallel or perpendicular to the direction of strain. Cells aligned with the direction of the collagen fibers regardless of the orientation to strain. Cells aligned with the direction of strain showed an increased number of proliferative Ki67 positive cells, while those strained perpendicular to the direction of cell alignment showed no change in cell proliferation. A bioreactor system was designed to simulate the indentation of a single, wire stent strut. After 10 days of cyclic loading to 10% strain, MVSC showed regions of densely packed, highly proliferative cells. Therefore, MVSC may play a significant role in in-stent restenosis, and this proliferative response could potentially be controlled by controlling MVSC orientation relative to applied strain.     

Gas transfer characteristics of a novel membrane bioreactor

We show the design features of a membrane bioreactor based on pulsatile flow across dimpled membranes. Results show an enhanced mass transfer of air of at least five-fold magnitude as compared with flat membranes. An increased working volume form 20 mL to 120 mL reduced the k(L)A at a given Reynolds number because of axial mixing of fluid from the deoxygenated end chamber. The bioreactor was used to supply air to a hybridoma mammalian cell line, and the calculated oxygen uptake showed that high-density cultures could be maintained in a 20mL, single-dimpled cultures could be maintained in a 20 mL, single-dimpled membrane system. Indirect aeration of a 2 L continuous stirred tank reactor, by a double-membrane system, showed that air could be supplied to mammalian cells at cell densities of approximately 4 x 10(6) /mL.     

Dynamic cell stretching increases human osteoblast proliferation and CICP synthesis but decreases osteocalcin synthesis and alkaline phosphatase activity

The cell activity of human-bone-derived cell cultures was studied after mechanical stimulation by cyclic strain at a magnitude occurring in physiologically loaded bone tissue. Monolayers of subconfluently grown human-bone-derived cells were stretched in rectangular silicone dishes with cyclic predominantly uniaxial movement along their longitudinal axes. Strain was applied over two days for 30 min per day with a frequency of 1 Hz and a strain magnitude of 1000 microstrain. Cyclic stretching of the cells resulted in an increased proliferation (10-48%) and carboxyterminal collagen type I propeptide release (7-49%) of human-cancellous bone-derived osteoblasts while alkaline phosphatase activity and osteocalcin release were significantly reduced by 9-25 and 5-32%, respectively. These results demonstrate that cyclic strain at physiologic magnitude leads to an increase of osteoblast activities related to matrix production while those activities which are characteristic for the differentiated osteoblast and relevant for matrix mineralization are decreased.     

Design and characterization of a new bioreactor for continuous ultra-slow uniaxial distraction of a three-dimensional scaffold-free stem cell culture

A computer controlled dynamic bioreactor for continuous ultra-slow uniaxial distraction of a scaffold-free three-dimensional (3D) mesenchymal stem cell pellet culture was designed to investigate the influence of stepless tensile strain on behavior of distinct primary cells like osteoblasts, chondroblasts, or stem cells without the influence of an artificial culture matrix. The main advantages of this device include the following capabilities: (1) Application of uniaxial ultra-slow stepless distraction within a range of 0.5-250 μm/h and real-time control of the distraction distance with high accuracy (mean error -3.4%); (2) tension strain can be applied on a 3D cell culture within a standard CO(2) -incubator without use of an artificial culture matrix; (3) possibility of histological investigation without loss of distraction; (4) feasibility of molecular analysis on RNA and protein level. This is the first report on a distraction device capable of applying continuous tensile strain to a scaffold-free 3D cell culture within physiological ranges of motion comparable to distraction ostegenesis in vivo. We expect the newly designed microdistraction device to increase our understanding on the regulatory mechanisms of mechanical strains on the metabolism of stem cells.     

A novel culture morphology resulting from applied mechanical strain

Summary To demonstrate that cells both perceive and respond to external force, a strain/relaxation regimen was applied to normal human fetal and aged dermal fibroblasts cultured as monolayers on flexible membranes. The precisely controlled protocol of stretch (20% elongation of the culture membrane) at 6.67 cycles/min caused a progressive change in the monolayers, such that the original randomly distributed pattern of cells became a symmetric, radial distribution as the cell bodies aligned parallel to the applied force. High cell density interfered with the success of re-alignment in the fetal cell cultures observed, which may reflect a preference in this cell strain for cell-cell over cell-matrix contacts. The chronologically aged cells observed did not demonstrate this feature, aligning efficiently at all seeding densities examined. The role of microfilaments in force perception and transmission was investigated through the addition of cytochalasin D in graded doses. Both intercellular interactions and cytoskeletal integrity mediate the morphological response to mechanical strain.     

