Design and Construction of an Equibiaxial Cell Stretching System That Is Improved for Biochemical Analysis

Abstract

We describe the design and validation of an equibiaxial stretching device in which cells are confined to regions of homogeneous strain. Using this device, we seek to overcome a significant limitation of existing equibiaxial stretching devices, in which strains are not homogeneous over the entire region of cell culture. We cast PDMS in a mold to produce a membrane with a cylindrical wall incorporated in the center, which was used to confine cell monolayers to the central membrane region subjected to homogeneous equibiaxial strain. We demonstrated that the presence of the wall to hold the culture medium did not affect strain homogeneity over the majority of the culture surface and also showed that cells adhered well onto the PDMS membranes. We used our device in cyclic strain experiments and demonstrated strain-dependent changes in extracellular signal-regulated kinase (ERK) and tyrosine phosphorylation upon cell stretching. Furthermore, we examined cell responses to very small magnitudes of strain ranging from 1% to 6% and were able to observe a graduated increase in ERK phosphorylation in response to these strains. Collectively, we were able to study cellular biochemical response with a high degree of accuracy and sensitivity to fine changes in substrate strain. Because we have designed our device along the lines of existing equibiaxial stretching technologies, we believe that our innovations can be incorporated into existing systems. This device would provide a useful addition to the set of tools applied for in vitro studies of cell mechanobiology.

MEKK1-dependent phosphorylation of calponin-3 tunes cell contractility

“…These results suggest that CNN3 phosphorylation at Thr288 is attenuated under the conditions where development of actomyosin-based cytoskeletal tension is hampered. By contrast, sustained equibiaxial stretching (3%, 5 min) of substrates to which cells adhered (Ursekar et al, 2014) caused an increase in CNN3 phosphorylation (Fig. 5F).…”

“…Cells were equibiaxially stretched using the device reported elsewhere (Ursekar et al, 2014). In brief, cells were grown overnight on polydimethylsiloxane (PDMS) stretch chambers coated with 10 µg/ml collagen type I.…”

Device-Based In Vitro Techniques for Mechanical Stimulation of Vascular Cells: A Review

“…With any stretch modality (uniaxial, equiaxial, or biaxial), validation of strain is important to determine whether cells in different regions are receiving a consistent stimulus. This may be accomplished by video tracking of visible markers or fluorescent dots on the substrate during stretch, sometimes in conjunction with a displacement transducer [91][92][93][94][95][96][97][98][99]. Tension on the membrane can be measured using a load cell [96].…”

“…To produce equiaxial strains, a circular clamped membrane is stretched by use of an indenter [90,93,114,[116][117][118] or vacuum [91,119]. Systems may be modified to provide additional device features, such as live-cell imaging, protein analysis, and higher throughput [98,[114][115][116][117]119]. Also of note are commercially available systems that use a vacuum to stretch the membrane (Flexcell International, Hillsborough, NC), which can be used for equiaxial strain studies [120][121][122].…”

“…A major difficulty in strain studies lies in producing a homogeneous strain field. This is easier to achieve in a biaxial or equiaxial strain system compared to uniaxial systems [98,118]. However, this is only true for the central region of the membrane; the outer region may experience a different strain field.…”