Development of a cell culture system loading cyclic mechanical strain to chondrogenic cells

Mechanical stimulation is considered to be one of the major epigenetic factors regulating the metabolism, proliferation, survival and differentiation of cells in the skeletal tissues. It is generally accepted that the cytoskeleton can undergo remodeling in response to mechanical stimuli such as tensile strain or fluid flow. Mechanically induced cell deformation is one of the possible mechanotransduction pathways by which chondrocytes sense and respond to changes in their mechanical environment. Mechanical strain has a variety of effects on the structure and function of their cells in the skeletal tissues, such as chondrocytes, osteoblasts and fibroblasts. However, little is known about the effect of the quality and quantity of mechanical strain and the timing of mechanical loading on the differentiation of these cells. The present study was designed to investigate the effect of the deformation of chondrogenic cells, and cyclic compression using a newly developed culture device, by analyzing mechanobiological response to the differentiating chondrocytes. Cyclic compression between 0 and 22% strains, at 23 microHz was loaded on chondrogenic cell line ATDC5 by seeding in a mass mode on PDMS membrane, assuming direct transfer of cyclic deformation from the membrane to the cells at the same frequency. The compressive strain, induced within the membrane, was characterized based on the analysis of the finite element modeling (FEM). The results showed that the tensile strain inhibits the chondrogenic differentiation of ATDC5 cells, whereas the compressive strain enhances the chondrogenic differentiation, suggesting that the differentiation of the chondrogenic cells could be controlled by the amount and the mode of strain. In conclusion, we have developed a unique strain loading culture system to analyze the effect of various types of mechanical stimulation on various cellular activities.     

An analysis of the complete strain field within Flexercell membranes

The purpose of the current investigation was to use finite element analysis to quantify the complete strain field for the membranes of the Flexercell^TM apparatus, a device extensively used to study the effects of mechanical loading on cultured cells. Four vacuum pressure simulations were run for the membrane for both the uniaxial and biaxial loading post, yielding the distribution of longitudinal (E_xx) and transverse (E_yy) strain for the uniaxial post, and the radial (E_rr) and the circumferential (E_θθ) strain for the biaxial post. The discrete values of each strain were evaluated at the center of the loading post as well as the region off of the post. Experimental measurements were made for both types of loading posts in order to validate our simulations. The biaxial post simulation was found to provide a central circular region of equal and constant E_rr and E_θθ in the membrane on the post. Likewise, the uniaxial post simulation provided a definitive region of constant E_xx for a central rectangular region on the post. For the uniaxial simulation, the region on the post resulted in small compressive E_yy, while the region off the post resulted in tensile E_yy. The biaxial simulation resulted in large tensile E_rr and E_θθ on the post, while the region off the post resulted in large E_rr and smaller E_θθ. Our simulations were reasonably consistent with the experimental measurements made for both types of loading posts. We believe that the results of this study will allow scientists to more accurately describe the response of cells to known strains on all portions of the membrane, thus increasing the range of known strain regions for investigation in the Flexercell^TM apparatus.     

Primary cilium mechanotransduction of tensile strain in 3D culture: Finite element analyses of strain amplification caused by tensile strain applied to a primary cilium embedded in a collagen matrix

Human adipose-derived stem cells (hASC) exhibit multilineage differentiation potential with lineage specification that is dictated by both the chemical and mechanical stimuli to which they are exposed. We have previously shown that 10% cyclic tensile strain increases hASC osteogenesis and cell-mediated calcium accretion. We have also recently shown that primary cilia are present on hASC and that chemically-induced lineage specification of hASC concurrently results in length and conformation changes of the primary cilia. Further, we have observed cilia length changes in hASC cultured within a collagen I gel in response to 10% cyclic tensile strain. We therefore hypothesize that primary cilia may play a key mechanotransduction role for hASC exposed to tensile strain. The goal of this study was to use finite element analysis (FEA) to determine strains occurring within the ciliary membrane in response to 10% tensile strain applied parallel, or perpendicular, to cilia orientation. To elucidate the mechanical environment experienced by the cilium, several lengths were modeled and evaluated based on cilia lengths measured on hASC grown under varied culture conditions. Principal tensile strains in both hASC and ciliary membranes were calculated using FEA, and the magnitude and location of maximum principal tensile strain determined. We found that maximum principal tensile strain was concentrated at the base of the cilium. In the linear elastic model, applying strain perpendicular to the cilium resulted in maximum strains within the ciliary membrane from 150% to 200%, while applying strain parallel to the cilium resulted in much higher strains, approximately 400%. In the hyperelastic model, applying strain perpendicular to the cilium resulted in maximum strains within the ciliary membrane around 30%, while applying strain parallel to the cilium resulted in much higher strains ranging from 50% to 70%. Interestingly, FEA results indicated that primary cilium length was not directly related to ciliary membrane strain. Rather, it appears that cilium orientation may be more important than cilium length in determining sensitivity of hASC to tensile strain. This is the first study to model the effects of tensile strain on the primary cilium and provides newfound insight into the potential role of the primary cilium as a mechanosensor, particularly in tensile strain and potentially a multitude of other mechanical stimuli beyond fluid shear.     

In Vitro Experimental Study for the Determination of Cellular Axial Strain Threshold and Preferential Axial Strain from Cell Orientation Behavior in a Non-uniform Deformation Field

Cells within connective tissues are routinely subjected to a wide range of non-uniform mechanical loads that regulate many cell behaviors. In the present study, the relationship between cell orientation angle and strain value of the membrane was comprehensively investigated using an inhomogeneous strain field. Additionally, the cellular axial strain threshold, which corresponds to the launching of cell reorientation response, was elucidated. Human bone marrow mesenchymal stem cells were used for these experiments. In this study, an inhomogeneous strain distribution was easily created by removing one side holes of an elastic chamber in a commonly used uniaxial stretching device. The strains of 2D stretched membranes were quantified on a position-by-position basis using the digital image correlation method. The normal strain in the direction of stretch was changed continuously from 2.0 to 15.0 %. A 3D histogram of the cell frequency, which was correlated with the cell orientation angle and normal strain of the membrane, made it possible to determine the axial strain threshold accurately. The value of the axial strain threshold was 4.4 ± 0.3 %, which was reasonable compared with previous studies based on cyclic uniaxial stretch stimulation (homogeneous strain field). Additionally, preferential axial strain of cells, which was a cell property firstly introduced, was also achieved and the value was ?2.0 ± 0.1 %. This study is novel in three respects: (i) it precisely and easily determined the axial strain threshold of cells; (ii) it is the first to suggest preferential axial strain of cells; and (iii) it methodically investigated cell behavior in an inhomogeneous strain field.     

A system to impose prescribed homogenous strains on cultured cells.

There is presently significant interest in cellular responses to physical forces, and numerous devices have been developed to apply stretch to cultured cells. Many of the early devices were limited by the heterogeneity of deformation of cells in different locations and by the high degree of anisotropy at a particular location. We have therefore developed a system to impose cyclic, large-strain, homogeneous stretch on a multiwell surface-treated silicone elastomer substrate plated with pulmonary epithelial cells. The pneumatically driven mechanism consists of four plates each with a clamp to fix one edge of the cruciform elastomer substrate. Four linear bearings set at predetermined angles between the plates ensure a constant ratio of principal strains throughout the stretch cycle. We present the design of the device and membrane shape, the surface modifications of the membrane to promote cell adhesion, predicted and experimental measurements of the strain field, and new data using cultured airway epithelial cells. We present for the first time the relationship between the magnitude of cyclic mechanical strain and the extent of wound closure and cell spreading.     

Effect of a mechanical stimulation bioreactor on tissue engineered, scaffold-free cartilage

Achieving sufficient functional properties prior to implantation remains a significant challenge for the development of tissue engineered cartilage. Many studies have shown chondrocytes respond well to various mechanical stimuli, resulting in the development of bioreactors capable of transmitting forces to articular cartilage in vitro. In this study, we describe the production of sizeable, tissue engineered cartilage using a novel scaffold-free approach, and determine the effect of perfusion and mechanical stimulation from a C9-x Cartigen bioreactor on the properties of the tissue engineered cartilage. We created sizable tissue engineered cartilage from porcine chondrocytes using a scaffold-free approach by centrifuging a high-density chondrocyte cell-suspension onto an agarose layer in a 50 mL tube. The gross and histological appearances, biochemical content, and mechanical properties of constructs cultured in the bioreactor for 4 weeks were compared to constructs cultured statically. Mechanical properties were determined from unconfined uniaxial compression tests. Constructs cultured in the bioreactor exhibited an increase in total GAG content, equilibrium compressive modulus, and dynamic modulus versus static constructs. Our study demonstrates the C9-x CartiGen bioreactor is able to enhance the biomechanical and biochemical properties of scaffold-free tissue engineered cartilage; however, no additional enhancement was seen between loaded and perfused groups.     

Microtubule dynamics regulate cyclic stretch-induced cell alignment in human airway smooth muscle cells

Microtubules are structural components of the cytoskeleton that determine cell shape, polarity, and motility in cooperation with the actin filaments. In order to determine the role of microtubules in cell alignment, human airway smooth muscle cells were exposed to cyclic uniaxial stretch. Human airway smooth muscle cells, cultured on type I collagen-coated elastic silicone membranes, were stretched uniaxially (20% in strain, 30 cycles/min) for 2 h. The population of airway smooth muscle cells which were originally oriented randomly aligned near perpendicular to the stretch axis in a time-dependent manner. However, when the cells treated with microtubule disruptors, nocodazole and colchicine, were subjected to the same cyclic uniaxial stretch, the cells failed to align. Lack of alignment was also observed for airway smooth muscle cells treated with a microtubule stabilizer, paclitaxel. To understand the intracellular mechanisms involved, we developed a computational model in which microtubule polymerization and attachment to focal adhesions were regulated by the preexisting tensile stress, pre-stress, on actin stress fibers. We demonstrate that microtubules play a central role in cell re-orientation when cells experience cyclic uniaxial stretching. Our findings further suggest that cell alignment and cytoskeletal reorganization in response to cyclic stretch results from the ability of the microtubule-stress fiber assembly to maintain a homeostatic strain on the stress fiber at focal adhesions. The mechanism of stretch-induced alignment we uncovered is likely involved in various airway functions as well as in the pathophysiology of airway remodeling in asthma.